December 2007

• Cdc42 and proton efflux: A positive loop to move forward

  Positive feedback loops that generate asymmetric amplification of signaling molecules are important for the establishment and maintenance of cell polarity, and for actin assembly at the front of moving cells. The Rho GTPase Cdc42 plays a crucial role in signaling to the cytoskeleton and establishing cell polarity. In The Journal of Cell Biology, Frantz et al. now report a positive feedback loop between Cdc42 and H+ efflux in migrating fibroblasts. Previous studies have shown that H+ efflux at the leading edge by Na-H+ exchanger 1 (NHE1) is required for polarity and directional movement, and that Cdc42 acts upstream of NHE1 to promote H+ efflux. This work shows that NHE1 function is necessary for Cdc42 activation and for the localization of active Cdc42-GTP to the leading edge of migrating cells. Cdc42 activation occurs in three steps: release of Cdc42-GDP from Rho GDP dissociation inhibitor; recruitment to the plasma membrane; and exchange of GDP for GTP by a guanine nucleotide exchange factor (GEF). Whereas the first two steps were not affected by NHE1 activity, the findings of Frantz et al. show that GEF activity is pH-sensitive and GEF-induced nucleotide exchange requires NHE1. This work opens the question of whether other signaling pathways requiring GEFs may also be regulated by proton flux.

  
  

  Frantz C, Karydis A, Nalbant P, Hahn KM, Barber DL. Positive feedback between Cdc42 activity and H+ efflux by the Na-H exchanger NHE1 for polarity of migrating cells. J Cell Biol 2007; 179 (3):403-10 PubMed

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• Cancer cell migration and chemotaxis: Cofilin to set new directions

  The small protein cofilin plays a key role in regulating actin cytoskeleton organization in migrating cells. It functions at the leading edge, where, by severing F-actin, it increases the number of filament ends available for polymerization and depolymerization. A study published in The Journal of Cell Biology now sheds light on how cofilin affects protrusion dynamics in cancer cells. Sidani et al. observed that upon cofilin knockdown, metastatic mammary tumour cells switched from an amoeboid to an elongated shape, and altered their migration properties. The turning frequency was reduced and cells followed a more linear path. Metastatic cells lacking cofilin showed reduced actin polymerization, and produced fewer and more stable protrusions. Cells also lost the capacity to efficiently respond to chemotactic stimulation as they could protrude only towards the front of the cell rather than in any direction. Thus, cofilin enables cells to protrude around their complete periphery, respond to chemoattractants and change direction of movement accordingly. Interestingly, the authors also show that cofilin affects the localisation of the Arp2/3 complex, a known actin nucleator.

  
  

  Sidani M, Wessels D, Mouneimne G, Ghosh M, Goswami S, Sarmiento C, Wang W, Kuhl S, El-Sibai M, Backer JM, Eddy R, Soll D, Condeelis J. Cofilin determines the migration behavior and turning frequency of metastatic cancer cells. 2007 JCB 179(4):777-91.PubMed

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• Other CMC Publications

  • Keeping control of protrusion efficiency: One of this month's featured articles in the Cell Migration Updates section of the Gateway highlights cofilin's role in the control of protrusion. Delorme V, Machacek M, Dermardirossian C, Anderson KL, Wittmann T, Hanein D, Waterman-Storer C, Danuser G, Bokoch GM. Cofilin activity downstream of pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks. Dev Cell 2007; 13 (5):646-62. Click here to read the article review

  • Ena/VASP Is Required for Neuritogenesis in the Developing Cortex. Kwiatkowski AV, Rubinson DA, Dent EW, van Veen JE, Leslie JD, Zhang J, Mebane LM, Philippar U, Pinheiro EM, Burds AA, Bronson RT, Mori S, Fässler R, Gertler FB. 2007 Neuron 56(3):441-55. PubMed

  • Identification of phosphorylation sites in betaPIX and PAK1. Mayhew MW, Jeffery ED, Sherman NE, Nelson K, Polefrone JM, Pratt SJ, Shabanowitz J, Parsons JT, Fox JW, Hunt DF, Horwitz AF. J Cell Sci. 2007 Nov 15;120(Pt 22):3911-8. PubMed

  • GEF-H1 modulates localized RhoA activation during cytokinesis under the control of mitotic kinases. Birkenfeld J, Nalbant P, Bohl BP, Pertz OP, Hahn KM, Bokoch GM. Dev. Cell 12(5): 699-712, 2007. PubMed

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• New Data

  Phosphoproteomics data for alpha-actinin, vinculin & p115 RhoGEF: Data showing the phosphorylation sites present in three new migration-related proteins, alpha-actinin, vinculin and p115 RhoGEF, have recently been made available through the proteomics initiative activity table.

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November 2007

• Breaking the symmetry: A back start

  Migrating cells are both morphologically and functionally polarized, and their movement is largely dependent upon a polarized actin network. Reports that different cell types are able to spontaneously polarize to initiate motility in the absence of any external cues suggest that the initiation of polarization may rely on intrinsic mechanisms. In a recent study published in the Journal of Cell Biology, Yam et al. report that spontaneous polarization and motility initiation in fish keratocytes result from changes in F-actin flow velocity and that the direction of motility begins at the cell rear and perinuclear region. Changes in F-actin dynamics coincided with rear retraction and morphological polarization, suggesting that F-actin network movement triggers the initiation of motility. Conversely, flow changes at the cell front and front protrusion occurred at later stages as a consequence of motility initiation. Yam et al. also report that motility initiation required the activity of myosin II and its regulator Rho kinase. Altogether, these experiments suggest that the breaking of symmetry in fish keratocytes begins with myosin-mediated changes in actin network dynamics at the rear of cells, and that polarity is then propagated towards the front.

  
  

  Yam PT, Wilson CA, Ji L, Hebert B, Barnhart EL, Dye NA, Wiseman PW, Danuser G, Theriot JA. Actin-myosin network reorganization breaks symmetry at the cell rear to spontaneously initiate polarized cell motility. J Cell Biol 2007; 178 (7):1207-21 PubMed

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• EGF-stimulated motility in carcinoma cells: A Cdc42 response

  Cell motility requires actin nucleation at the leading edge, which results in the production of a highly branched actin network that provides protrusive force to the cell membrane and allows lamellipodial extension. In a recent study published in the Journal of Cell Science, El-Sibai et al. report that the small GTPase Cdc42 plays a crucial role in the regulation of actin polymerization, protrusion and motility in epidermal growth factor (EGF)-stimulated MTLn3 carcinoma cells. The authors observed that in EGF-stimulated cells Cdc42 was activated at the edge of actively protruding lamellipods where actin is newly polymerized. Knockdown of Cdc42 by RNA silencing inhibited the formation of persistent protrusions. Furthermore, Cdc42-depleted cells showed a defect in actin branching and the accumulation of F-actin due to failure of Arp2/3 complex recruitment to the protruding edge. Activation of Arp2/3-mediated actin branching involves the interaction between Arp2/3 and actin nucleation factors of the WASP-family. Importantly, the findings of El-Sibai et al. suggest that Cdc42 is required for the activation of WASP proteins, in particular WAVE2, and that, in contrast to existing models, this occurs independently from the Rac1 GTPase.

  
  

  El-Sibai M, Nalbant P, Pang H, Flinn RJ, Sarmiento C, Macaluso F, Cammer M, Condeelis JS, Hahn KM, Backer JM. Cdc42 is required for EGF-stimulated protrusion and motility in MTLn3 carcinoma cells. J Cell Sci 2007; 120 (Pt 19):3465-74 PubMed

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• Other CMC Publications

  • Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode. Dalal RB, Digman MA, Horwitz AF, Vetri V, Gratton E. Microsc Res Tech Epub 2007 Oct 15 PubMed

  • Experimental and DFT Studies: Novel Strucutral Modifications Greatly Enhance the Solvent Sensitivity of Live Cell Imaging Dyes. Toutchkine A, Han WG, Ullmann M, Liu T, Bashford D, Noodleman L, Hahn KM. J Phys Chem A. 2007 111 (42) 10849-60. PubMed

  • Phosphoblast: A computational tool for comparing phosphoprotein signatures among large datasets. Wang Y, Klemke RL. Mol Cell Proteomics Epub 2007 Oct 13 PubMed

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• Addition of New Data to the site

  Geiger Lab siRNA screen: Data showing the effects of siRNA-induced gene knockdown on the formation and morphogenesis of focal adhesions is now available http://www.cellmigration.org/resource/discovery/geiger/view_geiger_rnai.cgi.

  Three siRNA libraries targeting 196 phosphatases, 580 kinases and 318 additional genes have been used in conjunction with a high-throughput screening strategies to elucidate the function of the knocked-down proteins on the formation and morphogenesis of focal adhesions. Effects on cell viability, morphology and focal adhesion quantity, size, shape, intensity and organization have been evaluated. Results include cell rounding, cell elongation, decrease in focal adhesion number and change in distribution throughout the cell.

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• Upcoming Conferences & Workshops

  • 1-5 December 2007 - 47th Annual meeting of the ASCB, Washington DC:

    Members of the Consortium's Biosensor and Imaging & Photomanipulation Initiatives will be amongst the presenters at a workshop entitled "High Performance Image Analysis and Photomanipulation Techniques for Cell Biology". Organizers Michael A. Mancini and Ken Jacobson have gathered together an impressive list of speakers to present talks ranging from high throughput imaging efforts that capitalize on automated image acquisition and image analyses to photomanipulative studies on the subcellular scale.

    
    

    Advances light microscopic imaging of cells have ushered in a new era in cell biology, and have increased awareness and development of systems level biology at the single cell level. One focus of this workshop will be on the utilization of these technologies to quantitatively investigate gene regulation, mRNA splicing, stem cell differentiation, and the extracellular matrix. A second focus will be on biosensors that provide subcellular readouts of key signaling molecules and on photomanipulative techniques, including the photoactivation of caged compounds or chromophore assisted laser inactivation (CALI or FALI) of selected components, as an important complement to traditional genetic manipulations.

    
    

    Speaker list:

    Jeffrey H. Price, M.D., Ph.D., "Burnham MLSCN Screening Center Update and Automated Stem Cell Tracking", Burnham Institute for Medical Research, La Jolla, CA

    Michael A. Mancini, Ph.D., "High throughput image analyses of nuclear receptor function", Baylor College of Medicine, Houston, TX

    Jeffrey A. Nickerson, Ph.D., "In Vitro FRAP and the Study of mRNA Export", University of Massachusetts Medical School, Worcester, MA

    Gordon L. Hager, Ph.D. "Gene Targeting by Nuclear Receptors in Living Cells" National Cancer Institute, NIH, Bethesda, MD

    Klaus Hahn, Ph.D., "New tools for multiplex manipulation and visualization of protein activities in living cells", University of North Carolina-Chapel Hill, Chapel Hill, NC

    David Lawrence, Ph.D., "Light-sensitive sensors and modulators of intracellular biochemistry", Albert Einstein College of Medicine, New York, NY

    Dan Larson, Ph.D., "Spatio-temporal control of protein activity and expression in living cells", Albert Einstein College of Medicine, New York, NY

    Ken Jacobson, Ph.D., "On the use of EGFP as a CALI agent to induce loss of function in actin binding proteins", University of North Carolina-Chapel Hill, Chapel Hill, NC

    
    

    For more details and to register (early registration deadline is October 1st) go to http://www.ascb.org/meetings/

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October 2007

• Actin dynamics and VASP: The shape of cells to come

  Cytoskeletal rearrangements play a major role in cell shaping and migration, but how such small-scale rearrangements result in large-scale morphological changes remains unclear. By combining experimental and mathematical modelling approaches, Julie Theriot and colleagues now report how local changes in F-actin dynamics affect the morphology and motility of epithelial keratocytes in fish. Primary keratocyte populations are heterogeneous in size, shape and motile behaviours. Cells predominantly have a canoe-like shape with a smooth lamellipodial leading edge, although they can also exhibit an irregular shape with a rough leading edge. Smooth cells migrate faster and along straighter trajectories than rough cells. By modulating the activity of vasodilator-stimulated phosphoprotein (VASP) - known to promote the formation of long actin filaments - the authors showed that the smooth, fast, straight-moving phenotype requires high levels of VASP at the leading edge. The authors went on to measure the morphological variations in a keratocyte population showing a spectrum of shapes from smooth to rough, and developed a mathematical model correlating shape changes to both the levels of VASP at the leading edge and F-actin distribution. The model suggests that high F-actin density at the cell front minimises the cell membrane resistance per growing filament, thus allowing rapid VASP-mediated F-actin growth that pushes the membrane forward. Conversely, low F-actin density due to low VASP activity results in high membrane resistance per filament, limiting membrane protrusion and causing slower cell migration.

  
  

  Lacayo CI, Pincus Z, VanDuijn MM, Wilson CA, Fletcher DA, Gertler FB, Mogilner A and Theriot JA. Emergence of large-scale cell morphology and movement from local actin filament growth dynamics. PLoS Biol. 2007;5(9):e233. PubMed

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• Biomaterials: Tether length and ligand spacing affect cell spreading

  Synthetic substrates help illuminate how cell migration-related processes are affected by cues in the extracellular environment. For example, integrin-mediated adhesion is regulated by multiple factors pertaining to the adhesive surface, including its chemical composition, physical properties and topography. In a recent study published in Biomacromolecules, Anne Mayes and colleagues investigated how variations in the length of tethers used to attach RGD (Arg–Gly–Asp) adhesion peptides to a polymer brush — as well as the spacing between the adhesive RGD ligands — affect integrin-mediated cell attachment and cell spreading. The authors used surfaces bearing PEO (poly(ethylene oxide))-tethered RGD ligands displaying PEO side chains that varied in length, with either 10 or 22 ethylene oxide segments. They demonstrated that increasing the length of PEO tethers increases the rate of cell spreading, thus allowing focal adhesions to form at shorter incubation times. Furthermore, the studies suggested that cell adhesion occurred faster because of the higher mobility allowed by the longer tethers. This facilitates the rearrangement of surface-bound peptides and thus promotes the formation of integrin clusters at the cell membrane. These results have implications for the design of biomaterials for tissue engineering and regenerative medicine, as they reveal how the local nanoscale presentation mode of adhesive ligands influences dynamic cell behaviours.

  
  

  Kuhlman W, Taniguchi I, Griffith LG and Mayes AM. Interplay Between PEO Tether Length and Ligand Spacing Governs Cell Spreading on RGD-Modified PMMA-g-PEO Comb Copolymers. Biomacromolecules 2007; Sep 18 PubMed

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• Other CMC Publications

  • Membrane-binding/modification model of signaling protein activation and analysis of its control by cell morphology.
    Haugh JM.. Biophys J 2007; 92 (11):L93-5 PubMed

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• Improved Protein Constructs page now has protocols available

  • Constructs for migration-related proteins, or portions thereof, are available through the Consortium’s Structure Initiative and the protocols associated with the expression and purification of these proteins are now easily available through the reworked Constructs page http://www.cellmigration.org/resource/structure/structure_resources_constructs.shtml and the Initiative's resource page http://www.cellmigration.org/resource/structure/structure_resources.shtml#protocols

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• Upcoming Conferences & Workshops

  • 1-5 December 2007 - 47th Annual meeting of the ASCB, Washington DC:

    Members of the Consortium's Biosensor and Imaging & Photomanipulation Initiatives will be amongst the presenters at a workshop entitled "High Performance Image Analysis and Photomanipulation Techniques for Cell Biology". Organizers Michael A. Mancini and Ken Jacobson have gathered together an impressive list of speakers to present talks ranging from high throughput imaging efforts that capitalize on automated image acquisition and image analyses to photomanipulative studies on the subcellular scale.

    
    

    Advances light microscopic imaging of cells have ushered in a new era in cell biology, and have increased awareness and development of systems level biology at the single cell level. One focus of this workshop will be on the utilization of these technologies to quantitatively investigate gene regulation, mRNA splicing, stem cell differentiation, and the extracellular matrix. A second focus will be on biosensors that provide subcellular readouts of key signaling molecules and on photomanipulative techniques, including the photoactivation of caged compounds or chromophore assisted laser inactivation (CALI or FALI) of selected components, as an important complement to traditional genetic manipulations.

    
    

    Speaker list:

    Jeffrey H. Price, M.D., Ph.D., "Burnham MLSCN Screening Center Update and Automated Stem Cell Tracking", Burnham Institute for Medical Research, La Jolla, CA

    Michael A. Mancini, Ph.D., "High throughput image analyses of nuclear receptor function", Baylor College of Medicine, Houston, TX

    Jeffrey A. Nickerson, Ph.D., "In Vitro FRAP and the Study of mRNA Export", University of Massachusetts Medical School, Worcester, MA

    Gordon L. Hager, Ph.D. "Gene Targeting by Nuclear Receptors in Living Cells" National Cancer Institute, NIH, Bethesda, MD

    Klaus Hahn, Ph.D., "New tools for multiplex manipulation and visualization of protein activities in living cells", University of North Carolina-Chapel Hill, Chapel Hill, NC

    David Lawrence, Ph.D., "Light-sensitive sensors and modulators of intracellular biochemistry", Albert Einstein College of Medicine, New York, NY

    Dan Larson, Ph.D., "Spatio-temporal control of protein activity and expression in living cells", Albert Einstein College of Medicine, New York, NY

    Ken Jacobson, Ph.D., "On the use of EGFP as a CALI agent to induce loss of function in actin binding proteins", University of North Carolina-Chapel Hill, Chapel Hill, NC

    
    

    For more details and to register (early registration deadline is October 1st) go to http://www.ascb.org/meetings/

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September 2007

• Integrin adhesome: The complex world of cell-extracellular matrix interactions

  Specialized adhesion complexes, which contain integrin receptors, cytoskeletal elements and a wide range of adaptor proteins, mediate cell-extracellular matrix (ECM) interactions and so allow cells to sense extracellular signals. As a result, cells respond differently when exposed to different adhesive ligand density, surface rigidity, ECM components or when cultured on two- or three-dimensional matrices.

  
  

  To understand how the adhesion machinery senses environmental cues and responds, Geiger and colleagues provide a detailed description of the in silico integrin adhesome network (http://www.adhesome.org/), now reported in Nature Cell Biology. The authors used data derived from published experimental studies to outline the molecular basis for integrin-mediated adhesion and signaling. A total of 156 components were assembled, and an interaction map is presented as a network in which the different molecular components are considered as nodes, and their direct interactions are considered as links, with a distinction made between 'binding' and 'signaling' interactions.

  
  

  Zaidel-Bar R, Itzkovitz S, Ma'ayan A, Iyengar R, Geiger B. Functional atlas of the integrin adhesome. Nat Cell Biol 2007; 9 (8):858-67 PubMed

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• Methods: How to identify compartmentalized proteins in migrating cells

  Directional cell migration, which occurs in response to a gradient of chemoattractant or extracellular matrix (ECM) protein, is characterized by the formation of a dominant pseudopodium. Upon sensing the gradient, cells respond by locally amplifying the signals on the side facing the gradient. As a result, cells polarize, the actin-myosin cytoskeleton reorganizes and a pseudopodium extends in the direction of movement. Although extensively studied, the molecular mechanisms underlying this cellular response are still poorly defined. In Science STKE, Klemke and colleagues now report a detailed protocol for purifying pseudopodia and cell body compartments of migrating cells, which then allows analysis of their protein and phosphoprotein components. An in vitro system that uses porous filters and mimics the physiological events associated with pseudopodial protrusion through small openings of the ECM was developed, allowing separation of the pseudopodium from the cell body. Methods for large-scale quantitative proteomic and phosphoproteomic analyses, using immobilized metal affinity chromatography (IMAC) to enrich for phosphopeptides, and ¹⁸O/¹⁶O labeling for quantitation, are described in detail. These techniques should allow the systematic analysis of proteins differentially localized or differentially phosphorylated in the front and back of polarized migrating cells.

  
  

  Wang Y, Ding SJ, Wang W, Yang F, Jacobs JM, Camp D, 2nd, Smith RD, Klemke RL. Methods for pseudopodia purification and proteomic analysis. Sci STKE 2007; 2007 (400):pl4 PubMed

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• New Postings to the CMC Activity Center

  • CM Knowledgebase is New and Improved

    This month sees the launch of the new and improved Cell Migration Knowledgebase (CMKB) which now includes links to a host of additional information about; genes (Entrez, OMIM), proteins (UniProt) orthologs (Homologene, Signaling Gateway, WormBase, FlyBase), motifs (Pfam, Scansite, NCBI CDD), structures (PDB), interactions (BioGRID, IntAct, BIND) and pathways (KEGG, BioCarta, NCI ZPathway DB). Details about available reagents (plasmids, antibodies, transgenic mice, biosensors etc) both commercial and those generated through the Consortium's efforts are also available from these pages. We encourage you to visit the site and search for information on you favorite migration-related protein. Please give us your feedback so we can make these pages as useful to you, our users, as possible. http://cmckb.cellmigration.org/

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• Other CMC Publications

  • Model of polarization and bi-stability of cell fragments. Kozlov MM, Mogilner A. Biophys J 2007 Aug 17; [Epub ahead of print]. PubMed

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• Upcoming Conferences & Workshops

  • 22–26 October 2007 - 2nd LFD Workshop in Advanced Fluorescence Imaging and Dynamics:

    The Laboratory for Fluorescence Dynamics (LFD) is organizing its 2nd advanced workshop, which will consist of theoretical lectures, a hands on laboratory course at the LFD facility, and computer based training on data analysis and simulations. It will outline the advanced concepts of fluorescence correlation spectroscopy and fluorescence lifetime resolved imaging techniques. Student registrants are encouraged to submit an abstract for the registration waiver award competition. For more details and to register visit http://www.lfd.uci.edu/workshop/

  • 1-5 December 2007 - 47th Annual meeting of the ASCB, Washington DC:

    Members of the Consortium's Biosensor and Imaging & Photomanipulation Initiatives will be amongst the presenters at a workshop entitled "High Performance Image Analysis and Photomanipulation Techniques for Cell Biology". Organizers Michael A. Mancini and Ken Jacobson have gathered together an impressive list of speakers to present talks ranging from high throughput imaging efforts that capitalize on automated image acquisition and image analyses to photomanipulative studies on the subcellular scale.

    
    

    Advances light microscopic imaging of cells have ushered in a new era in cell biology, and have increased awareness and development of systems level biology at the single cell level. One focus of this workshop will be on the utilization of these technologies to quantitatively investigate gene regulation, mRNA splicing, stem cell differentiation, and the extracellular matrix. A second focus will be on biosensors that provide subcellular readouts of key signaling molecules and on photomanipulative techniques, including the photoactivation of caged compounds or chromophore assisted laser inactivation (CALI or FALI) of selected components, as an important complement to traditional genetic manipulations.

    
    

    Speaker list:

    Jeffrey H. Price, M.D., Ph.D., "Burnham MLSCN Screening Center Update and Automated Stem Cell Tracking", Burnham Institute for Medical Research, La Jolla, CA

    Michael A. Mancini, Ph.D., "High throughput image analyses of nuclear receptor function", Baylor College of Medicine, Houston, TX

    Jeffrey A. Nickerson, Ph.D., "In Vitro FRAP and the Study of mRNA Export", University of Massachusetts Medical School, Worcester, MA

    Gordon L. Hager, Ph.D. "Gene Targeting by Nuclear Receptors in Living Cells" National Cancer Institute, NIH, Bethesda, MD

    Klaus Hahn, Ph.D., "New tools for multiplex manipulation and visualization of protein activities in living cells", University of North Carolina-Chapel Hill, Chapel Hill, NC

    David Lawrence, Ph.D., "Light-sensitive sensors and modulators of intracellular biochemistry", Albert Einstein College of Medicine, New York, NY

    Dan Larson, Ph.D., "Spatio-temporal control of protein activity and expression in living cells", Albert Einstein College of Medicine, New York, NY

    Ken Jacobson, Ph.D., "On the use of EGFP as a CALI agent to induce loss of function in actin binding proteins", University of North Carolina-Chapel Hill, Chapel Hill, NC

    
    

    For more details and to register (early registration deadline is October 1st) go to http://www.ascb.org/meetings/

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August 2007

• Talin-integrin interaction: An antithrombosis target
  Haemostasis occurs upon vascular injury, and platelets aggregate to form a plug at the site of damage. Abnormal platelet aggregation can occur in arterial disease causing thrombosis, where blood clots within the vessel and impairs the flow of blood through the circulatory system. This requires ligand-induced activation of platelet integrin alpha IIb beta 3 (platelet GPIIb-IIIa). Ginsberg and colleagues now report in The Journal of Clinical Investigation the first in vivo evidence that binding of the integrin beta cytoplasmic domain to talin is critical for integrin activation. Whereas mutations at integrin beta 3 Tyrosine 747 (Y747A) blocked binding of talin and other proteins, mutations at Leucine 746 (L746A) exclusively blocked talin binding. Although both mutant mouse strains bearing these mutations showed a disruption in integrin activation, platelets from beta 3(L746A) mutants were still able to spread on fibrinogen in response to outside-in signals. Reduced thrombosis was also observed in both strains. beta 3(Y747A) mice showed pathological bleeding akin to treatment with agents that prevent ligand binding, and selectively blocking the talin-beta 3 interaction reduced bleeding and increased mice survival. These results highlight the talin-beta 3 interaction as an important therapeutic target for pathological thrombosis.
  
  Petrich BG, Fogelstrand P, Partridge AW, Yousefi N, Ablooglu AJ, Shattil SJ, Ginsberg MH. The antithrombotic potential of selective blockade of talin-dependent integrin αIIbβ3 (platelet GPIIb–IIIa) activation. J. Clin. Invest. 2007 Jul 12; [Epub ahead of print] PubMed

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• Inverse dynamics: Reconstructing the forces for cell movement

  Motor-based contraction, turnover, and cytoskeletal interaction with the plasma membrane and extracellular matrix are highly regulated in time and space during cell migration,. This regulation depends on local structural reorganizations and the precise coordination of intracellular force-generating and force-transducing machinery. In Methods in Cell Biology, Danuser and colleagues describe a new method that allows the mapping of intracellular forces at high spatial and temporal resolution. High-resolution live cell movies were generated using quantitative fluorescent speckle microscopy. These were used to measure cytoskeletal flows, which were then used to mathematically reconstruct a map of intracellular forces. The principle relies upon assessing the deformations of subcellular structures with high precision and then mathematically deducing the forces required to obtain the observed deformation. This indirect reconstruction of physical quantities is termed ‘inverse dynamics’. Danuser and colleagues used this method to analyse deformations of the F-actin cytoskeleton and reconstruct the underlying forces in migrating epithelial cells, highlighting its potential for studying intracellular force regulation in numerous cell functions.
  
  Ji L, Loerke D, Gardel M, Danuser G. Probing intracellular force distributions by high-resolution live cell imaging and inverse dynamics. Methods Cell Biol. 2007;83:199-235 Pubmed

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July 2007

• Mapping the Pseudopodial Proteome of Moving Cells
  Directed cell migration is a highly polarized process involving the protrusion of a leading pseudopodium at the front, and the establishment of a trailing rear compartment at the back of the cell. An increasing number of specific signaling proteins and pathways controlling cell polarity and directed migration have been identified, but it is still largely unknown how these signals integrate into spatial networks. In PNAS, Klemke and colleagues (http://klemkelab.ucsd.edu/) now report the proteome profiling and phosphoproteomic analysis of the pseudopodium and cell body compartments of migrating cells. Of the 5266 proteins that were identified in the pseudopodium and cell body fractions from polarized cells, 1348 localized in both compartments, 1073 were enriched only in the pseudopodium, and 1088 were enriched only in the cell body. Bioinformatic analysis identified putative pathways within these protein data sets, revealing a high degree of signaling pathway spatial separation between the front and back of polarized cells. Quantitative comparison of 228 protein phosphorylation sites identified in the pseudopodium and cell body also showed that both phosphorylation levels and specific phosphorylation signatures are differentially regulated depending on the subcellular localization of the protein. Overall, this work represents a valuable database resource for the investigation of signal polarity and the identification of signaling networks in the migrating cell. The data can be accessed on the Consortium web site at http://www.cellmigration.org/resource/proteomics/klemke.shtm
  
  Wang Y, Ding S-J, Wang W, Jacobs JM, Qian W-J, Moore RJ, Feng Y, Camp DG, Smith RD, Klemke RL. Profiling signaling polarity in chemotactic cells. Proc Natl Acad Sci U S A 2007; 104 (20): 8328-8333.PubMed

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• Fluorescent live cell imaging dyes with greatly improved photostability

  Merocyanine dyes are high performance fluorophores used because they are bright, fluoresce at long wavelengths, and most importantly because they change their fluorescence spectra in response to environment. They have been used with great success to report protein conformational changes in individual, living cells. Unfortunately, many otherwise excellent merocyanine dye have limited use because they photobleach rapidly. In Organic Letters, Toutchkine et al. describe a broadly applicable modification of merocyanine dyes that greatly improves photostability. The approach can lead to improvemets in many existing dyes and bring into reach new dyes of extraordinary brightness. Attachment of an electron withdrawing group at a specific position reduces reaction with singlet oxygen, a major photobleaching pathway.
  
  Toutchkine A, Nguyen DV, Hahn KM. Merocyanine Dyes with Improved Photostability. Org Lett 2007 Jun 21; [Epub ahead of print] PubMed

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• Dyes from one pot: A new synthesis method for cyanines and merocyanines Merocyanine and cyanine dyes are high performance fluorophores used in protein labelling. In Bioconjugate Chemistry, Toutchkine et al. now describe a method for the synthesis of cyanine and merocyanine dyes carrying chloro- and iodoacetamido side chains for site-selective attachment to protiens. Time consuming and difficult steps from earlier syntheses are eliminated and substituted with a simple sequence that can be performed in only one receptacle. Synthesis of both dye families starts with a common intermediate — 1 (3-ammoniopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate bromide. This method delivers bright dyes that have excellent absorption and fluorescence properties. Furthermore, labelling of extracellular regulated kinase 2 (EGFP-Erk2) fusion proteins was successful, confirming the practicality of the new dyes.
  
  Toutchkine A, Nguyen DV, Hahn KM. Simple One-Pot Preparation of Water-Soluble, Cysteine-Reactive Cyanine and Merocyanine Dyes for Biological Imaging. Bioconjug Chem 2007 Jun 2; [Epub ahead of print]PubMed

• Other CMC Publications

  • On the freezing and identification of lipid monolayer 2-D arrays for cryoelectron microscopy. Taylor DW, Kelly DF, Cheng A, Taylor KA. J Struct Biol 2007 May 6; [Epub ahead of print] PubMed

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June 2007

• Dating Ras: Identification of putative Ras-interacting partners

  Closely related members of the Ras family of small GTPases regulate different cellular functions by signaling through different pathways. Functional diversity is achieved through their interaction with specific effectors, but only a few of these have been identified so far. Reporting in Journal of Proteome Research, Goldfinger et al. have isolated candidate Ras-specific effectors in mammalian cells, using a modified tandem affinity purification tag (TAP tag) technique. The authors expressed N-terminal TAP fusions of different Ras family members in fibroblasts and showed that these fusions do not interfere with Ras functions in vivo. Using TAP tag combined with nanoflow high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry, the authors identified 21, 28 and 222 proteins in complex with activated R-Ras, Rap1A and H-Ras, respectively. Although some proteins were found in complex with R-Ras and H-Ras, and some proteins were common to Rap1A and H-Ras, there was no overlap observed between R-Ras and Rap1A. Rap1A and R-Ras interactors included signaling molecules, many of which are membrane-targeted, whereas H-Ras associated with cytoskeletal proteins, including talin-1. As the talin domain that interacts with activated H-Ras also binds and activates integrins, this finding suggests a functional connection between H-Ras, talin-1 and integrin activation. The data obtained in this study constitute a database of putative specific and shared effectors of Ras GTPases that may be a valuable resource in the characterization of signaling pathways. These data are available on the Consortium's web site at: http://www.cellmigration.org/resource/proteomics/data/ginsberg_ras_data.shtml
  
  Goldfinger LE, Ptak C, Jeffery ED, Shabanowitz J, Han J, Haling JR, Sherman NE, Fox JW, Hunt DF, Ginsberg MH. An Experimentally Derived Database of Candidate Ras-Interacting Proteins. Journal of Proteome Research 2007 May;6(5):1806-11. PubMed

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• Tensin1: Recruiting integrins to fibrillar adhesions

  Extracellular matrix (ECM) cell attachment is mediated by integrins and plasma membrane receptors that have a crucial role in cell migration and signaling from the ECM. Whereas the integrin extracellular domain binds directly to the ECM, the intracellular domain binds to the actin cytoskeleton through adaptor proteins such as tensin1. Liddington and colleagues now describe the structural basis of the tensin1–integrin interaction and its implications for the recruitment of tensin1 to fibrillar adhesions.

  The authors describe the crystal structure of the C-terminal tensin1 PTB domain and show that it interacts with the NPxY motif in integrin beta‘s cytoplasmic tail. Phosphorylation of the NPxY tyrosine residue has been shown to modulate interaction between integrin and PTB domain-containing proteins, and inhibits the interaction of integrin with the adaptor protein talin. Liddington and colleagues found that the NPxY tyrosine residue is also required for the interaction of integrin with tensin1, but that binding affinity is unaffected by the state of phosphorylation. The dynamics of cell–ECM interaction maturation — from focal contacts to focal adhesions and then fibrillar adhesions — are poorly understood. The authors propose that integrin tail phosphorylation occurs during focal contact maturation to release talin, making the binding site available to tensin, which is involved in fibrillar adhesion establishment. The interaction between integrins and tensin1 then facilitates the translocation of integrins to fibrillar adhesions.
  
  McCleverty CJ, Lin DC, Liddington RC. Structure of the PTB domain of tensin1 and a model for its recruitment to fibrillar adhesions. Protein Science 2007; 16:1-7. PubMed

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• Other CMC Publications

  • GEF-H1 Modulates Localized RhoA Activation during Cytokinesis under the Control of Mitotic Kinases. Birkenfeld J, Nalbant P, Bohl BP, Pertz O, Hahn KM, Bokoch GM. Dev Cell 2007; 12 (5):699-712 PubMed

  • Microtubule-targeting-dependent reorganization of filopodia. Schober JM, Komarova YA, Chaga OY, Akhmanova A, Borisy GG. J Cell Sci 2007; 120 (Pt 7):1235-44 PubMed

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May 2007

• Light-induced protein inactivation: On and off in one step

  In PNAS, Jacobson and colleagues now report the development of a new system to inactivate specific proteins using chromophore-assisted laser inactivation (CALI). CALI is a technique whereby a target protein is labeled with a chromophore — often a fluorescent protein — and its activity then inhibited by reactive photoproducts following light treatment.

  The authors constructed a modified lentiviral vector containing a short hairpin RNA (shRNA) and a functional Enhanced Green Fluorescent Protein (EGFP)-target protein fusion. The shRNA is used to silence the gene of interest, whereas the EGFP-fusion leads to the production of a functional copy of the protein, rescuing the knockdown. The only functional copy in the cells is the EGFP-fusion, which can in turn be inactivated by CALI.  Insertion of a light-sensitive version of the target protein, and knockdown of the endogenous copy — which eliminates any residual activity — can be achieved in a single step, allowing accurate control of protein function.

  The knockdown/rescue system was used to test the function of Capping protein (CP) and Mena in fibroblasts. CP and Mena localize to the barbed ends of F-actin at the leading edge, and regulate cell motility and migration. CALI of CP resulted in a local increase in the number of barbed ends, an increase in F-actin levels and the production of numerous protrusions. CALI of Mena induced large stable lamellipodia. These loss-of-function phenotypes indicate that specific protein inactivation was successful.
  
  Vitriol EA, Uetrecht AC, Shen F, Jacobson K, Bear JE. Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins. Proc Natl Acad Sci U S A 2007; 104 (16):6702-7 PubMed

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• Predicting effects of drugging motility signaling network targets

  Although a myriad of potential molecular targets for therapeutic intervention have been and are continuously identified, a daunting challenge is how to predict the effects of drugs that interfere with their activity. The main problem in this challenge lies in the need to quantitatively integrate the contributions to governing phenotypic cell behavior from multiple signaling pathways. This challenge is especially difficult for cell migration applications, such as tissue regeneration and tumour metastasis, because the dominant rate-limiting biophysical process regulated by the signaling pathways is likely to be different under diverse environmental conditions. Thus, a model aimed at predicting how inhibiting a given target will affect migration behavior must be capable of incorporating multi-pathway activities in different conditions. The work by Kharait et al recently published in BMC Systems Biology describes a successful demonstration of a priori prediction of the effects of a small molecule inhibitor of myosin light-chain kinase on fibronectin migration across a range of substratum fibronectin levels. This prediction was accomplished using a decision-tree model trained on measurements of five phospho-protein levels (p-EGFR, p-ERK, p-PLCgamma, p-PKCdelta, p-MLCK) downstream of EGF stimulation, on four different fibronectin levels. An especially important validated prediction is that sub-maximal inhibition of p-MLCK activity can lead to either enhanced or reduced migration, depending on fibronectin levels, presumably due to consequently altered balances among underlying biophysical processes.
  
  Kharait S, Hautaniemi S, Wu S, Iwabu A, Lauffenburger DA, Wells A. Decision tree modeling predicts effects of inhibiting contractility signaling on cell motility. BMC Syst Biol 2007; 1:9 PubMed

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• Aquaporins: Promoters of filopodia formation

  Aquaporins (AQPs) are bidirectional water channels that allow rapid fluid transport across lipid membranes. The function of these water fluxes is mostly unknown; however, AQP9 has recently been implicated in directed motility. In Experimental Cell Research, Jacobson and colleagues now show that AQP9 induces the formation of filopodia and alters cell migration.

  Transfection of cultured fibroblasts with AQP9 resulted in reduced polarization, appearance of numerous filopodia and increased water transport across the membrane. Filopodia formation and enhanced permeability required phosphorylation of AQP9 at Ser 11 and Ser 222. AQP9 Ser 11 was phosphorylated by protein kinase C (PKC), suggesting that PKC may regulate the gating of AQP9. Higher levels of active CDC42, a Rho GTPase involved in actin polymerization, were observed upon AQP9 expression and were shown to be required for filopodia formation.

  These results show that ectopic expression of AQP9 can induce filopodia formation in a process likely to involve increased water transport, AQP9 phosphorylation and CDC42 signaling. The authors also speculate that local microdilutions and changes in hydrostatic pressure may facilitate actin polymerization to promote filopodia extension.
  
  Loitto VM, Huang C, Sigal YJ, Jacobson K. Filopodia are induced by aquaporin-9 expression. Exp Cell Res 2007; 313 (7):1295-306 PubMed
  

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• Other CMC Publications

  • Biomaterial scaffold promotes adult stem cells survival: Griffith and colleagues recent publication in Nature Cell Biology demonstrates increased survival and proliferation of bone marrow mesenchymal stem cells grown outside of the body on a ceramic and polymer biomaterial scaffold associated with the appropriate growth factors. Click here (http://web.mit.edu/newsoffice/2007/stem-cells.html) to read an MIT press release on this publication - Tethered EGF Provides a Survival Advantage to Mesenchymal Stem Cells. Fan VH, Tamama K, Au A, Littrell R, Richardson LB, Wright JW, Wells A, Griffith LG. Nat Cell Biol 2007; 9 (4):415-21 PubMed

  • Formation of osteogenic colonies on well-defined adhesion peptides by freshly isolated human marrow cells. Au A, Boehm CA, Mayes AM, Muschler GF, Griffith LG. Biomaterials. 2007 Apr;28(10):1847-61. Epub 2007 Jan 11. PubMed

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April 2007

• Osteoclasts: Sealing zone structure

  Physiological bone remodelling requires the activity of both bone-degrading cells, or osteoclasts, and bone-depositing cells, known as osteoblasts. The adhesive apparatus of osteoclasts is key to the degradation process and previous studies have shown that the re-organization of adhesion structures into 'sealing zones' is necessary to delimit bone resorption sites. By combining high-resolution scanning electron microscopy and fluorescence microscopy, Chen Luxenburg et al. have uncovered the molecular architecture of the sealing zone at extraordinary resolution. Their results, published in PLOS One, show that the reorganizationi of individual podosomes into a stable ring, a sealing zone hallmark, correlates with a 10-fold increase in the amount of actin associated with the podosome unit. These findings suggest that podosomes are structural units able to sense the properties of the substrate through adhesion receptors, and via associated proteins and radial actin filaments connnect with other podosomes leading to the formation of a sealing zone. The precise mechanism by which the sealing zone assembles and disassembles is currently the subject of ongoing investigations.

  
  

  Luxenburg C, Geblinger D, Klein E, Anderson K, Hanein D, Geiger B, Addadi L. The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly. PLoS ONE 2007; 2:e179 PubMed

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• Electron tomography: Noise filtering made simple(r)

  Electron tomography is a powerful method for elucidating biological structures, but the resulting data require noise filtering. The currently available filtering algorithms are laborious as they need to be tweaked for individual applications, and have high performance requirements on computer hardware. Van der Heide et al. now report in the Journal of Structural Biology the development of a comparatively simple and fast noise-reduction algorithm based on iterative median filtering. They compared the performance of the new med3 filter to two other available noise-reduction methods. The authors tested med3 with synthetic and experimental data including that from actin filaments, HIV virons and cell sections. The evaluation showed that the med3 filter matches or even surpasses other filter procedures; furthermore, it can be used for automatic de-noising of tomograms. The software application based on the med3 filter is available upon request from Niels Volkmann (niels@burnham.org).

  
  

  van der Heide P, Xu XP, Marsh BJ, Hanein D, Volkmann N. Efficient automatic noise reduction of electron tomographic reconstructions based on iterative median filtering. J Struct Biol. 2006 Nov 21; [Epub ahead of print] PubMed

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• Alpha-Actinin: More than a linker

  alpha-Actinin cross-links and bundles actin filaments, as well as interacting with numerous other proteins involved in mechanosensing, signalling and transcription. However, the structure of alpha-actinin crosslinking is not yet completely understood. In the Journal of Molecular Biology, Hampton et al. report an analysis of electron micrographs of two-dimensional F-actin:alpha-actinin rafts electron micrographs to further elucidate alpha-actinin crosslinking. Their analysis reveals that the polarity preference of F-actin:alpha-actinin binding is mainly determined by extrinsic factors and not by the complex itself. Furthermore, the authors observed that alpha-actinin is able to bind a single actin filament simultaneously with the actin binding domains at each end. They hypothesize that this feature might block actin from interacting with other proteins or that it might connect individual actin filaments to membrane-associated adhesion factors or channels. Contrary to previous studies, alpha-actinin length varied by more than 5.5 nm, which could facilitate alpha-actinin's role in sensing tension. In conclusion, the study by Hampton et al. reveals novel features of alpha-actinin-actin binding that are important for the understanding of cytoskeleton dynamics.

  
  Hampton C.M., Taylor D.W., Taylor K.A. Novel Structures for alpha-Actinin:F-Actin Interactions and their Implications for Actin-Membrane Attachment and Tension Sensing in the Cytoskeleton. J Mol Biol. 386, 92-104 (2007).PubMed
  

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March 2007

• Paxillin phosphorylation: A switch in adhesion

  The focal adhesion-associated adaptor protein paxillin is phosphorylated upon integrin activation or growth factor stimulation. Many paxillin phosphorylation sites have been mapped but in most cases, their effect on the protein’s function has not been characterised. In a study published in the Journal of Cell Science, Geiger and colleagues have investigated the role of paxillin tyrosine phosphorylation at residues 31 and 118 on endothelial cell adhesion. Using phosphospecific antibodies, they show that tyrosine phosphorylated paxillin localizes to focal complexes and focal adhesions but not to fibrillar adhesions. Expression of a phosphomimetic paxillin mutant not only increased adhesion assembly and turnover, but also stimulated the formation of lamellipodial protrusions. Conversely, a non-phosphorylatable paxillin mutant was found to stabilise adhesion sites. The authors also show that paxillin phosphorylation is negatively regulated by mechanical force and that focal adhesion kinase (FAK) is a key mediator of the phosphopaxillin-induced disassembly of adhesions. Together, this data has enabled the creation of a mathematical model which suggests that tyrosine phosphorylation of paxillin acts as a crucial switch in the regulation adhesion dynamics.

  
  

  Zaidel-Bar R, Milo R, Kam Z, Geiger B. A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions. J Cell Sci 2007; 120 (Pt 1):137-48 PubMed

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• Uncaging migration

  In Protein Science, Vogel and Imperiali report the development and characterization of a new probe for investigating the effects of paxillin phosphorylation on cell migration. The semisynthesis of a 61 kDa paxillin construct that contains a caged phosphotyrosine residue at position 31 was accomplished by using native chemical ligation. In vitro, the probe interacts with the same partners as wild-type paxillin and when expressed in cells, removal of the caging moiety by photolysis, enables researchers to examine the local effects of Tyr31-paxillin on motility.

  
  

  Vogel EM, Imperiali B. Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies. Protein Sci. 2007 Jan 22: [Epub ahead of print] PubMed

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February 2007

• Actin and the ECM: Transmitting the motion

  The transmission of actin motion to the extracellular matrix (ECM) through focal adhesions (FAs) is necessary for cells to migrate. Waterman-Storer, Danuser and colleagues have analyzed the dynamic interactions between FA components and actin filaments by combining total internal reflection fluorescence microscopy (TIRFM) and fluorescent speckle microscopy (FSM), and tracking fluorescently tagged F-actin and FA protein speckles. Their results, published in Science, show that FA proteins are differentially coupled to F-actin motion. Whereas FA proteins with no known actin-binding activity, such as integrin, moved slowly and had a low degree of motion correlation with actin, FA actin-binding proteins exhibit a variety of behaviors. GFP-alpha-actinin and GFP-vinculin speckles for example, moved a lot more coherently than GFP-talin speckles. Differences in motion correlation were not only observed between proteins within individual FAs but also transiently between FAs in protruding and retracting sectors of the cell leading edge. The authors propose that the differential coupling of FA proteins to actin filaments and thus, the diverse efficiency of motion transmission, is necessary for protrusion and retraction. These differences are likely to result from tightly regulated, transient interactions with other binding partners in FAs. Their data identified vinculin as one of the major candidate components within this molecular clutch.

  
  

  Hu K, Ji L, Applegate KT, Danuser G, Waterman-Storer CM. Differential transmission of actin motion within focal adhesions. Science. 2007 Jan 5;315(5808):111-5 PubMed

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• Other CMC Publications

  Integrin activation. Talin's tale: This month’s featured article in the Cell Migration Updates section of the Gateway highlights Campbell and colleagues’ recent paper in Cell, describing the structural basis of integrin activation by talin. Click here to read the article review in the Cell Migration Updates section of the Gateway.

  
  

  Wegener KL, Partridge AW, Han J, Pickford AR, Liddington RC, Ginsberg MH, Campbell ID. Structural basis of integrin activation by talin. Cell. 2007 Jan 12;128(1):171-8 PubMed

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• Data postings & improvements to the CMC Activity Center

  Initiative Activity Tables now sortable: In the Activities section of the Gateway, the tables, which outline the different projects and their status, now have a sort function. This enables viewers to select the manner by which the activities are displayed. By clicking on any of the column headings the tables can be sorted on the fly. Do you want to know what projects have been completed and published or which are still in progress? Click on the Status column and the information is sorted on this basis. Do you want to view all the projects currently underway in a particular investigators group? Click on the contact column in the far right and all the information is re-sorted and presented by investigator in either ascending or descending alphabetical order. E.g.; http://www.cellmigration.org/resource/structure/

  
  

  PAK1 Phosphoproteome data posted: Based on these Consortium data a total of 20 phosphorylation sites have been identified in PAK. Click here to see the data.
  

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• Forthcoming Conferences & Workshops

  3-7 March, 2007 - 51st Annual meeting of the Biophysical Society, Baltimore MD. One of the symposia at the 51st Annual meeting of the Biophysical Society will provide a glimpse into the Cell Migration Consortium. Symposium 10 entitled "Molecular Mechanisms of Cell Migration" will be held between 10:45 am - 12:45 pm on Monday March 5th. Click here for more details on the symposium. Click here to register for the meeting

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January 2007

• Podosomes: Maturation and turnover

  Osteoclasts are multinucleated cells that originate from monocyte/macrophage lineage cells and play a critical role in bone resorption. At resorption sites osteoclasts polarize and undergo profound morphological changes including the formation of actin rings surrounding the resorptive area and the appearance of ruffled borders. In the Journal of Cell Science, Geiger and colleagues shed new light on the mechanisms involved in the reorganization of osteoclast adhesions, known as podosomes. They show that the catalytic activity of the tyrosine kinase Src is essential for podosome maturation and turnover. Furthermore, Src-mediated phosphorylation of the actin-binding protein cortactin is critical for ensuring that actin polymerisation is reduced in the stable, mature sealing-zone-like (SZL) structures that form during osteoclast polarization. Overexpression of a non-phosphorylatable cortactin mutant increased podosome dynamics and prevented the formation of SZL structures. These findings suggest that the ability of osteoclasts to dynamically remodel their adhesions is regulated, at least in part, by cortactin phosphorylation. The involvement of other Src substrates in podosome dynamics awaits to be discovered.

  
  

  Luxenburg C, Parsons JT, Addadi L, Geiger B. Involvement of the Src-cortactin pathway in podosome formation and turnover during polarization of cultured osteoclasts J Cell Sci 2006 119: 4878-4888 PubMed

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• Light microscopy screening

  Kam and colleagues describe in Methods in Enzymology a novel high-resolution automated plate-screening microscope system that acquires large sets of data at light microscope resolution. The authors have designed a multiwell plate-scanning microscope platform that can acquire up to two autofocused images per second using up to x100 objectives. Along with bespoke image analysis software, this system has been employed to measure biological responses and has greatly facilitated screening for chemical compounds or genes that affect cell migration.

  
  

  Paran Y, Lavelin I, Naffar-Abu-Amara S, Winograd-Katz S, Liron Y, Geiger B, Kam Z. Development and Application of Automatic HighResolution Light Microscopy for CellBased Screens Methods in Enzymology 2006 414: 228-247 PubMed

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• Other CMC Publications

  Velocity Mapping: This month's featured article in the Cell Migration Updates section of the Gateway highlights a new approach to mapping the velocities of cytoskeletal and adhesion molecules in migrating cells. Click here to read the article review.
  
  Brown CM, Hebert B, Kolin DL, Zareno J, Whitmore L, Horwitz AR, Wiseman PW. Probing the integrin-actin linkage using high-resolution protein velocity mapping. J Cell Sci. 2006 Dec 15;119(Pt 24):5204-14.PubMed

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• Forthcoming Conferences & Workshops

  3-7 March, 2007 - 51st Annual meeting of the Biophysical Society, Baltimore MD. One of the symposia at the 51st Annual meeting of the Biophysical Society will provide a glimpse into the Cell Migration Consortium. Symposium 10 entitled "Molecular Mechanisms of Cell Migration" will be held between 10:45 am - 12:45 pm on Monday March 5th. Click here for more details on the symposium. Click here to register for the meeting

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