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Drax Biomass invests in greenhouse gas efficiencies
Truck dumps, a new train line and use of sawmill residues are helping our own biomass producer to lower its carbon emissions.
Why we need the whole country on the same frequency
Riding the fluctuations of Britain's electricity network.
Sale of gas assets for £193.3 million and trading update
Everything you need to know about biomass and biomass energy
The inside of a cooling tower looks like no place on earth
Step into the vast, eerie structures at the heart of Drax Power Station.
We have increased the capacity at Drax Biomass Amite and Morehouse pellet plants to increase capacity and made them more greenhouse gas (GHG) efficient. Central to the projects was the addition of storage silos and handling equipment to allow increased use of dry shavings and other mill residuals. The developments included the addition of an extra truck dump at each facility to allow delivery of increased volumes of these feedstocks.
Use of mill residuals and dry shavings reduces the energy required to make a pellet, as such material does not need to be de-barked, chipped and re-sized in the same way as roundwood. Some of the material has a low moisture content and is therefore able to enter the process after the dryer, which effectively increases the capacity of each plant. This drives down the average GHG emission per tonne of pellets produced. A key measure of this is the KWh of electricity per tonne of pellets, and we saw this reduce by about 10% in the final months of the year compared with the start of the year, with further savings anticipated.
LaSalle BioEnergy in Louisiana
At LaSalle, a significant amount of our investment is going into allowing pellets to be transported to the port by rail, rather than truck. For the 250 km trip to Baton Rouge, a significant carbon saving compared to trucks will be achieved when LaSalle reaches its capacity of 450,000 tonnes per year. Moving pellets by rail should start in the next year.
Charge. Recharge. The evolution of batteries
Batteries have been useful for over 200 years, but the ways we use them are ever-changing
What is renewable energy?
Renewable energy is produced from a resource that is infinite or can be replenished on a human timescale, such as the sun, wind, water or sustainably managed forests.
Energy white paper "recognises negative emissions and sustainable biomass as a vital technology to achieve net zero" - Drax Group CEO
Will Gardiner responds to 'Energy white paper: Powering our net zero future'


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The loan process at the bank put people through a vetting process where they had to explain why they needed the loan and how they would go about paying it.
Then came along credit cards. The credit card companies quickly learned that they could make large amounts of money from giving away easy credit without making people go through the banking vetting process.
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The information provided within this website about consumer proposals is protected by copyright law. Any distribution or reproduction in any way, shape, means or manner is expressly prohibited by law without the the prior written consent of the owner(s) of the copyright.
This site is for information only and is not meant to replace qualified legal advice by a Licensed Insolvency Trustee the writers shall not be held responsible for any information that may be incorrect or out of date in regards to consumer proposals. The owners of this website recommend that anyone wishing to learn about how a trustee can deal with your financial issues should contact our office.


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Chatter sets final 2019 performance
By Kathaleen Roberts / Journal Staff Writer
Sunday, December 22nd, 2019 at 12:02am
James Shields
ALBUQUERQUE, N.M. — For its final performance of 2019, Chatter will breathe life into masterworks by Mozart and Igor Stravinsky.
Fifteen musicians will tackle Stravinsky's "Dumbarton Oaks" concerto, as well as Mozart's Concerto in A Major, the first major concerto written for clarinet.
Stravinsky is perhaps best known for his ballet trio: "Petrouschka," "The Firebird" and "The Rite of Spring."
Chatter associate artistic director and clarinetist James Shields has long been captivated by the Dumbarton concerto, written in the neoclassical style in 1837-38. It was the last piece the composer penned in Europe; he finished it in Paris. It was heavily inspired by Bach's Brandenburg Concertos.
"It's a really vibrant, fun and rhythmically exacting piece," Shields said in a telephone interview from Portland, where he plays principal clarinet in the Oregon Symphony. "It's got that bigger, virtuosic style, but the harmony is a little off. So it has these echoes of Baroque rhythms with modern harmonies.
"It's echoes of Bach with Cubist harmonies."
The Concerto in A Major was Mozart's last. At the time of his death in 1791, the clarinet was still a relatively new instrument. Mozart wrote the piece for his clarinetist friend Anton Stadler. The work was intended for an extended instrument known as the basset clarinet. The basset could play four extra semi-tones lower than our modern instrument. Mozart took advantage of its acrobatic and dramatic possibilities.
"It's a very sublime concerto," Shields said.
Its slow movement – the adagio – has been used in films such as "Out of Africa."
"It's a major test piece for clarinetists and students," Shields added. "You have to have grace and style in all the movements, even the faster technical stuff."
Shields will be playing a custom-made instrument produced in Burnaby, Canada.
"Mozart really took advantage of this extended low range," he said. "It's almost operatic."
WHAT: Chatter Sunday
WHEN: 10:30 a.m. Sunday, Dec. 29
WHERE: Las Puertas Event Center, 1512 First NW
HOW MUCH: $16 adults; $9 under 30; $5 children at chatterabq.org/boxoffice.
Life in NM

redpajama_set_name: RedPajamaCommonCrawl


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Exclusive: Take A Look Inside A Riverside County Field Hospital Set Up To Treat Coronavirus Patients
Filed Under:Coronavirus, indio, Riverside County
INDIO (CBSLA) — When the call went out for retired medical professionals and students to volunteer their services in the wake of the coronavirus pandemic, thousands stepped up.
The first wave of volunteers will be headed to a field hospital set up at the Riverside County Fairgrounds in Indio where they will be testing and treating patients.
"We've got some challenges," Shane Reichardt, with Riverside County's Emergency Management Department, said.
Reichardt said health care workers there, like many across the nation, are dealing with a limited supply of personal protective equipment and testing kits.
"These are things they've never seen before in their careers," he said.
But, fortunately, the workers on the frontline of the crisis were not dealing with a limited supply of help manning new field hospitals that have begun popping up across the state.
RELATED: Riverside County Announces 80 New Coronavirus Cases, 4 Additional Deaths
The California Health Corps put out a call for volunteers to prepare for the anticipated surge in coronavirus cases. The new volunteers will care for less severe patients at field hospitals like the one in Indio.
And, across the state, 35,000 people have already answered the call to help including Thomas Rood.
"Been an EMT since I was 18, so right out of high school," Rood said.
Rood was supposed to ship out to Georgia this week to join the United States Army, but he said all new recruits have been delayed due to the pandemic, so he decided to dedicate his medical training to the biggest public health crisis his generation has ever seen.
"I can just sit at home and self-quarantine, or I can actually get out and help out with the situation we have," he said.
He will soon begin his new assignment at the Riverside County Fairgrounds where up to 125 patients can be quarantined and treated in two buildings to relieve the strain on local hospitals as they treat those in need of critical, life-saving care.
"We've been so amazed by the outpouring of support," Michael Osur, Riverside County Public Health assistant director, said.
Osur said that while it's difficult to predict when the number of cases will peak in each county, the surge will be here soon.
"It's like a tsunami wave," he said. "It's coming, and we want to be prepared."


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search filter All ContentAll JournalsJournal of Cell Biology
Articles| September 23 2002
T cell receptor ligation induces the formation of dynamically regulated signaling assemblies
Stephen C. Bunnell,
Stephen C. Bunnell
1Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
David I. Hong,
David I. Hong
Julia R. Kardon,
Julia R. Kardon
Tetsuo Yamazaki,
Tetsuo Yamazaki
C. Jane McGlade,
C. Jane McGlade
2Department of Medical Biophysics and Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
Valarie A. Barr,
Valarie A. Barr
Lawrence E. Samelson
Address correspondence to Lawrence Samelson, Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bldg. 37, Rm. 1E24, Bethesda, MD 20892. Tel.: (301) 496-9683. Fax: (301) 496-8479. E-mail: samelson@helix.nih.gov
Received: March 08 2002
Revision Received: August 23 2002
Accepted: August 27 2002
The Rockefeller University Press
J Cell Biol (2002) 158 (7): 1263–1275.
https://doi.org/10.1083/jcb.200203043
Revision Received:
Stephen C. Bunnell, David I. Hong, Julia R. Kardon, Tetsuo Yamazaki, C. Jane McGlade, Valarie A. Barr, Lawrence E. Samelson; T cell receptor ligation induces the formation of dynamically regulated signaling assemblies . J Cell Biol 30 September 2002; 158 (7): 1263–1275. doi: https://doi.org/10.1083/jcb.200203043
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.
Signaling pathways activated after engagement of the T cell antigen receptor (TCR)* have been studied using biochemical techniques for over a decade (for review see Koretzky and Myung, 2001; Yablonski and Weiss, 2001; Samelson, 2002). Signaling downstream of the TCR is initiated when the signal-transducing subunits (CD3 and TCRζ) of engaged TCRs are phosphorylated by the Src-family protein tyrosine kinases (PTKs) Lck and Fyn. These modifications occur within immunoreceptor tyrosine-based activation motifs and direct the recruitment of ZAP-70, a Syk-family PTK, to the TCR via its tandem Src-homology 2 domains. Once recruited, ZAP-70 is phosphorylated and activated by Lck and Fyn. Activated ZAP-70 phosphorylates several substrates, including LAT and SLP-76, adapter proteins required for optimal T cell activation and development (Finco et al., 1998; Yablonski et al., 1998; Zhang et al., 1999). The phosphorylation of LAT, a lipid raft-associated integral membrane protein, creates docking sites for various Src-homology 2 domains. PLC-γ1 and the adapters Grb2, Grap, and Gads bind to LAT in this manner (Zhang et al., 1998a). SLP-76 is also recruited into LAT-nucleated complexes through its interaction with the COOH-terminal SH3 domain of Gads (Liu et al., 1999). SLP-76 provides an additional platform for the recruitment of signaling molecules, including PLC-γ1, the Rho-family GTPase nucleotide exchange factor Vav, the adapter Nck, and the Tec-family tyrosine kinase Itk (Wu et al., 1996; Bubeck Wardenburg et al., 1998; Bunnell et al., 2000; Yablonski et al., 2001). c-Cbl, an adapter and ubiquitin ligase, also associates with LAT, although by an undefined mechanism (Zhang et al., 1998a). Because these associations have primarily been defined in coimmunoprecipitation studies, relatively little is known about the initiation, maturation, dynamic composition, and stoichiometry of these complexes.
Various imaging studies have established that T cells that engage antigen-presenting cells (APCs) bearing stimulatory MHC-peptide complexes undergo macromolecular rearrangements that result in the formation of an immune synapse. This structure develops over 5–30 min as a result of active cytoskeletal processes and subdivides the T cell–APC interface into two concentric zones: the central and peripheral supramolecular activation clusters (cSMAC and pSMAC) (for review see Delon and Germain, 2000). The cSMAC is enriched in the TCR, CD2, and CD28, whereas the pSMAC contains integrins and cytoskeletal proteins. Larger glycoproteins, such as CD43 and CD45, are initially excluded from the entire synapse (Sperling et al., 1998; Johnson et al., 2000). Despite the dramatic nature of these rearrangements, the function of the immune synapse remains elusive.
Initial reports describing the immune synapse emphasized the correlation between SMAC formation and productive T cell activation (Monks et al., 1998; Grakoui et al., 1999). However, neither thymocytes nor T helper 2 CD4-positive T cells (Th2) cells develop immune synapses during their activation (Balamuth et al., 2001; Richie et al., 2002). Additionally, increases in cellular phosphotyrosine, intracellular calcium elevations, the dephosphorylation of moesin, and cytoskeletal rearrangements are all initiated within seconds of receptor engagement and peak within 2 to 3 min of TCR engagement (Negulescu et al., 1996; Wulfing and Davis, 1998; Bunnell et al., 2001; Delon et al., 2001). Therefore, the signals driving these events must be derived from TCRs ligated before the formation of a cSMAC (Monks et al., 1998; Grakoui et al., 1999; Dustin and Cooper, 2000; Lee et al., 2002). Several reports have indicated that TCRs are initially engaged at the periphery of the T cell–APC contact or in small clusters within the maturing synapse (Grakoui et al., 1999; Dustin and Cooper, 2000; Krummel and Davis, 2002). More recently, peak levels of activated Lck and ZAP-70 were found with the TCR in a peripheral zone before the coalescence of the engaged TCRs into a cSMAC (Lee et al., 2002), suggesting that signal transduction is initiated in these small, TCR-rich clusters, before the formation of a mature SMAC.
Dynamic studies of high temporal resolution are needed to relate the biochemical events described above to the complex morphological changes associated with T cell activation. To this end, we adapted a method developed in order to simultaneously visualize T cell contacts and the rearrangement of enhanced GFP (EGFP)-tagged actin in live, activated T cells (Bunnell et al., 2001). This approach constrains responding T cells to the plane of a stimulatory coverslip, allowing the rapid collection of high-resolution images of chimeric proteins participating in signaling complexes within developing contacts. Our results establish that fully functional signaling complexes form rapidly within membrane domains tightly associated with the stimulatory surface, that components of these complexes are continuously dissociating and reassociating, and that the components of these complexes exit by distinct mechanisms. These results reveal both the speed of TCR-induced signal initiation and the dynamically changing composition of the resulting signaling complexes.
Actin-rich protrusions initiate T cell coverslip contacts
T cells engaging TCR-specific stimulatory antibodies on a coverslip establish contact through protruding filopodia and lamellipodia (Bunnell et al., 2001). These contacts trigger dramatic cytoskeletal alterations which result in the spreading of the T cells across the coverslip and the development of a peripheral actin-rich zone (Video 1). To determine if structures rich in filamentous actin initiate T cell coverslip contacts, we imaged EGFP-actin within the contact while collecting interference reflection microscopy (IRM) images of the contact. As shown in Fig. 1, the tips of projecting lamellipodia (top) are clearly associated with darker structures corresponding to points of close contact (bottom). Thus, actin-dependent protrusive structures may promote the formation of tight contacts between the T cell and coverslip.
The TCR is specifically clustered within T cell coverslip contacts
To determine whether TCR-dependent signals are initiated in these close contacts, we tracked the sub-cellular localization of the subunits of the TCR. T cells were introduced into chambered stimulatory coverslips, fixed at various times after plating, and processed for immunofluorescence. TCRζ and Vβ8, the TCRβ chain present in Jurkat T cells, are observed in clusters 0.5–1.0 μm in size shortly after T cell coverslip contact (Fig. 2 A, left). These clusters colocalize with the tight contacts (darker spots) observed by IRM (Fig. 2 A, center and right, overlaid images). Cluster-containing conjugates begin to appear within seconds of plating the T cells onto the coverslip, and the number of detectable clusters in an individual cell reaches a maximum within 3 min, when the T cell coverslip contact reaches its maximum extent (Bunnell et al., 2001). These TCR-containing clusters persist in T cells plated for up to 20 min before fixation (unpublished data). The recruitment of the TCR into these tight contacts is specific, as a control protein composed of the extracellular and transmembrane domains CD25 joined to a cytoplasmic EGFP tag (Tac-EGFP), is not clustered under these conditions (Fig. 2 B, top). Similarly, the recruitment of the TCR into tight contacts requires TCR engagement, as the TCR is not clustered when Tac-EGFP–expressing Jurkat T cells are plated on coverslips coated with antibodies to CD25 (Fig. 2 B, bottom). These observations establish that ligated TCRs are rapidly and specifically clustered in close contacts.
Sites of TCR clustering are sites of signal initiation
Next, we tested whether ZAP-70–EGFP was recruited into these TCR-rich clusters. Like the TCR, ZAP-70 is recruited into clusters that coincide with contact sites (Fig. 3 A; Video 2). This process is extremely rapid, and occurs within 15 s of the initial formation of a contact. The immature contacts into which ZAP-70 is initially recruited are the size of individual TCR-rich clusters (Fig. 3 A, top row, left; note the contact marked by the white arrowhead). The formation of new ZAP-70–containing clusters continues until the contact reaches its maximum extent, ∼3 min into the spreading process (Video 2). TCR-induced tyrosine phosphorylation parallels the spreading process. Phosphotyrosine is detectable within 30 s of the initiation of these assays (unpublished data), and reaches a maximum after ∼3 min (Fig. S1). These observations suggest that PTK-dependent signals are initiated in newly formed TCR clusters. To confirm the tyrosine phosphorylation of critical signaling molecules in these tight contacts, we imaged ZAP-70–EGFP, phosphotyrosine, and TCRζ in T cell coverslip contacts (Fig. 3 B). As expected, the ZAP-70 clusters (green) colocalized extensively with the clusters of TCRζ (red). Phosphotyrosine-containing proteins (blue) were specifically observed in regions of extensive colocalization between TCRζ and ZAP-70 (compare overlays 1 and 2). Therefore, PTK-dependent signal transduction is initiated in TCR-rich clusters closely apposed to the stimulatory coverslip. Treatment with PP2, an inhibitor of Lck and Fyn, drastically reduces total tyrosine phosphorylation and prevents the recruitment of ZAP-70 into TCR-containing clusters without inhibiting the clustering of the TCR itself (Fig. 3 C). Thus, the organization of the TCR into clusters preceeds the tyrosine phosphorylation required for the initiation of downstream signals.
Signaling adapters and effectors are selectively recruited to phosphotyrosine-rich signaling assemblies
To test whether the adapters and effectors required for downstream signal transduction are also recruited into signaling clusters, we examined T cells fixed 2 min after plating. Due to delays in the settling of the cells onto the stimulatory coverslips, the cells shown here have been spreading for 1 to 2 min. Two representative cases are shown in Fig. 4, where ZAP-70 and Grb2 (left), and LAT and Gads (right) colocalized with one another in clusters within tight contacts (dark points). More extensive studies have shown that ZAP-70, LAT, Grb2, Gads, SLP-76, and Cbl all transiently colocalize in TCR-containing, phosphotyrosine-rich signaling complexes tightly associated with the stimulatory coverslip (Fig. S2; unpublished data). Several control surface proteins fail to accumulate in the clusters (Fig. S2). Tac-EGFP neither accumulated in nor was excluded from the signaling complexes. In contrast, CD43 was excluded from the entire contact interface, and CD45 was specifically excluded from ZAP-70–rich signaling clusters. These studies demonstrate that TCR-containing signaling assemblies form at sites of close contact between the T cell and coverslip and that adapters critical to T cell activation are recruited to these assemblies quickly and specifically.
Contacts the size of individual clusters are sufficient to initiate calcium influxes
To determine whether these signaling assemblies could induce calcium elevations, we monitored contact formation and intracellular calcium levels in parallel. Two representative cells are shown in Fig. 5, A and B. Here, intracellular calcium levels in the responding cells jump from low (blue) to high (red) levels within 24 s. In fact, as shown in the corresponding movies and calcium traces (Fig. 5, C and D; Videos 3 and 4, available at http://www.jcb.org/cgi/content/ full/jcb.200203043/DC1), calcium levels frequently go from basal to maximal levels within one frame, or 12 s. These calcium elevations are typically triggered in response to the formation of one or two point contacts the size of individual signaling clusters (Fig. 5, A and B, white arrows). During the subsequent spreading response, continued oscillations in intracellular calcium were observed. These results suggest that biochemically competent signaling complexes can be assembled quickly within individual point contacts.
Cluster components exhibit distinct patterns of movement
To dynamically observe the formation and dissipation of these signaling assemblies, we tracked the redistribution of individual chimeric signaling proteins during T cell spreading and activation. ZAP-70–EGFP is present in clusters which are stable over time (Fig. 6 A; Video 5). Studies carried to later timepoints demonstrate that clusters containing ZAP-70 persist for at least 20 min without moving laterally (unpublished data). Quantitative analyses show that ZAP-70 is incorporated into clusters forming near the leading edge of the contact (Fig. S3). LAT and Grb2 are incorporated into clusters in a similar manner and with similar kinetics. In contrast to ZAP-70, LAT-EGFP gradually disappears from the contact over 2 to 3 min (Fig. 6 B; Video 6). Although the mechanisms governing the exit of LAT remain obscure, LAT may leave these signaling complexes in small vesicular intermediates (Video 6). Grb2-containing clusters also persist for ∼2 to 3 min before fading from the contact (Fig. 6 C; Video 7). Enhanced green fluorescent protein (EYFP)-Gads is also recruited to the T cell coverslip contact, where it forms clusters that persist for no more than 1 min (Fig. 6 D; Video 8). In contrast, SLP-76–EYFP behaves uniquely. After its rapid recruitment into clusters, SLP-76 translocates towards the center of the contact in large cohesive structures (Fig. 6 E; Video 9). This pattern of movement results in the accumulation of SLP-76 in the center of the contact, near the coverslip. Therefore, although signaling molecules are coordinately recruited into signaling assemblies at the leading edges of contacts, these proteins depart by distinct mechanisms.
ZAP-70 initially equilibrates between signaling complexes
As shown above, signaling clusters containing ZAP-70 are laterally immobile within the plane of the coverslip. To test whether or not individual ZAP-70 molecules are stably bound to these immobile clusters, we monitored fluorescence recovery in photobleached and control regions. The recovery of fluorescence in the bleached region was rapid and nearly complete when the responding cells had been plated for 2 min before bleaching (Fig. 7 A, red box; Video 10). The recovered fluorescence retained the pattern of clustering observed prior to the bleach, indicating that ZAP-70 equilibrates freely between bleached complexes and an unbleached pool of ZAP-70 elsewhere in the cell. The recovery of fluorescence in older complexes, present in cells plated for 20 min before bleaching, was much less complete (Fig. 7 B, red box; Video 11). The same trends can be observed in plots showing the recovery of fluorescence after bleaching (Fig. 7, C and D). The extent of fluorescence recovery drops with increasing time of plating, as shown in Fig. 7 E. Nevertheless, the time to half-maximal fluorescence recovery is independent of the time at which the cells are bleached, and remains between 7.5 and 10 s (Fig. 7 E). Therefore, although ZAP-70–containing clusters appear to be static, these clusters rapidly exchange ZAP-70 molecules for several minutes. At present, we do not know whether additional cluster components equilibrate in this manner; these studies have been hindered by the transient nature of the clustering displayed by LAT, Grb2, and Gads.
A nonphosphorylated lipid raft marker is not retained in signaling complexes
Several reports have suggested that TCR ligation results in the coordinated accumulation of lipid rafts with the aggregated TCR (Janes et al., 1999). As a lipid raft component, LAT could be passively stabilized in signaling complexes through interactions with rafts associated with the activated TCR. To test this hypothesis we tracked the localization of LAT-EGFP and EYFP-GPI, a lipid raft marker (Keller et al., 2001). As shown above, LAT-EGFP transiently clusters upon T cell activation (Fig. 8 A; Video 12). In contrast, EYFP-GPI does not cluster in this manner, suggesting that raft association is insufficient to direct clustering (Fig. 8 B; Video 13). Fixed cell studies confirmed that LAT accumulates within signaling clusters identified by immunofluorescent staining for phosphotyrosine (Fig. 8 C). YFP-GPI, in contrast, does not colocalize with these signaling clusters, and appears to form aggregates alongside the phosphotyrosine-rich signaling clusters (Fig. 8 D). These data indicate that lipid raft components do not nonspecifically accumulate in signaling clusters. Furthermore, the inhibition of Src-family kinases by PP2 revealed that the clustering of LAT is strictly tyrosine kinase-dependent (Fig. 8, E and F). These data indicate that the accumulation of LAT in signaling clusters is unlikely to be stabilized solely through interactions with lipid rafts, but does require phosphotyrosine-dependent interactions.
Characterizing the formation and movement of SLP-76–rich structures
To demonstrate that the unique movement of SLP-76 requires activation of the TCR, we plated SLP-76-YFP–expressing cells on coverslips coated with nonstimulatory antibodies. SLP-76 is recruited into moving clusters in response to TCR-ligation (see above; Fig. 9 A; Video 14), but not in response to either CD45 (Fig. 9 B; Video 15) or CD43 ligation (Fig. 9 C; Video 16). Furthermore, resting SLP-76-YFP–expressing cells contain few or no detectable vesicle-like accumulations of SLP-76 (unpublished data). One property of the movement of SLP-76–rich structures observed in Videos 9 and 14 is striking: entire SLP-76–containing clusters arising at the periphery of the contact translocate radially towards the center of the contact within 1 to 2 min of their formation. This centrally directed movement is inhibited by colchicine treatment, and is therefore dependent on the microtubule cytoskeleton (Fig. 10, A and B; Movies 17 and 18). Although several vesicular compartments display directed movement along microtubules, we have not yet been able to conclusively identify SLP-76–containing structures with any known intracellular compartments. As shown in Fig. 10 C, SLP-76 does not colocalize with transferrin, a marker of the endosomal recycling pathway (Video 19); EEA1, a marker of early endosomes; Rab11, a marker of recycling endosomes; or TGN46, a marker for the trans-Golgi network (Hopkins and Trowbridge, 1983; Gruenberg and Maxfield, 1995; Mu et al., 1995; Ullrich et al., 1996; Prescott et al., 1997). SLP-76 also failed to colocalize with GM130, a marker for the stacks of the Golgi apparatus (unpublished data; Nakamura et al., 1997). The identification of the SLP-76–containing compartment is obviously of significant interest, but will require further study.
The visualization of signaling complexes in live T cells
To date, microscopic studies of live T cells undergoing activation have primarily focused on the movements of the transmembrane proteins that define the immunological synapse (TCR, CD4, CD28, LFA-1, CD43, CD45) (Monks et al., 1998; Grakoui et al., 1999; Dustin and Cooper, 2000; Johnson et al., 2000; Krummel et al., 2000; Allenspach et al., 2001; Delon et al., 2001; Lee et al., 2002; Zal et al., 2002). With the exception of Lck, little is known about the movements of proteins recruited to signaling complexes in response to receptor engagement (Richie et al., 2002). Using a novel assay system, we have monitored the dynamic redistribution of ZAP-70, LAT, Grb2, Gads, and SLP-76 in response to TCR ligation, significantly extending the number of intracellular signaling molecules studied in living T cells. In this manner, we have observed the formation of tyrosine-phosphorylated signaling complexes 0.5–1.0 μm in size and enriched in the TCR, ZAP-70, LAT, Grb2, Gads, SLP-76, and Cbl. These signaling clusters develop rapidly within domains of the plasma membrane tightly associated with the stimulatory coverslip and are individually competent to induce intracellular calcium elevations. Strikingly, these structures are continuously assembled and disassembled throughout the process of contact formation. Finally, we have shown that recruited molecules depart from these signaling complexes by distinct mechanisms. SLP-76, in particular, exits the TCR-rich clusters in a unique manner that suggests an ongoing role in signal transduction.
The assembly of TCR-containing complexes
The most critical event in the initiation of antigen receptor-dependent signaling in T cells is the aggregation of the TCR (Boniface et al., 1998). In our system, the TCR is recruited into clusters even though the TCR-specific stimulatory antibodies we use are uniformly distributed across our coverslips (unpublished data). The clustering of the TCR is PP2-insensitive, and is therefore independent of downstream PTK-driven signaling pathways. Strikingly, TCR-rich clusters preferentially form in membrane domains tightly apposed to the stimulatory coverslip. These observations are consistent with models of immune synapse formation in which the TCR is preferentially engaged within membrane domains separated by a narrow gap compatible with TCR-ligand engagement (Shaw and Dustin, 1997; Anton van der Merwe et al., 2000). Because we rarely observe TCR-rich clusters that are not tyrosine phosphorylated, PTK-dependent phosphorylation must be rapidly triggered after the clustering of the TCR. ZAP-70, which binds directly to the signal-transducing subunits of the TCR upon their phosphorylation, is recruited into clusters within 6–15 s of contact initiation, supporting this conclusion. As expected, the clustering of ZAP-70 is sensitive to PP2. Thus, the formation of functional signaling complexes requires phosphotyrosine-dependent interactions, whereas the initial clustering of the TCR does not.
The assembly of LAT-nucleated complexes
LAT nucleates the formation of signaling complexes critical to T cell activation. Our live-cell studies have shown that LAT is specifically recruited into clusters within 15 s of TCR ligation. Several components of the LAT-nucleated complex, including Grb2, Gads, and SLP-76, are recruited into clusters within a similar time frame. Furthermore, our fixed cell studies have shown these LAT-containing complexes are coincident with the ZAP-70–containing clusters described above. This observation confirms that ZAP-70 is poised to phosphorylate two of its most prominent substrates, LAT and SLP-76, in intact T cells. Previously, these interactions had only been observed in purified membranes (Harder and Kuhn, 2000; Wilson et al., 2001).
Lipid rafts and complex assembly
Normal T cell activation requires the association of LAT with lipid rafts (Zhang et al., 1998b). Because lipid raft components have been reported to accumulate with crosslinked antigen receptors, TCR ligation may govern the clustering of LAT by directing the local accumulation of lipid rafts (Xavier et al., 1998; Janes et al., 1999; Holowka et al., 2000). However, the extensive accumulation of lipid rafts at sites of receptor ligation does not appear to be an essential prerequisite of T cell activation. For example, Th2 cells responding to antigen-loaded APCs do not significantly accumulate lipid raft components in their contacts (Balamuth et al., 2001). Additionally, the direct crosslinking of the TCR at 37°C does not necessarily result in the enrichment of either cholesterol or GM1 in receptor-associated membranes (Harder and Kuhn, 2000). Here, we were unable to observe the recruitment of a lipid raft marker, EYFP-GPI, into signaling clusters. Therefore, interactions between LAT and receptor-associated raft components are unlikely to explain the clustering of LAT with the TCR. In contrast, the recruitment of LAT into clusters is PP2-sensitive, indicating that PTK-dependent scaffolds play a dominant role in the clustering of LAT. Nevertheless, lipid rafts could be required for the initiation, rather than the maintenance, of interactions between LAT and TCR-associated signaling proteins. Given the basal association of LAT with lipid rafts, these observations suggest that LAT is either abstracted from lipid rafts during cluster formation, or present in a subset of lipid rafts distinct from that containing EYFP-GPI. The induced segregation of LAT from other lipid raft components could contribute to T cell activation by limiting the recruitment of lipid raft-associated negative regulators of activation, such as Cbp/PAG, to the TCR (Brdicka et al., 2000; Kawabuchi et al., 2000).
Signal initiation by minimal complexes
Calcium elevations typically begin within 12 s of contact initiation, when the T cell coverslip contact consists of a few small points. This lag between the initiation of contact and the elevation of calcium is similar to the time required for complex formation. Because the point contacts present at this time are the size of a single TCR-rich cluster the minimal functional unit required for calcium elevations may be a single cluster. The early onset of these calcium elevations is consistent with the permissive role of intracellular calcium in T cell spreading (Bunnell et al., 2001). Strikingly, this behavior parallels that of T cells engaging APCs, which also elevate intracellular calcium before initiating more active contact with their targets, but do so after a variable lag which may correspond to the time required to form a single functional signaling complex (Negulescu et al., 1996; Delon et al., 1998).
The dynamic composition of signaling complexes
Signaling complexes quickly disassemble, reassemble, and change in composition as specific proteins dissociate. ZAP-70 achieves a steady state where it remains associated with laterally immobile clusters for 30 min or more. In contrast, LAT, Grb2, Gads, and SLP-76 are transiently recruited to signaling complexes, and disappear from these structures within 1–3 min. Our FRAP studies have shown that early in the spreading process ZAP-70, which appears to be immobile, disassociates from and reassociates with signaling complexes quickly enough to half maximally repopulate bleached complexes within 7.5–10 s. This rate of recovery is consistent with that observed in a previous FRAP study that examined the reassociation of ZAP-70 with the activated TCR in a non–T cell model (Sloan-Lancaster et al., 1998). Cells bleached later in the spreading process displayed similar rates of fluorescence recovery, but recovered to a lesser extent. Because the rate of recovery remains constant, the dissociation and reassociation rates are probably unchanged at these later timepoints. The reduced extent of fluorescence recovery could be explained by three mechanisms: TCRs are rendered incapable of rebinding ZAP-70, free ZAP-70 becomes incapable of rebinding the TCR, or existing receptor-kinase complexes are prohibited from dissociating. Because the kinetics with which fluorescence recovery declines are similar to the kinetics with which receptor phosphorylation is lost, the time-dependent reduction in fluorescence recovery may be caused by the dephosphorylation of the TCR.
Signaling complexes as protosynapses
Multiple signals are triggered by TCR engagement prior to the formation of a central TCR-rich cluster, or cSMAC (Negulescu et al., 1996; Delon et al., 1998; Grakoui et al., 1999; Lee et al., 2002). These signals may be initiated by TCR-rich clusters, or protosynapses, that develop in advance of the mature cSMAC (Grakoui et al., 1999; Dustin and Cooper, 2000; Krummel et al., 2000). The TCR-rich clusters observed here resemble these protosynapses in several respects, including their size, kinetics of formation, and ability to exclude CD43 and CD45 (Sperling et al., 1998; Johnson et al., 2000). Although interactions between engaged TCRs and coverslip-bound stimulatory antibodies preclude the translocation of TCRs into a central cluster, we observe most of the biochemical outputs induced by antigen-loaded APCs. Our results suggest that protosynapses are capable of directing T cell activation in the absence of cSMAC formation and could drive the activation of thymocytes and Th2 cells, which do not form well-defined SMACs (Balamuth et al., 2001; Richie et al., 2002). We also observed the continuous dissociation of components from these signaling complexes, suggesting that the generation of new complexes throughout contact formation is required to maintain the levels of tyrosine phosphorylation observed when T cell contacts and synapses reach their greatest extent (Lee et al., 2002). In addition, the formation of these new clusters at the leading edge may facilitate the coupling of the TCR to the active remodeling of the actin cytoskeleton through downstream adaptors, such as SLP-76 (Krause et al., 2000).
Molecular fates postclustering
The molecular clusters observed here function as both signaling and sorting structures. These complexes are assembled from proteins residing in multiple cellular compartments. ZAP-70 remains associated with signaling complexes, LAT departs the complexes by undefined mechanisms, and both Grb2 and Gads dissociate into the cytosol. In contrast, SLP-76 departs TCR-rich signaling complexes in large structures specifically induced by TCR ligation. These structures are transported along microtubules as discrete units and accumulate in an undefined perinuclear structure not labeled with markers specific for early endosomes, recycling endosomes, the Golgi apparatus, or the TGN. The association of SLP-76 with these structures persists to timepoints at which neither clusters of Grb2 nor Gads can be detected. Although amounts of LAT and Gads beneath the threshold for visualization may direct the movement and localization of SLP-76, we believe that SLP-76 dissociates from LAT-nucleated complexes and enters these novel structures by a distinct and as of yet undefined mechanism.
Potential consequences of late trafficking events
The termination of signal transduction through the targeting of signaling molecules into degradative compartments has been well described (Strous and Govers, 1999). In non–T cells, internalization has also been shown to direct signaling molecules into intracellular compartments from which they can initiate novel signals (Daaka et al., 1998). The consequences of the protein trafficking described here are not yet clear. The sustained recruitment of SLP-76 to a perinuclear vesicular compartment might in some way contribute to T cell activation. Many questions remain regarding the functional consequences of these molecular sorting events and the identities of the structures that contain sorted signaling molecules. In future studies we will image the redistribution of multiple chimeric proteins, lipid markers, and labeled proteins during T cell activation, helping to resolve these issues.
The CD3ε-specific monoclonal antibodies used to induce T cell spreading (HIT3a and UCHT1) were obtained from Pharmingen. Anti-CD25 (anti-TAC) was obtained from UBI. Primary staining antibodies were from the following suppliers: Vβ8, CD43, and CD45 were from Pharmingen; Gads and phosphotyrosine (4G.10) were from UBI; TCRζ, and Cbl were from Santa Cruz Biotechnology, Inc.; SLP-76 was from Antibody Solutions; and Grb2, EEA1, Rab11, TGN46, and GP130 were from Transduction Labs. Alexa-conjugated isotype-specific secondary antibodies, Alexa-conjugated transferrin, Fluo-4, and Fura-Red were purchased from Molecular Probes. The expression vectors pEGFP-n1, pEYFP-n1, and pEYFP-c3 were obtained from CLONTECH Laboratories, Inc. The SLP-76–deficient Jurkat T cell line J14 was provided by Arthur Weiss (University of California San Francisco, San Francisco, CA). RPMI 1640 and G418 were obtained from GIBCO BRL. All other tissue culture supplies were from Biofluids. All remaining chemicals are from Sigma-Aldrich. Jurkat T cells and derivatives were maintained as described previously (Bunnell et al., 2001).
Constructs and the generation of stable cell lines
The ZAP-70–EGFP, Grb2-EYFP, and EYFP–GL-GPI chimeras have been described (Sloan-Lancaster et al., 1998; Keller et al., 2001; Yamazaki et al., 2002). The TAC-EGFP chimera was provided by Dr. J. Donaldson (National Institutes of Health, Bethesda, MD). The remaining chimeric proteins were derived by standard methods. All chimeras were fully functional, as assessed by biochemical complex formation, and, where possible, by the reconstitution of deficient cell lines. Jurkat E6.1 cells stably expressing EGFP-actin have been described. Jurkat E6.1 cells expressing either TAC-EGFP, ZAP-70–EGFP, LAT-EGFP, Grb2-EYFP, or EYFP-Gads, and J14 cells expressing SLP-76–EYFP were generated as described (Bunnell et al., 2001). The stable lines used in these studies express the chimeric signaling proteins at approximately physiological levels. EYFP–GL-GPI was transiently transfected into Jurkat E6.1 cells using the AMAXA electroporation system.
Spreading assays
Spreading assays were performed as described, with the following modifications for either immunofluorescent staining or for biochemical analyses (Bunnell et al., 2001). For inhibitor studies, cells were preincubated with the corresponding drugs for 1 h, and then plated and imaged in the presence of the drug. For staining procedures, four-chambered coverglasses (Lab-Tek II; Nunc/Nalgene) were coated with either UCHT1 or HIT3a, as required, warmed, and preloaded with 300 μl ml of normal culture media supplemented with 25 mM Hepes. Cells (2 × 105) were injected into the bottoms of the chambers in 100 μl of normal media and fixed at the indicated times by the addition of 600 μl 4% paraformaldehyde in PBS. After 30 min of fixation at 37°C, the chambers were rinsed three times in PFN buffer (PBS, 10% FBS, and 0.02% sodium azide), permeabilized for 5 min with 0.1% Triton X-100 in PBS, rinsed three times in PFN, and blocked for 1 h in PFN-G (PFN supplemented with 2% normal goat serum). Blocked chambers were incubated for 1 h with the indicated primary antibodies diluted in PFN-G, rinsed three times in PFN, and stained for 1 h with isotype-specific Alexa-conjugated goat antisera diluted in PFN-G. Stained cells were rinsed three times in PBS and imaged as below. For the immunofluorescent visualization of TCR subunits cells were fixed in a final paraformaldehyde concentration of 0.25% and 500 μg/ml digitonin was used for permeabilization.
All IRM and corresponding fluorescent images were collected on a Zeiss LSM 410 confocal microscope, as described (Bunnell et al., 2001). Ratiometric calcium measurements correlated with IRM images were collected by alternately visualizing Fluo-4 and Fura-Red in the cell body with a wide pinhole, and taking images of the contact interface under normal conditions. Jurkat E6.1 cells were loaded with calcium indicators, as described, and maintained in the presence of 0.5 mM probenecid (Liu et al., 1998; Jung et al., 2001). The dynamic rearrangements of the actin cytoskeleton and of chimeric signaling molecules during spreading responses were monitored using a Perkin-Elmer Ultraview spinning wheel confocal system equipped with an Orca-ERII CCD camera (Hamamatsu), and filters suitable for the visualization of both EGFP and red dyes. For these live studies, samples were illuminated with either the 488- or 568-nm laser lines from a krypton/argon laser. Live images were collected as vertical Z-stacks which were projected in three dimensions or subsampled for the plane of the coverslip. All photobleaching studies were performed using a Zeiss LSM 510. Cells were imaged for 20 s, photobleached within a specific region of interest (ROI) using the combined 458- and 488-nm laser lines, and then imaged over time. All images were collected using a 63× Plan-Apochromat objective (Carl Zeiss). For live studies, the temperature of the sample was maintained at 37°C using a hot air blower (Nevtek) and an objective heater (Bioptechs).
Image processing and quantitation
IP Lab 3.5 was used for most image processing. Adobe Photoshop (v. 5.0) was used to prepare composite images and for all annotations. Calculations for the photobleaching studies were performed in Excel. Mean fluorescence recovery traces were calculated by monitoring fluorescence intensity over time in the bleached region and in a control region containing similar total fluorescence, normalizing fluorescence in these regions by the total cell fluorescence; and setting the normalized fluorescence during the prebleach interval to 100% before averaging. The percent recovery of fluorescence in bleached regions is calculated by taking the differential between the normalized mean fluorescence intensities within the control and bleached regions, and scaling so that the maximum differential corresponds to a 100% loss of fluorescence.
Online supplemental materials
Supplemental materials are available online. These materials include movies of IRM-correlated calcium elevations, the localization of EGFP-actin, ZAP-70–EGFP, LAT-EGFP, Grb2-EGFP, EYFP-Gads, and SLP-76–EYFP during TCR-induced spreading, as well as FRAP and inhibitor studies.
We thank Susan Garfield for her help in the operation of the LSM 510, Ms. V. Kapoor for help in the establishment of stable cells lines, Dr. J. Donaldson for providing the Tac-EGFP chimera, and Drs. J. Lippincott-Schwartz, P. Schwartzberg, and C. Sommers for reading the manuscript.
S.C. Bunnell is a Fellow of the Cancer Research Institute. D.I. Hong was a Howard Hughes Medical Institute–National Institutes of Health Research Scholar.
The online version of this paper contains supplemental material.
Tetsuo Yamazaki's present address is Dept. of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan.
Abbreviations used in this paper: APC, antigen-presenting cell; cSMAC, central supramolecular activation cluster; EGFP, enhanced GFP; EYFP, enhanced yellow fluorescent protein; IRM, interference reflection microscopy; pSMAC, peripheral SMAC; PTK, protein tyrosine kinase; TCR, T cell receptor; Th2 cells, T helper 2 CD4-positive T cells.
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Figure S1
Tyrosine phosphorylation during contact formation. Jurkat E6.1 cells were plated on stimulatory coverslips and lysed in situ at the indicated times. Chambers were prepared normally, and assays were initiated by plating 1 x106 in 100 µl of normal culture media without FCS and buffered with 25 mM Hepes. Cells were lysed at the indicated time points by the direct addition of 100 µl 2x lysis buffer (100 mM Tris 6.8, 2% SDS, 2 mM sodium orthovanadate, 2 mM EDTA, and a 2x protease inhibitor cocktail [Cømplete; Roche]). After 5 min, lysates were harvested, sonicated, and analyzed for tyrosine phosphorylation by Western blotting. Prominent phosphorylated substrates have been labeled. Tyrosine phosphorylation peaks with 3 min of plating.
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. The identification of signaling complex components. Jurkat cells stably expressing EGFP-containing fusion proteins were plated on stimulatory coverslips, fixed after 2 min, and stained for either phosphotyrosine or intracellular signaling adapters. In each case, three panels are shown: the EGFP-containing chimera (green, left), the immunofluorescent stain (red, center), and an overlay emphasizing the colocalization of these proteins (right). Of the proteins shown in green, ZAP-70, LAT, and TAC were expressed as EGFP fusion proteins in Jurkat E6.1 cells, whereas SLP-76 was expressed as an EYFP fusion protein in the Jurkat-derived, SLP-76-deficient J14 line. Although shown in green, Cbl was detected by antibody staining.
. Cluster formation is favored in new areas of contact. The positions of the indicated clusters relative to the initial point of T cell-coverslip contact were plotted as a function of time. The values shown here were derived from the ZAP-70, LAT, and Grb2 image sequences in Fig. 6. Similar results were obtained in several runs (data not depicted). For simplicity, individual data points have been replaced by bars spanning the range of distances from the center of the contact at which clusters formed at a given time point. Cluster formation is favored in a relatively narrow zone near the leading edge of the contact.
The rearrangement of the actin cytoskeleton is coupled to contact formation. Jurkat T cells expressing EGFP-actin were plated on stimulatory coverslips and imaged using a Zeiss LSM 410 confocal system. EGFP-actin and IRM images of the contact were collected every 45 s. Arrowheads emphasize the correspondence between the actin rich structures (top) and the developing contacts (bottom).
The TCR is specifically clustered within tight contacts. (A) Jurkat cells were either plated on HIT3a-coated coverslips, fixed after 1 min and stained for TCRζ (top), or plated on UCHT1-coated coverslips, fixed after 2 min and stained for Vβ8 (bottom). Cells were then permeabilized and stained for TCR components (left). Contacts between stained T cells and the coverslip were also imaged by IRM, where tight contacts appear darker than loose contacts (middle). Overlays reveal that receptor engagement occurs preferentially in the zones of tightest contact (right). (B) TAC–EGFP-expressing Jurkat T cells were plated on coverslips and fixed after 5 min. In the top panel, cells were plated on stimulatory HIT3a-coated coverslips and imaged for EGFP. In contrast, in the bottom panel, cells were plated on anti–TAC-coated coverslips and stained for TCRζ. No clustering is apparent in either sample.
ZAP-70 is specifically recruited to activated TCRs in tight contacts. (A) Jurkat T cells expressing ZAP-70–EGFP were plated on coverslips. ZAP-70–EGFP and IRM images were collected every 15 s using the Zeiss LSM 410. The white arrow marks the earliest observed signaling cluster and contact point. (B) Jurkat T cells expressing ZAP-70–EGFP were plated on stimulatory coverslips, fixed after 5 min, and stained for both TCRζ and phosphotyrosine. ZAP-70–EGFP, TCRζ, and phosphotyrosine were pseudocolored green, red, and blue, respectively. The TCRζ staining pattern shows a large cluster in the center of the contact; this strong signal results from the staining of a large, perinuclear pool of TCRζ. (C) Cells were processed and imaged as in B after pretreatment and stimulation in the presence of 10 μM PP2.
Signal transducing complexes form at tight contacts. Jurkat T cells stably expressing either ZAP-EGFP or LAT-EGFP (green) were plated on stimulatory coverslips, fixed after 2 min, permeabilized, and stained for either Grb2 or Gads (red). IRM images (left) and fluorescent overlays on the IRM images (right) are shown. Note the strong correspondence between tight contacts and clusters of signaling proteins.
Small contacts are sufficient to initiate calcium influxes. (A and B) Jurkat T cells loaded with the calcium responsive dyes Fluo-4 and Fura-Red were plated on coverslips and dynamically imaged for calcium content and T cell-coverslip contact. In the bottom panels the bodies of responding T cells are pseudocolored to indicate their calcium content. The pseudocolored calcium scale is inset in the leftmost panels. White arrows mark the earliest observed contact points. (C and D) Traces of calcium over time for the two cells shown in A and B. The time axis is marked in frames; one frame corresponds to 12 s. These traces are representative of those obtained from >30 cells imaged in three separate experiments.
Signaling adaptors are dynamically redistributed throughout contact formation. (A–E) Live Jurkat E6.1 cells expressing EGFP- or EYFP-chimeras were plated on stimulatory coverslips and observed dynamically using the Ultraview spinning wheel confocal system. Seven-image, 3.5 μm deep Z stacks were collected every 10 s for ZAP-70, every 15 s for the remaining proteins.
Photobleaching reveals that ZAP-70 equilibrates between signaling complexes. (A and B) ZAP-70–EGFP-expressing Jurkat T cells were plated on coverslips and allowed to develop contacts for either 2 or 20 min. Contacts were imaged for 20 s, photobleached within a specific region of interest (ROI, red boxes), and monitored for the recovery of fluorescence in the bleached ROI. Images were obtained every 2.5 s. (C) Mean fluorescence recovery traces from cells plated for 2 min before photobleaching (n = 6). (D) Mean fluorescence recovery traces from cells plated for 20 min before photobleaching (n = 6). (E) The extent of fluorescence recovery in bleached ROIs as a function of the age of the observed contacts at the time of photobleaching. In each case, half-maximal recovery is observed within 7.5 to 10 s.
LAT is selectively retained in signaling complexes. (A and B) Jurkat cells expressing either LAT-EGFP or EYFP-GPI were plated on stimulatory coverslips and imaged using the Ultraview system. Five-image, 2.5 μm deep Z stacks were collected every 20 s. The bright spot observed in the EYFP-GPI–expressing cells corresponds to a membrane fold, not a cluster. Similar structures are also occasionally observed with Tac-EGFP, a nonraft membrane protein. (C and D) Jurkat cells expressing either LAT-EGFP or EYFP-GPI (green) were plated on stimulatory coverslips, fixed after 2 min, and stained for phosphotyrosine (red). (E and F) Jurkat cells expressing either LAT-EGFP or EYFP-GPI were treated with 10 μM PP2, and then plated and imaged in C and D.
SLP-76 clustering is selectively induced by TCR ligation. (A and C) SLP-76–EYFP expressing J14 cells were plated on antibody-coated coverslips and imaged using the Ultraview system. Five-image, 2.5 μm deep Z stacks were collected every 10 s. The plate-bound antibodies were specific for (A) CD3ε, (B) CD45, or (C) CD43.
SLP-76–containing clusters move on microtubules to an undefined perinuclear compartment. (A and B) Jurkat J14 cells expressing SLP-76–EYFP were plated on stimulatory coverslips and imaged as in Fig. 9 after 1 h pretreatment with either DMSO carrier (A) or 100 μM colchicine (B). Drugs were also present throughout imaging. (C) The colocalization of SLP-76–EYFP with various intracellular markers was tested in both live and fixed cells. Jurkat J14 cells expressing SLP-EGFP (green) were serum starved for 40 min, incubated with 100 μM transferrin-Alexa 594 (red), plated on stimulatory coverslips and imaged using the Ultraview system. Every 20 s, individual red and green image stacks were collected. Each image stack is five images (2.5 μm) deep. The image shown here was collected 2 min into the spreading process (panel 1). Jurkat J14 cells expressing SLP-76–EYFP (green) were also plated on stimulatory coverslips, fixed after 10 min, and stained for intracellular markers (red): (panel 2) EEA1; (panel 3) rab11; or (panel 4) TGN46.
About JCB


text:
Q: Why are the quotes necessary in this code? Hi sorry if this is a dumb question, probably is... this is from codingbat (exercise: everNth)
why is it that this works?
public String everyNth(String str, int n) {
String characters = "";

for (int i = 0; i < str.length(); i = i + n) {
characters = characters + Character.toString(str.charAt(i));
}

return characters;
}

but this one doesn't?
public String everyNth(String str, int n) {

for (int i = 0; i < str.length(); i = i + n) {
return Character.toString(str.charAt(i));
}

}

^^ gives me the error: "This method must return a result of type String"
However, doesn't the Character.string() method already create a string?
Why do I have to add in additional quotation marks? Thanks guys!!~~ peace and love

A: It has nothing to do with the quotation marks.  The problem is that you don't have a return statement after the for loop.
The compiler is warning you that if the for loop is never entered, then you will never return anything.  You need to make sure your method returns a string value under all circumstances, not just when the loop is entered.
To better understand, walk through your method, and consider what would happen if str has a length of zero.  What would your method return then?
EDIT
However, as pointed out by WalterM, keep in mind that, even if you fix the compiler error, your 2nd method's logic is different from your first one.  The 2nd method only ever returns the 1st character of the string.

redpajama_set_name: RedPajamaStackExchange


text:
Jenna Dewan Just Responded To Those Jessie J Comparisons
Jenna Dewan Tatum Speaks Out For First Time Since Channing Tatum Split
Channing Tatum Asked His Wife Out Wearing Nothing But Uggs And A Sombrero... At 2AM
Channing Tatum's Emotional Birthday Message To Wife Jenna Dewan Will Move You To Tears!
Jenna Dewan Tatum Opens Up About Having More Children With Channing Tatum
WATCH: Channing Tatum Tells His Daughter He Ate All Her Halloween Candy
Channing Tatum Has Already Won Halloween With This Magical Unicorn Costume
Courteney Cox Has Re-Created Her Iconic 'Friends' Routine 21 Years Later
Channing Tatum And Jessie J Have Called It Quits
Anna Faris Reportedly Confirms Engagement To Michael Barrett
Céline Dion Shared Rare Photos of Her Twin Sons
Jenna Dewan Has Revealed The Real Reason She And Channing Tatum Ended Their Marriage
5 Of Angelina Jolie's 6 Children Joined Her On The Red Carpet For The 'Maleficent 2' Premiere
Channing Tatum Just Deleted Instagram Because It's Blocking His Inspiration
Jenna Dewan Just Shared The First Photo Of Her Baby Bump
Her boyfriend wrote the sweetest message - by Isabelle Truman
Yesterday, Jenna Dewan and her boyfriend Steve Kazee announced they are expecting their first child together in a joint statement to PEOPLE. Shortly after, Jenna took to Instagram to share a sweet photo of herself lying beside her six-year-old daughter, Everly, whom she shares with her ex-husband, Channing Tatum.
"Becoming a mother is quite simply the absolute best most incredible thing that has ever happened to me 💕
@stevekazee you are a gift from above and I couldn't be more excited to be expanding our family together...! Thank you guys for all the love!!," she captioned the image, which gave followers the first look at her growing baby bump.
jennadewan View Profile
119.3k likes - View Post on Instagram
Becoming a mother is quite simply the absolute best most incredible thing that has ever happened to me 💕 @stevekazee you are a gift from above and i couldn't be more excited to be expanding our family together...! Thank you guys for all the love!!
But that's not all. Steve also took to Instagram to share a photo of Jenna cradling her baby bump alongside the sweetest message. "Well...the news is out," he wrote. "I can't even begin to describe the feelings that I have had since we first learned that Jenna was pregnant! I have waited my entire life for this moment and couldn't have found a better person to build a family with."
He continued: "The past year and a half has brought so many things into my life that have filled me with the deepest of gratitude. A partner who radiates love, warmth, and kindness. A partner who has faced some of the hardest moments life can deal you with grace, humility, and a desire to learn from the ups and downs. A partner who is continuously growing from those lessons and who supports my growth as a person with love, unwavering support, and understanding. Jenna also brought with her one of the greatest gifts one could ever imagine...her beautiful daughter. Evie burst into my life like a supernova and has continued to burn so brightly in it. I have learned so much about parenting already from watching how amazing Jenna is with her and Evie has taught me so much about what it means to love and communicate with a child and I can't wait to give her a sibling and make our little family a little bit bigger."
stevekazee View Profile
View Post on Instagram
Well...the news is out. I can't even begin to describe the feelings that I have had since we first learned that Jenna was pregnant! I have waited my entire life for this moment and couldn't have found a better person to build a family with. The past year and a half has brought so many things into my life that have filled me with the deepest of gratitude. A partner who radiates love, warmth, and kindness. A partner who has faced some of the hardest moments life can deal you with grace, humility, and a desire to learn from the ups and downs. A partner who is continuously growing from those lessons and who supports my growth as a person with love, unwavering support, and understanding. Jenna also brought with her one of the greatest gifts one could ever imagine...her beautiful daughter. Evie burst into my life like a supernova and has continued to burn so brightly in it. I have learned so much about parenting already from watching how amazing Jenna is with her and Evie has taught me so much about what it means to love and communicate with a child and I can't wait to give her a sibling and make our little family a little bit bigger.
As far as how Channing Tatum feels about the news, the actor, who now lives in London with British singer Jessie J, reportedly took it really well. According to E! News, Jenna made sure she told Channing about her pregnancy before it became public knowledge and he was really excited for her.
"Jenna told Channing that she was pregnant and wanted to make sure he knew before she announced it. He is happy for her and was very supportive," a source told the site.
Isabelle Truman
Isabelle Truman is a freelance writer based out of London, UK. She specialises in fashion, lifestyle and travel and knows a thing or two (read: everything) about both Meghan Markle and 'Sex and the City'.


TheChieftain added the Revell German Submarine Deutsches U-Boot VII C "Wolf Pack/Die grauen Wölfe" (1:72) to his stash.
TheChieftain added the Hobby Boss PzKpfw I Ausf C (VK 601) (1:35) to his stash.
TheChieftain added the Tamiya Panzerkampfwagen III - Ausf. L (Sd.Kfz. 141/1) (1:35) to his stash.
TheChieftain added the Tamiya Panzer Kampfwagen II Ausf. F/G (1:35) to his stash.
TheChieftain marked this item as on order.
TheChieftain added the Airfix BAe Harrier GR9A (1:72) to his stash.


text:
Q: Change special letter to normal
Possible Duplicate:
JavaScript: Efficiently replace all accented characters in a string?

HI,
I want to change letter selected letters: "Ą" to "A", "ć" to C etc... in jQuery
currently my code changes letter to *
how to modify it to change given letters?
var finishedslug = slugcontent_hyphens.replace(/[^a-zA-Z0-9\-]/g,'*');


text:
Haghill police incident: teenagers rushed to hospital after disturbance involving weapons
Teenagers were found with injuries on Aberfeldy Street after an incident involving up to eight people on Cumbernauld Road.
Gary ArmstrongJournalist
Aberfeldy Street in Glasgow's east end (Image: Google Maps)
Two teenagers were rushed to hospital after a 'large scale disturbance involving weapons' involving up to eight people in Haghill.
Emergency services raced to the east end of Glasgow on Sunday evening after a 17-year-old and 18-year-old were found injured on Aberfeldy Street, following the incident on Cumbernauld Road.
The 17-year-old remains in Glasgow Royal Infirmary, however the 18-year-old has since been discharged.
Person rushed to hospital following large scale disturbance involving weapons in Glasgow's east end
Glasgow sex creep on Tinder weeks after prison release for attacking woman on first date
Eyewitnesses last night told Glasgow Live of a 'running battle' between youngsters in the area and police have subsequently launched an investigation.
A Police Scotland statement this morning confirmed: "Around 5.40 pm last night, Sunday, 13 September, 2020, police were called after two men were found injured on Aberfeldy Street near to Walter Street, Haghill, Glasgow.
"The two men, aged 18 and 17 years, had been injured during a disturbance involving up to 8 people in nearby Cumbernauld Road. Both were taken by ambulance to Glasgow Royal Infirmary where the 18 year old was treated and has since been discharged.
Coronavirus Scotland: 'rule of six' comes into force today
Nicola Sturgeon coronavirus updates LIVE: social gatherings limited to six from today
"The 17 year old man remains in the hospital for observations.
"Enquiries in to the incident are ongoing."
Police last night advised: "I can confirm there was a large scale disturbance on Walter Street in the Haghill area earlier this evening involving weapons. At least one person was injured and was taken to hospital. Enquiries are ongoing at the locus and elsewhere."
Royal Infirmary
Glasgow Royal Infirmary
Celtic FCCeltic line-up vs Livingston predicted as David Turnbull handed unorthodox roleCeltic will have to make do with a threadbare team once again as 13 first team players isolate.


There will be a post-secondary Financial Aid Night on Monday, Jan. 11 at 7 p.m. in the Mraz Center, hosted by the Burnsville High School Guidance Department. There will be information provided about the FAFSA (Free Application for Federal Student Aid), as well as grants, loans and scholarships. Students and parents who have questions about these application processes should plan on attending.
If you have any questions about this event, please contact the Guidance Department at 952-707-2108.


I want to attach the kicker on my Ent to the boom with a snap shackle, so I can take it off easily during launching, as it fouls the centreboard and has already sheared it off twice (don't ask).
What is the maximum load that the shackle should be able to withstand? The one I'm looking at currently comes in three sizes, with loads of between 1000kg to 3000kg.
I'd never thought of it in that way, but I assume that there can potentially be quite a lot of pressure, and I wouldn't want the shackle to break just at the wrong moment. Would the 1000kg one be big enough?
What is the purchase on the kicker? If it's 8:1, you can probably pull 100kg so that's approaching 800kg after friction losses. The 1000kg one will probably be rated as SWL so actual max load will be at least double. I think that might get exceeded in a high wind gybe so I'd be inclined to go for the 2000kg one.
However, I don't recall either of my Ents kickers fouling the CB head? Fittings are pretty strictly controlled in the Ent so are the kicker attachments in the correct places?
Also it will be easy enough to reattach the kicker when the wind is light/moderate but in a blow it might be difficult on the water so a kicker that can be attached before launching might be a better choice if it can done.
The shock loads can be far higher than you can pull. However, I'd think a couple of tonnes would be plenty.
I think the kicker is 16:1, so pulling 60kg would get me almost to 1000kg if I worked that out correctly.
The grip at the front of the centreboard is just in the wrong place for the kicker; it's fine once the board is down even a little bit. I had problems on the shore with the boat still on the trolley with the wind gusting and shifty; the boom moved across, kicker got stuck on the handle, and off broke the top part (the bit that was out of the case at the top).
Rupert: so you think the 2000kg should suffice?
The weakest point in an older Ent kicker system is probably the lower fixing. Not sure how your's is fixed but mine were both screwed into the hog (early wooden boats, I can't remember but I imagine the kicker was no more than 4:1 TBH).
I think that 1000kg will be more than enough. Sure there is a lot of purchase, but do you think you could feasibly lift 60kg, equivalent to a small adult with one hand using 4mm control line? Also think about the strength of other components in the system. Look at the final line strength, or final block type they will give you a good steer for strength of the system.
Enterprise 20361: When new, that had the lower kicker attachment as a U-bolt, bolted through the hull, with a backing plate underneath. It would take a lot of rotten wood and close to a complete boat failure for that to pull out!
And if the boom is still the original, then the fitting on the boom could easily be in the wrong place. When new, it had a complex highfield lever system on the boom for the top of the kicker, to allow a coarse as well as a fine adjustment on the kicker lever. I suspect this was all replaced with a proper cascade system years ago!
I've bent 1 tonne ones before,(they distort enough that they no longer meet) but never a 2 tonne one, and the pressures cannot be that different between Ents and Minisails in the great scheme of things. My other boats have always used either closed shackles or keys.
Moving the boom take off point forward would reduce leverage, but mostly take out the forward component. It would create more crew room, too. But the big draw back it it shortens the length of travel for the last purchase. Do Ents have strict rules on the length of handle the centreboard needs?
Good point about the block types, they will indeed be the weakest link (most likely) but I reckon I could pull 60 kg onto my kicker line so, with a 16:1 cascade 960kg less friction losses (which will be a fair percentage) but it's the additional shock loads in a windy gybe that will be the issue and they could easily be double or triple the static load.


text:
Where does OLAIS rank in the most common names in the U.S.?
OLAIS is identified by the U.S. Bureau of the Census as a surname with more than 100 occurrences in the United States for the year-2000 U.S. Census. In "Demographic Aspects of Surnames from Census 2000", the Census Bureau tabulated the surnames of all people who had obtained Social Security Numbers by the year 2000.
OLAIS ranks # 116835 in terms of the most common surnames in America for 2000.
OLAIS had 138 occurrences in the 2000 Census, according the U.S. government records.
Out of a sample of 100,000 people in the United States, OLAIS would occur an average of 0.05 times.
For the last name of OLAIS the Census Bureau reports the following race / ethnic origin breakdown:
7.97 percent, or 11 total occurrences, were "Non-Hispanic White Only"
16.67 percent, or 23 total occurrences, were "Non-Hispanic Asian and Pacific Islander Only"
3.62 percent, or 5 total occurrences, were "Non-Hispanic American Indian and Alaskan Native"
71.01 percent, or 98 total occurrences, were "Hispanic Origin"
Search the web for more on the name OLAIS :