text:
Grese, Irma, Ida Ilse "The Beautiful…
Czech, Alfred "Hitler boy".
Peiper, Joachim "Jochen".
Koch, Ilse-Kohler, "The Bitch of…
Kaltenbrunner, Ernst.
Horton, Oliver Martin,
Heydrich-von Osten, Lina Mathilde.
Volkenrath, Elisabeth.
Himmler, Heinrich Luitpold…
Dietrich, Josef "Sepp".
Günsche, Otto.
Goeth, Amon Leopold.
Timoshenko, Semyon Konstantinovich
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- Nationality
Russia.
- Ranking
Russian Field Marshal, defender Moscow.
18-02-1895, Furmanca, Ukraine.
- Died
31-03-1970, age 75, Moscow
- Buried
Kremlin Wall, Moscow.
- Medals
russiaArmyFieldmarschal
Timoshenko, Semyon Konstantinovich, born 18-02-1895, into a peasant family in Furmanivka, Bessarabia, Russian Empire (now in Ukraine). In 1915, he was drafted into the army and served as a cavalry soldier during World War 1. In 1917, during the Russian civil war, he sympathized with the communist revolutionaries, joining the communist forces in 1918 and the Bolshevik Party in 1919. During the civil war, he met Joseph Stalin in Tsaritsyn (later Stalingrad and then Vogograd), which was to be the foundation for his steady rise in the chain of command as Stalin rose to power later. In the 1920s, Timoshenko between the commander of all cavalry forces in the Russian Army. In 1933, he was named the commanding officer of the Russian Army in Byelorussia. In 1935, he became the commanding officer of the forces in Kiev, Ukraine. In 1937, he was named the commanding officer of forces in the northern Caucasus, and then of Kharkov, Ukraine. In 1938, he returned to Kiev as the commander. In 1939, he was given command of the entire western region of Russia. When the European War began in Sep 1939, Timoshenko led Russian forces in the invasion of Poland. In Jan 1940, he was assigned to the north to join the already commenced Winter War with Finland; Russian troops turned the tide and pushed through the Finnish defensive Carl Gustav Mannerheim
Line and led to the Russian victory. In May, he was made a Marshal of the Soviet Union for the victory against Finland, and was made a Hero of the Soviet Union. Between 1940 and 1941, as the People's Commissar for Defence, he was responsible for the introduction of the harsh discipline reminiscent of the Tsarist Army into the military, and also for the modernization of the army by mechanization. When German invaded Russia in Jun 1941, Stalin took over the role of People's Commissar for Defence and sent Timoshenko to the front lines to conduct a fighting retreat toward Smolensk, Russia, where he was able to lead a bulk of the routed army for the defence of Moscow. In September, he was transferred to Ukraine to hold the lines against the German onslaught, which he was able to do rather successfully. In May 1942, he led a counter-offensive at Kharkov, Ukraine which saw initial success, but ultimately was defeated with extremely high casualties (at least 200,000 wounded, killed, or captured); he was blamed for the defeat, although some secretly thought that the defeat should have been attributed to Stalin, who underestimated German strength in the region when he ordered the offensive. He was transferred to Stalingrad in June 1942; his previous role as the commanding officer of the west was taken over by Marshal, Georgi Zhukov
. In October 1942, he was named the commanding officer of the northwest, followed by an appointment to Leningrad, Russia in June 1943. He returned to the Caucasus in June 1944, and then served in the Russian-occupied Baltic states in Aug 1944.
Death and burial ground of Timoshenko, Semyon Konstantinovich.
After the war, Timoshenko became the commanding officer in Byelorussia between March and May 1946. In June 1946, he served in the southern Ural Mountains. He returned to Byelorussia in Mar 1949. In 1960, he was appointed the Inspector General of the Defence Ministry, which was largely a ceremonial post without much responsibility. In 1961, he became the State Committee Chairman for War Veterans. He was named Hero of the Soviet Union a second time in 1965. He passed away in Moscow, Russia on 31-03-1970, age 75 and is buried next to Josef Stalin along the Kremlin Wall in Moscow, next to Marshal Konstantin Rokossovski.
Röhm, Ernst Julius.
Roth, Ernst August
Fick, Roderich.
Peregory, Frank Dabney.
Swing, Joseph May
Bausewein, Alfred
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Kette regulates actin dynamics and genetically interacts with Wave and Wasp
Sven Bogdan, Christian Klämbt
Sven Bogdan
Christian Klämbt
During development of the Drosophila nervous system, kette is required for axonal growth and pathfinding. It encodes a highly conserved homolog of the Nck-associated protein 1 (NAP1) that genetically interacts with the Drosophila homolog of Nck, dock. We show that in vivo as well as in tissue culture models most of the Kette protein is found in the cytoplasm where it colocalizes with F-actin to which it can bind via its N-terminal domain. Some Kette protein is localized at the membrane and accumulates at focal contact sites. Loss of Kette protein results in the accumulation of cytosolic F-actin. The kette mutant phenotype can be suppressed by reducing the wave gene dose, demonstrating that kette antagonizes wave function. Overexpression of the wild-type Kette protein does not interfere with normal development, whereas expression of an activated, membrane-tethered Kette protein induces the formation of large F-actin bundles in both, tissue culture cells and in vivo. This gain-of-function phenotype is independent of wave but can be suppressed by reducing the wasp gene dose, indicating that Kette is able to regulate Wasp, to which it is linked via the Abelson interactor (Abi). Our data suggest a model where Kette fulfils a novel role in regulating F-actin organization by antagonizing Wave and activating Wasp-dependent actin polymerization.
F-actin
NAP1/Kette
The actin cytoskeleton plays a central role in cell motility, morphology, phagocytosis and cytokinesis. It is spatially and dynamically reorganized, providing force for the shape change and surface movement in most eukaryotic cells (Borisy and Svitkina, 2000; Pantaloni et al., 2001). Rearrangement of actin is evoked rapidly by extracellular stimuli, and sets of actin-associated proteins are thought to act cooperatively in the polymerization, crosslinking and anchorage of actin filaments. Although the central dynamic nature of the actin system is known, actin organization and regulation are less well understood.
Actin dynamics crucially depend on the ability of the protein to switch from a monomeric (G-actin) to a filamentous form (F-actin). Polymerization of F-actin starts with the de novo nucleation of an actin trimer, a process that occurs relatively slowly and requires the action of the Arp2/3 complex (Machesky et al., 1994; May, 2001; Robinson et al., 2001; Welch et al., 1997). Subsequent elongation is fast and cells have to prevent spontaneous actin polymerization by expressing a variety of actin-binding proteins such as profilin (Schafer and Cooper, 1995). The nucleation activity of the Arp2/3 complex in turn is regulated by a set of activators, such as the members of the Wasp (Wiskott-Aldrich syndrome protein) and Wave (Scar - FlyBase) families (Higgs and Pollard, 2001; Suetsugu et al., 2002b; Takenawa and Miki, 2001). Wasp proteins are usually self-inhibiting and require small G proteins of the Rho family for activation (Kim et al., 2000; Miki et al., 1998a). In its activated GTP-bound form, Cdc42 can bind to the Crib (Cdc42/Rac Interactive Binding) domain of Wasp, releasing the auto-inhibition and thereby leading to the activation of the Arp2/3 complex (Higgs and Pollard, 2001; Rohatgi et al., 1999). By contrast, Wave, which does not bind Cdc42, is trans-inhibited through its association with members of the Kette (Hem - FlyBase) family, Sra1 (specifically Rac associated 1) and Abi (Abelson-interactor). Rac1 binding, presumably to Sra1, relieves the inhibitory function of this complex (Eden et al., 2002; Kobayashi et al., 1998; Miki et al., 2000).
Both, Cdc42 and Rac1 exert distinct functions in rearranging the F-actin cytoskeleton, GTP-bound Rac1 promoting lamellipodia and activated Cdc42 promoting filopodia formation (Hall, 1998). This matches the finding that Wasp enhances the generation of filopodia, whereas Wave activation near the cell membrane results in an increase of lamellipodia (Miki et al., 1998a; Takenawa and Miki, 2001).
Drosophila possesses only two genes, wasp and scar, that encode a Wasp and Wave homolog, respectively (Zallen et al., 2002; Ben-Yaacov et al., 2001). Both genes act largely independently of each other and have a strong maternal component. Only embryos lacking both the zygotic and the maternal gene functions display severe embryonic nervous system phenotypes. In the adult, wasp is required for formation of external mechanosensory organs, whereas scar/wave is - unlike wasp - required for normal formation of the compound eye. Recent structure function analyses demonstrated that Drosophila Wasp can perform at least part of its function independent of Cdc42 (Tal et al., 2002). Alternative modes of Wasp activation such as phosphorylation or SH3 (Src homology 3) domain binding have been described (Cory et al., 2002; Scott et al., 2002; Suetsugu et al., 2002a).
Obviously, regulation of the dynamic F-actin cytoskeleton is pivotal especially during cell movement and thus requires a close link to the plasma membrane and guidance receptors involved in the perception of extracellular signals. The latter generally induce conformational changes in the cytoplasmic domain of membrane-anchored receptors, which recruits other proteins to the ligand-receptor complex. In the case of receptor tyrosine kinases (Rtk), autophosphorylation of tyrosine residues leads to the recruitment of SH2 domain containing adapter proteins. One of these is the SH2 SH3 adapter protein Nck, which is able to link several Rtks as well as guidance receptors like the Netrin-receptor DCC to the actin cytoskeleton (Li et al., 2001; Li et al., 2002). Nck can recruit additional proteins to the cell membrane via its three SH3 domains. Among the highly conserved Nck-interacting proteins are Wasp (Rivero-Lezcano et al., 1995), the non-receptor tyrosine kinase Abl (Adler et al., 2000) and Nap1 (Nck-associated protein 1), which is a member of the evolutionary conserved Kette family (Baumgartner et al., 1995; Hummel et al., 2000; Kitamura et al., 1996; Soto et al., 2002).
Based on its requirement for axonal growth, we have previously identified mutations in the Drosophila kette gene (Hummel et al., 2000). Loss of kette function primarily affects neurite growth and subsequently causes glial migration defects leading to a characteristic commissure phenotype in the embryonic CNS similar to the one observed in waspmat/zyg mutants (Zallen et al., 2002) and Drosophila Nck (dock) mutants (Desai et al., 1999; Garrity et al., 1996). Furthermore, kette genetically interacts with mutations in the Nck homolog dock as well as with the small GTPase Rac1, supporting the notion that Kette may provide a novel mechanism linking extracellular signals to the actin cytoskeleton (Hummel et al., 2000). Recently, the Kette homolog Nap1 was found in a large 500 kDa protein complex comprising PIR121/Sra1, Abi, HSPC300 and Wave that keeps Wave in an inactive state in vitro (Eden et al., 2002).
However, to date it is still unclear how Kette could regulate the organization of the actin cytoskeleton in vivo. As Kette has been predicted to be an integral membrane protein with six transmembrane domains (Baumgartner et al., 1995), it might serve as a receptor recruiting Nck/Dock or Rho-GTPases to the membrane. We present biochemical and genetic evidence revealing that Kette is found predominantly in the cytosol. Only a small amount of Kette is recruited to the plasma membrane. In vivo as well as in tissue culture models, Kette protein colocalizes with F-actin and co-sedimentation assays revealed a direct interaction with F-actin. Within the membrane, Kette accumulates at the insertion sites of large F-actin bundles, suggesting that targeted localization of Kette may be required for its function. Loss of Kette protein leads to a Scar/Wave-dependent accumulation of F-actin within the cell. Ectopic expression of wild-type or different truncated Kette proteins in tissue culture cells or during Drosophila development does not affect F-actin formation or viability. However, expression of membrane-tethered Kette efficiently induces ectopic bundles of F-actin in a process depending on Wasp but not on Scar/Wave. These data indicate that Kette fulfils a novel role in regulating F-actin organization by antagonizing Wave and activating Wasp-dependent actin polymerization.
All crosses were performed at 25°C unless otherwise indicated. The following strains were used: wasp1/TM6 wasp3/TM6 (Ben-Yaacov et al., 2001); scark13811/CyO and scarΔ37/CyO (Zallen et al., 2002); and ketteΔ2-6/TM6 and ketteJ1-70/TM6 (Hummel et al., 2000). To determine the gain-of-function phenotype of KetteMyr, we used the GAL4 system (Brand and Perrimon, 1993). Transgenic flies carrying the following constructs were generated following standard procedures: UAS-kette, UAS-ketteMyc; UAS-kette1-374; UAS-kette375-907; UAS-kette908-1126 and UAS-ketteMyr. The KetteMyr construct was made by fusing DNA encoding the first 88 amino acids from Drosophila Src1 (Simon et al., 1985) to the first codon of Kette. Several independent transgenic lines were analyzed in each case.
Two hybrid assay
To isolate proteins interacting with Kette protein, we inserted the kette as well as the wasp ORF in frame with the GAL4 DNA binding domain into the GBK-T7 vector (Clontech). With pGBK-Kette we screened a matchmaker Drosophila embryo cDNA library (1×106 clones tested) using the GAL4-based Two-hybrid System 3 from Clontech. pGAD-Abi contains the entire Abi-ORF.
The rabbit anti-Kette antibody (97/82), directed against a peptide derived from the middle region of Kette (652-666), KHFDDIRKPGDESYR, was made by Eurogentec (Belgium). In addition, polyclonal antibodies were generated against parts of Kette fused with a His6-tag (Qiagen). pQE plasmids express amino acid regions 1-374, 375-906 and 907-1126 of Kette, respectively. Using these expression constructs, His6-Kette fusion proteins were expressed in E. coli and purified with Ni-NTA resin (Qiagen) under denaturing conditions. Rabbits were immunized with purified proteins by Davids Biotechnologie (Germany).
Cell culture, transfection and immunofluorescence
Drosophila S2 cells were propagated in 1× Schneider's Drosophila media (Gibco) supplemented with 10% FBS, 50 units/ml penicillin and 50 μg/ml streptomycin in 75 cm3 T-flasks (Sarstedt) at 25°C. For transfections, 5×105 cells were plated on glass cover slips (pretreated with fibronectin) in 24-well plates, cultured for 24 hours and transfected with Fugene 6 (Roche) at a transfection reagent:DNA ratio of 3:1. Cells were incubated 24 hours after transfection, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, stained with Drosophila anti-Kette antibody 97/82 (1:2000 dilution) followed by Alexa-fluorophore-conjugated goat anti-rabbit IgG antibody (Molecular Probes, 1:1000 dilution). For filamentous actin and nucleic acid staining, cells were prepared as described above and incubated in 1-2 units Alexa-fluorophore-conjugated phalloidin (Molecular Probes) and in 300 nM DAPI (Molecular Probes), respectively. Tyrosine phosphorylated proteins were detected using the PT-66 anti-phosphotyrosine antibody (Sigma). The samples were mounted in a 25% (w/v) Mowiol (Sigma) solution containing DABCO (Sigma), and visualized on a Leica LSM.
dsRNA production and RNAi treatment
570 bp of the kette ORF and 750 bp of the wasp and scar/wave ORF were inserted into the vector pLITMUS-28i (BioLabs) flanked by T7 promoters. After linearization, dsRNA was generated using a HiScribe RNAi Transcription Kit (BioLabs). dsRNA products were resuspended in water and annealed by incubation at 65°C for 30 minutes followed by slow cooling to room temperature and stored at -20°C. For RNAi treatment, 5×105 Drosophila S2 cells in 0.5 ml serum-free medium were plated on fibronectin coated cover slips in 24-well plates (Sarstedt). dsRNA (5 μg) were added directly to the media followed by vigorous agitation. The cells were incubated for 30 minutes at 25°C followed by addition of 1 ml medium containing 15% FCS. dsRNA treated cells were incubated for additional 2-3 days and analyzed by immunofluorescence and western blot analysis.
Actin binding assay
Actin was purified from rabbit skeletal muscle following the methods of Spudich and Watt (Spudich and Watt, 1971) and stored as G-actin at -172°C (Spudich and Watt, 1971). Actin was polymerized in a buffer of 100 mM KCl, 2 mM MgCl2, 20 mM HEPES, pH 7.4, 0.5 mM β-mercaptoethanol and 2 mM NaN3. Prior to use, MBP, MBPKette and actin were centrifuged at 200,000 g for 30 minutes in a tabletop ultracentrifuge (Beckmann Instruments) to pellet any aggregated protein. For binding assays, increasing amounts of full-length or truncated Kette proteins were incubated with 3 μM F-actin for 30 minutes at room temperature in polymerization buffer. The mixtures were centrifuged at 200,000 g for 30 minutes at 4°C. Equal amounts of supernatant and pellet were separated by SDS-PAGE followed by Coomassie blue staining.
Fractionation experiments
Fractionation of Drosophila embryonic and S2 cell extracts were performed as described (Zhang and Hsieh, 2000). The pellets were solubilized in SDS sample buffer and supernatants were precipitated in trichloroacetic acid, followed by solubilization in SDS sample buffer. Western blot analysis was carried out as described previously (Bogdan et al., 2001).
Kette is a cytosolic actin-binding protein
Kette was predicted to be an integral membrane protein (Baumgartner et al., 1995). To test this, we generated polyclonal antisera against the Kette protein (Fig. 1A). Western blot analyses were performed on protein extracts of wild-type embryos and embryos overexpressing the full-length Kette protein under the control of the GAL4 system. All antisera recognized a 120 kDa protein, which is in good agreement with the predicted molecular mass of 125 kDa for Kette (Fig. 1B). The specificity of the antisera was further supported by the detection of a recombinant Myc-tagged Kette protein recognized in vivo by both anti-Kette and anti-Myc antibodies (Fig. 1B) and RNAi experiments (see Fig. 4A). No evidence for post-translational proteolytic cleavage was obtained.
Kette associates with membranes. (A) Schematic view of the Kette protein; hydrophobic domains are indicated. Different antisera were generated. N, N-terminal domain, amino acids 1-374; the M-serum directed amino acids 375-907 is not indicated; C, C-terminal domain, amino acids 908-1126; P, peptide antibodies, amino acids 652-666. (B) Western blot analyses of protein extracts prepared from wild-type, rhoGAL4/UAS-Kette full-length or rhoGAL4/UAS-Kettemyc embryos were probed either with the anti-Kette P antiserum or anti-Myc antibodies (Mab 9E10) as indicated. The Kette protein is ∼120 kDa. Anti-Myc antibodies recognize a similar sized protein confirming the specificity of the antisera. (C) Differential centrifugation reveals that Kette is located primarily in the cytosol. Western blots were probed with anti-Kette antisera (top), anti-actin antibodies (middle) and anti Na+/K+ ATPase antibodies (bottom) to monitor a typical transmembrane protein. The different lanes show: total, total protein extracts of S2 cells; PNS, supernatant following 1,000 g centrifugation. The supernatant was subjected to 25,000 g centrifugation and subsequently to 200,000 g centrifugation. The pellet was resuspended in either PBS at high pH or 1% NP-40 and subjected to 200,000 g centrifugation (S, supernatant; P, pellet). For each lane, equal amounts of total proteins were loaded on SDS-PAGE. (D) Post-nuclear supernatant (PNS) of wild-type embryos was subjected to equilibrium sedimentation on a discontinuous sucrose density gradient. Only small amounts of the Kette protein were detectable in the membrane fraction, while most of the Kette protein remains in high dense sucrose containing cytosolic proteins. Western blot analysis was performed using anti-Kette antisera (top) and anti Fas3 antibodies (bottom) to monitor a typical transmembrane protein. Equal amounts of total proteins in the post nuclear supernatant (PNS), membrane and cytosolic fraction were loaded for SDS-PAGE.
Kette expression. (A) Top panel shows a western Blot probed for Kette expression (P-antiserum), equal amounts of protein were loaded on the gel (Coomassie stain). (B) Western blot showing Kette expression in S2 cells and similarly increased levels of Kette expression following transfection with act5c-GAL4 and UAS-ketteMyc or act5c-GAL4 and UAS-ketteMyr.
To examine whether Kette is an integral membrane protein, we first analyzed its distribution in Drosophila cell extracts prepared from embryos or Drosophila S2-tissue culture cells by differential ultracentrifugation and flotation assays (Fig. 1C,D). In both cases the results were identical. After ultracentrifugation at 200,000 g Kette was found predominantly in the cytosolic supernatant, whereas smaller amounts could be detected in the pellet containing membranes and large protein complexes. The particulate fraction of Kette was efficiently extractable by sodium carbonate treatment (0.1 M, pH 11.5), but it was retained in the 1% NP-40 insoluble cytoskeletal fraction (Fig. 1C). To further investigate how much Kette protein really binds to membranes, a flotation assay was performed. We fractionated post-nuclear supernatants from Drosophila embryo lysates by isopycnic centrifugation on a discontinuous sucrose density gradient. Unlike the integral membrane protein FasIII (Patel et al., 1987), some of the Kette protein could float out of the most dense sucrose during centrifugation to equilibrium, while most of the Kette protein remains in high dense sucrose (Fig. 1D). Taken together, these data show that Kette is not an integral membrane protein but small amounts are associated with membranes.
The recovery of Kette protein in the 1% NP-40 insoluble fraction containing F-actin and other cytoskeletal proteins suggest an association between Kette and F-actin. To test this directly, we determined a possible interaction of Kette with F-actin in a co-sedimentation assay. Soluble, maltose-binding protein (MBP) fusion proteins containing full-length Kette or its N-terminal third (Kette1-374) were generated. Both fusion proteins, and unaltered MBP as a control, were subjected to ultracentrifugation either alone or in a mixture with F-actin. Only the fusion proteins pelleted with F-actin (Fig. 2A,B), indicating that actin binding is mediated by the N terminus of Kette, which, however, lacks known F-actin binding sequence motifs. Furthermore, we identified three different actin isoforms (Act42A, Act5C and Act57B) as putative interaction partners of Kette in a yeast two-hybrid screen using full-length Kette as bait (Fig. 2C). Based on sequence analyses of these functionally redundant actin isoforms (Wagner et al., 2002), Kette is able to bind to the C-terminal 110 amino acids of G-actin.
Kette binds to actin. (A) Soluble Kette protein was prepared as a maltose-binding-protein (MBP) fusion and stayed in the supernatant (S) when centrifuged at 200,000 g for 30 minutes. By contrast, F-actin was found in the pellet (P). When soluble Kette is added to F-actin, about 50% of Kette associates with the F-actin. Proteins were separated by SDS-PAGE followed by Coomassie blue staining. (B) MBP does not bind to F-actin. (C) Kette interacts with G-actin. Four clones of highly related actin sequences were identified in a yeast two hybrid screen. Sequence analyses indicate that interaction with Kette occurs at the C terminus.
Subcellular localization of the Kette protein in vivo
Unfortunately, none of the antisera generated detects endogenous Kette protein in whole-mount preparations of wild-type embryos. As many epitopes are sensitive to methanol treatment required to prepare Drosophila whole-mount embryos, we studied the subcellular localization of the endogenous Kette protein in freshly dissociated cells from Drosophila embryos as well as in Drosophila S2 tissue culture cells (Fig. 3). In neuronal cells, which can be identified by expression of the HRP epitope (Jan and Jan, 1982), Kette is localized in the cytosol as well as in a punctuated pattern along the emerging neurites (Fig. 3A). In muscle cells, Kette is expressed in long fibers that resemble F-actin fibers. Indeed double staining experiments using phalloidin showed prominent co-localization with F-Actin (Fig. 3B). Both cell types are affected by the kette mutation (Hummel et al., 2000).
Subcellular localization of endogenous Kette. (A,B) Primary tissue culture cells of Drosophila embryos were plated on fibronectin-coated cover slips and were allowed to differentiate over night. (C,D) Schneider S2 cells. Endogenous Kette protein was detected using anti-Kette antisera in a dilution of 1/1000. Anti-HRP antibodies recognize a carbohydrate present on all neuronal membranes. Anti-phosphotyrosine antibodies were used to detect focal contact sites. Phalloidin staining was used to visualize the F-actin cytoskeleton. A-C are projections of several confocal sections, D shows a single focal plane. Scale bar: 5 μm. (A) In neurons, Kette is expressed in an often punctuated pattern throughout the cell and can be detected in dendrites as well as axons. Higher levels of Kette are found at sites where the axon turns or branches (arrowheads). (B) Muscle cells are characterized by a highly regular F-actin cytoskeleton. Kette and F-actin expression largely overlap in these cells. (C) Schneider S2 cells endogenously express Kette. The majority of Kette is found in the perinuclear region (star). Small amounts of Kette are recruited to the leading edge of lamellipodia-like cell processes (arrowheads). Several Kette rich spokes extend from the nucleus to the membrane with higher levels of Kette at their end points close to the cell membrane (arrows). F-actin largely follows the Kette localization and can be detected surrounding the nucleus and in spokes extending to the membrane. In addition, a subcortical F-actin mesh can be detected in the lamellipodia-like structures just underneath Kette. (D) Kette expression as in C. Note the punctate appearance of Kette at the membrane. The distribution of tyrosine phosphorylated proteins frequently overlaps with Kette expression. Merged images are shown on the right.
As shown by western blot analyses, Drosophila Schneider S2 tissue cells express high levels of Kette (Fig. 4A). When S2 cells are grown on fibronectin coated substrates, they adhere to the culture dish and adopt a characteristic pancake shape (Fig. 3C,D). The majority of the Kette protein is found in the cytosol around the nucleus; however, small amounts of Kette can be detected at the leading edge of lamellipodia-like extensions (Fig. 3C, arrowheads). Increased levels of Kette expression were noted on spoke-like structures extending from the nucleus to the membrane (Fig. 3C, arrows). Highest Kette concentration is generally found close to the cell membrane overlapping with tyrosine-phosphorylated proteins, which mark focal contact sites (Fig. 3D) (Parsons et al., 2000). F-actin is similarly distributed as Kette and the spokes showing high Kette expression correspond to thick F-actin bundles. At the cell margin, however, Kette appears to be localized closer to the membrane than the cortical F-actin network. In line with the above mentioned fractionation results this again indicates that some Kette protein is associated with the membrane.
Loss of kette function leads to an excess of F-actin in the cytosol
The above data showed that Kette can be detected at the membrane and it is able to bind to F-actin. First evidence for a regulatory function of Kette in F-actin organization stems from our phenotypic analysis of kette mutants (Hummel et al., 2000). Loss of kette function affects the organization of the F-actin cytoskeleton and is characterized by an excess of disorganized F-actin bundles. To better analyze the function of Kette in F-actin dynamics we reduced kette expression in Drosophila S2 cells by RNA interference (see Materials and Methods). As judged by western blot and immunofluorescence analyses, treatment of S2 cells with a 570 bp dsRNA fragment generated from the kette-coding region resulted in a marked reduction of Kette protein expression (compare Fig. 4A with Fig. 5A,B). No interference was seen with sense and antisense RNAs (data not shown). With increasing time after dsRNA treatment, cells with reduced levels of Kette show dramatic alterations in cell morphology and concomitant changes of the F-actin cytoskeleton. Within 3 days, cells appear to collapse and accumulate large amounts of F-actin. Two days after RNAi treatment, intense granular F-actin structures are observed which are similar to those in kette-null mutants (Hummel et al., 2000) (Fig. 5B, arrowhead).
Kette regulates F-actin organization. Kette expression (top), the F-actin cytoskeleton (middle) and the merge (bottom) are shown in Schneider S2 cells using anti-Kette antisera. All images represent stacks of confocal sections. (A) In wild-type cells, endogenous Kette protein largely colocalizes with F-actin. (B) Disruption of Kette expression by RNA interference leads to a concomitant disruption of the F-actin cytoskeleton. Forty-eight hours after treatment with kette dsRNA, aggregates of F-actin are found surrounding the nucleus (arrowhead). (C) Depletion of Scar/Wave by treatment with scar/wave dsRNA leads to a marked reduction in F-actin formation and alterations in cell morphology. Interestingly, in the absence of Wave, Kette appears to be uniformly distributed throughout the cell. (D) After RNAi for both kette and scar/wave, the formation of F-actin is not further reduced. (E) Overexpression of high levels of a Myc-tagged Kette protein does not lead to any changes in the organization of the F-actin cytoskeleton. The high dilution of the anti-Kette antiserum (1:50,000 compared with 1:2000 in A does not allow the detection of the endogenous Kette protein). (E) After expression of a membrane-tethered Kette protein, the F-actin cytoskeleton is rearranged. Large clumps of F-actin can be detected close to the membrane at sites that also show high levels of Kette expression. Scale bar: 5 μm.
Kette represses Scar/Wave activity in vivo
These data led to the question how Kette influences actin dynamics. F-actin formation is controlled by the action of the Arp2/3 complex, whose activity is in turn regulated by members of the Wasp/Wave family. Drosophila has a single Wasp as well as a single Wave homolog. Recently, the Kette homolog Nap1 was found in a large 500 kDa protein complex comprising PIR121/Sra1, Abi and HSPC300 that keep Wave inactive in vitro (Eden et al., 2002). In agreement with this work disruption of kette function leads to an excess formation of cytoplasmic F-actin. In contrast to kette depletion, treatment of S2 cells with scar dsRNA leads to a reduced F-actin formation in the cytosol. Only some cortical F-actin is detected (Fig. 5C). Interestingly, disruption of Scar/Wave function resulted in a fairly uniform distribution of Kette in the cytosol and spoke-like F-actin structures cannot be recognized any more (Fig. 5C). This suggests an intact actin cytoskeleton is required to position Kette in the cell and may point to the functional relevance of the F-actin binding properties of Kette. Treatment of S2 cells with both scar/wave and kette dsRNA suppresses the excess formation of cytoplasmic F-actin induced by kette dsRNA alone. In all cell shape changes appeared slightly more severe compared with those evoked via scar dsRNA alone (Fig. 5C,D).
Further support for a Kette mediated repression of Scar/Wave activity stems from genetic analyses. Wild-type embryos are characterized by clearly separated anterior and posterior commissures, which is due to the migrating midline glia (Fig. 6A). Embryos that lack zygotic kette function display a characteristic CNS phenotype and commissures appeared fused (Fig. 6B). By contrast, loss of zygotic Scar/Wave expression does not affect embryonic nervous system development (Zallen et al., 2002) (Fig. 6D). To test whether the kette mutant phenotype might due to an upregulation of scar/wave activity we removed one copy of the scar/wave locus in a kette background, which indeed suppressed the homozygous kette phenotype and distinct commissures are now recognizable (Fig. 6C). Taken together, these data suggest that in vivo Kette represses scar/wave activity.
kette antagonizes scar/wave function. Frontal views of embryonic nerve cords of stage 16 embryos. Axon tracts are labeled using Mab BP102 and HRP immunohistochemistry (brown), midline glia cells are labeled in blue (enhancer trap insertion AA142). (A) In a wild-type nerve cord, two commissures are found in each neuromere (anterior commissure, ac; posterior commissure, pc). They are clearly separated by the midline glia (star). (B) In homozygous mutant ketteC3-20 embryos the segmental commissures are not separated into distinct axon bundles and instead appear fused. (C) Removal of one copy of the scar/wave gene in a homozygous mutant ketteC3-20 embryo significantly restores CNS development and commissures are recognizable as distinct axon bundles. (D) Homozygous mutant scar/wave embryos display no mutant CNS phenotype. (E) Reduction of wasp function in a kette mutant embryo (wasp kette double mutant) does not modify the mutant kette phenotype. (F) Expression of a membrane-tethered Kette protein can rescue the kette mutant phenotype. A rho-GAL/UAS-ketteMyr; ketteC3-20/ketteC3-20 embryo is shown. Scale bar: 30 μm.
Kette functions at the membrane
To further understand how Kette influences the dynamics of the F-actin cytoskeleton, we first conducted overexpression studies. In contrast to the kette loss of function phenotype, overexpression of full-length protein as well as the expression of several deletion constructs did not lead to any phenotype in wild-type flies or S2 cells (Fig. 4B, Fig. 5E; data not shown). As the overexpressed Kette protein was found in the cytosol, it appeared likely that membrane-recruitment of Kette is crucial for its function. In addition, biochemical as well as genetic data suggest that Kette - like the human homolog Nap1, which binds to the membrane-associated SH2SH3 adapter NCK/Dock - acts at the membrane (Hummel et al., 2000; Kitamura et al., 1996).
To test this possibility directly, we generated a membrane-tethered form of Kette by fusing a myristyolation signal derived from the Drosophila Src1 protein to the N terminus of Kette (KetteMyr). Overexpression of comparable amounts of KetteMyr in S2 cells also led to a dramatic rearrangement of the cytoskeleton and F-actin accumulates close to the membrane at sites of Kette expression (Fig. 4B, Fig. 5F). Thus, membrane tethered Kette protein is able to induce local actin polymerization.
In order to assess whether the membrane-tethered Kette protein performs similar to the wild-type protein we established UAS-ketteMyr transgenic flies and conducted genetic rescue experiments. Expression of KetteMyr in the rhomboid pattern in all CNS midline cells of mutant kette embryos suppresses the mutant phenotype in such that the segmental commissures can be clearly identified again which is not possible in mutant kette embryos (Fig. 6F). This indicates that the KetteMyr protein performs wild-type functions.
Increased expression of membrane-tethered Kette reorganizes the actin cytoskeleton
Elevated expression of the activated, membrane-tethered, Kette protein is able to induce a dominant phenotype in a dose-dependent manner. Flies carrying a scabrous-GAL4 driver (Klaes et al., 1994) and three copies of the UAS-KetteMyr transgene are characterized by pronounced alterations in bristle morphology (Fig. 7). Whereas in wild-type flies macrochaete and microchaete form as thin and relatively straight cuticular structures, expression of KetteMyr leads to thicker and often forked bristles (Fig. 7A,B,E,G). In addition to the bristle phenotypes we observed defects in the epidermal hairs. In the wild-type, each epithelial cell will develop one fine hair, whereas KetteMyr expression results in multiple hairs emerging from one cell (Fig. 7B,C, arrows). When we expressed only two copies of the KetteMyr transgene, bristle development and epidermal hair formation was affected more moderately (Fig. 7C,F).
Kette regulates F-actin formation in vivo. (A-D) Top row, dorsal view of a Drosophila notum. Bottom row, higher magnification showing the morphology of microchaete and epidermal hairs (scale bar: 10 μm). (A) Wild-type flies are characterized by an ordered array of macro- and microchaete, which are normally thin, straight and with a pointed end (E). All epidermal cells generate a small hair. (B) After expression of three copies of a UAS-KetteMyr transgene in the scabrous pattern, bristle morphology is severely disrupted. KetteMyr expression results in shorter, branched and thicker bristles (G). In addition, epidermal cells generate more than one hair (arrow). The area boxed in red is shown enlarged. (C) After expression of two copies the UAS-KetteMyr transgene in the scabrous pattern results in a weaker phenotype; however, bristles are still forked and shorter (star and F) and epidermal cells develop more than one hair (arrow). The area boxed in black is shown enlarged. (D) Same genetic background as in C but lacking one copy of the wasp gene. The bristle phenotype evoked by KetteMyr expression is suppressed. (H) During pupal development, bristle morphology is prefigured by an apical F-actin extension. (I) After KetteMyr expression (three copies), F-actin formation is initiated in a broad region of the apical cell surface. In addition, the F-actin at the cell boundary appears to have a fuzzier organization after KetteMyr expression. Scale bars: 100 μm in A-D; 2 μm in E-G; 10 μm in H,I.
The bristle and hair phenotypes induced by KetteMyr expression, reflect alterations in the organization of the apical F-actin cytoskeleton during pupal development when apical F-actin bundles prefigure bristles (Fig. 7H). In flies overexpressing KetteMyr, a rearrangement of the F-actin cytoskeleton is apparent. In contrast to wild type, large and irregular F-actin bundles emerge. In addition, cell morphology is slightly altered, cells exhibit more irregular shapes and the cortical F-actin network appears fuzzy (Fig. 7I).
Membrane recruitment of Kette induces F-actin formation via Wasp
Treatment of S2 cells expressing the membrane-bound KetteMyr protein with wave dsRNA did not suppress the KetteMyr induced phenotype, suggesting the Kette may induce F-actin formation independent of Wave (data not shown). Another important regulator of F-actin formation is Wasp, which has a single Drosophila homolog. Removal of one copy of the wasp gene significantly restores the bristle phenotype evoked by overexpression of KetteMyr (Fig. 7D). If Kette can act via activating Wasp, similar phenotypes might be expected following disruption of either gene. This is indeed the case, and loss of zygotic and maternal Wasp function results in a kette-like embryonic CNS phenotype (Tal et al., 2002; Zallen et al., 2002). To test whether a reduction in the gene dose of wasp may also affect the mutant kette phenotype, we generated a ketteC3-20 wasp3 double mutant. However, the kette wasp phenotype appeared identical to the kette mutant phenotype (Fig. 6E), which is in agreement with the notion that Kette and Wasp act positively in the same pathway.
In summary, Kette appears to repress Wave and, after membrane association, is able to activate Wasp. In the cytosol Kette is found in a complex with Wave but to date no interaction between Kette and Wasp has been reported. To elucidate further how Kette may regulate Wasp activity, we determined possible protein-protein interactions between Kette and Wasp in a yeast two hybrid assay (Fig. 8). Although no direct interaction was found between Kette and Wasp, it could be demonstrated between Kette and Abi as well as between Abi and Wasp, which thus is able to link Kette and Wasp.
Abi binds Kette and Wasp. The yeast two hybrid system was used to determine the interaction between Kette and Wasp. The indicated constructs were tested for their ability to induce three distinct selection genes (-Ade, -His andα -Gal reporters). The interactions of Kette and Abi and Wasp and Abi reconstitute functional GAL4 proteins, which enable yeast cells (AH109 strain) to grow on plates lacking adenine and histidine (Ade-, His-). In addition, these interactions activated α-Gal expression as demonstrated by the blue color of the colonies. Yeast cells co-transformed with the Gal4 fusions pGAD-Kette and pGBK-Kette; pGAD-Kette and pGBK-Wasp failed to grow on selection plates.
Regulation of growth cone and cell motility involves coordinated control of cytoskeleton dynamics (Baum and Perrimon, 2001; Mitchison and Cramer, 1996). We present biochemical and genetic evidence that the Drosophila Nap1 homolog, Kette, is able to modulate the activity of both Wasp and Scar/Wave and thus contribute to the regulation of F-actin dynamics. Wasp and Wave together with Cdc42 and Rac1 control different aspects of cortical actin dynamics (Hall, 1998; Takenawa and Miki, 2001). Cdc42 and Wasp are required for filopodia formation, whereas dominant-negative Wave disrupts the Rac1-dependent formation of branched network of F-actin bundles required to form lamellipodia (Fukuoka et al., 2001; Hall, 1998; Miki et al., 1998a). Wave localizes to membrane ruffles induced by activated Rac1 and Wasp accumulates in microspikes containing bundled F-actin (Miki et al., 1998b; Nakagawa et al., 2001).
Regulation of Scar/Wave activity
Recent work from the Kirschner laboratory showed that a large 500 kDa complex comprising Nap1/Kette, PIR121/Sra1, Abi, HSPC300 and Wave silences the otherwise constitutive activity of Wave in stimulating actin polymerization (Eden et al., 2002). The 500 kDa complex is stabilized by direct protein-protein interactions that have been demonstrated between Nap1/Kette and Abi (Tsuboi et al., 2002; Yamamoto et al., 2001), and Nap1/Kette and PIR121/Sra1 (also called Gex2) (Soto et al., 2002) (see Fig. 9 for a model). The exact binding partners of Wave are presently unknown.
Model of Kette function. Kette negatively regulates the activity of Wave in the cytosol but can activate Wasp at the membrane via Abi. Known interacting proteins are indicated. Kette also binds to F-actin, which helps to keep the Wave complex in a good position to stimulate formation of a meshed F-actin network. For further details see text.
Two modes of Wave activation have been demonstrated in vitro. On the one hand, activated Rac1 is able to bind to PIR121/Sra1 (Kobayashi et al., 1998) and can relieve Wave inhibition by dissociating the Nap1/Kette, PIR121/Sra1 and Abi sub-complex (Eden et al., 2002). On the other hand, PIR121/Sra1 is able to bind to the first SH3 domain of SH2SH3 adapter Nck (Kitamura et al., 1996; Kitamura et al., 1997), which is sufficient to activate the Wave complex (Eden et al., 2002). In vivo these two mechanisms may work at the same time to fully activate Wave.
In agreement with the work of Eden et al. (Eden et al., 2002) disruption of Kette function leads to an excess formation of cytoplasmic F-actin. However, expression of even very high levels of wild-type Kette protein do not evoke any mutant phenotype. Thus, in wild type cells the inactive Wave complexes are already formed and Kette overexpression does not result in an additional sequestering of Wave into the silencing complexes and/or an incorporation of additional Kette into these complexes. In addition, as Kette requires Sra1 function to bind to membrane-associated adapters such as Nck, excess cytosolic Kette will not be able to reorganize subcellular Wave distribution.
Further support for the notion that Kette mediates repression of Scar/Wave activity stems from genetic analyses. Embryos lacking zygotic kette function display a characteristic CNS phenotype, whereas loss of zygotic Scar/Wave expression does not affect embryonic nervous system development (Zallen et al., 2002). The kette mutant phenotype, which is due to defects in neurite outgrowth (Hummel et al., 2000), could significantly be suppressed by reducing the dose of Scar/Wave expression. This demonstrates that in wild-type embryos, Kette acts as a negative regulator of Scar/Wave. Similar results were obtained when we reduced Kette and or Scar/Wave expression in Drosophila S2 cells. These experiments also revealed that Scar/Wave is required for the normal subcellular distribution of Kette, which may, however, be an indirect effect caused the disruption of the F-actin actin cytoskeleton.
Kette function at the membrane
Kette protein localizes to the plasma membrane where it accumulates in focal adhesion contacts. A prime candidate that may mediate recruitment of Kette to the membrane is the SH2 SH3 adapter protein Nck which, besides binding of the Sra1/Kette/Abi complex, also recruits numerous other proteins to focal contact sites (Li et al., 2001). Among these is Wasp, which binds to the third SH3 domain of Nck (Quilliam et al., 1996; Rivero-Lezcano et al., 1995) (Fig. 9).
The genetic interaction of kette and dock which encodes the Drosophila Nck homolog has recently been shown (Hummel et al., 2000). We have demonstrated that membrane recruitment of Kette is sufficient to activate actin polymerization in the cell cortex mediated by Wasp. How is this brought about? One explanation might be that recruitment of Kette to the membrane disintegrates the inhibitory Wave complex - independent of the Nck/Sra1 association. This would then lead to an excess of Wave activity and subsequently to an excess of actin polymerization. However, the genetic data clearly show that membrane bound Kette functions independent of Scar/Wave but depends on Wasp.
Genetic interaction between kette and wasp
Wasp usually adopts an auto-inhibited conformation and is activated after Cdc42, Nck binding or phosphorylation (Kim et al., 2000; Miki et al., 1998a). A structure-function analysis of the Drosophila Wasp demonstrated that the Cdc42-binding domain is not necessary for function, suggesting that alternative pathways, such as phosphorylation can activate Wasp (Tal et al., 2002). Kette might be a part of such an alternative pathway, as we could demonstrate a genetic interaction between kette and wasp in the regulation of actin dynamics. Regulation of Wasp by Kette is not mediated by direct protein-protein interaction, but probably involves Abi that is able to link Kette and Wasp. The Abl interactor (Abi) protein localizes to sites of actin polymerization at the tips of lamellipodia and filopodia and has been implicated in the cytoskeletal reorganization in response to growth factor stimulation (Stradal et al., 2001). As a positive regulator of the non-receptor tyrosine-kinase Abelson (Abl) Abi may bring Abl into position to phosphorylate and thus activate Wasp. Abl is known to phosphorylate many proteins regulating focal adhesion and F-actin dynamics and overexpression of activated Abl induces F-Actin formation in Cdc42-independent manner (Woodring et al., 2002). Some tyrosine kinases activate by phosphorylation of Wasp (Cory et al., 2002; Scott et al., 2002; Suetsugu et al., 2002a); however, direct phosphorylation of Wasp by Abl remains to be demonstrated.
Further support of the model (Fig. 9) that in vivo Kette activates Wasp but suppresses Wave are the phenotypic analyses of Drosophila kette, wasp and scar/wave mutants. Mutations in kette have been isolated due to defects in commissure formation in the embryonic CNS (Hummel et al., 1999). If Kette acts via activating Wasp, similar phenotypes are expected following disruption of either gene. This is indeed the case and loss of zygotic and maternal Wasp function results in a kette-like embryonic CNS phenotype (Hummel et al., 2000; Zallen et al., 2002).
In agreement with the proposed function of Kette in regulating both, Wasp and Wave, is its subcellular distribution. Whereas the majority of Kette is present in the cytoplasm to keep Wave in its inactive state (Eden et al., 2002) some is present leading edge of lamellipodia-like structures. However, highest amounts of Kette are present at the insertion points of large F-actin bundles where N-Wasp is also present (Nakagawa et al., 2001). Kette might be recruited to these focal adhesion sites via Sra1/Nck (Goicoechea et al., 2002) and via Wasp could enhance the formation of F-actin bundles.
How Wasp activity results in straight F-actin bundles, whereas Wave stimulates the formation of a meshed F-actin network is presently unclear (Takenawa and Miki, 2001). In the cytosol, Kette may act as a scaffold protein that keeps Wave close to F-actin and recruits additional factors to F-actin such as Profilin, which not only binds to Kette but also enhances actin nucleation (Tsuboi et al., 2002; Witke et al., 1998; Yang et al., 2000). Thus, Kette could promote the formation of a meshed F-actin network characteristic for lamellipodia. At the membrane other proteins may interact with Kette and in this respect it is interesting to note that the F-actin crosslinking protein Filamin, which plays an important role in filopodia formation, also binds to Kette (S.B., unpublished). This suggests that Kette, in addition to regulating Wasp and Wave, may also contribute to the decision whether filopodia or lamellipodia are formed.
We thank E. Schejter for sending wave and scar mutants; M. Bähler for help during the co-sedimentation experiments and reagents; M. Fröhlich and K. Krukkert for excellent technical assistance; M. Bähler, G. Edenfeld, V. Gerke and A. Püschel for many helpful discussions and comments on the manuscript; and members of the Klämbt laboratory for help throughout the project. This work was funded through a grant of the DFG to C.K. and S.B.
Accepted June 12, 2003.
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You are going to email the following Kette regulates actin dynamics and genetically interacts with Wave and Wasp

redpajama_set_name: RedPajamaCommonCrawl


Lives and works in Chicago, Illinois, USA.
Doug Stapleton is an artist, curator and educator. He is an Assistant Curator of Art with the Illinois State Museum Chicago Gallery and an adjunct faculty in the Interdisciplinary Arts graduate program at Columbia College Chicago. Stapleton's work has been the subject of two solo exhibitions at the Chicago Cultural Center and Loyola University Museum of Art, Chicago in 2012.
A former Artistic Associate with the Chicago based contemporary dance company "The Seldoms", he had worked on eleven evening length dance performances in the capacity of dramaturg and as a performer. Prior to his work with the Seldoms, he has performed in over forty solo and collaborative performances since 1989. He received his MFA from the School of the Art Institute of Chicago in 1989, and a BA in Anthropology from University of Delaware with a minor concentration in Art History and Ceramics.
"In my collages, I re-configure found images into ambiguous narratives that often refer to the source, but mess around with the original message. Visually I respond to the operatic sparkle and complexity—the grandly narrated spectacle—that is the history of western art. I want to continue that high key drama in my work.


"I had my car booked in yesterday for a service. Have always serviced at Hyundai but this was my first visit to Noosa dealer after moving to the area this year. After initial mix up with booking date and without any hesitation it was no problem at all for Annie to take in for service on the day. I received a text msg when my car was ready and at the collection, Olivia greeted us and was so lovely explaining what had been done in the service and what may need attention at next service. My car was waiting outside washed and vacuumed. Couldn't believe it. You guys are awesome. Will be back for my next service. Staff should be commended for there great customer service. Great job, have not had an experience like this from service visits to three other Hyundai dealers."


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The new psychology of awkward moments
The fascination of socially awkward moments certainly hasn't been missed by comedy writers. Millions of us have cringed our way through series like Curb Your Enthusiasm and The Office. In contrast, psychology before now has largely neglected to study this fundamental part of social life.
In a new exploratory study, Johsua Clegg proposes a model. Social awkwardness, he posits, is what we feel when the situation threatens our goal of being accepted by others. The feeling prompts us to direct our attention inwards, to monitor our behaviour and attempt to behave in a way that will improve our chances of achieving acceptance. There's been a lot of research before on embarrassment, but that's tended to focus on embarrassed individuals, their feelings and dispositions. This new study is less personal, being more about the situations that reliably trigger everyday feelings of social awkwardness in most people.
Clegg invited 30 undergrad participants (13 men) into a carefully prepared room in groups of three. Each trio sat facing each other on chairs arranged in a triangle. They knew they were being filmed through a two-way mirror. There was also a table with a microphone and five cookies on it.
For the first three minutes, the participants were given no instructions. Then another participant (actually a stooge working for Clegg) arrived with a chair and sat down with them. Three more minutes passed, a researcher appeared and instructed the trio to begin an ice-breaker task (the stooge exited at this point). After three minutes discussion he would ask each of them to introduce each other to the group. Once this was done, the participants left the room and moved to another where they watched back the footage of themselves. They used a slider box, like the kind used in audience research, to indicate how awkward they were feeling during the social interactions on a moment by moment basis.
Clegg noted those moments that participants recorded a dramatic increase in social awkwardness and he cross-checked with the videos to see what was happening at the time. Moments of feeling awkward fell into distinct situational categories, which we can probably all relate to. These included times when participants didn't know what was expected of them or what the social rules were (such as when they first sat down in the room without instructions); when a social norm was broken (e.g. one person interrupted another; someone infringed on another's personal space); a social standard wasn't obtained (e.g. a person stumbled with their speech, there was a long silence); norms around eating were broken (e.g. spilling food from mouth while eating); negative social judgements were made by one person towards another, either explicitly or implicitly (e.g. by pulling a face); when names were forgotten or people weren't recognised; and when social processes were made explicit, such as during the ice-breaker task.
There were also five kinds of moment when social awkwardness plunged. This included: when people were sharing common interests, when one person helped another, when one person was positive about another, and humour. It's notable that a lot of the humour was actually about social awkwardness - joking about it seemed to make it go away.
The study is a tentative first step towards cataloguing when and why people feel socially awkward. It has obvious limitations, foremost that the participants were being filmed and the study is US-centric. But as Clegg argues, it raises all sorts of interesting avenues for future investigation. Perhaps most significant is the similarity of participants' descriptions of social awkwardness to typical accounts of full-blown social anxiety - they talked about feeling "pressured", "anxious", "nervous" and "crazy". In attempting to understand problematic social anxiety, Clegg said psychology has tended to focus on the individual, on traits like shyness and attention to the self. His new psychology of awkward moments focuses attention on the situations that trigger social discomfort in all of us. Understanding more about everyday social awkwardness, and how people deal with it, could provide new insight into why and how socially anxious people come to feel awkward nearly all of the time.
How often do you experience social awkwardness? Are there any specific social situations that trigger the feeling for you?
Clegg, J. (2012). Stranger situations: Examining a self-regulatory model of socially awkward encounters. Group Processes and Intergroup Relations, 15 (6), 693-712 DOI: 10.1177/1368430212441637
--Further reading--
The new science of "Phew!"
The new psychology of everyday playing cards
Post written by Christian Jarrett for the BPS Research Digest.
Psychology categories: Emotion, Mental health, Social
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I'm writing this blog post from Puerto Vallarta, Mexico — A place dear to my heart as I was able to spend a month here with my mom & sister while growing up! Tomorrow, this place becomes even more special as the site of the first destination wedding of my life. Obviously I'm bursting with excitement over the scenery, but the main thing that has me all fired up to be here is not this most gorgeous location — it is the couple that is uniting tomorrow! Not only are John & Faeben quite possibly one of the most stunning couples I've ever photographed, they could be the sweetest two people around. Literally. If you don't believe me, watch their proposal video, here: https://www.youtube.com/watch?v=naW7TuwgF94 Tender alert.
On the eve of their wedding day, I'm happy to share the complete set of their engagement photo session, which we shot out in the North Carolina countryside earlier this summer. These two just radiate happiness. Can you see it?! I can't wait to see the smiles on their friends & families' faces as they are wed on the beach this weekend! Congrats Faeben & John!! Thank you for having me here :) #YorksInLove!


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Administrative Policies Administrative Policies
86-5 Patent Administration and Procedures
99-1 University Policy-Making Processes
99-2 Delegation Of Executive Authority In The Absence Of The President
99-3 Workplace Violence
99-6 Cancellation Of University Activities During Severe Weather Conditions
99-7 Releasing Employees During Business Hours
00-3 Smoke-Free and Tobacco-Free Campus Policy
01-4 Freedom Of Information Act
01-5 Sexual Assault
01-6 Publication and Use of Alumni Directories
01-7 Administrative Designees Under The Agreement Between WSU And The WSU Chapter Of The AAUP-AFT Local 6075
01-8 University Symbols
01-9 Private Support For Facilities
01-10 Administration of Institutional and Departmental Nongovernmental Financial Aid (Second Release)
01-11 Service and Sale of Alcoholic Beverages on Campus
02-3 Consulting By Executive Officers
03-1 University Policy On Printed Matter
02-7 University Risk Management Policy
03-3 Tuition And Fee Appeals Board Policy
03-5 Facilities And Administrative Costs Distribution Policy
04-1 University Art Collection Management Policy
04-2 Supplier Diversity Program
04-3 Space Planning, Allocation, And Alteration
04-5 Approval, Stewardship, And Reporting Of Gifts, Grants And Contracts
04-6 Contract Signatories
04-7 Approval of Educational Policies
05-1 Class and Room Scheduling
05-3 Discrimination And Harassment Complaint Process
05-4 Contracts Between Wayne State University And External Organizations In Which University Employees Participate
05-6 Distribution and Sales of Literature, Food and Goods
05-7 Receipt, Deposit, and Management of University Funds
06-1 University Awards
06-1 University Awards - Attachment
06-2 University Centers and Institutes: Policy On Creation, Operation, Review And Discontinuance
07-2 Confidential Information Policy
08-1 Conflict Of Interest Disclosure
08-2 Investigator Disclosure
09-1 Mandatory Immunization Records
09-2 Tuberculosis Testing
10-1 Wayne State University Policy and Procedure Regarding Research Misconduct
11-1 Video Surveillance Policy
14-2 Contacts with Covered Federal and State Government Officials
15-1 Internal Financial Documents and Transaction Approvals
16-1 University Donations and Sponsorships to External Organizations
16-2 International Travel
16-4 Family Educational Rights and Privacy
18-2 Prevailing Wage Rate Requirements
19-1 Missing Student Notifications
1.1 The Board of Governors has delegated to the president, in section 2.2 of the bylaws, full authority as chief executive officer of the university and has, in addition, authorized the president to designate the duties of other executive officers.
1.2 The purpose of this executive order is to delegate to the vice-president for finance and facilities management principal responsibility for space planning, allocation, and alteration within the university.
2.1 Space is defined as the volume contained within the envelope of a building. All space constructed or acquired by the University belongs to the University and not to the respective schools, colleges, divisions, or programs even though such space may have been constructed or acquired specifically for those schools, colleges, divisions, or programs.
2.2 "Allocation" and "reallocation" are defined as the assigned custody of space to a specific school, college, division, or unit.
2.3 "Space use," "room use," and "room use categories" are defined in accordance with the "Higher Education Facilities Planning and Management Manuals" (Western Interstate Commission on Higher Education) and the state of Michigan "Capital Outlay Manual."
2.4 "Program" and "program classification" are defined in accordance with the "Higher Education Facilities Planning and Management Manuals."
"Alteration" is defined as the reconfiguration of the current building plan, change in room use, or change in program classification.
3.0 Delegation of Authority
3.1 The vice president for finance and business operations is hereby delegated principal responsibility for all matters concerning space planning, allocation (excluding classroom scheduling), alteration and all matters concerning the development of a space database and a space inventory.
4.0 Committee on Space Planning and Space Allocation
4.1 The vice president for finance and business operations will establish and chair a committee on space planning and space allocation. The committee will consist of the chair and up to three other members of the President's Cabinet, including the provost and senior vice president for academic affairs.
4.2 The responsibility of the committee on Space Planning and Space Allocation is to assist and advise the vice president for finance and business operations on major renovations that require permanent reassignment of space or substantial modification of space. The committee will also assist and advise on the planning for the overall allocation and utilization of space across the campus.
4.3 For major modification of space and/or major changes in space assignments, the vice president for finance and business operations and the Space Committee will seek advice from and review the proposed changes with the affected users including:
The senior officer(s) of the division(s) whose space assignment(s) is/are affected and or his/her/their designee(s).
The dean(s) of the affected academic unit(s), if any.
The unit head (if different from the dean) of the actual and/or potential user(s) group(s).
5.0 Liaison with the Academic Senate
5.1 The vice president for finance and business operations will periodically review the status of projects with the Academic Senate. The Academic Senate will direct its comments regarding academic space needs to the provost and senior vice president for academic affairs.
6.0 Scope of Responsibility
6.1 All matters concerning space planning, allocation or reallocation, alteration, and purpose must be approved by the vice president for finance and business operations. Schools, colleges, divisions, and units within the University must submit requests, through the appropriate vice president for preliminary approval, and then to the associate vice president for facilities planning and management, who will review the request for feasibility, code compliance, preliminary cost estimates, and consistency with the long-range plan.
6.2 Principal responsibility for the development, accuracy, and maintenance of a space database and space inventory is delegated by the vice president for finance and business operations to the associate vice president for facilities planning and management, to assure that their contents will serve the purposes of all users of that inventory for planning, reporting, and management purposes.
All actions resulting in alterations, room use changes, space allocation or reallocation, or program changes shall be reported to the associate vice president for facilities planning and management.
7.0 Supersession
7.1 This university policy supersedes all previous administrative policies and procedures concerning space allocation and reallocation, including Executive Order 85-3 Third Release.
8.1 This executive order is revocable at any time at the discretion of the president and without notice.
9.1 This executive order is effective upon issuance.
9.2 Executive Order 85-3, Third Release, is hereby revoked.
Signed by President Irvin D. Reid April 29, 2004


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