Podocytes are motile and migrate in the presence of injury.

Summary

Glomerular basement membrane (GBM) and podocalyxin are essential for podocyte morphology. We provide evidence of functional interconnections between basement membrane components (collagen IV and laminin), the expression of podocalyxin and the morphology of human glomerular epithelial cells (podocytes). We demonstrated that GBM and laminin, but not collagen IV, up-regulated the expression of podocalyxin. Scanning electron microscopy revealed that laminin induced a modified morphology of podocytes with process formation, which was more extensive in the presence of GBM. Under high magnification, podocytes appeared ruffled. Using transmission electron microscopy we observed that raised areas occurred in the basal cell surface. Furthermore, the presence of anti-podocalyxin antibody increased the extent of adhesion and spreading of podocytes to both collagen IV and laminin, thus podocalyxin apparently inhibits cell-matrix interactions. We also performed adhesion and spreading assays on podocytes grown under increased glucose concentration (25 mM). Under these conditions, the expression of podocalyxin was almost totally suppressed. The cells adhered and spread to basement membrane components but there was no increase in the extent of adhesion and spreading in the presence of anti-podocalyxin antibody, or ruffling of the cell edges. Additionally, in podocytes expressing podocalyxin, the presence of anti-podocalyxin antibody partially reversed the inhibition of adhesion to collagen IV provoked by anti-beta1 integrin antibody, thus podocalyxin should compete with beta1-related cell adhesion. We suggest that the observed podocalyxin-mediated inhibition of binding to the matrix could be in part responsible for the specialized conformation of the basal surface of podocytes.

Tracking the fate of glomerular epithelial cells in vivo using serial multiphoton imaging in novel mouse models with fluorescent lineage tags

Podocytes are critical in the maintenance of a healthy glomerular filter; however, they have been difficult to study in the intact kidney because of technical limitations. Here we report the development of serial multiphoton microscopy (MPM) of the same glomeruli over several days to visualize the motility of podocytes and parietal epithelial cells (PECs) in vivo. In podocin-GFP mice, podocytes formed sporadic multicellular clusters after unilateral ureteral ligation and migrated into the parietal Bowman's capsule. The tracking of single cells in podocin-confetti mice featuring cell-specific expression of CFP, GFP, YFP or RFP revealed the simultaneous migration of multiple podocytes. In phosphoenolpyruvate carboxykinase (PEPCK)-GFP mice, serial MPM found PEC-to-podocyte migration and nanotubule connections. Our data support a highly dynamic rather than a static nature of the glomerular environment and cellular composition. Future application of this new approach should advance our understanding of the mechanisms of glomerular injury and regeneration.

Role of boundary conditions in an experimental model of epithelial wound healing.

Coordinated cell movements in epithelial layers are essential for proper tissue morphogenesis and homeostasis, but our understanding of the mechanisms that coordinate the behavior of multiple cells in these processes is far from complete. Recent experiments with Madin-Darby canine kidney epithelial monolayers revealed a wave-like pattern of injury-induced MAPK activation and showed that it is essential for collective cell migration after wounding. To investigate the effects of the different aspects of wounding on cell sheet migration, we engineered a system that allowed us to dissect the classic wound healing assay. We studied Madin-Darby canine kidney sheet migration under three different conditions: 1) the classic wound healing assay, 2) empty space induction, where a confluent monolayer is grown adjacent to a slab of polydimethylsiloxane and the monolayer is not injured but allowed to migrate upon removal of the slab, and 3) injury via polydimethylsiloxane membrane peel-off, where an injured monolayer migrates onto plain tissue culture surface, as in the case of empty space induction allowing for direct comparison. By tracking the motion of individual cells within the sheet under these three conditions, we show how the dynamics of the individual cells' motion is responsible for the coordinated migration of the sheet and is coordinated with the activation of ERK1/2 MAPK. In addition, we demonstrate that the propagation of the waves of MAPK activation depends on the generation of reactive oxygen species at the wound edge.

EMT, the cytoskeleton, and cancer cell invasion

The metastatic process, i.e. the dissemination of cancer cells throughout the body to seed secondary tumors at distant sites, requires cancer cells to leave the primary tumor and to acquire migratory and invasive capabilities. In a process of epithelial-mesenchymal transition (EMT), besides changing their adhesive repertoire, cancer cells employ developmental processes to gain migratory and invasive properties that involve a dramatic reorganization of the actin cytoskeleton and the concomitant formation of membrane protrusions required for invasive growth. The molecular processes underlying such cellular changes are still only poorly understood, and the various migratory organelles, including lamellipodia, filopodia, invadopodia and podosomes, still require a better functional and molecular characterization. Notably, direct experimental evidence linking the formation of migratory membrane protrusions and the process of EMT and tumor metastasis is still lacking. In this review, we have summarized recent novel insights into the molecular processes and players underlying EMT on one side and the formation of invasive membrane protrusions on the other side.

Podoplanin-Rich Stromal Networks Induce Dendritic Cell Motility via Activation of the C-type Lectin Receptor CLEC-2

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function.

Metastatic tumor cells that actively migrate and invade surrounding tissues rely on invadopodia to degrade extracellular matrix (ECM) barriers. Invadopodia are membrane protrusions that localize enzymes required for ECM degradation. Little is known about the formation, function, and regulation of invadopodia. Here, we show that invadopodia have two distinct aspects: (a) structural for organizing the cellular actin cytoskeleton to form membrane protrusions and (b) functional for using proteolytic enzyme(s) for ECM degradation. Small interfering RNA (siRNA) inhibition established that organization of invadopodia structure requires cortactin, whereas protease inhibitor studies identified membrane type 1 matrix metalloproteinase (MT1-MMP) as the key invadopodial enzyme responsible for gelatin matrix degradation in the breast carcinoma cell line MDA-MB-231. The inhibition of invadopodial structure assembly by cortactin depletion resulted in a block of matrix degradation due to failure of invadopodia formation. Either protease inhibition or MT1-MMP siRNA depletion moderately decreased the formation of invadopodial structures that were identified as actin-cortactin accumulations at the ventral cell membrane adherent to matrix. The invadopodia that were able to form upon MT1-MMP inhibition or depletion retained actin-cortactin accumulations but were unable to degrade matrix. Examination of cells at different time points as well as live-cell imaging revealed four distinct invadopodial stages: membrane cortactin aggregation at membranes adherent to matrix, MT1-MMP accumulation at the region of cortactin accumulation, matrix degradation at the invadopodia region, and subsequent cortactin dissociation from the area of continued MT1-MMP accumulation associated with foci of degraded matrix. Based on these results, we propose a stepwise model of invadopodia formation and function.

Visualizing the function and fate of neutrophils in sterile injury and repair

Neutrophils have been implicated as harmful cells in a variety of inappropriate inflammatory conditions where they injure the host, leading to the death of the neutrophils and their subsequent phagocytosis by monocytes and macrophages. Here we show that in a fully repairing sterile thermal hepatic injury, neutrophils also penetrate the injury site and perform the critical tasks of dismantling injured vessels and creating channels for new vascular regrowth. Upon completion of these tasks, they neither die at the injury site nor are phagocytosed. Instead, many of these neutrophils reenter the vasculature and have a preprogrammed journey that entails a sojourn in the lungs to up-regulate CXCR4 (C-X-C motif chemokine receptor 4) before entering the bone marrow, where they undergo apoptosis.

Sequential signals toward podosome formation in NIH-src cells

Podosomes (also termed invadopodia in cancer cells) are actin-rich adhesion structures with matrix degradation activity that develop in various cell types. Despite their significant physiological importance, the molecular mechanism of podosome formation is largely unknown. In this study, we investigated the molecular mechanisms of podosome formation. The expression of various phosphoinositide-binding domains revealed that the podosomes in Src-transformed NIH3T3 (NIH-src) cells are enriched with PtdIns(3,4)P2, suggesting an important role of this phosphoinositide in podosome formation. Live-cell imaging analysis revealed that Src-expression stimulated podosome formation at focal adhesions of NIH3T3 cells after PtdIns(3,4)P2 accumulation. The adaptor protein Tks5/FISH, which is essential for podosome formation, was found to form a complex with Grb2 at adhesion sites in an Src-dependent manner. Further, it was found that N-WASP bound all SH3 domains of Tks5/FISH, which facilitated circular podosome formation. These results indicate that augmentation of the N-WASP-Arp2/3 signal was accomplished on the platform of Tks5/FISH-Grb2 complex at focal adhesions, which is stabilized by PtdIns(3,4)P2.

Excitotoxic brain injury in adult zebrafish stimulates neurogenesis and long-distance neuronal integration.

Zebrafish maintain a greater capacity than mammals for central nervous system repair after injury. Understanding differences in regenerative responses between different vertebrate species may shed light on mechanisms to improve repair in humans. Quinolinic acid is an excitotoxin that has been used to induce brain injury in rodents for modeling Huntington's disease and stroke. When injected into the adult rodent striatum, this toxin stimulates subventricular zone neurogenesis and neuroblast migration to injury. However, most new neurons fail to survive and lesion repair is minimal. We used quinolinic acid to lesion the adult zebrafish telencephalon to study reparative processes. We also used conditional transgenic lineage mapping of adult radial glial stem cells to explore survival and integration of neurons generated after injury. Telencephalic lesioning with quinolinic acid, and to a lesser extent vehicle injection, produced cell death, microglial infiltration, increased cell proliferation, and enhanced neurogenesis in the injured hemisphere. Lesion repair was more complete with quinolinic acid injection than after vehicle injection. Fate mapping of her4-expressing radial glia showed injury-induced expansion of radial glial stem cells that gave rise to neurons which migrated to injury, survived at least 8 weeks and formed long-distance projections that crossed the anterior commissure and synapsed in the contralateral hemisphere. These findings suggest that quinolinic acid lesioning of the zebrafish brain stimulates adult neural stem cells to produce robust regeneration with long-distance integration of new neurons. This model should prove useful for elucidating reparative mechanisms that can be applied to restorative therapies for mammalian brain injury.

Identification of splenic reservoir monocytes and their deployment to inflammatory sites.

A current paradigm states that monocytes circulate freely and patrol blood vessels but differentiate irreversibly into dendritic cells (DCs) or macrophages upon tissue entry. Here we show that bona fide undifferentiated monocytes reside in the spleen and outnumber their equivalents in circulation. The reservoir monocytes assemble in clusters in the cords of the subcapsular red pulp and are distinct from macrophages and DCs. In response to ischemic myocardial injury, splenic monocytes increase their motility, exit the spleen en masse, accumulate in injured tissue, and participate in wound healing. These observations uncover a role for the spleen as a site for storage and rapid deployment of monocytes and identify splenic monocytes as a resource that the body exploits to regulate inflammation.

Stresses at the cell-to-substrate interface during locomotion of fibroblasts

Recent technological improvements in the elastic substrate method make it possible to produce spatially resolved measurements of the tractions exerted by single motile cells. In this study we have applied these developments to produce maps of the tractions exerted by 3T3 fibroblasts during steady locomotion. The resulting images have a spatial resolution of approximately 5 micrometers and a maximum intensity of approximately 10(2) kdyn/cm2 (10(4) pN/micrometers2). We find that the propulsive thrust for fibroblast locomotion, approximately 0.2 dyn, is imparted to the substratum within 15 micrometers of the leading edge. These observations demonstrate that the lamellipodium of the fibroblast is able to generate intense traction stress. The cell body and posterior seem to be mechanically passive structures pulled forward entirely by this action.

Rho GTPase signalling in cell migration

Cells migrate in multiple different ways depending on their environment, which includes the extracellular matrix composition, interactions with other cells, and chemical stimuli. For all types of cell migration, Rho GTPases play a central role, although the relative contribution of each Rho GTPase depends on the environment and cell type. Here, I review recent advances in our understanding of how Rho GTPases contribute to different types of migration, comparing lamellipodium-driven versus bleb-driven migration modes. I also describe how cells migrate across the endothelium. In addition to Rho, Rac and Cdc42, which are well known to regulate migration, I discuss the roles of other less-well characterized members of the Rho family.

Matrix Metalloproteinase 1 Is Necessary for the Migration of Human Bone Marrow-Derived Mesenchymal Stem Cells Toward Human Glioma

Human mesenchymal stem cells (MSCs) have increasingly been used as cellular vectors for the delivery of therapeutic genes to tumors. However, the precise mechanism of mobilization remains poorly defined. In this study, MSCs that expressed similar cell surface markers and exhibited multilineage differentiation potentials were isolated from various donors. Interestingly, different MSC isolates displayed differential migration ability toward human glioma cells. We hypothesized that distinct molecular signals may be involved in the varied tumor tropisms exhibited by different MSC isolates. To test this hypothesis, gene expression profiles of tumor-trophic MSCs were compared with those of non-tumor-trophic MSCs. Among the various differentially regulated genes, matrix metalloproteinase one (MMP1) gene expression and its protein activities were enhanced by 27-fold and 21-fold, respectively, in highly migrating MSCs compared with poorly migrating MSCs. By contrast, there was no change in the transcriptional levels of other MMPs. Functional inactivation of MMP1 abrogated the migratory potential of MSCs toward glioma-conditioned medium. Conversely, the nonmigratory phenotype of poorly migrating MSC could be rescued in the presence of either recombinant MMP1 or conditioned medium from the highly migrating MSCs. Ectopic expression of MMP1 in these poorly migrating cells also rendered the cells responsive to the signaling cues from the glioma cells in vivo. However, blocking the interaction of MMP1 and its cognate receptor PAR1 effectively diminished the migratory ability of MSCs. Taken together, this study provides, for the first time, supporting evidence that MMP1 is critically involved in the migration capacity of MSCs, acting through the MMP1/PAR1 axis.

Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms.

In patients with spinal cord injury, the primary or mechanical trauma seldom causes total transection, even though the functional loss may be complete. In addition, biochemical and pathological changes in the cord may worsen after injury. To explain these phenomena, the concept of the secondary injury has evolved for which numerous pathophysiological mechanisms have been postulated. This paper reviews the concept of secondary injury with special emphasis on vascular mechanisms. Evidence is presented to support the theory of secondary injury and the hypothesis that a key mechanism is posttraumatic ischemia with resultant infarction of the spinal cord. Evidence for the role of vascular mechanisms has been obtained from a variety of models of acute spinal cord injury in several species. Many different angiographic methods have been used for assessing microcirculation of the cord and for measuring spinal cord blood flow after trauma. With these techniques, the major systemic and local vascular effects of acute spinal cord injury have been identified and implicated in the etiology of secondary injury. The systemic effects of acute spinal cord injury include hypotension and reduced cardiac output. The local effects include loss of autoregulation in the injured segment of the spinal cord and a marked reduction of the microcirculation in both gray and white matter, especially in hemorrhagic regions and in adjacent zones. The microcirculatory loss extends for a considerable distance proximal and distal to the site of injury. Many studies have shown a dose-dependent reduction of spinal cord blood flow varying with the severity of injury, and a reduction of spinal cord blood flow which worsens with time after injury. The functional deficits due to acute spinal cord injury have been measured electrophysiologically with techniques such as motor and somatosensory evoked potentials and have been found proportional to the degree of posttraumatic ischemia. The histological effects include early hemorrhagic necrosis leading to major infarction at the injury site. These posttraumatic vascular effects can be treated. Systemic normotension can be restored with volume expansion or vasopressors, and spinal cord blood flow can be improved with dopamine, steroids, nimodipine, or volume expansion. The combination of nimodipine and volume expansion improves posttraumatic spinal cord blood flow and spinal cord function measured by evoked potentials. These results provide strong evidence that posttraumatic ischemia is an important secondary mechanism of injury, and that it can be counteracted.

Cell death: the significance of apoptosis.

Publisher Summary The classification of cell death can be based on morphological or biochemical criteria or on the circumstances of its occurrence. Currently, irreversible structural alteration provides the only unequivocal evidence of death; biochemical indicators of cell death that are universally applicable have to be precisely defined and studies of cell function or of reproductive capacity do not necessarily differentiate between death and dormant states from which recovery may be possible. It has also proved feasible to categorize most if not all dying cells into one or the other of two discrete and distinctive patterns of morphological change, which have, generally, been found to occur under disparate but individually characteristic circumstances. One of these patterns is the swelling proceeding to rupture of plasma and organelle membranes and dissolution of organized structure—termed “coagulative necrosis. ” It results from injury by agents, such as toxins and ischemia, affects cells in groups rather than singly, and evokes exudative inflammation when it develops in vivo. The other morphological pattern is characterized by condensation of the cell with maintenance of organelle integrity and the formation of surface protuberances that separate as membrane-bounded globules; in tissues, these are phagocytosed and digested by resident cells, there being no associated inflammation.

THE ORIGIN AND DIFFERENTIATION OF MICROGLIAL CELLS DURING DEVELOPMENT

Some authors claim that microglia originate from the neuroepithelium, although most now believe that microglial cells are of mesodermal origin, and probably belong to the monocyte/macrophage cell line. These cells must enter the developing central nervous system (CNS) from the blood stream, the ventricular space or the meninges. Afterward microglial cells are distributed more or less homogeneously through the entire nervous parenchyma. Stereotyped patterns of migration have been recognized during development, in which long-distance tangential migration precedes radial migration of individual cells. Microglial cells moving through the nervous parenchyma are ameboid microglia, which apparently differentiate into ramified microglia after reaching their definitive location. This is supported by the presence of cells showing intermediate features between those of ameboid and ramified microglia. The factors that control the invasion of the nervous parenchyma, migration within the developing CNS and differentiation of microglial cells are not well known. These phenomena apparently depend on environmental factors such as soluble or cell-surface bound molecules and components of the extracellular matrix. Microglial cells within the developing CNS are involved in clearing cell debris and withdrawing misdirected or transitory axons, and presumably support cell survival and neurite growth.

Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding

The development and maintenance of an epithelium requires finely balanced rates of growth and cell death. However, the mechanical and biochemical mechanisms that ensure proper feedback control of tissue growth, which when deregulated contribute to tumorigenesis, are poorly understood. Here we use the fly notum as a model system to identify a novel process of crowding-induced cell delamination that balances growth to ensure the development of well-ordered cell packing. In crowded regions of the tissue, a proportion of cells undergo a serial loss of cell–cell junctions and a progressive loss of apical area, before being squeezed out by their neighbours. This path of delamination is recapitulated by a simple computational model of epithelial mechanics, in which stochastic cell loss relieves overcrowding as the system tends towards equilibrium. We show that this process of delamination is mechanistically distinct from apoptosis-mediated cell extrusion and precedes the first signs of cell death. Overall, this analysis reveals a simple mechanism that buffers epithelia against variations in growth. Because live-cell delamination constitutes a mechanistic link between epithelial hyperplasia and cell invasion, this is likely to have important implications for our understanding of the early stages of cancer development.

A talin-dependent LFA-1 focal zone is formed by rapidly migrating T lymphocytes

Cells such as fibroblasts and endothelial cells migrate through the coordinated responses of discrete integrin-containing focal adhesions and complexes. In contrast, little is known about the organization of integrins on the highly motile T lymphocyte. We have investigated the distribution, activity, and cytoskeletal linkage of the integrin lymphocyte function associated antigen-1 (LFA-1) on human T lymphocytes migrating on endothelial cells and on ligand intercellular adhesion molecule-1 (ICAM-1). The pattern of total LFA-1 varies from low expression in the lamellipodia to high expression in the uropod. However, high affinity, clustered LFA-1 is restricted to a mid-cell zone that remains stable over time and over a range of ICAM-1 densities. Talin is essential for the stability and formation of the LFA-1 zone. Disruption of the talin–integrin link leads to loss of zone integrity and a substantial decrease in speed of migration on ICAM-1. This adhesive structure, which differs from the previously described integrin-containing attachments displayed by many other cell types, we have termed the “focal zone. ”

The Ly49Q receptor plays a crucial role in neutrophil polarization and migration by regulating raft trafficking.

Neutrophils rapidly undergo polarization and directional movement to infiltrate the sites of infection and inflammation. Here, we show that an inhibitory MHC I receptor, Ly49Q, was crucial for the swift polarization of and tissue infiltration by neutrophils. During the steady state, Ly49Q inhibited neutrophil adhesion by preventing focal-complex formation, likely by inhibiting Src and PI3 kinases. However, in the presence of inflammatory stimuli, Ly49Q mediated rapid neutrophil polarization and tissue infiltration in an ITIM-domain-dependent manner. These opposite functions appeared to be mediated by distinct use of effector phosphatase SHP-1 and SHP-2. Ly49Q-dependent polarization and migration were affected by Ly49Q regulation of membrane raft functions. We propose that Ly49Q is pivotal in switching neutrophils to their polarized morphology and rapid migration upon inflammation, through its spatiotemporal regulation of membrane rafts and raft-associated signaling molecules.

From Cell Differentiation to Cell Collectives: Bacillus subtilis Uses Division of Labor to Migrate

The organization of cells, emerging from cell-cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called "van Gogh bundles") of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.

Getting to the site of inflammation: the leukocyte adhesion cascade updated

Neutrophil recruitment, lymphocyte recirculation and monocyte trafficking all require adhesion and transmigration through blood-vessel walls. The traditional three steps of rolling, activation and firm adhesion have recently been augmented and refined. Slow rolling, adhesion strengthening, intraluminal crawling and paracellular and transcellular migration are now recognized as separate, additional steps. In neutrophils, a second activation pathway has been discovered that does not require signalling through G-protein-coupled receptors and the signalling steps leading to integrin activation are beginning to emerge. This Review focuses on new aspects of one of the central paradigms of inflammation and immunity — the leukocyte adhesion cascade.

Modification of kidney barrier function by the urokinase receptor

Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of αvβ3 integrin. Mice lacking uPAR (Plaur−/−) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active β3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate αvβ3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of αvβ3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.

Myeloperoxidase: friend and foe.

Neutrophilic polymorphonuclear leukocytes (neutrophils) are highly specialized for their primary function, the phagocytosis and destruction of microorganisms. When coated with opsonins (generally complement and/or antibody), microorganisms bind to specific receptors on the surface of the phagocyte and invagination of the cell membrane occurs with the incorporation of the microorganism into an intracellular phagosome. There follows a burst of oxygen consumption, and much, if not all, of the extra oxygen consumed is converted to highly reactive oxygen species. In addition, the cytoplasmic granules discharge their contents into the phagosome, and death of the ingested microorganism soon follows. Among the antimicrobial systems formed in the phagosome is one consisting of myeloperoxidase (MPO), released into the phagosome during the degranulation process, hydrogen peroxide (H2O2), formed by the respiratory burst and a halide, particularly chloride. The initial product of the MPO-H2O2-chloride system is hypochlorous acid, and subsequent formation of chlorine, chloramines, hydroxyl radicals, singlet oxygen, and ozone has been proposed. These same toxic agents can be released to the outside of the cell, where they may attack normal tissue and thus contribute to the pathogenesis of disease. This review will consider the potential sources of H2O2 for the MPO-H2O2-halide system; the toxic products of the MPO system; the evidence for MPO involvement in the microbicidal activity of neutrophils; the involvement of MPO-independent antimicrobial systems; and the role of the MPO system in tissue injury. It is concluded that the MPO system plays an important role in the microbicidal activity of phagocytes.

T cell receptor ligation induces the formation of dynamically regulated signaling assemblies

Tcell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein–GPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70–containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.

CD64 expression distinguishes monocyte-derived and conventional dendritic cells and reveals their distinct role during intramuscular immunization.

Although most vaccines are administered i.m., little is known about the dendritic cells (DCs) that are present within skeletal muscles. In this article, we show that expression of CD64, the high-affinity IgG receptor FcγRI, distinguishes conventional DCs from monocyte-derived DCs (Mo-DCs). By using such a discriminatory marker, we defined the distinct DC subsets that reside in skeletal muscles and identified their migratory counterparts in draining lymph nodes (LNs). We further used this capability to analyze the functional specialization that exists among muscle DCs. After i.m. administration of Ag adsorbed to alum, we showed that alum-injected muscles contained large numbers of conventional DCs that belong to the CD8α(+)- and CD11b(+)-type DCs. Both conventional DC types were capable of capturing Ag and of migrating to draining LNs, where they efficiently activated naive T cells. In alum-injected muscles, Mo-DCs were as numerous as conventional DCs, but only a small fraction migrated to draining LNs. Therefore, alum by itself poorly induces Mo-DCs to migrate to draining LNs. We showed that addition of small amounts of LPS to alum enhanced Mo-DC migration. Considering that migratory Mo-DCs had, on a per cell basis, a higher capacity to induce IFN-γ-producing T cells than conventional DCs, the addition of LPS to alum enhanced the overall immunogenicity of Ags presented by muscle-derived DCs. Therefore, a full understanding of the role of adjuvants during i.m. vaccination needs to take into account the heterogeneous migratory and functional behavior of muscle DCs and Mo-DCs revealed in this study.

Adhesion disengagement uncouples intrinsic and extrinsic forces to drive cytokinesis in epithelial tissues.

Cytokinesis entails cell invagination by a contractile actomyosin ring. In epithelia, E-cadherin-mediated adhesion connects the cortices of contacting cells; thus, it is unclear how invagination occurs, how the new junction forms, and how tissue integrity is preserved. Investigations in Drosophila embryos first show that apicobasal cleavage is polarized: invagination is faster from the basal than from the apical side. Ring contraction but not its polarized constriction is controlled by septin filaments and Anillin. Polarized cleavage is due instead to mechanical anchorage of the ring to E-cadherin complexes. Formation of the new junction requires local adhesion disengagement in the cleavage furrow, followed by new E-cadherin complex formation at the new interface. E-cadherin disengagement depends on the tension exerted by the cytokinetic ring and by neighboring cells. We uncover intrinsic and extrinsic forces necessary for cytokinesis and present a framework for understanding how tissue cohesion is preserved during epithelial division.

Microfluidic platform to evaluate migration of cells from patients with DYT1 dystonia.

BACKGROUND Microfluidic platforms for quantitative evaluation of cell biologic processes allow low cost and time efficient research studies of biological and pathological events, such as monitoring cell migration by real-time imaging. In healthy and disease states, cell migration is crucial in development and wound healing, as well as to maintain the body's homeostasis. NEW METHOD The microfluidic chambers allow precise measurements to investigate whether fibroblasts carrying a mutation in the TOR1A gene, underlying the hereditary neurologic disease--DYT1 dystonia, have decreased migration properties when compared to control cells. RESULTS We observed that fibroblasts from DYT1 patients showed abnormalities in basic features of cell migration, such as reduced velocity and persistence of movement. COMPARISON WITH EXISTING METHOD The microfluidic method enabled us to demonstrate reduced polarization of the nucleus and abnormal orientation of nuclei and Golgi inside the moving DYT1 patient cells compared to control cells, as well as vectorial movement of single cells. CONCLUSION We report here different assays useful in determining various parameters of cell migration in DYT1 patient cells as a consequence of the TOR1A gene mutation, including a microfluidic platform, which provides a means to evaluate real-time vectorial movement with single cell resolution in a three-dimensional environment.

Membrane lipids in invadopodia and podosomes: Key structures for cancer invasion and metastasis

Invadopodia are extracellular matrix (ECM)-degrading protrusions formed by invasive cancer cells. Podosomes are structures functionally similar to invadopodia that are found in oncogene-transformed fibroblasts and monocyte-derived cells, including macrophages and osteoclasts. These structures are thought to play important roles in the pericellular remodeling of ECM during cancer invasion and metastasis. Much effort has been directed toward identification of the molecular components and regulators of invadopodia/podosomes, which could be therapeutic targets in the treatment of malignant cancers. However, it remains largely unknown how these components are assembled into invadopodia/podosomes and how the assembly process is spatially and temporally regulated. This review will summarize recent progress on the molecular mechanisms of invadopodia/podosome formation, with strong emphasis on the roles of lipid rafts and phosphoinositides.

The role of free radicals in cold injuries.

Cold injury is a tissue trauma produced by exposure to freezing temperatures and even brief exposure to a severely cold and windy environment. Rewarming of frozen tissue is associated with blood reperfusion and the simultaneous generation of free oxygen radicals. In this review is discussed the current understanding of the mechanism of action of free oxygen radicals as related to cold injury during rewarming. Decreased energy stores during ischaemia lead to the accumulation of adenine nucleotides and liberation of free fatty acids due to the breakdown of lipid membranes. On rewarming, free fatty acids are metabolized via cyclo-oxygenase and adenine nucleotides are metabolized via the xanthine oxidase pathway. These may be the source of free oxygen radicals. Leukocytes may also play a major role in the pathogenesis of cold injury. Oxygen radical scavengers, such as superoxide dismutase and catalase, may help to reduce the cold induced injury but their action is limited due to the inability readily to cross the plasma membrane. Lipid soluble antioxidants are likely to be more effective scavengers because of their presence in membranes where peroxidative reactions can be arrested.

Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels.

To understand how cells sense and adapt to mechanical stress, we applied tensional forces to magnetic microbeads bound to cell-surface integrin receptors and measured changes in bead displacement with sub-micrometer resolution using optical microscopy. Cells exhibited four types of mechanical responses: (1) an immediate viscoelastic response; (2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces; (3) later adaptive cell stiffening with sustained (>15 second) static stresses; and (4) a large-scale repositioning response with prolonged (>1 minute) stress. Importantly, these adaptation responses differed biochemically. The immediate and early responses were affected by chemically dissipating cytoskeletal prestress (isometric tension), whereas the later adaptive response was not. The repositioning response was prevented by inhibiting tension through interference with Rho signaling, similar to the case of the immediate and early responses, but it was also prevented by blocking mechanosensitive ion channels or by inhibiting Src tyrosine kinases. All adaptive responses were suppressed by cooling cells to 4 degrees C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.

CNS repair requires both effector and regulatory T cells with distinct temporal and spatial profiles.

Monocyte-derived macrophages (mo-MΦs) and T cells have been shown to contribute to spinal cord repair. Recently, the remote brain choroid plexus epithelium (CP) was identified as a portal for monocyte recruitment, and its activation for leukocyte trafficking was found to be IFN-γ-dependent. Here, we addressed how the need for effector T cells can be reconciled with the role of inflammation-resolving immune cells in the repair process. Using an acute spinal cord injury model, we show that in mice deficient in IFN-γ-producing T cells, the CP was not activated, and recruitment of inflammation-resolving mo-MΦ to the spinal cord parenchyma was limited. We further demonstrate that mo-MΦ locally regulated recruitment of thymic-derived Foxp3(+) regulatory T (Treg) cells to the injured spinal cord parenchyma at the subacute/chronic phase. Importantly, an ablation protocol that resulted in reduced Tregs at this stage interfered with tissue remodeling, in contrast to Treg transient ablation, restricted to the 4 d period before the injury, which favored repair. The enhanced functional recovery observed following such a controlled decrease of Tregs suggests that reduced systemic immunosuppression at the time of the insult can enhance CNS repair. Overall, our data highlight a dynamic immune cell network needed for repair, acting in discrete compartments and stages, and involving effector and regulatory T cells, interconnected by mo-MΦ. Any of these populations may be detrimental to the repair process if their level or activity become dysregulated. Accordingly, therapeutic interventions must be both temporally and spatially controlled.

Molecular mechanisms of invadopodium formation: the role of the N-WASP–Arp2/3 complex pathway and cofilin

Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP–Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.

Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia

For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration.

Mesenchymal stem cells (MSCs) commonly defined by in vitro functions have entered clinical application despite little definition of their function in residence. Here, we report genetic pulse-chase experiments that define osteoblastic cells as short-lived and nonreplicative, requiring replenishment from bone-marrow-derived, Mx1(+) stromal cells with "MSC" features. These cells respond to tissue stress and migrate to sites of injury, supplying new osteoblasts during fracture healing. Single cell transplantation yielded progeny that both preserve progenitor function and differentiate into osteoblasts, producing new bone. They are capable of local and systemic translocation and serial transplantation. While these cells meet current definitions of MSCs in vitro, they are osteolineage restricted in vivo in growing and adult animals. Therefore, bone-marrow-derived MSCs may be a heterogeneous population with the Mx1(+) population, representing a highly dynamic and stress responsive stem/progenitor cell population of fate-restricted potential that feeds the high cell replacement demands of the adult skeleton.

Origin and functions of tissue macrophages.

Macrophages are distributed in tissues throughout the body and contribute to both homeostasis and disease. Recently, it has become evident that most adult tissue macrophages originate during embryonic development and not from circulating monocytes. Each tissue has its own composition of embryonically derived and adult-derived macrophages, but it is unclear whether macrophages of distinct origins are functionally interchangeable or have unique roles at steady state. This new understanding also prompts reconsideration of the function of circulating monocytes. Classical Ly6c(hi) monocytes patrol the extravascular space in resting organs, and Ly6c(lo) nonclassical monocytes patrol the vasculature. Inflammation triggers monocytes to differentiate into macrophages, but whether resident and newly recruited macrophages possess similar functions during inflammation is unclear. Here, we define the tools used for identifying the complex origin of tissue macrophages and discuss the relative contributions of tissue niche versus ontological origin to the regulation of macrophage functions during steady state and inflammation.

Pericyte differentiation.

Pericytes are defined in vivo by their location: They are embedded within the basement membrane of microvessels. They form an integral part of the microvascular wall and are believed to participate in angiogenesis, although their precise role is not clear. Pericytes derived from the retinal microvasculature have been cultured and identified by a series of phenotypic characteristics that clearly distinguishes them from other stromal cells such as smooth muscle cells. Pericytes in vitro form multicellular nodules rich in extracellular matrix. This matrix becomes mineralized in the presence of growth medium containing serum, without exogenous beta-glycerophosphate. These results indicate that pericytes represent primitive mesenchymal cells able to differentiate into an osteogenic phenotype. Pericyte differentiation also is defined by alterations in their response to transforming growth factor beta 1 and changes in the synthesis and/or deposition of various extracellular matrix proteins such as laminin, Type IV collagen, tenascin, Type X collagen osteonectin, and thrombospondin-1. Angiogenesis is associated commonly with mineralization. These data suggest that pericytes may contribute to mineralization in vivo.

Skeletal Myogenic Progenitors Originating from Embryonic Dorsal Aorta Coexpress Endothelial and Myogenic Markers and Contribute to Postnatal Muscle Growth and Regeneration

Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met-/- embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells. The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system.

Immobilized chemokine fields and soluble chemokine gradients cooperatively shape migration patterns of dendritic cells.

Chemokines orchestrate immune cell trafficking by eliciting either directed or random migration and by activating integrins in order to induce cell adhesion. Analyzing dendritic cell (DC) migration, we showed that these distinct cellular responses depended on the mode of chemokine presentation within tissues. The surface-immobilized form of the chemokine CCL21, the heparan sulfate-anchoring ligand of the CC-chemokine receptor 7 (CCR7), caused random movement of DCs that was confined to the chemokine-presenting surface because it triggered integrin-mediated adhesion. Upon direct contact with CCL21, DCs truncated the anchoring residues of CCL21, thereby releasing it from the solid phase. Soluble CCL21 functionally resembles the second CCR7 ligand, CCL19, which lacks anchoring residues and forms soluble gradients. Both soluble CCR7 ligands triggered chemotactic movement, but not surface adhesion. Adhesive random migration and directional steering cooperate to produce dynamic but spatially restricted locomotion patterns closely resembling the cellular dynamics observed in secondary lymphoid organs.

Cytomegalovirus US2 destroys two components of the MHC class II pathway, preventing recognition by CD4+ T cells

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes life-threatening disease in patients who are immunosuppressed for bone marrow or tissue transplantation or who have AIDS (ref. 1). HCMV establishes lifelong latent infections and, after periodic reactivation from latency, uses a panel of immune evasion proteins to survive and replicate in the face of robust, fully primed host immunity. Monocyte/macrophages are important host cells for HCMV, serving as a latent reservoir and as a means of dissemination throughout the body. Macrophages and other HCMV-permissive cells, such as endothelial and glial cells, can express MHC class II proteins and present antigens to CD4+ T lymphocytes. Here, we show that the HCMV protein US2 causes degradation of two essential proteins in the MHC class II antigen presentation pathway: HLA-DR-α and DM-α. This was unexpected, as US2 has been shown to cause degradation of MHC class I (refs. 5,6), which has only limited homology with class II proteins. Expression of US2 in cells reduced or abolished their ability to present antigen to CD4+ T lymphocytes. Thus, US2 may allow HCMV-infected macrophages to remain relatively 'invisible' to CD4+ T cells, a property that would be important after virus reactivation.

Catch and release: how do kinetochores hook the right microtubules during mitosis?

Sport fishermen keep tension on their lines to prevent hooked fish from releasing. A molecular version of this angler's trick, operating at kinetochores, ensures accuracy during mitosis: the mitotic spindle attaches randomly to chromosomes and then correctly bioriented attachments are stabilized due to the tension exerted on them by opposing microtubules. Incorrect attachments, which lack tension, are unstable and release quickly, allowing another chance for biorientation. Stabilization of molecular interactions by tension also occurs in other physiological contexts, such as cell adhesion, motility, hemostasis, and tissue morphogenesis. Here, we review models for the stabilization of kinetochore attachments with an eye toward emerging models for other force-activated systems. Although attention in the mitosis field has focused mainly on one kinase-based mechanism, multiple mechanisms may act together to stabilize properly bioriented kinetochores and some principles governing other tension-sensitive systems may also apply to kinetochores.

Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages.

Rho family proteins are known to regulate actin organization in fibroblasts, but their functions in cells of haematopoietic origin have not been studied in detail. Bac1.2F5 cells are a colony-stimulating factor-1 (CSF-1)-dependent murine macrophage cell line; CSF-1 stimulates their proliferation and motility, and acts as a chemoattractant. CSF-1 rapidly induced actin reorganization in Bac1 cells: it stimulated the formation of filopodia, lamellipodia and membrane ruffles at the plasma membrane, as well as the appearance of fine actin cables within the cell interior. Microinjection of constitutively activated (V12)Rac1 stimulated lamellipodium formation and membrane ruffling. The dominant inhibitory Rac mutant, N17Rac1, inhibited CSF-1-induced lamellipodium formation, and also induced cell rounding. V12Cdc42 induced the formation of long filopodia, while the dominant inhibitory mutant N17Cdc42 prevented CSF-1-induced formation of filopodia but not lamellipodia. V14RhoA stimulated actin cable assembly and cell contraction, while the Rho inhibitor, C3 transferase, induced the loss of actin cables. Bac1 cells had cell-to-substratum adhesion sites containing beta1 integrin, pp125FAK, paxillin, vinculin, and tyrosine phosphorylated proteins. These 'focal complexes' were present in growing and CSF-1-starved cells, but were disassembled in cells injected with N17Cdc42 or N17Rac1. Interestingly, beta1 integrin did not disperse until long after focal phosphotyrosine and vinculin staining had disappeared. We conclude that in Bac1 macrophages Cdc42, Rac and Rho regulate the formation of distinct actin filament-based structures, and that Cdc42 and Rac are also required for the assembly of adhesion sites to the extracellular matrix.

Another Barrier to Regeneration in the CNS: Activated Macrophages Induce Extensive Retraction of Dystrophic Axons through Direct Physical Interactions

Injured axons of the adult CNS undergo lengthy retraction from the initial site of axotomy after spinal cord injury. Macrophage infiltration correlates spatiotemporally with this deleterious phenomenon, but the direct involvement of these inflammatory cells has not been demonstrated. In the present study, we examined the role of macrophages in axonal retraction within the dorsal columns after spinal cord injury in vivo and found that retraction occurred between days 2 and 28 after lesion and that the ends of injured axons were associated with ED-1+ cells. Clodronate liposome-mediated depletion of infiltrating macrophages resulted in a significant reduction in axonal retraction; however, we saw no evidence of regeneration. We used time-lapse imaging of adult dorsal root ganglion neurons in an in vitro model of the glial scar to examine macrophage-axon interactions and observed that adhesive contacts and considerable physical interplay between macrophages and dystrophic axons led to extensive axonal retraction. The induction of retraction was dependent on both the growth state of the axon and the activation state of the macrophage. Only dystrophic adult axons were susceptible to macrophage "attack. " Unlike intrinsically active cell line macrophages, both primary macrophages and microglia required activation to induce axonal retraction. Contact with astrocytes had no deleterious effect on adult dystrophic axons, suggesting that the induction of extensive retraction was specific to phagocytic cells. Our data are the first to indicate a direct role of activated macrophages in axonal retraction by physical cell-cell interactions with injured axons.

Regeneration, tissue injury and the immune response.

The involvement of the immune system in the response to tissue injury has raised the possibility that it might influence tissue, organ or appendage regeneration following injury. One hypothesis that has been discussed is that inflammatory aspects may preclude the occurrence of regeneration, but there is also evidence for more positive roles of immune components. The vertebrate eye is an immunoprivileged site where inflammatory aspects are inhibited by several immunomodulatory mechanisms. In various newt species the ocular tissues such as the lens are regenerative and it has recently been shown that the response to local injury of the lens involves activation of antigen-presenting cells which traffic to the spleen and return to displace and engulf the lens, thereby inducing regeneration from the dorsal iris. The activation of thrombin from prothrombin in the dorsal iris is one aspect of the injury response that is important in the initiation of regeneration. The possible relationships between the immune response and the regenerative response are considered with respect to phylogenetic variation of regeneration in general, and lens regeneration in particular.

Cell death and immune privilege.

The host response to pathogens involves complex inflammatory responses and immune reactions. While these are central to host defense and vital to clearing infections, they are often accompanied by injury to surrounding tissue. Most organ systems can tolerate these responses without permanent consequences. However, there are sites that limit the spread of inflammation because it can threaten organ function. The most prominent examples of these are the eye, brain, and reproductive organs (testis, ovary), where even minor bouts of inflammation can have long-term consequences for the survival of the organism. In these organs immune responses either do not proceed, or proceed in a manner different from other areas; thus, they are called "immunologically privileged. " Here a functioning immune response can be the culprit that leads to disease.

Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells

The mechanism of B cell–antigen encounter in lymphoid tissues is incompletely understood. It is also unclear how immune complexes are transported to follicular dendritic cells. Here, using real-time two-photon microscopy we noted rapid delivery of immune complexes through the lymph to macrophages in the lymph node subcapsular sinus. B cells captured immune complexes by a complement receptor–dependent mechanism from macrophage processes that penetrated the follicle and transported the complexes to follicular dendritic cells. Furthermore, cognate B cells captured antigen-containing immune complexes from macrophage processes and migrated to the T zone. Our findings identify macrophages lining the subcapsular sinus as an important site of B cell encounter with immune complexes and show that intrafollicular B cell migration facilitates the transport of immune complexes as well as encounters with cognate antigen.

Neural crest cell lineage segregation in the mouse neural tube.

Neural crest (NC) cells arise in the dorsal neural tube (NT) and migrate into the embryo to develop into many different cell types. A major unresolved question is when and how the fate of NC cells is decided. There is widespread evidence for multipotential NC cells, whose fates are decided during or after migration. There is also some evidence that the NC is already divided into subpopulations of discrete precursors within the NT. We have investigated this question in the mouse embryo. We find that a subpopulation of cells on the most dorsomedial aspect of the NT express the receptor tyrosine kinase Kit (previously known as c-kit), emigrate exclusively into the developing dermis, and then express definitive markers of the melanocyte lineage. These are thus melanocyte progenitor cells. They are generated predominantly at the midbrain-hindbrain junction and cervical trunk, with significant numbers also in lower trunk. Other cells within the dorsal NT are Kit-, migrate ventrally, and, from embryonic day 9.5, express the neurotrophin receptor p75. These cells most likely only give rise to ventral NC derivatives such as neurons and glia. The p75+ cells are located ventrolateral to the Kit+ cells in areas of the NT where these two cell types are found. These data provide direct in vivo evidence for NC lineage segregation within the mouse neural tube.

A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth

Cell growth, an increase in mass and size, is a highly regulated cellular event. The Akt/mTOR (mammalian target of rapamycin) signalling pathway has a central role in the control of protein synthesis and thus the growth of cells, tissues and organisms. A striking example of a physiological context requiring rapid cell growth is tissue repair in response to injury. Here we show that keratin 17, an intermediate filament protein rapidly induced in wounded stratified epithelia, regulates cell growth through binding to the adaptor protein 14-3-3σ. Mouse skin keratinocytes lacking keratin 17 (ref. 4) show depressed protein translation and are of smaller size, correlating with decreased Akt/mTOR signalling activity. Other signalling kinases have normal activity, pointing to the specificity of this defect. Two amino acid residues located in the amino-terminal head domain of keratin 17 are required for the serum-dependent relocalization of 14-3-3σ from the nucleus to the cytoplasm, and for the concomitant stimulation of mTOR activity and cell growth. These findings reveal a new and unexpected role for the intermediate filament cytoskeleton in influencing cell growth and size by regulating protein synthesis.

Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus.

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte beta1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin beta1 (podocin-Cre beta1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre beta1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin beta1 activity in epithelial cells. To further explore whether integrin beta1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte beta1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.

YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration

Tissue regeneration requires dynamic cellular adaptation to the wound environment. It is currently unclear how this is orchestrated at the cellular level and how cell fate is affected by severe tissue damage. Here we dissect cell fate transitions during colonic regeneration in a mouse dextran sulfate sodium (DSS) colitis model, and we demonstrate that the epithelium is transiently reprogrammed into a primitive state. This is characterized by de novo expression of fetal markers as well as suppression of markers for adult stem and differentiated cells. The fate change is orchestrated by remodeling the extracellular matrix (ECM), increased FAK/Src signaling, and ultimately YAP/TAZ activation. In a defined cell culture system recapitulating the extracellular matrix remodeling observed in vivo, we show that a collagen 3D matrix supplemented with Wnt ligands is sufficient to sustain endogenous YAP/TAZ and induce conversion of cell fate. This provides a simple model for tissue regeneration, implicating cellular reprogramming as an essential element.

An actomyosin-based barrier inhibits cell mixing at compartmental boundaries in Drosophila embryos

Partitioning tissues into compartments that do not intermix is essential for the correct morphogenesis of animal embryos and organs. Several hypotheses have been proposed to explain compartmental cell sorting, mainly differential adhesion, but also regulation of the cytoskeleton or of cell proliferation. Nevertheless, the molecular and cellular mechanisms that keep cells apart at boundaries remain unclear. Here we demonstrate, in early Drosophila melanogaster embryos, that actomyosin-based barriers stop cells from invading neighbouring compartments. Our analysis shows that cells can transiently invade neighbouring compartments, especially when they divide, but are then pushed back into their compartment of origin. Actomyosin cytoskeletal components are enriched at compartmental boundaries, forming cable-like structures when the epidermis is mitotically active. When MyoII (non-muscle myosin II) function is inhibited, including locally at the cable by chromophore-assisted laser inactivation (CALI), in live embryos, dividing cells are no longer pushed back, leading to compartmental cell mixing. We propose that local regulation of actomyosin contractibility, rather than differential adhesion, is the primary mechanism sorting cells at compartmental boundaries.

AIR-2: An Aurora/Ipl1-related Protein Kinase Associated with Chromosomes and Midbody Microtubules Is  Required for Polar Body Extrusion and Cytokinesis  in Caenorhabditis elegans Embryos

An emerging family of kinases related to the Drosophila Aurora and budding yeast Ipl1 proteins has been implicated in chromosome segregation and mitotic spindle formation in a number of organisms. Unlike other Aurora/Ipl1-related kinases, the Caenorhabditis elegans orthologue, AIR-2, is associated with meiotic and mitotic chromosomes. AIR-2 is initially localized to the chromosomes of the most mature prophase I–arrested oocyte residing next to the spermatheca. This localization is dependent on the presence of sperm in the spermatheca. After fertilization, AIR-2 remains associated with chromosomes during each meiotic division. However, during both meiotic anaphases, AIR-2 is present between the separating chromosomes. AIR-2 also remains associated with both extruded polar bodies. In the embryo, AIR-2 is found on metaphase chromosomes, moves to midbody microtubules at anaphase, and then persists at the cytokinesis remnant. Disruption of AIR-2 expression by RNA- mediated interference produces entire broods of one-cell embryos that have executed multiple cell cycles in the complete absence of cytokinesis. The embryos accumulate large amounts of DNA and microtubule asters. Polar bodies are not extruded, but remain in the embryo where they continue to replicate. The cytokinesis defect appears to be late in the cell cycle because transient cleavage furrows initiate at the proper location, but regress before the division is complete. Additionally, staining with a marker of midbody microtubules revealed that at least some of the components of the midbody are not well localized in the absence of AIR-2 activity. Our results suggest that during each meiotic and mitotic division, AIR-2 may coordinate the congression of metaphase chromosomes with the subsequent events of polar body extrusion and cytokinesis.

Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease

Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.

Mechanical modulation of receptor-ligand interactions at cell-cell interfaces.

Cell surface receptors have been extensively studied because they initiate and regulate signal transduction cascades leading to a variety of functional cellular outcomes. An important class of immune receptors (e.g., T-cell antigen receptors) whose ligands are anchored to the surfaces of other cells remain poorly understood. The mechanism by which ligand binding initiates receptor phosphorylation, a process termed "receptor triggering", remains controversial. Recently, direct measurements of the (two-dimensional) receptor-ligand complex lifetimes at cell-cell interface were found to be smaller than (three-dimensional) lifetimes in solution but the underlying mechanism is unknown. At the cell-cell interface, the receptor-ligand complex spans a short intermembrane distance (15 nm) compared to long surface molecules (LSMs) whose ectodomains span >40 nm and these LSMs include phosphatases (e.g., CD45) that dephosphorylate the receptor. It has been proposed that size-based segregation of LSMs from a receptor-ligand complex is a mechanism of receptor triggering but it is unclear whether the mechanochemistry supports such small-scale segregation. Here we present a nanometer-scale mathematical model that couples membrane elasticity with the compressional stiffness and lateral mobility of LSMs. We find robust supradiffusive segregation of LSMs from a single receptor-ligand complex. The model predicts that LSM redistribution will result in a time-dependent tension on the complex leading to a decreased two-dimensional lifetime. Interestingly, the model predicts a nonlinear relationship between the three- and two-dimensional lifetimes, which can enhance the ability of receptors to discriminate between similar ligands.

Neural crest-derived stem cells migrate and differentiate into cardiomyocytes after myocardial infarction.

OBJECTIVE We recently demonstrated that primitive neural crest-derived (NC) cells migrate from the cardiac neural crest during embryonic development and remain in the heart as dormant stem cells, with the capacity to differentiate into various cell types, including cardiomyocytes. Here, we examined the migration and differentiation potential of these cells on myocardial infarction (MI). METHODS AND RESULTS We obtained double-transgenic mice by crossing protein-0 promoter-Cre mice with Floxed-enhanced green fluorescent protein mice, in which the NC cells express enhanced green fluorescent protein. In the neonatal heart, NC stem cells (NCSCs) were localized predominantly in the outflow tract, but they were also distributed in a gradient from base to apex throughout the ventricular myocardium. Time-lapse video analysis revealed that the NCSCs were migratory. Some NCSCs persisted in the adult heart. On MI, NCSCs accumulated at the ischemic border zone area (BZA), which expresses monocyte chemoattractant protein-1 (MCP-1). Ex vivo cell migration assays demonstrated that MCP-1 induced NCSC migration and that this chemotactic effect was significantly depressed by an anti-MCP-1 antibody. Small NC cardiomyocytes first appeared in the BZA 2 weeks post-MI and gradually increased in number thereafter. CONCLUSIONS These results suggested that NCSCs migrate into the BZA via MCP-1/CCR2 signaling and contribute to the provision of cardiomyocytes for cardiac regeneration after MI.

Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell–, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0–Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells—and under the right conditions—differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.

CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis.

Cancer stem cells drive tumor formation and metastasis, but how they acquire metastatic traits is not well understood. Here, we show that all colorectal cancer stem cells (CR-CSCs) express CD44v6, which is required for their migration and generation of metastatic tumors. CD44v6 expression is low in primary tumors but demarcated clonogenic CR-CSC populations. Cytokines hepatocyte growth factor (HGF), osteopontin (OPN), and stromal-derived factor 1α (SDF-1), secreted from tumor associated cells, increase CD44v6 expression in CR-CSCs by activating the Wnt/β-catenin pathway, which promotes migration and metastasis. CD44v6(-) progenitor cells do not give rise to metastatic lesions but, when treated with cytokines, acquire CD44v6 expression and metastatic capacity. Importantly, phosphatidylinositol 3-kinase (PI3K) inhibition selectively killed CD44v6 CR-CSCs and reduced metastatic growth. In patient cohorts, low levels of CD44v6 predict increased probability of survival. Thus, the metastatic process in colorectal cancer is initiated by CSCs through the expression of CD44v6, which is both a functional biomarker and therapeutic target.

Large deformation of red blood cell ghosts in a simple shear flow.

Red blood cells are known to change shape in response to local flow conditions. Deformability affects red blood cell physiological function and the hydrodynamic properties of blood. The immersed boundary method is used to simulate three-dimensional membrane-fluid flow interactions for cells with the same internal and external fluid viscosities. The method has been validated for small deformations of an initially spherical capsule in simple shear flow for both neo-Hookean and the Evans-Skalak membrane models. Initially oblate spheroidal capsules are simulated and it is shown that the red blood cell membrane exhibits asymptotic behavior as the ratio of the dilation modulus to the extensional modulus is increased and a good approximation of local area conservation is obtained. Tank treading behavior is observed and its period calculated.

Dynein pulls microtubules without rotating its stalk.

Dynein is a microtubule motor that powers motility of cilia and flagella. There is evidence that the relative sliding of the doublet microtubules is due to a conformational change in the motor domain that moves a microtubule bound to the end of an extension known as the stalk. A predominant model for the movement involves a rotation of the head domain, with its stalk, toward the microtubule plus end. However, stalks bound to microtubules have been difficult to observe. Here, we present the clearest views so far of stalks in action, by observing sea urchin, outer arm dynein molecules bound to microtubules, with a new method, "cryo-positive stain" electron microscopy. The dynein molecules in the complex were shown to be active in in vitro motility assays. Analysis of the electron micrographs shows that the stalk angles relative to microtubules do not change significantly between the ADP.vanadate and no-nucleotide states, but the heads, together with their stalks, shift with respect to their A-tubule attachments. Our results disagree with models in which the stalk acts as a lever arm to amplify structural changes. The observed movement of the head and stalk relative to the tail indicates a new plausible mechanism, in which dynein uses its stalk as a grappling hook, catching a tubulin subunit 8 nm ahead and pulling on it by retracting a part of the tail (linker).

Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo

Neutrophil extracellular traps (NETs) are released as neutrophils die in vitro in a process requiring hours, leaving a temporal gap that invasive microbes may exploit. Neutrophils capable of migration and phagocytosis while undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination. NETosis occurred during crawling, thereby casting large areas of NETs. NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA. Cells with abnormal nuclei showed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release. Additionally, live human PMNs injected into mouse skin developed decondensed nuclei and formed NETS in vivo, and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection NETosis involves neutrophils that do not undergo lysis and retain the ability to multitask.

Acute kidney injury and chronic kidney disease: an integrated clinical syndrome.

The previous conventional wisdom that survivors of acute kidney injury (AKI) tend to do well and fully recover renal function appears to be flawed. AKI can cause end-stage renal disease (ESRD) directly, and increase the risk of developing incident chronic kidney disease (CKD) and worsening of underlying CKD. In addition, severity, duration, and frequency of AKI appear to be important predictors of poor patient outcomes. CKD is an important risk factor for the development and ascertainment of AKI. Experimental data support the clinical observations and the bidirectional nature of the relationships between AKI and CKD. Reductions in renal mass and nephron number, vascular insufficiency, cell cycle disruption, and maladaptive repair mechanisms appear to be important modulators of progression in patients with and without coexistent CKD. Distinction between AKI and CKD may be artificial. Consideration should be given to the integrated clinical syndrome of diminished GFR, with acute and chronic stages, where spectrum of disease state and outcome is determined by host factors, including the balance of adaptive and maladaptive repair mechanisms over time. Physicians must provide long-term follow-up to patients with first episodes of AKI, even if they presented with normal renal function.

Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCKindependent mechanism

The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

Compensatory anti-inflammatory response syndrome.

The concept of 'Compensatory anti-inflammatory response syndrome' (CARS) was proposed in 1997 by Roger Bone (1941-1997) to qualify the consequences of the counter-regulatory mechanisms initiated to limit the overzealous inflammatory process in patients with infectious (sepsis) or non-infectious systemic inflammatory response syndrome (SIRS). One major consequence of CARS is the modification of the immune status that could favour the enhanced susceptibility of intensive care patients to nosocomial infections. Indeed, most animal 'two-hit' models illustrate an enhanced sensitivity to infection after a first insult. However, this observation is highly dependent on the experimental procedure. Numerous functions of circulating leukocytes are altered in sepsis and SIRS patients, as well as in animal models of sepsis or SIRS. However, this is rather a reprogramming of circulating leukocytes, since there is not a global defect of the immune cells functions. Furthermore, within tissues, leukocytes are rather primed or activated than immunosuppressed. Thus, CARS may be considered as an adapted compartmentalized response with the aim to silence some acute proinflammatory genes, and to maintain the possible expression of certain genes involved in the anti-infectious process.

The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans.

Studies of the mutant gene in Huntington's disease, and for eight related neurodegenerative disorders, have identified polyglutamine (polyQ) expansions as a basis for cellular toxicity. This finding has led to a disease hypothesis that protein aggregation and cellular dysfunction can occur at a threshold of approximately 40 glutamine residues. Here, we test this hypothesis by expression of fluorescently tagged polyQ proteins (Q29, Q33, Q35, Q40, and Q44) in the body wall muscle cells of Caenorhabditis elegans and show that young adults exhibit a sharp boundary at 35-40 glutamines associated with the appearance of protein aggregates and loss of motility. Surprisingly, genetically identical animals expressing near-threshold polyQ repeats exhibited a high degree of variation in the appearance of protein aggregates and cellular toxicity that was dependent on repeat length and exacerbated during aging. The role of genetically determined aging pathways in the progression of age-dependent polyQ-mediated aggregation and cellular toxicity was tested by expressing Q82 in the background of age-1 mutant animals that exhibit an extended lifespan. We observed a dramatic delay of polyQ toxicity and appearance of protein aggregates. These data provide experimental support for the threshold hypothesis of polyQ-mediated toxicity in an experimental organism and emphasize the importance of the threshold as a point at which genetic modifiers and aging influence biochemical environment and protein homeostasis in the cell.

Drosophila oocyte localization is mediated by differential cadherin-based adhesion.

In a Drosophila follicle the oocyte always occupies a posterior position among a group of sixteen germline cells. Although the importance of this cell arrangement for the subsequent formation of the anterior-posterior axis of the embryo is well documented, the molecular mechanism responsible for the posterior localization of the oocyte was unknown. Here we show that the homophilic adhesion molecule DE-cadherin mediates oocyte positioning. During follicle biogenesis, DE-cadherin is expressed in germline (including oocyte) and surrounding follicle cells, with the highest concentration of DE-cadherin being found at the interface between oocyte and posterior follicle cells. Mosaic analysis shows that DE-cadherin is required in both germline and follicle cells for correct oocyte localization, indicating that germline-soma interactions may be involved in this process. By analysing the behaviour of the oocyte in follicles with a chimaeric follicular epithelium, we find that the position of the oocyte is determined by the position of DE-cadherin-expressing follicle cells, to which the oocyte attaches itself selectively. Among the DE-cadherin positive follicle cells, the oocyte preferentially contacts those cells that express higher levels of DE-cadherin. On the basis of these data, we propose that in wild-type follicles the oocyte competes successfully with its sister germline cells for contact to the posterior follicle cells, a sorting process driven by different concentrations of DE-cadherin. This is, to our knowledge, the first in vivo example of a cell-sorting process that depends on differential adhesion mediated by a cadherin.

Kindlin-3 is essential for integrin activation and platelet aggregation

Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel. The shift is mediated through binding of the cytoskeletal protein Talin to the β subunit cytoplasmic tail. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of β-integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.

Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1.

Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.

Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin

Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, postnatally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.

A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4

S100A4 is implicated in metastasis and chronic inflammation, but its function remains uncertain. Here we establish an S100A4-dependent link between inflammation and metastatic tumor progression. We found that the acute-phase response proteins serum amyloid A (SAA) 1 and SAA3 are transcriptional targets of S100A4 via Toll-like receptor 4 (TLR4)/nuclear factor-κB signaling. SAA proteins stimulated the transcription of RANTES (regulated upon activation normal T-cell expressed and presumably secreted), G-CSF (granulocyte-colony-stimulating factor) and MMP2 (matrix metalloproteinase 2), MMP3, MMP9 and MMP13. We have also shown for the first time that SAA stimulate their own transcription as well as that of proinflammatory S100A8 and S100A9 proteins. Moreover, they strongly enhanced tumor cell adhesion to fibronectin, and stimulated migration and invasion of human and mouse tumor cells. Intravenously injected S100A4 protein induced expression of SAA proteins and cytokines in an organ-specific manner. In a breast cancer animal model, ectopic expression of SAA1 or SAA3 in tumor cells potently promoted widespread metastasis formation accompanied by a massive infiltration of immune cells. Furthermore, coordinate expression of S100A4 and SAA in tumor samples from colorectal carcinoma patients significantly correlated with reduced overall survival. These data show that SAA proteins are effectors for the metastasis-promoting functions of S100A4, and serve as a link between inflammation and tumor progression.

Blood stem cells emerge from aortic endothelium by a novel type of cell transition

The ontogeny of haematopoietic stem cells (HSCs) during embryonic development is still highly debated, especially their possible lineage relationship to vascular endothelial cells. The first anatomical site from which cells with long-term HSC potential have been isolated is the aorta-gonad-mesonephros (AGM), more specifically the vicinity of the dorsal aortic floor. But although some authors have presented evidence that HSCs may arise directly from the aortic floor into the dorsal aortic lumen, others support the notion that HSCs first emerge within the underlying mesenchyme. Here we show by non-invasive, high-resolution imaging of live zebrafish embryos, that HSCs emerge directly from the aortic floor, through a stereotyped process that does not involve cell division but a strong bending then egress of single endothelial cells from the aortic ventral wall into the sub-aortic space, and their concomitant transformation into haematopoietic cells. The process is polarized not only in the dorso-ventral but also in the rostro-caudal versus medio-lateral direction, and depends on Runx1 expression: in Runx1-deficient embryos, the exit events are initially similar, but much rarer, and abort into violent death of the exiting cell. These results demonstrate that the aortic floor is haemogenic and that HSCs emerge from it into the sub-aortic space, not by asymmetric cell division but through a new type of cell behaviour, which we call an endothelial haematopoietic transition.

Lpd depletion reveals that SRF specifies radial versus tangential migration of pyramidal neurons

During corticogenesis, pyramidal neurons (∼80% of cortical neurons) arise from the ventricular zone, pass through a multipolar stage to become bipolar and attach to radial glia, and then migrate to their proper position within the cortex. As pyramidal neurons migrate radially, they remain attached to their glial substrate as they pass through the subventricular and intermediate zones, regions rich in tangentially migrating interneurons and axon fibre tracts. We examined the role of lamellipodin (Lpd), a homologue of a key regulator of neuronal migration and polarization in Caenorhabditis elegans, in corticogenesis. Lpd depletion caused bipolar pyramidal neurons to adopt a tangential, rather than radial-glial, migration mode without affecting cell fate. Mechanistically, Lpd depletion reduced the activity of SRF, a transcription factor regulated by changes in the ratio of polymerized to unpolymerized actin. Therefore, Lpd depletion exposes a role for SRF in directing pyramidal neurons to select a radial migration pathway along glia rather than a tangential migration mode.

A kinetochore-independent mechanism drives anaphase chromosome separation during acentrosomal meiosis

Although assembly of acentrosomal meiotic spindles has been extensively studied, little is known about the segregation of chromosomes on these spindles. Here, we show in Caenorhabditis elegans oocytes that the kinetochore protein, KNL-1, directs assembly of meiotic kinetochores that orient chromosomes. However, in contrast to mitosis, chromosome separation during meiotic anaphase is kinetochore-independent. Before anaphase, meiotic kinetochores and spindle poles disassemble along with the microtubules on the poleward side of chromosomes. During anaphase, microtubules then form between the separating chromosomes. Functional analysis implicated a set of proteins that localize to a ring-shaped domain between kinetochores during pre-anaphase spindle assembly and anaphase separation. These proteins are localized by the chromosomal passenger complex, which regulates the loss of meiotic chromosome cohesion. Thus, meiotic segregation in C. elegans is a two-stage process, where kinetochores orient chromosomes, but are then dispensable for their separation. We suggest that separation is controlled by a meiosis-specific chromosomal domain to coordinate cohesin removal and chromosome segregation.

Endothelial cell membranes contain podocalyxin--the major sialoprotein of visceral glomerular epithelial cells

Podocalyxin is the major sialoprotein in the glycocalyx of glomerular podocytes. Here we report on its extraglomerular localization, using a monospecific antibody which was obtained by affinity purification of IgG on nitrocellulose transfers of glomerular podocalyxin. By indirect immunofluorescence, podocalyxin was found in the blood vessels of several organs (lung, heart, kidney, small intestine, brain, pancreas, aorta, the periportal blood vessels in liver, and the central arteries of follicles of the spleen, but not in the endothelia that line the sinusoids of the latter organs). By immunoelectron microscopy--using immunogold conjugates in diffusion ("pre-embedding") and surface ("postembedding") procedures--podocalyxin was localized on the luminal membrane domain of endothelial cells, in a patchy distribution. The presence of podocalyxin was confirmed in SDS extracts of lung tissue by immunoblotting. We conclude that (a) podocalyxin is a widespread component of endothelial plasma membranes, (b) it is restricted to the luminal membrane domain, and (c) it is distributed unevenly on the endothelial cell surface.

Reduced osteoblastic population and defective mineralization in osteopetrotic (op/op) mice.

Osteopetrotic (op/op) mice fail to exhibit bone remodeling because of a defective osteoclast formation due to a lack of macrophage colony-stimulating factor. In this study, we investigated the femora of op/op mice to clarify whether the osteoblastic population and bone mineralization are involved in osteoclasts or their bone resorption. The op/op mice extended the meshwork of trabecular bones from the chondro-osseous junction to the diaphyseal region. In the femoral metaphyses of op/op mice, intense alkaline phosphatase (ALPase)-positive osteoblasts were observed on the metaphyseal bone in close proximity to the erosion zone of the growth plates. Von Kossa's staining revealed scattered mineralized nodules and a fine meshwork of mineralized bone matrices while the wild-type littermates developed well-mineralized trabeculae parallel to the longitudinal axis. In contrast to the metaphysis, some op/op diaphyses showed flattened osteoblasts with weak ALPase-positivity, and the other diaphyses displayed bone surfaces without a covering by osteoblasts. It is likely, therefore, that the osteoblastic population and activity were lessened in the op/op diaphyses. Despite the osteopetrotic model, von Kossa's staining demonstrated patchy unmineralized areas in the op/op diaphyses, indicating that a lower population and/or the activity of osteoblasts resulted in defective mineralization in the bone. Transmission electron microscopy disclosed few osteoblasts on the diaphyseal bones, and instead, bone marrow cells and vascular endothelial cells were often attached to the unmineralized bone. Osteocytes were embedded in the unmineralized bone matrix. Thus, osteoclasts appear to be involved in the osteoblastic population and activity as well as subsequent bone mineralization.

VEGF and podocytes in diabetic nephropathy.

Vascular endothelial growth factor-A (VEGF-A) is a protein secreted by podocytes that is necessary for survival of endothelial cells, podocytes, and mesangial cells. VEGF-A regulates slit-diaphragm signaling and podocyte shape via VEGF-receptor 2-nephrin-nck-actin interactions. Chronic hyperglycemia-induced excess podocyte VEGF-A and low endothelial nitric oxide drive the development and the progression of diabetic nephropathy. The abnormal cross-talk between VEGF-A and nitric oxide pathways is fueled by the diabetic milieu, resulting in increased oxidative stress. Recent findings on these pathogenic molecular mechanisms provide new potential targets for therapy for diabetic renal disease.

Endocytic Trafficking of Rac Is Required for the Spatial Restriction of Signaling in Cell Migration

The small GTPases, Rab5 and Rac, are essential for endocytosis and actin remodeling, respectively. Coordination of these processes is critical to achieve spatial restriction of intracellular signaling, which is essential for a variety of polarized functions. Here, we show that clathrin- and Rab5-mediated endocytosis are required for the activation of Rac induced by motogenic stimuli. Rac activation occurs on early endosomes, where the RacGEF Tiam1 is also recruited. Subsequent recycling of Rac to the plasma membrane ensures localized signaling, leading to the formation of actin-based migratory protrusions. Thus, membrane trafficking of Rac is required for the spatial resolution of Rac-dependent motogenic signals. We further demonstrate that a Rab5-to-Rac circuitry controls the morphology of motile mammalian tumor cells and primordial germinal cells during zebrafish development, suggesting that this circuitry is relevant for the regulation of migratory programs in various cells, in both in vitro settings and whole organisms.

Thrombopoietin-induced Polyploidization of Bone Marrow Megakaryocytes Is Due to a Unique Regulatory Mechanism in Late Mitosis

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. However, the mechanism underlying this polyploidization remains totally unknown. It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication. We carried out immunohistochemical studies on mouse bone marrow megakaryocytes during thrombopoietin- induced polyploidization and found that during this process megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. It was clearly observed that multiple spindle poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B. Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to a skipping of mitosis, and that the megakaryocytes must have a unique regulatory mechanism in anaphase, e.g., factors regulating anaphase such as microtubule motor proteins might be involved in this polyploidization process.

Neuroinflammation in spinal cord injury: therapeutic targets for neuroprotection and regeneration.

Traumatic spinal cord injury triggers a complex local inflammatory reaction capable of enhancing repair and exacerbating pathology. The composition and effector potential of the post-injury cellular and molecular immune cascade changes as a function of time and distance from the lesion. Production along this time-space continuum of cytokines, proteases, and growth factors establishes dynamic environments that lead to the death, damage, repair or growth of affected neurons and glia. Microenvironmental cues, therefore, generated by the cells therein, may determine these distinct fates of repair versus pathology. To harness repair, it is necessary to manipulate the assembly and phenotype of cells that comprise the neuroinflammatory response to injury. Here, the potential of the neuroinflammatory response to cause outcomes such as pain, regeneration, and functional recovery is reviewed.

Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.

Blood monocytes are well-characterized precursors for macrophages and dendritic cells. Subsets of human monocytes with differential representation in various disease states are well known. In contrast, mouse monocyte subsets have been characterized minimally. In this study we identify three subpopulations of mouse monocytes that can be distinguished by differential expression of Ly-6C, CD43, CD11c, MBR, and CD62L. The subsets share the characteristics of extensive phagocytosis, similar expression of M-CSF receptor (CD115), and development into macrophages upon M-CSF stimulation. By eliminating blood monocytes with dichloromethylene-bisphosphonate-loaded liposomes and monitoring their repopulation, we showed a developmental relationship between the subsets. Monocytes were maximally depleted 18 h after liposome application and subsequently reappeared in the circulation. These cells were exclusively of the Ly-6C(high) subset, resembling bone marrow monocytes. Serial flow cytometric analyses of newly released Ly-6C(high) monocytes showed that Ly-6C expression on these cells was down-regulated while in circulation. Under inflammatory conditions elicited either by acute infection with Listeria monocytogenes or chronic infection with Leishmania major, there was a significant increase in immature Ly-6C(high) monocytes, resembling the inflammatory left shift of granulocytes. In addition, acute peritoneal inflammation recruited preferentially Ly-6C(med-high) monocytes. Taken together, these data identify distinct subpopulations of mouse blood monocytes that differ in maturation stage and capacity to become recruited to inflammatory sites.

Directed Actin Polymerization Is the Driving Force for Epithelial Cell–Cell Adhesion

We have found that epithelial cells engage in a process of cadherin-mediated intercellular adhesion that utilizes calcium and actin polymerization in unexpected ways. Calcium stimulates filopodia, which penetrate and embed into neighboring cells. E-cadherin complexes cluster at filopodia tips, generating a two-rowed zipper of embedded puncta. Opposing cell surfaces are clamped by desmosomes, while vinculin, zyxin, VASP, and Mena are recruited to adhesion zippers by a mechanism that requires alpha-catenin. Actin reorganizes and polymerizes to merge puncta into a single row and seal cell borders. In keratinocytes either null for alpha-catenin or blocked in VASP/Mena function, filopodia embed, but actin reorganization/polymerization is prevented, and membranes cannot seal. Taken together, a dynamic mechanism for intercellular adhesion is unveiled involving calcium-activated filopodia penetration and VASP/Mena-dependent actin reorganization/polymerization.

Emergent structures and dynamics of cell colonies by contact inhibition of locomotion

Cells in tissues can organize into a broad spectrum of structures according to their function. Drastic changes of organization, such as epithelial-mesenchymal transitions or the formation of spheroidal aggregates, are often associated either to tissue morphogenesis or to cancer progression. Here, we study the organization of cell colonies by means of simulations of self-propelled particles with generic cell-like interactions. The interplay between cell softness, cell-cell adhesion, and contact inhibition of locomotion (CIL) yields structures and collective dynamics observed in several existing tissue phenotypes. These include regular distributions of cells, dynamic cell clusters, gel-like networks, collectively migrating monolayers, and 3D aggregates. We give analytical predictions for transitions between noncohesive, cohesive, and 3D cell arrangements. We explicitly show how CIL yields an effective repulsion that promotes cell dispersal, thereby hindering the formation of cohesive tissues. Yet, in continuous monolayers, CIL leads to collective cell motion, ensures tensile intercellular stresses, and opposes cell extrusion. Thus, our work highlights the prominent role of CIL in determining the emergent structures and dynamics of cell colonies.

Polo kinase and Asp are needed to promote the mitotic organizing activity of centrosomes

Interfering with the activity of polo-like kinases can lead to the formation of monopolar spindles. Polo-like kinases also regulate mitotic entry, activation of the anaphase-promoting complex and the necessary preconditions for cytokinesis. Similarities between the phenotypes of the Drosophila mutants asp and polo point towards a common role in spindle pole function. The abnormal spindles of asp mutants are bipolar but have disorganized broad poles at which γ-tubulin has an abnormal distribution. Moreover, the synergism or of polo1 aspE3 double mutants indicates a possible involvement of these genes in a common process. Asp is a microtubule-associated protein of relative molecular mass 220,000 (Mr 220K) found at the face of the centrosome that contacts spindle microtubules. In partially purified centrosomes, it is required with γ-tubulin to organize microtubule asters. Here, we show that Asp is the previously identified Mr 220K substrate of Polo kinase. Polo phosphorylates Asp in vitro, converting it into an MPM2 epitope. Polo and Asp proteins immunoprecipitate together and exist as part of a 25–38S complex. Extracts of polo-derived embryos are unable to restore the ability of salt-stripped centrosomes to nucleate microtubule asters. This can be rescued by addition of phosphorylated Asp or active Polo kinase.

Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines

The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin-coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain.

Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.

Despite accumulating evidence suggesting local self-maintenance of tissue macrophages in the steady state, the dogma remains that tissue macrophages derive from monocytes. Using parabiosis and fate-mapping approaches, we confirmed that monocytes do not show significant contribution to tissue macrophages in the steady state. Similarly, we found that after depletion of lung macrophages, the majority of repopulation occurred by stochastic cellular proliferation in situ in a macrophage colony-stimulating factor (M-Csf)- and granulocyte macrophage (GM)-CSF-dependent manner but independently of interleukin-4. We also found that after bone marrow transplantation, host macrophages retained the capacity to expand when the development of donor macrophages was compromised. Expansion of host macrophages was functional and prevented the development of alveolar proteinosis in mice transplanted with GM-Csf-receptor-deficient progenitors. Collectively, these results indicate that tissue-resident macrophages and circulating monocytes should be classified as mononuclear phagocyte lineages that are independently maintained in the steady state.

Cell and molecular mechanisms of insulin-induced angiogenesis

Angiogenesis, the development of new blood vessel from pre-existing vessels, is a key process in the formation of the granulation tissue during wound healing. The appropriate development of new blood vessels, along with their subsequent maturation and differentiation, establishes the foundation for functional wound neovasculature. We performed studies in vivo and used a variety of cellular and molecular approaches in vitro to show that insulin stimulates angiogenesis and to elucidate the signalling mechanisms by which this protein stimulates microvessel development. Mice skin injected with insulin shows longer vessels with more branches, along with increased numbers of associated alpha-smooth muscle actin-expressing cells, suggesting the appropriate differentiation and maturation of the new vessels. We also found that insulin stimulates human microvascular endothelial cell migration and tube formation, and that these effects occur independently of VEGF/VEGFR signalling, but are dependent upon the insulin receptor itself. Downstream signalling pathways involve PI3K, Akt, sterol regulatory element-binding protein 1 (SREBP-1) and Rac1; inhibition of these pathways results in elimination of endothelial cell migration and tube formation and significantly decreases the development of microvessels. Our findings strongly suggest that insulin is a good candidate for the treatment of ischaemic wounds and other conditions in which blood vessel development is impaired.

Nitrite protects against morbidity and mortality associated with TNF- or LPS-induced shock in a soluble guanylate cyclase–dependent manner

Nitrite (NO(2)(-)), previously viewed as a physiologically inert metabolite and biomarker of the endogenous vasodilator NO, was recently identified as an important biological NO reservoir in vasculature and tissues, where it contributes to hypoxic signaling, vasodilation, and cytoprotection after ischemia-reperfusion injury. Reduction of nitrite to NO may occur enzymatically at low pH and oxygen tension by deoxyhemoglobin, deoxymyoglobin, xanthine oxidase, mitochondrial complexes, or NO synthase (NOS). We show that nitrite treatment, in sharp contrast with the worsening effect of NOS inhibition, significantly attenuates hypothermia, mitochondrial damage, oxidative stress and dysfunction, tissue infarction, and mortality in a mouse shock model induced by a lethal tumor necrosis factor challenge. Mechanistically, nitrite-dependent protection was not associated with inhibition of mitochondrial complex I activity, as previously demonstrated for ischemia-reperfusion, but was largely abolished in mice deficient for the soluble guanylate cyclase (sGC) alpha1 subunit, one of the principal intracellular NO receptors and signal transducers in the cardiovasculature. Nitrite could also provide protection against toxicity induced by Gram-negative lipopolysaccharide, although higher doses were required. In conclusion, we show that nitrite can protect against toxicity in shock via sGC-dependent signaling, which may include hypoxic vasodilation necessary to maintain microcirculation and organ function, and cardioprotection.

Steroid receptor interactions with heat shock protein and immunophilin chaperones.

We have provided a historical perspective on a body of steroid receptor research dealing with the structure and physiological significance of the untransformed 9S receptor that has often confused both novice and expert investigators. The frequent controversies and equivocations of earlier studies were due to the fact that the native, hormone-free state of these receptors is a large multiprotein complex that resisted description for many years because of its unstable and dynamic nature. The untransformed 9S state of the steroid and dioxin receptors has provided a unique system for studying the function of the ubiquitous, abundant, and conserved heat shock protein, hsp90. The hormonal control of receptor association with hsp90 provided a method of manipulating the receptor heterocomplex in a manner that was physiologically meaningful. For several steroid receptors, binding to hsp90 was required for the receptor to be in a native hormone-binding state, and for all of the receptors, hormone binding promoted dissociation of the receptor from hsp90 and conversion of the receptor to the DNA-binding state. Although the complexes between tyrosine kinases and hsp90 were discovered earlier, the hormonal regulation or steroid receptor association with hsp90 permitted much more rapid and facile study of hsp90 function. The observations that hsp90 binds to the receptors through their HBDs and that these domains can be fused to structurally different proteins bringing their function under hormonal control provided a powerful linkage between the hormonal regulation of receptor binding to hsp90 and the initial step in steroid hormone action. Because the 9S receptor hsp90 heterocomplexes could be physically stabilized by molybdate, their protein composition could be readily studied, and it became clear that these complexes are multiprotein structures containing a number of unique proteins, such as FKBP51, FKBP52, CyP-40, and p23, that were discovered because of their presence in these structures. Further analysis showed that hsp90 itself exists in a variety of native multiprotein heterocomplexes independent of steroid receptors and other 'substrate' proteins. Cell-free systems can now be used to study the formation of receptor heterocomplexes. As we outlined in the scheme of Fig. 1, the multicomponent receptor-hsp90 heterocomplex assembly system is being reconstituted, and the importance of individual proteins, such as hsp70, p60, and p23, in the assembly process is becoming recognized. It should be noted that our understanding of the mechanism and purpose of steroid receptor heterocomplex assembly is still at an early stage. We can now speculate on the roles of receptor-associated proteins in receptor action, both as individuals and as a group, but their actual functions are still vague or unknown. We can make realistic models about the chaperoning and trafficking of steroid receptors, but we don't yet know how these processes occur, we don't know where chaperoning occurs in the cell (e.g. Is it limited to the cytoplasm? Is it a diffuse process or does chaperoning occur in association with structural elements?), and, with the exception of the requirement for hormone binding, we don't know the extent to which the hsp90-based chaperone system impacts on steroid hormone action. It is not yet clear how far the discovery of this hsp90 heterocomplex assembly system will be extended to the development of a general understanding of protein processing in the cell. Because this assembly system is apparently present in all eukaryotic cells, it probably performs an essential function for many proteins. The bacterial homolog of hsp90 is not an essential protein, but hsp90 is essential in eukaryotes, and recent studies indicate that the development of the cell nucleus from prokaryotic progenitors was accompanied by the duplication of genes for hsp90 and hsp70 (698). (ABSTRACT TRUNCATED)

The Histone H3 Lysine-27 Demethylase Jmjd3 Links Inflammation to Inhibition of Polycomb-Mediated Gene Silencing

Epigenetic chromatin marks restrict the ability of differentiated cells to change gene expression programs in response to environmental cues and to transdifferentiate. Polycomb group (PcG) proteins mediate gene silencing and repress transdifferentiation in a manner dependent on histone H3 lysine 27 trimethylation (H3K27me3). However, macrophages migrated into inflamed tissues can transdifferentiate, but it is unknown whether inflammation alters PcG-dependent silencing. Here we show that the JmjC-domain protein Jmjd3 is a H3K27me demethylase expressed in macrophages in response to bacterial products and inflammatory cytokines. Jmjd3 binds PcG target genes and regulates their H3K27me3 levels and transcriptional activity. The discovery of an inducible enzyme that erases a histone mark controlling differentiation and cell identity provides a link between inflammation and reprogramming of the epigenome, which could be the basis for macrophage plasticity and might explain the differentiation abnormalities in chronic inflammation.

Electron microscopic studies of macrophages in early human yolk sacs.

Distribution and fine structure of macrophages were studied in 10 human embryos in the 6th and 7th week of gestation, 5.5 to 12 mm in crown-rump length. The yolk sac macrophages were found in the extravascular mesenchymal tissues and intravascular spaces long before the first appearance of bone marrow and lymphatic tissues in the embryos. In addition to the macrophages, the fibroblastic cells and the cells of erythropoietic series were also present in the extravascular space. The macrophages showed a variety of cellular structures suggesting transition from immature cell type with no heterophagolysosomes to mature cell type in phagocytosis. The mature macrophages avidly phagocytized the primitive erythroblasts and occasionally platelets. They were positively stained for lysosomal enzymes and were characterized by numerous pleomorphic heterophagolysosomes which exhibited various stages of digestion of phagocytized blood cells. The origin of intravascular macrophages may be in either migrated extravascular macrophages or phagocytic endothelial cells. The phagocytosis and degradation of erythroblasts appear to be one of the main functions of yolk sac macrophages. The presence of the macrophages in mitosis indicates their proliferation in situ.

Actin dynamics and cell-cell adhesion in epithelia.

Recent advances in the field of intercellular adhesion highlight the importance of adherens junction association with the underlying actin cytoskeleton. In skin epithelial cells a dynamic feature of adherens junction formation involves filopodia, which physically project into the membrane of adjacent cells, catalyzing the clustering of adherens junction protein complexes at their tips. In turn, actin polymerization is stimulated at the cytoplasmic interface of these complexes. Although the mechanism remains unclear, the VASP/Mena family of proteins seems to be involved in organizing actin polymerization at these sites. In vivo, adherens junction formation appears to rely upon filopodia in processes where epithelial sheets must be physically moved closer to form stable intercellular connections, for example, in ventral closure in embryonic development or wound healing in the postnatal animal.

Inflammatory Reaction as Determinant of Foreign Body Reaction Is an Early and Susceptible Event after Mesh Implantation

PURPOSE To investigate and relate the ultrashort-term and long-term courses of determinants for foreign body reaction as biocompatibility predictors for meshes in an animal model. MATERIALS AND METHODS Three different meshes (TVT, UltraPro, and PVDF) were implanted in sheep. Native and plasma coated meshes were placed bilaterally: (a) interaperitoneally, (b) as fascia onlay, and (c) as muscle onlay (fascia sublay). At 5 min, 20 min, 60 min, and 120 min meshes were explanted and histochemically investigated for inflammatory infiltrate, macrophage infiltration, vessel formation, myofibroblast invasion, and connective tissue accumulation. The results were related to long-term values over 24 months. RESULTS Macrophage invasion reached highest extents with up to 60% in short-term and decreased within 24 months to about 30%. Inflammatory infiltrate increased within the first 2 hours, the reached levels and the different extents and ranking among the investigated meshes remained stable during long-term follow up. For myofibroblasts, connective tissue, and CD31+ cells, no activity was detected during the first 120 min. CONCLUSION The local inflammatory reaction is an early and susceptible event after mesh implantation. It cannot be influenced by prior plasma coating and does not depend on the localisation of implantation.

Conditional Kif3a ablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis.

The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain ill-defined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and micro-computed tomography (microCT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.

A role for mitochondria in NLRP3 inflammasome activation

An inflammatory response initiated by the NLRP3 inflammasome is triggered by a variety of situations of host ‘danger’, including infection and metabolic dysregulation. Previous studies suggested that NLRP3 inflammasome activity is negatively regulated by autophagy and positively regulated by reactive oxygen species (ROS) derived from an uncharacterized organelle. Here we show that mitophagy/autophagy blockade leads to the accumulation of damaged, ROS-generating mitochondria, and this in turn activates the NLRP3 inflammasome. Resting NLRP3 localizes to endoplasmic reticulum structures, whereas on inflammasome activation both NLRP3 and its adaptor ASC redistribute to the perinuclear space where they co-localize with endoplasmic reticulum and mitochondria organelle clusters. Notably, both ROS generation and inflammasome activation are suppressed when mitochondrial activity is dysregulated by inhibition of the voltage-dependent anion channel. This indicates that NLRP3 inflammasome senses mitochondrial dysfunction and may explain the frequent association of mitochondrial damage with inflammatory diseases.

Abi1 is essential for the formation and activation of a WAVE2 signalling complex

WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2–Abi1–Nap1–PIR121 complex. The WAVE2–Abi1–Nap1–PIR121 complex is as active as the WAVE2–Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.

N- and E-cadherins in Xenopus are specifically required in the neural and non-neural ectoderm, respectively, for F-actin assembly and morphogenetic movements.

Transmembrane cadherins are calcium-dependent intercellular adhesion molecules. Recently, they have also been shown to be sites of actin assembly during adhesive contact formation. However, the roles of actin assembly on transmembrane cadherins during development are not fully understood. We show here, using the developing ectoderm of the Xenopus embryo as a model, that F-actin assembly is a primary function of both N-cadherin in the neural ectoderm and E-cadherin in the non-neural (epidermal) ectoderm, and that each cadherin is essential for the characteristic morphogenetic movements of these two tissues. However, depletion of N-cadherin and E-cadherin did not cause dissociation in these tissues at the neurula stage, probably owing to the expression of C-cadherin in each tissue. Depletion of each of these cadherins is not rescued by the other, nor by the expression of C-cadherin, which is expressed in both tissues. One possible reason for this is that each cadherin is expressed in a different domain of the cell membrane. These data indicate the combinatorial nature of cadherin function, the fact that N- and E-cadherin play primary roles in F-actin assembly in addition to roles in cell adhesion, and that this function is specific to individual cadherins. They also show how cell adhesion and motility can be combined in morphogenetic tissue movements that generate the form and shape of the embryonic organs.

Immune complex relay by subcapsular sinus macrophages and non-cognate B cells drives antibody affinity maturation

Subcapsular sinus (SCS) macrophages capture antigens from lymph and present them intact for B cell encounter and follicular delivery. However, the properties of SCS macrophages are poorly defined. Here we show SCS macrophage development depended on lymphotoxin-alpha1beta2, and the cells had low lysosomal enzyme expression and retained opsonized antigens on their surface. Intravital imaging revealed immune complexes moving along macrophage processes into the follicle. Moreover, noncognate B cells relayed antigen opsonized by newly produced antibodies from the subcapsular region to the germinal center, and affinity maturation was impaired when this transport process was disrupted. Thus, we characterize SCS macrophages as specialized antigen-presenting cells functioning at the apex of an antigen transport chain that promotes humoral immunity.

Estrogen receptor-alpha promotes alternative macrophage activation during cutaneous repair.

Efficient local monocyte/macrophage recruitment is critical for tissue repair. Recruited macrophages are polarized toward classical (proinflammatory) or alternative (prohealing) activation in response to cytokines, with tight temporal regulation crucial for efficient wound repair. Estrogen acts as a potent anti-inflammatory regulator of cutaneous healing. However, an understanding of estrogen/estrogen receptor (ER) contribution to macrophage polarization and subsequent local effects on wound healing is lacking. Here we identify, to our knowledge previously unreported, a role whereby estrogen receptor α (ERα) signaling preferentially polarizes macrophages from a range of sources to an alternative phenotype. Cell-specific ER ablation studies confirm an in vivo role for inflammatory cell ERα, but not ERβ, in poor healing associated with an altered cytokine profile and fewer alternatively activated macrophages. Furthermore, we reveal intrinsic changes in ERα-deficient macrophages, which are unable to respond to alternative activation signals in vitro. Collectively, our data reveal that inflammatory cell-expressed ERα promotes alternative macrophage polarization, which is beneficial for timely healing. Given the diverse physiological roles of ERs, these findings will likely be of relevance to many pathologies involving excessive inflammation.

A C-Type Lectin Collaborates with a CD45 Phosphatase Homolog to Facilitate West Nile Virus Infection of Mosquitoes

West Nile virus (WNV) is the most common arthropod-borne flavivirus in the United States; however, the vector ligand(s) that participate in infection are not known. We now show that an Aedes aegypti C-type lectin, mosGCTL-1, is induced by WNV, interacts with WNV in a calcium-dependent manner, and facilitates infection in vivo and in vitro. A mosquito homolog of human CD45 in A. aegypti, designated mosPTP-1, recruits mosGCTL-1 to enable viral attachment to cells and to enhance viral entry. In vivo experiments show that mosGCTL-1 and mosPTP-1 function as part of the same pathway and are critical for WNV infection of mosquitoes. A similar phenomenon was also observed in Culex quinquefasciatus, a natural vector of WNV, further demonstrating that these genes participate in WNV infection. During the mosquito blood-feeding process, WNV infection was blocked in vivo with mosGCTL-1 antibodies. A molecular understanding of flaviviral-arthropod interactions may lead to strategies to control viral dissemination in nature.

Recovery of visual response of injured adult rat optic nerves treated with transglutaminase.

Failure of axons of the central nervous system in adult mammals to regenerate spontaneously after injury is attributed in part to inhibitory molecules associated with oligodendrocytes. Regeneration of central nervous system axons in fish is correlated with the presence of a transglutaminase. This enzyme dimerizes interleukin-2, and the product is cytotoxic to oligodendrocytes in vitro. Application of this nerve-derived transglutaminase to rat optic nerves, in which the injury had caused the loss of visual evoked potential response to light, promoted the recovery of that response within 6 weeks after injury. Transmission electron microscopy analysis revealed the concomitant appearance of axons in the distal stump of the optic nerve.

A Whole Brain Staining, Embedding, and Clearing Pipeline for Adult Zebrafish to Visualize Cell Proliferation and Morphology in 3-Dimensions

The field of macro-imaging has grown considerably with the appearance of innovative clearing methods and confocal microscopes with lasers capable of penetrating increasing tissue depths. The ability to visualize and model the growth of whole organs as they develop from birth, or with manipulation, disease or injury, provides new ways of thinking about development, tissue-wide signaling, and cell-to-cell interactions. The zebrafish (Danio rerio) has ascended from a predominantly developmental model to a leading adult model of tissue regeneration. The unmatched neurogenic and regenerative capacity of the mature central nervous system, in particular, has received much attention, however tools to interrogate the adult brain are sparse. At present there exists no straightforward methods of visualizing changes in the whole adult brain in 3-dimensions (3-D) to examine systemic patterns of cell proliferation or cell populations of interest under physiological, injury, or diseased conditions. The method presented here is the first of its kind to offer an efficient step-by-step pipeline from intraperitoneal injections of the proliferative marker, 5-ethynyl-2'-deoxyuridine (EdU), to whole brain labeling, to a final embedded and cleared brain sample suitable for 3-D imaging using optical projection tomography (OPT). Moreover, this method allows potential for imaging GFP-reporter lines and cell-specific antibodies in the presence or absence of EdU. The small size of the adult zebrafish brain, the highly consistent degree of EdU labeling, and the use of basic clearing agents, benzyl benzoate, and benzyl alcohol, makes this method highly tractable for most laboratories interested in understanding the vertebrate central nervous system in health and disease. Post-processing of OPT-imaged adult zebrafish brains injected with EdU illustrate that proliferative patterns in EdU can readily be observed and analyzed using IMARIS and/or FIJI/IMAGEJ software. This protocol will be a valuable tool to unlock new ways of understanding systemic patterns in cell proliferation in the healthy and injured brain, brain-wide cellular interactions, stem cell niche development, and changes in brain morphology.

Speed Limits for Nonvesicular Intracellular Sterol Transport.

Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by nonvesicular mechanisms requiring sterol transport proteins (STPs). Here we examine the idea that transport is enhanced at membrane contact sites where the ER is closely apposed to the PM. We conclude that sterol desorption from the membrane, rather than STP-mediated diffusion, is rate limiting in the cellular context, so there is no apparent kinetic benefit to having STP-mediated sterol transfer occur at contact sites. Contact sites may instead compartmentalize lipid synthesis or transport machinery, providing opportunities for regulation.