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Consortium Updates

Welcome to the Cell Migration Consortium's updates page, where we highlight major additions of data and information, and outline some of the publications appearing as a result of the Consortium's activities.

  • December 2008

    1. Intravital imaging: Illuminating metastasis
    2. Modeling: Coupling intracellular forces and protrusion
    3. Other CMC Publications
    4. Upcoming Conferences & Workshops
    • Intravital imaging: Illuminating metastasis

      Metastatic dissemination of breast tumors begins with tumour cells invading the surrounding stroma and entering the blood. Cell behaviour has been reported to be heterogeneous during invasion and intravasation, depending upon the tumour microenvironment. However, quantifying the different cell behaviours in breast tumors requires tracking tumour cells over long periods of time in their natural environment (mammary glands) -- which has been technically unfeasible until now. Published in Nature Methods, Jacco van Rheenen and colleagues of the Gruss Lipper Biophotonics center at the Albert Einstein College of Medicine in New York have now developed an imaging system that allows the tracking and monitoring of selected tumour cell populations in different breast tumour microenvironments over a 24-hour period. Dmitriy Kedrin, Bojana Gligorijevic and Jacco van Rheenen in the labs of Jeff Segall and John Condeelis surgically implanted a mammary imaging window (MIW) on top of a mouse mammary gland or mammary tumour, and visualised the behaviour of tumour cell expressing the photoswitchable fluorescent protein Dendra2 in different tumor microenvironments. By analysing differentregions within the same tumor, the authors found that migration was limited in the absence of blood vessels, whereas invasion and intravasation were strongly favoured in a vascular microenvironment. Thus, this technique gives the spatial and temporal resolution required to study different microenvironments within the same tumour.

      • Kedrin D, Gligorijevic B, Wyckoff J, Verkhusha VV, Condeelis J, Segall JE, van Rheenen J. Intravital imaging of metastatic behavior through a mammary imaging window. Nat Methods. 2008; 5(12):1019-21. PubMed
    • Modeling: Coupling intracellular forces and protrusion

      Cell protrusion results from a balance between propulsive forces at the leading edge and adhesive forces behind the protruding area. It requires the polymerization of F-actin, the coupling of F-actin with the extracellular matrix, and contraction. Although the molecular mechanisms underlying these processes have been extensively studied, their spatial and time coordination, as well as their relationship with F-actin dynamics, remain poorly understood. In Nature Cell Biology, Gaudenz Danuser and colleagues report a mechanical model that couples fluctuations in F-actin network flow -- measured by fluorescence speckle microscopy (FSM) -- to variations in intracellular forces during cycles of protrusion-retraction in epithelial cells responding to wound healing. The authors show that force transmission at focal adhesions requires binding of vinculin to both F-actin and integrin, and that regulation occurs at the vinculin-integrin interface. Interestingly, the authors identified an inverse relationship between advancement of the cell edge and boundary force. Edge advancement stalled at a time-point of maximum boundary force and a new protrusion cycle could only start following a step of relaxation. Thus, it appears that tension at the plasma membrane limits cell protrusion and follows a cycle that is controlled by a feedback loop between increasing tension and F-actin polymerization.

      • Ji L, Lim J, Danuser G. Fluctuations of intracellular forces during cell protrusion. Nat Cell Biol. 2008 Nov 16. [Epub ahead of print] PubMed
    • Other CMC Publications

      • December issue of Science devoted to organ development

        The December issue of Science is a special issue devoted to organ development. Denise Montell has a review in this issue entitled "Morphogenetic Cell Movements: Diversity from Modular Mechanical Properties."

        Montell DJ. Morphogenetic cell movements: diversity from modular mechanical properties. Science. 2008 Dec 5;322(5907):1502-5. PubMed
      • October issue of Current Opinions in Cell Biology focuses on cell-cell contacts and extracellular matrix

        The October issue of Current Opinions in Cell Biology focuses on cell-cell contacts and extracellular matrix, and includes several articles by Consortium investigators.

        Ginsberg MH, Schwarzbauer JE. Related Articles, Cell-to-cell contact and extracellular matrix. Curr Opin Cell Biol. 2008 Oct;20(5):492-4. Epub 2008 Aug 3. PubMed

        Schwartz MA, DeSimone DW. Related Articles, Cell adhesion receptors in mechanotransduction. Curr Opin Cell Biol. 2008 Oct;20(5):551-6. Epub 2008 Jun 24. PubMed

        Sabouri-Ghomi M, Wu Y, Hahn K, Danuser G. Related Articles, Visualizing and quantifying adhesive signals. Curr Opin Cell Biol. 2008 Oct;20(5):541-50. Epub 2008 Jun 27. PubMed
    • Upcoming Conferences & Workshops

      • 48th ASCB Meeting, 13-17 December 2008:

        The 48th annual meeting of the American Society of Cell Biologists will be held in San Francisco December 13-17. The deadline for early registration is October 7th and more details on registration and abstract submission are available at the meeting web site at www.ascb.org/meetings

      • Beatson International Cancer Conference, 5-7 July 2009 - Microenvironment, Motility and Metastasis:

        The conference will focus on advances in imaging and model tumour systems which have enhanced the ability to study the mechanisms of motility, invasion and metastasis. Further information on registration and abstract submission may be found at the conference website at www.beatson.gla.ac.uk/Conference.html

      • Cell Biophysics Workshop, 30 - 31 May 2009 - From Motors to Morphogenesis: Oster-Inspired Research:

        This meeting will be held in Berkley California and will discuss new and exciting advances of cell biophysics - from molecular motors to cell division to morphogenesis - inspired and pioneered by George Oster. For more details and to view a list of speakers, visit the web site at http://www.math.ucdavis.edu/~mogilner/Berkeley.html

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  • November 2008

    1. Integrin structure: Let's get activated
    2. Upcoming Conferences & Workshops
    • Integrin structure: Let's get activated

      Integrins are cell surface adhesion receptors that connect the extracellular environment to the cell interior. They constitute both a structural connection and a bi-directional signalling pathway that crosses the cell membrane. Integrins are heterodimeric proteins comprised of an α and β subunit, which each contain a large extracellular domain, a single α-helix transmembrane domain and a short, unstructured cytoplasmic domain. In Molecular Membrane Biology, Kate Wegener and Iain Campbell now review the latest insights into the conformational changes within the membrane spanning and cytoplasmic regions of integrins. These changes are induced by protein-protein interactions, can be modulated by phosphorylation, and lead to integrin activation.

      • Transmembrane and cytoplasmic domains in integrin activation and protein-protein interactions. Wegener KL, Campbell ID. Mol Membr Biol. 2008 Aug;25(5):376-87. PubMed
    • Upcoming Conferences & Workshops

      • 48th ASCB Meeting, 13-17 December 2008:

        The 48th annual meeting of the American Society of Cell Biologists will be held in San Francisco December 13-17. More details on registration and abstract submission are available at the meeting web site at www.ascb.org/meetings

      • Beatson International Cancer Conference, 5-7 July 2009:

        Registration and abstract submissions, are now being accepted for the Beatson International Cancer Conference being held next summer. The conference will focus on advances in imaging and model tumour systems which have enhanced the ability to study the mechanisms of motility, invasion and metastasis. Further information on registration and abstract submission may be found at the conference website www.beatson.gla.ac.uk/Conference.html

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  • October 2008

    1. Actin bundling: Where shall we start?
    2. PKA: Activity at the leading edge
    3. Consortium Data Additions & Site Updates
    4. Upcoming Conferences & Workshops
    • Actin bundling: Where shall we start?

      F-actin meshwork structure and patterns change as filaments polymerize, branch and bundle. It is this bundling of rapidly polymerizing actin filaments that underlies the filopodial protrusion dynamics that are important for cell migration. It has been proposed that a bundle results from two filaments that are zipped up by linker proteins such as fascin, but the detailed mechanisms that initiate bundling remain unclear. Two possibilities have recently been proposed. One involves the binding of two filament tips into a complex that triggers zipping, whereas the other suggests that binding of one filament tip to the side of the other might be sufficient to initiate the bundling. However, both scenarios rely on the two filaments being separated by a small angle. In Physical Review Letters, Pavel Kraikivski and colleagues now report the theoretical analysis of these two bundling mechanisms and show that they can be distinguished experimentally. The authors propose an experimental approach that can determine which of the two mechanisms controls bundle formation in vitro.

      • Kraikivski P, Slepchenko BM, Novak IL. Phys Rev Lett. Epub 2008 Sep 17. [Epub ahead of print] link PubMed
    • PKA: Activity at the leading edge

      In order to understand cell migration it is crucial that we know how signaling events are restricted to certain regions of the cell. As well as mediating adhesion through their interaction with the extracellular matrix, integrins also initiate and coordinate the signaling events necessary for cell polarization and motility. Localized phosphorylation of the integrin α4 cytoplasmic domain by cAMP-dependent protein kinase (PKA) at the cells' leading edge is required for protrusion and efficient migration. Mark Ginsberg and colleagues have further characterized the function of PKA by analyzing the localization of its activity using a membrane-targeted Förster Resonance Energy Transfer (FRET)-based PKA kinase activity reporter. They show that selective and transient PKA activation at the leading edge is dependent upon integrin engagement and polymerized actin. Furthermore, accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) at the leading edge -- an early event in the establishment of migration directionality -- was shown to require localized PKA activity even though PKA localization was independent from PI3-kinase. Thus, this work shows that adhesion-mediated localized PKA activation is an early event in cell migration and that PKA contributes to the maintenance of cell polarity during movement.

      • Lim CJ, Kain KH, Tkachenko E, Goldfinger LE, Gutierrez E, Allen MD, Groisman A, Zhang J, Ginsberg MH. Integrin-mediated Protein Kinase A Activation at the Leading Edge of Migrating Cells. Mol Biol Cell. 2008 Sep 10; [Epub ahead of print] PubMed
    • Consortium Data Additions & Site Updates

      • Migration scientists converge on Bethesda, MD

        Over 400 participants from more than 20 countries participated in the Frontiers in Cell Migration conference held on the NIH campus in Bethesda, MD 16-18 September, 2008. Over 130 posters were presented during the course of the two and a half day conference at sessions that were alive with discussion and exchange. The Conference dinner on Wednesday evening provided a relaxed evening for participants to socialize and network, while listening to the three keynote presentations provided by John Condeelis, Iain Campbell and Doug Lauffenburger. Numerous inquiries were made about the plans for the next Cell Migration conference, which points to the success of this meeting. The answer to this question will have to be "watch this space". To view the conference program book and photos of the event visit the conference web site at visit the workshop web site at meeting2008.cellmigration.org

    • Upcoming Conferences & Workshops

      • 3rd LFD Workshop October 27-31:

        The 3rd workshop in Advanced Fluorescence Imaging and Dynamics will be held the University of California, Irvine October 27-31. For more details and to register, visit the workshop web site at www.lfd.uci.edu/workshop

      • 48th ASCB Meeting December 13-17:

        The 48th annual meeting of the American Society of Cell Biologists will be held in San Francisco December 13-17. The deadline for early registration is October 7th and more details on registration and abstract submission are available at the meeting web site at www.ascb.org/meetings

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  • September 2008

    1. Adhesion assembly and maturation: A dendritic actin task
    2. Cell Biology: Studying protein functions with CALI
    3. Consortium Data Additions & Site Updates
    4. Other CMC Publications
    5. Upcoming Conferences & Workshops
    • Adhesion assembly and maturation: A dendritic actin task

      Adhesions play a crucial role in the development of and response to traction forces, as well as functioning in the regulation of the signaling networks that control cell migration. Nevertheless, the mechanisms underlying adhesion assembly and turnover remain poorly understood. Reporting in Nature Cell Biology, Rick Horwitz and colleagues have now used high resolution TIRF microscopy and two-color imaging to define the early steps and mechanisms of nascent adhesion formation and maturation.

      Nascent adhesions were found to assemble in the lamellipodium at an assembly rate that was proportional to the local rate of protrusion. Treating the cells with cytochalasin-D -- a drug that caps actin barbed ends and prevents polymerization -- showed that the assembly process requires actin polymerization. Conversely, Myosin II knockdown showed that the formation and turnover of small dynamic adhesions near the leading edge do not require myosin II. When the lamellipodium moves past nascent adhesions, some adhesions disassemble whereas others mature. The authors conclude that maturation depends on F-actin reorganization, which requires the cross-linking activity of both α-actinin and myosin II.

      These results led the authors to develop a model for adhesion formation, turnover and maturation: driven by actin polymerization, adhesions form at discrete points in the lamellipodium and disassemble at its rear in conjunction with dendritic actin depolymerization. Alternatively, adhesions mature by centripetal elongation along an actin template -- a step that depends actin cross-linking.

      • Actin and alpha-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner. Choi CK, Vicente-Manzanares M, Zareno J, Whitmore LA, Mogilner A, Horwitz AR. Nat Cell Biol. 2008 Aug 17. [Epub ahead of print] PubMed
    • Cell Biology: Studying protein functions with CALI

      Chromophore-assisted laser inactivation (CALI) is a complement to conventional techniques such as genetic or pharmacological manipulation for the study of protein function. However, in contrast to gene knockdown and the use of pharmacological inhibitors, CALI allows protein inactivation in a temporally and spatially restricted manner. CALI introduces a small chromophore into the cell that is conjugated either to the protein of interest, an antibody or a fluorescent protein fused to the target protein. This is followed by irradiation with an intense beam of light, which causes the chromophores to absorb the light and produce highly reactive free radicals that inactivate the proximate proteins. The advantages of this technique are that inactivation can be achieved in less than 1 sec, the inactivating light beam can be directed to small regions within a single cell and the damage is limited to proteins that are immediately adjacent to the irradiated dye. Thus, CALI provides a spatially and temporally controlled loss-of-function tool. In a review published in Trends in Cell Biology, Ken Jacobsen and co-authors describe the development and the properties of this technique as well as its applications in Cell Biology.

      • Chromophore-assisted laser inactivation in cell biology. Jacobson K, Rajfur Z, Vitriol E, Hahn K. Trends Cell Biol. 2008 Sep;18(9):443-50. Epub 2008 Aug 14. PubMed
    • Consortium Data Additions & Site Updates

      • 66 Genes that regulate migration identified in siRNA screen

        An siRNA screen in mammalian epithelial cells (MCF-10A) identified 66 genes that positively or negatively regulate cell migration as measured using a wound healing assay. Time-lapse video microscopy analysis of the motility patterns induced by knockdown of these genes revealed significant alterations in cell-cell adhesion, cell polarity and protrusion dynamics. Informatics analysis found many genes are involved in beta-catenin, beta1-integrin and actin signaling and also identified 42 genes that were not previously implicated in migration or adhesion. A fully interactive database can be found at http://www.cellmigration.org/pubs/wound_rnai.htm

    • Other CMC Publications

      • Garduno E, Wong-Barnum M, Volkmann N, Ellisman MH. Segmentation of electron tomographic data sets using fuzzy set theory principles. J Struct Biol 2008; 162 (3):368-79 PubMed
      • siRNA screen: Showing migration-related genes

        One of this month's featured articles in the Cell Migration Updates section of the Gateway highlights an array of new data generated through siRNA work in the Brugge laboratory. Click here to read the article review.

        Simpson KJ, Selfors LM, Bui J, Reynolds A, Deake D, Khvorova A, Brugge JS. Identification of genes that regulate epithelial cell migration using an siRNA screening approach. Nature Cell Biology, Aug 10; [Epub ahead of print] PubMed
    • Upcoming Conferences & Workshops

      • 3rd LFD Workshop October 27-31:

        The 3rd workshop in Advanced Fluorescence Imaging and Dynamics will be held the University of California, Irvine October 27-31. For more details and to register, visit the workshop web site at www.lfd.uci.edu/workshop

      • 48th ASCB Meeting December 13-17:

        The 48th annual meeting of the American Society of Cell Biologists will be held in San Francisco December 13-17. The deadline for early registration is October 7th and more details on registration and abstract submission are available at the meeting web site at www.ascb.org/meetings

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  • August 2008

    1. Axon growth and guidance: Actin and microtubules at work
    2. The migration clutch: A kinetic model for actin-integrin dynamics
    3. Consortium Data Additions & Site Updates
    4. Other CMC Publications
    5. Upcoming Conferences & Workshops
    • Axon growth and guidance: Actin and microtubules at work

      Neuronal growth occurs in three steps: the growth cone peripheral domain protrudes, it fills with organelles from the central domain, and then the advancing central domain consolidates into the distal segment of the neurite shaft. Although recent studies have elucidated the signaling pathways that regulate axon growth and guidance, the basic cytoskeletal mechanisms involved in this process remain poorly understood. Using fluorescence speckle microscopy and automated adaptive fluorescent feature tracking, Paul Forscher and colleagues have now uncovered the F-actin and microtubule dynamics that underlie substrate-dependent neurite outgrowth using the model system Aplysia. It had been previously reported that Aplysia growth cones advance through an initial 'latency phase' followed by a 'growth phase' in which traction forces result from a strengthening of the linkage between the substrate and the underlying actin retrograde flow network. The authors describe how actin remodeling and recycling, as well as contractility of actin-myosin II structures called 'actin arcs', drive the advancement of the central domain and regulate underlying microtubule behaviour.

      • Coordination of Actin Filament and Microtubule Dynamics during Neurite Outgrowth. Schaefer AW, Schoonderwoert VTh.G, Ji L, Mederios N, Danuser G, Forscher P. Dev Cell. 2008; 15:146 - 160. PubMed
    • The migration clutch: A kinetic model for actin-integrin dynamics

      Cell migration relies on dynamic adhesive interactions between the cell and the substratum, which are highly regulated both at the cell front and rear. To be productive, cellular protrusions have to adhere stably. As stated in the 'clutch' hypothesis, stable attachment is obtained through the interaction between polymerised actin filaments and integrin receptors linked to the extracellular matrix. This interaction is mediated by a number of cytosolic factors, including paxillin; however, the interaction hierarchy, adaptor proteins and regulatory signalling events are yet to be fully elucidated. Increasing evidence shows that the balance between myosin II-mediated contractile force and integrin-mediated traction affects the stability of the adaptor complex -- either strengthening the interaction or increasing the rate of dissociation. The opposing effects of contractile forces and the effects of myosin contractility manipulation are difficult to predict. Thus, Douglas Lauffenburger and colleagues developed a simplified quantitative mathematical model that describes the actin-integrin linkage dynamics using a generic 'connector complex' to represent the intermediate interactions. This model was able to describe the effects of paxillin mutations on actin-integrin turnover and lays the ground for future more detailed predictive models.

      • Kinetic model for lamellipodal actin-integrin 'clutch' dynamics. Macdonald A, Horwitz AR, Lauffenburger DA. Cell Adhesion & Migration 2008;2(2)95 - 105 available here PubMed
    • Consortium Data Additions & Site Updates

      • New and Improved Cell Migration Knowledge Base

        The Cell Migration Knowledge Base (CMKB), a curated and informative resource for proteins involved in Cell Migration, has recently been restructured to include links to genes, protein domains, protein structure, interactions, phosphorylation sites and pathway information. Currently there are 800 families (6800 proteins) in the database. Please take a moment to visit the Knowledge Base and let us have your feedback on its utility.

    • Other CMC Publications

      • Kim HD, Guo TW, Wu AP, Wells A, Gertler FB, Lauffenburger DA. EGF-induced Enhancement of Glioblastoma Cell Migration in 3D Arises from Intrinsic Increase in Speed but Extrinsic Matrix- and Proteolysis-dependent Increase in Persistence. Mol Biol Cell 2008; Jul 16. [Epub ahead of print] PubMed
      • Harley BA, Kim HD, Zaman MH, Yannas IV, Lauffenburger DA, Gibson LJ. Micro-architecture of three-dimensional scaffolds influences cell migration behavior via junction interactions. Biophys J 2008; Jul 11. [Epub ahead of print] PubMed
      • Wegener KL, Basran J, Bagshaw CR, Campbell ID, Roberts GC, Critchley DR, Barsukov IL. Structural Basis for the Interaction between the Cytoplasmic Domain of the Hyaluronate Receptor Layilin and the Talin F3 Subdomain. J Mol Biol 2008; Jul 7. [Epub ahead of print] PubMed
      • Meighan CM, Schwarzbauer JE. Temporal and spatial regulation of integrins during development. Curr Opin Cell Biol 2008; Jul 4. [Epub ahead of print] PubMed
      • Wegener KL, Campbell ID. Transmembrane and cytoplasmic domains in integrin activation and protein-protein interactions Mol Membr Biol. 2008;25(5):376-87. PubMed
    • Upcoming Conferences & Workshops

      • 48th Annual Meeting of the American Society for Cell Biologists:

        The ASCB conference will be held in San Francisco December 13-17, 2008. Abstract deadline for Minisymposia talks is August 7th, and for posters is September 3rd. Early registration deadline is October 3rd. More details are available at the conference website ascb.org/meetings

      • Frontiers in Cell Migration, from mechanism to disease:

        The Conference will be held at the NIH Natcher Center, Bethesda MD 16 -18 September. Registration will close August 16th. For a detailed program and registration instructions please visit the conference website meeting2008.cellmigration.org

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  • July 2008

    1. Actin treadmilling: G-actin diffusion or local recycling?
    2. Signaling in space: Coupling at the plasma membrane
    3. Other CMC Publications
    4. Upcoming Conferences & Workshops
    • Actin treadmilling: G-actin diffusion or local recycling?

      Actin filaments are polarized structures with a barbed, fast growing end, and a pointed shortening end. Actin monomers assemble onto the barbed ends and disassemble from the pointed ends -- a process called treadmilling. During migration, the growing barbed ends push the leading edge forward whereas the pointed ends, located towards the rear, disassemble. This releases bound G-actin, which can then diffuse and assemble on the available uncapped barbed ends at the front. In the Biophysical Journal, Alex Mogilner and colleagues use a mathematical model to predict the distribution of G-actin and the resulting rates of diffusion in the context of a three-dimensional motile cell. Fish keratocytes were used as a representative system as they are characterized by rapid and persistent movements with barely any change in shape, speed or direction over several minutes. The author's calculations predict two possible scenarios: if F-actin assembly and disassembly are spatially separated at the front and rear of the cell respectively, then a G-actin gradient sufficient to support diffusion of G-actin forward is established. However, if F-actin assembly and disassembly occur throughout the lamellipod and partially co-localize, then the actin turnover becomes local and diffusion plays little role in the supply of actin monomers. Understanding which of the two scenarios applies to migrating cells will help elucidate how cell motility and polarity are regulated.

      • Quantitative analysis of G-actin transport in motile cells. Novak I, Slepchenko B, Mogilner A. Biophys J. 2008 May 23. [Epub ahead of print] PubMed
    • Signaling in space: Coupling at the plasma membrane

      Intracellular signaling pathways often depend on enzymes that are recruited by plasma membrane receptors and act upon membrane-associated substrates. Thus, the membrane acts as a physical platform for interactions taking place at the early stages of receptor-mediated signaling, bringing enzymes closer to their substrates. Existing models that describe enzyme-catalyzed reactions at cell membranes are based on the rate of lateral diffusion of membrane-associated molecules being the limiting factor. As reported in Biophysical Journal, Michael Monine and Jason Haugh have now developed a quantitative model that expands on the role of diffusion-controlled kinetics -- a two-dimensional Brownian dynamics kinetic model. The model introduces 'spatial coupling' whereby simultaneous recruitment of different enzymes to the same receptor facilitates cross-talk between different signaling pathways. The authors analyzed the specific case of phosphoinositide 3-kinase (PI3K), which is localized as a result of cooperative interactions between receptors and active Ras. The assembly of receptor/PI3K/Ras complexes is facilitated by the local action of a guanine exchange factor (GEF) bound to the same receptor. Spatial coupling between GEF and PI3K relies on Ras being first released by a receptor-bound GEF and then captured by a PI3K molecule associated with the same receptor. The authors were thus able to evaluate the probabilities of short- and long-range interactions to characterize spatial coupling.

      • Monine MI, Haugh JM. Signal Transduction at Point-blank Range: Analysis of a Spatial Coupling Mechanism for Pathway Crosstalk. Biophys J. 2008 May 23. [Epub ahead of print] PubMed
    • Other CMC Publications

      • Kapustina M, Weinreb GE, Costigliola N, Raifur Z, Jacobson K, Elston TC. Mechanical And Biochemical Modeling Of Cortical Oscillations In Spreading Cells. Biophys. J. 2008, 94(12):4605-20. PubMed
      • Sabouri-Ghomi M, Wu Y, Hahn K, Danuser G. Visualizing and quantifying adhesive signals. Curr Opin Cell Biol 2008;Jun 27; [Epub ahead of print] PubMed
    • Upcoming Conferences & Workshops

      • Frontiers in Cell Migration; from mechanism to disease:

        A conference co-sponsored by the Consortium and the National Institute of Health General Medical Sciences, will be held at the NIH Natcher Center, Bethesda MD 16 -18 September. More details are available at the meetings website at http://meeting2008.cellmigration.org.

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  • June 2008

    1. Crawling keratocytes: How to get in shape
    2. DLC-1: Lost in invasion
    3. Consortium Data Additions & Site Updates
    4. Other CMC Publications
    5. Upcoming Conferences & Workshops
    • Crawling keratocytes: How to get in shape

      The question of how cells acquire and maintain specific shapes has remained largely unresolved despite being researched for decades. However, as reported in Nature, Theriot and colleagues have now combined mathematical and cell biological approaches in order to provide a quantitative model for cell shaping during migration. As a model system the authors used motile fish epithelial keratocytes, which maintain nearly constant speed, direction and morphology while crawling. Analysis of the shape of keratocytes revealed that only a few parameters are needed to describe the natural phenotypic variability in a cell population. Moreover, cell area remains remarkably constant during migration. This observation, and serendipitous finding that the actin filaments' density is graded along the leading edge lead the authors to assume that actin treadmilling pushes forward an inextensible membrane. A quantitative physical model was then developed that correlated the variations in cell shape and movement to the distribution of actin filaments. In the model, cell shape and movement are caused by actin polymerization pushing from within as well as the counteracting forces imposed by the membrane. At the centre front, many actin filaments grow against membrane resistance, while at the sides fewer filaments are stalled by the membrane, and at the rear the membrane pushes forward the weakened actin network. By explaining the main features of keratocyte shape, and predicting how speed relates to cell morphology, this model contributes to our understanding of cell shaping principles. Furthermore, it highlights how actin dynamics -- which polymerize at the front and depolymerise at the rear -- are modulated by membrane tension.

      • Keren K, Pincus Z, Allen GM, Barnhart EL, Marriott G, Mogilner A, Theriot JA. Mechanism of shape determination in motile cells. Nature 2008;453(7194):475-80. PubMed
    • DLC-1: Lost in invasion

      Deleted in liver cancer-1 (DLC-1) is a multi-domain protein that contains an internal Rho GTPase-activating protein (RhoGAP) domain. RhoGAPs promote the transition of Rho GTPases from the active GTP-bound to the inactive GDP-bound state by stimulating their intrinsic GTPase activity. Human cancers have been associated with aberrant regulation of Rho GTPases --which is also known to transduce signals that control F-actin dynamics and cell movement. DLC-1 is a tumour suppressor whose expression is lost in several human cancers including the highly invasive non-small cell lung carcinomas (NSCLC). Although it has been hypothesised that loss of DCL-1 may promote carcinogenesis by causing RhoGTPase hyper-activation, it is unclear whether the RhoGAP domain is important for DCL-1 tumour suppression function. In Molecular Carcinogenesis, Healy et al. show that DLC-1 expression in NSCLC cell lines reduces both invasion and anchorage-dependent and -independent growth. The authors also provide evidence for the importance of DLC-1 for Rho GTPases activity, and shed light on the mechanisms that regulate such activity. Using a RhoA biosensor, the authors demonstrate DLC-1 inhibition of RhoA activity specifically at the cells' leading edge. Surprisingly, DCL-1 tumourigenic activity was neither associated with RhoGAP activity nor with another DLC-1 catalytic function -- the activation of phospholipase C delta.

      • Healy KD, Hodgson L, Kim TY, Shutes A, Maddileti S, Juliano RL, Hahn KM, Harden TK, Bang YJ, Der CJ. DLC-1 suppresses non-small cell lung cancer growth and invasion by RhoGAP-dependent and independent mechanisms. Mol Carcinog. 2008; 47(5):326-37. PubMed
    • Consortium Data Additions & Site Updates

      • New Data provides

        New data from Condeelis laboratory describes the pattern of gene expression in invasive and metastatic subpopulations of tumor cells isolated from two different mammary tumor types (PyMT and MTLn3) and reveals a characteristic "invasion signature". These data indicate that invasive cancer cells are a population that is neither proliferating nor apoptotic but highly chemotactic. To view these data go to discovery activity table.

      • Phosphoproteomics data for 4 new, migration-related proteins now available

        Data showing the phosphorylation sites present in Dynamin, Calpain, Parvin and N-Wasp are now available through the proteomics initiative activity table.

    • Other CMC Publications

      • Mogilner A. Mathematics of cell motility: have we got its number? J Math Biol 2008; [Epub ahead of print] PubMed
      • Monine MI, Haugh JM. Signal Transduction at Point-blank Range: Analysis of a Spatial Coupling Mechanism for Pathway Crosstalk. Biophys J 2008 May 23. [Epub ahead of print] PubMed
    • Upcoming Conferences & Workshops

      • Computational Cell Biology:

        Cold Spring Harbor Laboratory will be offering a three week course on Computational Cell Biology June 27-July 17, 2008. For further details go to: http://meetings.cshl.edu/courses/c-comp08.shtml.

      • Frontiers in Cell Migration from Mechanism to Disease:

        A conference co-sponsored by the Consortium and the National Institute of Health General Medical Sciences, will be held at the NIH Natcher Center, Bethesda MD 16 -18 September. More details are available at the meetings website at http://meeting2008.cellmigration.org.

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  • May 2008

    1. Integrin activation: A height complex
    2. Multicolour compositional imaging: Adhesions revealed
    3. Consortium Data Additions & Site Updates
    4. Other CMC Publications
    5. Upcoming Conferences & Workshops
    • Integrin activation: A height complex

      The large family of integrin cell surface receptors signal bidirectionally across the plasma membrane. They are comprised of a large extracellular domain that binds to other cell surface proteins or to extracellular matrix ligands, a single transmembrane α-helix, and a small cytoplasmic tail that binds to cytoplasmic components. During outside-in signaling, the integrin-ligand interaction is transduced to the cytoplasmic tail, which leads to the recruitment of cytoplasmic binding partners. Conversely, during inside-out signaling, binding of intracellular partners results in an increase of the extracellular domain binding affinity. Thus, integrin activation is accompanied by long-range conformational changes across the membrane, but these changes are subject of debate. Two existing models predict that integrins may either remain in a bent-over conformation (the 'deadbolt' model) or change to an extended conformation ('switchable' model) upon activation. Kenneth Taylor and colleagues used cryoelectron tomography to obtain 3D images of full length αIIbβ3 integrins in a membrane environment. These show that integrins remain the same height upon Mn2+-induced activation -- a result that is inconsistent with the switchable model.

      • Ye F, Liu J, Winkler H, Taylor KA. Integrin alpha(IIb)beta(3) in a Membrane Environment Remains the Same Height after Mn(2+) Activation when Observed by Cryoelectron Tomography. J Mol Biol 2008; 378 (5):976-86 PubMed
    • Multicolour compositional imaging: Adhesions revealed

      Cell-matrix adhesion sites, which consist of numerous cytoskeletal and signalling proteins, are variable in their morphology, dynamics and molecular composition. Studying the highly regulated process of adhesion site assembly requires tools that allow researchers to view both the location and quantity of relevant molecules at high resolution. In PloS One, Zamir et al. now report the development of a light-microscopy-based imaging approach for analysing the molecular reorganization of cell-matrix adhesions. Rat embryo fibroblasts were simultaneously labelled for different combinations of five focal-adhesion components, and the corresponding five-colour images were acquired. The image pixels were then analysed using a multi-dimensional clustering method, leading to the identification of unique "molecular signatures" in the focal adhesions and their sub-domains. Furthermore, the authors were able to identify variations in the abundance and distribution of these signatures along the focal adhesions and stress fibres in response to Rho-kinase inhibition. Thus, compositional imaging may provide a powerful tool for exploring the molecular diversity of complex cell structures and mapping their distribution at sub-micron resolution.

      • Zamir E, Geiger B, Kam Z. Quantitative multicolor compositional imaging resolves molecular domains in cell-matrix adhesions. PLoS ONE 2008 Apr 2;3(4):e1901. PubMed
    • Consortium Data Additions & Site Updates

      • New and Improved Cell Migration Knowledge Base

        The Cell Migration Knowledge Base (CMKB), a curated and informative resource for proteins involved in Cell Migration, has recently been restructured to include links to genes, protein domains, protein structure, interactions, phosphorylation sites and pathway information. Currently there are 800 families (6800 proteins) in the database. Please take a moment to visit the Knowledge Base to search for information on your favorite migration-related protein and let us have your feedback.

    • Other CMC Publications

      • Healy KD, Hodgson L, Kim TY, Shutes A, Maddileti S, Juliano RL, Hahn KM, Harden TK, Bang YJ, Der CJ. DLC-1 suppresses non-small cell lung cancer growth and invasion by RhoGAP-dependent and independent mechanisms. Mol Carcinog 2008; 47 (5):326-37 PubMed
      • Murase S, Cho C, White JM, Horwitz AF. ADAM2 promotes migration of neuroblasts in the rostral migratory stream to the olfactory bulb. Eur J Neurosci 2008; 27 (7):1585-95 PubMed
      • Shen F, Hodgson L, Price JH, Hahn KM. Digital differential interference contrast autofocus for high-resolution oil-immersion microscopy. Cytometry A 2008; Apr 7 [Epub ahead of print] PubMed
    • Upcoming Conferences & Workshops

      • 9th Annual Virtual Cell Short Course:

        This will be offered June 2-4, 2008 at the University of Connecticut Health Center, Farmington, CT. The Virtual Cell (http://vcell.org) is a computational software environment for modeling and simulating cell biological systems. The course is free, but requires an application that describes how prospective students plan to use the Virtual Cell system. For details on the course and how to apply, go to http://www.vcell.org/news/shortcourse_08.html

      • Computational Cell Biology:

        Cold Spring Harbor Laboratory will be offering a three week course on Computational Cell Biology June 27-July 17, 2008. For further details go to: http://meetings.cshl.edu/courses/c-comp08.shtml.

      • Frontiers in Cell Migration; from mechanism to disease:

        A Conference co-sponsored by the Consortium and the National Institute of Health General Medical Sciences, will be held at the NIH Natcher Center, Bethesda MD 16-18 September, 2008. More details are available at the meetings website at http://meeting2008.cellmigration.org.

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  • April 2008

    1. Collective migration: Modifying shape and adhesion in Hindsight
    2. Arp2/3 complex: Actin branching in 3D
    3. Consortium Data Additions & Site Updates
    4. Other CMC Publications
    5. Upcoming Conferences & Workshops
    • Collective migration: Modifying shape and adhesion in Hindsight

      Drosophila ovaries are composed of egg chambers, each of which contains approximately 650 follicular cells that surround 15 nurse cells and one oocyte. During development, anterior follicle cells switch from an adhesive cuboidal shape to a squamous cell shape and become capable both of remodeling cell-cell contact and spreading, thus lowering the cell density in the anterior part of the egg chamber. In Current Biology, Denise Montell and colleagues now report that the Zinc finger transcription factor Hindsight (HNT) regulates cell-cell adhesion and collective migration during Drosophila ovary development. The authors observed that anterior follicle cells from hnt mutants retained abnormally high levels of adhesion proteins at the plasma membrane and failed to spread. In addition, 4-6 follicle cells known as border cells delaminate from the anterior follicular epithelium and migrate between nurse cells, requiring even more complex regulation of cell-cell adhesions. Border cell migration was also impaired in the absence of HNT. The authors report that motility and cohesion among cells of a migratory group are separable features that are mediated by the distinct JNK and STAT signaling pathways, respectively. Silencing the HNT human homologue RREB1 (Ras Responsive Element Binding Protein 1) in mammary epithelial cell lines resulted in a reduction in cell protrusions, clustering and cell immobilization, thus confirming that the importance of these transcription factors in the regulation of morphological changes, cell-cell adhesion and collective migration has been conserved in evolution.

      • Melani M, Simpson K, Brugge J, Montell D. Regulation of cell adhesion and collective cell migration by Hindsight and its human homolog RREB1. Curr Biol. 2008 Apr 2 [Epub ahead of print] PubMed
    • Arp2/3 complex: Actin branching in 3D

      The actin-related protein 2/3 (Arp2/3) complex plays a key role in organising dynamic actin networks. In the presence of both ATP and nucleation-promoting factors, the complex binds to the side of a mother actin filament and initiates the growth of a daughter filament from its free barbed end. New filaments grow at an angle on the sides of pre-existing mother filaments, thus creating a branched F-actin network. The seven subunit Arp2/3 complex is comprised of five subunits called ARPC1-5 together with both Arp2 and Arp3, which are the first two subunits to be incorporated in the nascent filament. The architecture of the branch junction formed at the base of a new branch by the Arp2/3 complex has remained elusive. In The Journal of Cell Biology, Dorit Hanein, Niels Volkmann and colleagues now report a detailed 3D reconstruction of the branch at a 2.6 nm resolution using electron tomography and computational modelling. The structure reveals a surprisingly large, intricate interface between the mother filament and all seven subunits of the Arp2/3 complex. A large conformational change in the Arp2/3 complex is necessary during branch formation in order to allow Arp2 and Arp3 to form the first two units of the daughter filament template. In addition, conformational changes occur in the mother filament, indicating a more active role in forming the branch than previously anticipated.

      • Rouiller I, Xu XP, Amann KJ, Egile C, Nickell S, Nicastro D, Li R, Pollard TD, Volkmann N, Hanein D. The structural basis of actin filament branching by the Arp2/3 complex. J Cell Biol. 2008;180(5):887-95. PubMed
    • Consortium Data Additions & Site Updates

      • New and Improved Cell Migration Knowledge Base:

        The Cell Migration Knowledge Base (CMKB), a curated and informative resource for proteins involved in Cell Migration, has recently been restructured to include links to genes, protein domains, protein structure, interactions, phosphorylation sites and pathway information. Currently there are 800 families (6800 proteins) in the database. Please take a moment to visit the Knowledge Base and let us have your feedback on its utility.

      • Phosphoproteomics data for Filamin are now available

        Data showing the phosphorylation sites present in Filamin are now available through the proteomics initiative.

    • Other CMC Publications

      • Campbell ID. Studies of focal adhesion assembly. Biochem Soc Trans 2008; 36 (Pt 2):263-6 PubMed
      • El-Sibai M, Pertz O, Pang H, Yip SC, Lorenz M, Symons M, Condeelis JS, Hahn KM, Backer JM. RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells. Exp Cell Res 2008 Epub PubMed
      • Leone M, Yu EC, Liddington RC, Pasquale EB, Pellecchia M. The PTB domain of tensin: NMR solution structure and phosphoinositides binding studies. Biopolymers 2008; 89 (1):86-92 PubMed
    • Upcoming Conferences & Workshops

      • 9th Annual Virtual Cell Short Course:

        The Virtual Cell short course will be offered June 2-4, 2008 at the University of Connecticut Health Center, Farmington, CT. The Virtual Cell (http://vcell.org) is a computational software environment for modeling and simulating cell biological systems. The course is free, but requires an application that describes how prospective students plan to use the Virtual Cell system. For details on the course and how to apply, go to http://www.vcell.org/news/shortcourse_08.html

      • Frontiers in Cell Migration; from mechanism to disease:

        This Conference, co-sponsored by the Consortium and the National Institute of Health General Medical Sciences, will be held at the NIH Natcher Center, Bethesda MD 16-18 September. More details are available at the meetings website at meeting2008.cellmigration.org.

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  • March 2008

    1. Integrin phosphorylation: An ON and OFF switch
    2. Talin structure: How to bind actin
    3. Consortium Data Additions & Site Updates
    4. Other CMC Publications
    5. Upcoming Conferences & Workshops
    • Integrin phosphorylation: An ON and OFF switch

      Understanding how integrin activity is controlled is crucial to the understanding of cell adhesion and migration. Integrins are transmembrane heterodimers composed of an alpha and β subunit; ligands bind to their β cytoplasmic tails and control their state of activation. These cytoplasmic β tails contain two NPXY or NPXY-like motifs that bind to phosphotyrosine-binding (PTB) domains. Previous studies have shown that binding of the Talin PTB domain to the β3 tail constitutes the final step of integrin activation. The cytoplasmic protein Dok1 is involved in many signaling pathways and also binds to the β3 tail through its PTB domain, but in contrast to Talin it negatively regulates integrin activation. Using NMR and X-ray crystallography, Oxley et al. have now characterised the molecular basis of the Dok1-β3 interaction. They show Dok1 binds to the integrin NPLY motif and that the interaction is positively regulated by the phosphorylation of beta integrin at Tyr 747. As Tyr 747 phosphorylation inhibits Talin binding, the authors propose phosphorylation may function as a switch that controls integrin receptor activation.

      • Oxley CL, Anthis NJ, Lowe ED, Vakonakis I, Campbell ID, Wegener L. An Integrin Phosphorylation Switch: the effect of β3 integrin tail phosphorylation on Dok1 and Talin binding. J Biol Chem. 2008;283(9):5420-6. PubMed
    • Talin structure: How to bind actin

      Talin is a 2541 amino-acid dimeric protein that links integrin cell adhesion molecules to F-actin. It is composed of an N-terminal globular head, which binds β integrin cytoplasmic tails, connected to a C-terminal flexible rod by a short linker sequence. The Talin rod binds the cytoskeletal protein vinculin as well as β integrin, while the C-terminal end (residues 2300-2541) contains one of several binding sites for F-actin. This region is predicted to contain six helices that regulate actin binding and talin dimerization. Gingras et al. now report the NMR structure of talin residues 2300-2482, a five-helix bundle, and the crystal structure of helix 6. Using these two structures and small-angle X-ray scattering, the authors propose a model for the entire talin 2300-2541 dimer. They identify the residues that bind F-actin and also show that the Talin dimerization domain is important for binding. Furthermore, using electron microscopy, the authors provide direct evidence that the Talin dimer binds to three monomers along the long-pitch helix of F-actin.

      • Gingras AR, Bate N, Goult BT, Hazelwood L, Canestrelli I, Grossmann JG, Liu H, Putz NS, Roberts GC, Volkmann N, Hanein D, Barsukov IL, Critchley DR. The structure of the C-terminal actin-binding domain of talin. EMBO J. 2008; 27(2):458-69. PubMed
    • Consortium Data Additions & Site Updates

      • New and Improved Cell Migration Knowledge Base

        The Cell Migration Knowledge Base (CMKB) aims to provide a curated and informative resource for information on proteins involved in Cell Migration. Recently, the CMKB was restructured to provide ready access and more information on the proteins in the knowledge base. This includes links to genes, protein domains, protein structure, interactions, phosphorylation sites and pathway information. Currently there are 800 families (6800 proteins) in the database. Please take a moment to visit the Knowledge Base and let us have your feedback on its utility.

    • Other CMC Publications

      • Fass J, Pak C, Bamburg J, Mogilner A. Stochastic simulation of actin dynamics reveals the role of annealing and fragmentation. J Theor Biol 2008, Epub PubMed
      • Petrich BG, Marchese P, Ruggeri ZM, Spiess S, Weichert RA, Ye F, Tiedt R, Skoda RC, Monkley SJ, Critchley DR, Ginsberg MH. Talin is required for integrin-mediated platelet function in hemostasis and thrombosis. J Exp Med 2007; 204 (13):3103-11 PubMed
    • Upcoming Conferences & Workshops

      • Frontiers in Cell Migration; from mechanism to disease:

        This Conference is co-sponsored by the Consortium and the National Institute of Health General Medical Sciences and will be held at the NIH Natcher Center, Bethesda MD 16 -18 September. More details will be available at the meetings website meeting2008.cellmigration.org later this month.

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  • February 2008

    1. Live cell imaging: Bright ideas for measuring local concentration and aggregation
    2. Migration analysis: A modified phagokinetic track method
    3. Other CMC Publications
    4. Upcoming Conferences & Workshops
    • Live cell imaging: Bright ideas for measuring local concentration and aggregation

      A reductionist view of cell biology is the explanation of cellular activities in physical-chemical terms. To do this, one needs to quantify molecular concentrations and aggregation states in living cells while they execute and regulate various complex processes. For a complex cellular activity such as migration, which is characterized by highly localized, transient component processes, the need for high resolution spatial maps is essential. The techniques currently available only measure protein concentration and aggregation, either for the cell as a whole or for limited number of areas at relatively few time points, thus blurring both spatial and temporal information. The method described in publications by Digman et al. and Dalal et al., uses fluorescence fluctuations to map molecular interactions occurring at each pixel of an image. The novelty of this technique, termed the N and B (number and brightness) analysis, is that quantitative information about the number of molecules and their aggregation states are mapped pixel by pixel. This method is fast and can be applied to molecular signalling events to allow researcher to follow the aggregation states of receptors and downstream components as a function of time. Another advantage is that immobile or slowly moving features, such as cell edges and borders as well as contributions from photobleaching, are separated so that only the species that fluctuate more rapidly are analyzed. This technique is can be used with laser scanning microscopes and is a powerful, new tool for anyone interested in live cell imaging.

      • Digman M, Dalal RB, Horwitz AF, Gratton E. Mapping the number of molecules and brightness in the laser scanning microscope 2007, Biophys J Dec 20 [Epub] PubMed
      • Dalal RB, Digman MA, Horwitz AF, Vetri V, Gratton E. Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode. Microsc Res Tech 2008; 71 (1):69-81 PubMed
    • Migration analysis: A modified phagokinetic track method

      In Plos One, Geiger and colleagues present a new technique that -- by coupling the phagocytotic track (PKT) method to light microscopy -- is able to record the migration history and dynamic movement properties of cells concurrently. The authors tested various matrices and found that carboxylated beads with a 400nm diameter are optimal for the PKT assay. Furthermore, the authors used a cell-screening autofocus microscope in conjunction with an acquisition program that controls the visual parameters for each well of the assay. The number of automatically recorded and calculated track parameters, such as track area and lamellar activity, allowed the authors to distinguish between the migration patterns of cell types that appeared to move with equal velocity. In a further application of this technique, Geiger and colleagues attempted to identify genes that regulate breast cancer cell migration from a breast cancer gene library. Geiger and colleagues infected MCF7 breast cancer cells -- which are known to be poor migraters -- with 55 candidate genes from the library. Using their novel microscopy-based PKT method, the authors were able to identify pro-migratory genes such as HOVB7 and ERBB3, whose expression increases lamellipodal activity. In summary, the presented technique offers a promising method to identify and delineate the contribution of individual genes to cell migration.

      • Naffar-Abu-Amara S, Shay T, Galun M, Cohen N, Isakoff SJ, Kam Z, Geiger B. Identification of novel pro-migratory, cancer-associated genes using quantitative, microscopy-based screening. PLoS ONE 2008; 3 (1):e1457 PubMed
    • Other CMC Publications

      • Barua D, Faeder JR, Haugh JM. Computational models of tandem Src homology 2 domain interactions and application to phosphoinositide 3-kinase. J Biol Chem 2008; Jan 20; [Epub ahead of print] PubMed
      • Brown CM, Dalal RB, Hebert B, Digman MA, Horwitz AR, Gratton E. Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. J Microsc 2008; 229 (Pt 1):78-91 PubMed
      • Garrett SC, Hodgson L, Rybin A, Toutchkine A, Hahn KM, Lawrence DS, Bresnick AR. A Biosensor of S100A4 Metastasis Factor Activation: Inhibitor Screening and Cellular Activation Dynamics. Biochemistry 2008; 47 (3):986-96 PubMed
      • Hodgson L, Pertz O, Hahn KM. Design and optimization of genetically encoded fluorescent biosensors: GTPase biosensors. Methods Cell Biol 2008; 85:63-81 PubMed
    • Upcoming Conferences & Workshops

      • Migration meeting at the NIH Natcher Center, Bethesda MD:

        The Consortium will be holding a Migration meeting at the NIH Natcher Center, Bethesda MD 16 -18 September. More details will be posted at the meetings website at meeting2008.cellmigration.org, as they become available. In the meantime, please mark your calendars and check the site regularly for updates.

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  • January 2008

    1. The phasor approach: A lifetime method for fluorescence image analysis
    2. Biosensors: The life of FAK activation
    3. Consortium Data Additions & Site Updates
    4. Other CMC Publications
    • The phasor approach: A lifetime method for fluorescence image analysis

      An important new method has been developed by Gratton and colleagues at the University of California, Irvine, and the San Raffaele Scientific Institute, Milan, Italy for analyzing fluorescence energy transfer (FRET). The method is an application of the phasor approach and it provides a robust way to quantify FRET from fluorescence lifetime imaging microscopy (FLIM) measurements. In the phasor plot, FRET, which involves the change of lifetime of the donor molecule, can be easily separated from the combination of lifetimes for donor and autofluorescence.

      The analysis goes beyond FRET and greatly enhances analysis of any fluorescence lifetime measurement. FLIM allows imaging and characterization of a large variety of samples, from deep tissues to crystals, by measuring the difference in the rate of fluorescence decay (the lifetime) of fluorescent particles in the sample. One of the most difficult tasks in FLIM analysis is to discern between multiple molecular species which may be present in the same volume of observation (for example, a single pixel). The phasor approach tremendously simplifies FLIM analysis. It replaces the large number of calculations, assumptions and estimates of initial parameters that are required to fit the lifetime of the observed fluorescence excitation with a graphic plot of the phase and the modulation - the parameters used to determine the lifetime. The result is a far more accurate, reliable and robust analysis that will greatly increase the veracity of FRET and other FLIM based analyses.

      • Digman M, Caiolfa VR, Zamai M, Gratton E. The Phasor approach to fluorescence lifetime imaging analysis. 2007, Biophys J Nov 2; [Epub ahead of print] PubMed
    • Biosensors: The life of FAK activation

      Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase known to be important in the control of several developmental and pathological events. However, the molecular mechanisms regulating its activity have not yet been completely elucidated. In Molecular and Cellular Biology, Cai et al. now report the development of two FRET-based biosensors that measure FAK activation in living cells. Activation occurs through either phosphorylation or a conformational change. One of the probes measures phosphorylation at tyrosine 397 -- the major FAK autophosphorylation site that is crucial for FAK function and serves as a scaffold for the recruitment of interacting proteins. The other biosensor monitors the transition from an autoinhibitory conformation -- where the FAK FERM domain interacts with the FAK catalytic domain to impair its activity -- to an open active conformation. The autoinhibitory model of FAK regulation was confirmed in vivo by these two biosensors, which provided evidence for a conformational switch associated to FAK activation for the first time. FAK biosensors were used to study the spatial regulation of FAK in live cells, revealing a strong activation in peripheral focal adhesions and in cell protrusions. Furthermore, the conformational biosensor identified phosphatidylinositol 4,5-bisphosphate as a novel ligand that alters FAK conformation, providing mechanistic insight into how acidic phospholipids may regulate FAK activity.

      • Cai X, Lietha D, Ceccarelli DF, Karginov AV, Rajfur Z, Jacobson K, Hahn KM, Eck MJ, Schaller MD. Spatial and temporal regulation of focal adhesion kinase activity in living cells. Mol Cell Biol. 2008; 28(1):201-14. PubMed
    • Consortium Data Additions & Site Updates

      • Phosphoproteomics data for WASP now available

        Data showing the phosphorylation sites present in WASP are now available through the proteomics initiative activity table.

      • The Cell Migration Gateway is poised to make the Consortium's protocols more readily accessible through a joint venture with Nature Protocols. Existing protocols will be integrated into the Gateway over the course of the next few months and future protocol postings will be implemented using this approach. The Imperiali laboratory has recently submitted a series of protocols which were selected for publication in the peer reviewed, Nature Protocols journal. These detail the methodology for generating peptide-based environment-sensitive biosensors for the investigation of biological events such as peptide-protein interactions. The work presented here is divided into three parts: synthesis of the dimethylaminophthalimide-derived anhydrides used as precursors of the solvatochromic fluorophores 4-DMAP and 6-DMN and two complementary approaches for the subsequent insertion of these chromophores into peptides. One approach relies on a direct on-resin derivatization of fully synthesized peptides while the other proceeds via coupling of Fmoc-protected fluorescent amino acids using standard solid-phase peptide synthesis procedures. The 4-DMAP and 6-DMN fluorophores are used for their ability to report modification in the polarity of the peptide-based probe microenvironment via dramatic changes of their spectral properties (emission wavelength as well as intensity). The resulting sensors can be applied to study both qualitatively and quantitatively specific proteins interactions by fluorescence techniques. This general methodology constitutes a first step in the process of designing of full-length environment-sensitive proteins obtained by native chemical ligation.

        Tools for investigating peptide-protein interactions: peptide incorporation of environment-sensitive fluorophores via on-resin derivatization. Sainlos M, Imperiali B. Nature Protocols 2007: 2(12):3201-9. PubMed

        Tools for investigating peptide-protein interactions: peptide incorporation of environment-sensitive fluorophores through SPPS-based 'building block' approach. Sainlos M, Imperiali B. Nature Protocols 2007;2(12):3210-8. PubMed

        Synthesis of anhydride precursors of the environment-sensitive fluorophores 4-DMAP and 6-DMN. Sainlos M, Imperiali B. Nature Protocols 2007;2(12):3219-25. PubMed

    • Other CMC Publications

      • Digman M, Brown CM, Horwitz AF, Mantulin WW, Gratton E. Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy. 2007, Biophys J Nov 9; [Epub ahead of print] PubMed
      • Hall B, McLean MA, Davis K, Casanova JE, Sligar SG, Schwartz MA. A fluorescence resonance energy transfer activation sensor for Arf6. Anal Biochem. 2007 Dec 3; [Epub ahead of print] PubMed
      • Healy KD, Hodgson L, Kim TY, Shutes A, Maddileti S, Juliano RL, Hahn KM, Harden TK, Bang YJ, Der CJ. DLC-1 suppresses non-small cell lung cancer growth and invasion by RhoGAP-dependent and independent mechanisms. Mol Carcinog. 2007 Oct 11; [Epub ahead of print] PubMed
      • EGF and PIP2 hydrolysis: Cofilin set free

        One of this month's featured articles in the Cell Migration Updates section of the Gateway highlights Cofilin's regulation by EGF and its role in cell protrusion in mammary tumour cells. Click here to read the article review.

        van Rheenen J, Song X, van Roosmalen W, Cammer , Chen X, DesMarais V, Yip S-C, Backer JM, Eddy RJ, Condeelis JS. EGF-induced PIP2 hydrolysis releases and activates Cofilin locally in carcinoma cells. J Cell Biol. 2007: 179(6):1247-59. PubMed
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