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December 2005

• Correlation Microscopy

  New analysis methods allow determinations of molecular diffusion and transport in different parts of the cell

  Fluorescence correlation spectroscopy involves the analysis of rapid fluorescence intensity fluctuations at a single spot in a cell over time to determine molecular concentration and diffusion coefficients. To increase the spatial and temporal information gained, laser-scanning image time series can be used to simultaneously measure the concentration and diffusion of molecules in various cellular locations using Image Correlation Spectroscopy (ICS). However, the time taken to scan an entire image (~1sec) limits the temporal resolution of ICS, which means it is only useful for analysing slow moving (membrane) proteins. Gratton, Horwitz, Wiseman and their colleagues have now developed a new method Raster Image Correlation Spectroscopy (RICS), which exploits "hidden" temporal information on the millisecond time scale in scanned images providing high spatial and temporal resolution. Thus, RICS makes it possible to produce cellular maps of concentration, interactions, and dynamics on a time scale at which most physiological processes take place. Another development is the ability to map the velocities of directed, non-diffusive movement using Spatial-Temporal Image Correlation Spectroscopy (STICS). These technologies are generally accessible as they require only a laser scanning confocal microscope and analysis software, which will be available on the Laboratory for Fluorescence Dynamics and CMC websites.

  1. Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E. Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys J. 2005 Aug;89(2):1317-27. Epub 2005 May 20. PubMed
  2. Hebert B, Costantino S, Wiseman PW. Spatiotemporal Image Correlation Spectroscopy (STICS) Theory, Verification, and Application to Protein Velocity Mapping in Living CHO Cells. Biophys J. 2005 May;88(5):3601-3614. Epub 2005 Feb 18. PubMed
  3. Digman MA, Sengupta P, Wiseman PW, Brown CM, Horwitz AR, Gratton E. Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure. Biophys J. 2005 May;88(5):L33-6. Epub 2005 Mar 25. PubMed
  4. Wiseman PW, Brown CM, Webb DJ, Hebert B, Johnson NL, Squier JA, Ellisman MH, Horwitz AF. Spatial mapping of integrin interactions and dynamics during cell migration by image correlation microscopy. J Cell Sci. 2004 Nov 1;117(Pt 23):5521-34. Epub 2004 Oct 12. PubMed

• New Imaging Probes in Action

  Photorelease of caged phosphopeptides interrupts signal transduction pathways for migration.

  A major theme in cell migration is the transient activation of proteins at specific locations. To understand the function of these localized signals, methods that can detect them as well as locally activate or inactivate them are required. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that mediates integrin-based regulation of migration and adhesion turnover. To study the effects of local FAK activation, Imperiali, Jacobson, and colleagues have designed a peptide comprising residues 391-406 of human FAK with a caged 397 phosphotyrosine residue that can be photoactivated in situ. They find that local activation of the peptide in NBT-II cells leads to a temporary arrest of leading edge migration within approximately 1 min. This study demonstrates the power of the technology for synchronizing cellular events on the millisecond time scale and points to the next step, to control local activation of probes that are able to localize to the appropriate cellular compartment.

  1. Application described in... Humphrey D, Rajfur Z, Vazquez ME, Scheswohl D, Schaller MD, Jacobson K, Imperiali B. In situ photoactivation of a caged phosphotyrosine peptide derived from focal adhesion kinase temporarily halts lamellar extension of single migrating tumor cells. J Biol Chem. 2005 Jun 10;280(23):22091-101. Epub 2005 Apr 6. PubMed
  2. Methodology described in... Rothman DM, Vazquez ME, Vogel EM, Imperiali B. General method for the synthesis of caged phosphopeptides: tools for the exploration of signal transduction pathways. Org Lett. 2002 Aug 22;4(17):2865-8. PubMed and Rothman DM, Vazquez ME, Vogel EM, Imperiali B. Caged phospho-amino acid building blocks for solid-phase peptide synthesis. J Org Chem. 2003 Aug 22;68(17):6795-8. PubMed
  3. Rothman DM, Shults MD, Imperiali B. Chemical approaches for investigating phosphorylation in signal transduction networks. Trends Cell Biol. 2005 Sep;15(9):502-10. PubMed

• Phosphoproteomics

  CMC members collaborate to identify novel phosphorylation sites on FAK, paxillin and talin.

  Mass spectrometry can be used to identify protein post-translational modifications, including phosphorylation sites. However, these studies usually reveal only a limited number of sites - the most heavily phosphorylated ones. Recently, Don Hunt, Rick Horwitz and Tom Parsons at the University of Virginia, and Mark Ginsberg at the University of California at San Diego have collaborated to develop methods for a global analysis that reveals most of the utilized phosphorylation sites on proteins. In addition, methods for differential analyses have been developed to reveal sites that change kinetically in response to a stimulus.

  The key steps in these analyses include immobilized metal affinity chromatography (IMAC) to enrich for phosphopeptides, a broad spectrum of proteases to produce analysable peptides that cover the protein of interest, labelling with different mass reagents and Fourier transform techniques for differential analyses, and a new type of analysis method (electron transfer dissociation, ETD) to facilitate analysis of longer peptides. These methods have been applied to three key migration molecules: paxillin, FAK and talin and have uncovered many novel phosphorylation sites. The results of these analyses can also be found on the Consortium website and appear as capsule summaries in the October 27 issue of the Journal of Cell Science. These data should provide starting points for future studies on the regulation of paxillin, FAK and talin during cell migration.

  1. Ratnikov B, Ptak C, Han J, Shabanowitz J, Hunt DF, Ginsberg MH. Talin phosphorylation sites mapped by mass spectrometry. J Cell Sci. 2005 Nov 1;118(Pt 21):4921-3. PubMed
  2. Webb DJ, Schroeder MJ, Brame CJ, Whitmore L, Shabanowitz J, Hunt DF, Horwitz AR. Paxillin phosphorylation sites mapped by mass spectrometry. J Cell Sci. 2005 Nov 1;118(Pt 21):4925-9. PubMed
  3. Grigera PR, Jeffery ED, Martin KH, Shabanowitz J, Hunt DF, Parsons JT. FAK phosphorylation sites mapped by mass spectrometry. J Cell Sci. 2005 Nov 1;118(Pt 21):4931-5. PubMed
  4. Schroeder MJ, Webb DJ, Shabanowitz J, Horwitz AF, Hunt DF. Methods for the detection of paxillin post-translational modifications and interacting proteins by mass spectrometry. J Proteome Res. 2005 Sep-Oct;4(5):1832-41. PubMed

• Cell Migration at a Glance

  M. Vicente-Manzanares, D.J. Webb and R. Horwitz have developed a cartoon, with commentary, showing the proteins and mechanisms that regulate the cytoskeleton and cell adhesion during migration. This short piece can be found in the Journal of Cell Science 118, 4917-4919 (2005), and is accompanied by a poster illustrating the signaling pathways and multiprotein complexes involved.

  Vicente-Manzanares M, Webb DJ, Horwitz AR. Cell migration at a glance. J Cell Sci. 2005 Nov 1;118(Pt 21):4917-9. PubMed

November 2005

• Special Interest Subgroups feature CMC workshops at ASCB meeting

  Modeling and Structure Initiatives sponsor Special Interest Subgroup sessions at the ASCB meeting in San Francisco. A workshop on “Computational Modeling of Cell Migration” will be hosted by the Modeling Initiative in Room 232 at the Moscone Center at 1:00 pm on Dec. 10th. The Structure Initiative will host a workshop on “Structure-Function Studies of Cell-Matrix and Cell-Cell Interactions Involved in Cell Migration” in room 224 at the Moscone Center. For more details visit www.ascb.org.

• Micropatterning for Imaging studies

  Novel Micropatterning approaches advance imaging approaches

  The limitations of micropatterned substrates based on inert gold layers are avoided using an anisotropic solid microetching (ASOMIC) procedure that enables molecular dynamics imaging by wide-field and total internal reflection fluorescence (TIRF) microscopy of living mammalian cells and correlative platinum replica electron microscopy.

  Kandere-Grzybowska K, Campbell C, Komarova Y, Grzybowski BA, Borisy GG. Molecular dynamics imaging in micropatterned living cells. Nat Methods. 2005 Oct;2(10):739-41. PubMed

• New protocols added to the CMC Activity Center

  Signaling Initiative Protocols

  Information on making and using the dye-based Cdc42 biosensor (MeroCBD) including a link to free software which performs a new type of bleaching correction (Hodgson et al., 2005, Methods in Enzymology, in press) and protocols for Metamorph routines for Fluorescence Image Processing Steps in Ratio Imaging are now available on the Signaling Initiative's protocols page.

  Imaging & Photomanipulation Initiative Protocols

  Protocols for preparing substrates for quantitative traction assays (Damljanovic et al., 2005 in press) and for local inhibition of GFP CALI (chromophore assisted laser inactivation) are now available on the Imaging & Photomanipulation Initiative's protocol page.

October 2005

• New Mass Spectrometry method

  Methods for the identification of post-translational modifications and interacting proteins for paxillin and other adhesion molecules are presented in a new publication in the Sep-Oct issue of Journal of Proteomic Research. The new methods were used to identify 29 phosphorylation sites (19 novel and 10 previously reported) and a novel glycosylation site on Ser 74. Furthermore, 10 co-purifying proteins present in focal adhesion complexes are simultaneously detected.

  Schroeder MJ, Webb DJ, Shabanowitz J, Horwitz AF, Hunt DF. Methods for the detection of paxillin post-translational modifications and interacting proteins by mass spectrometry.J Proteome Res. 2005 Sep-Oct;4(5):1832-41. PubMed

September 2005

• Protocol posting to the CMC Activity Center

  RNAi protocols for C. elegans screens are posted

  RNAi protocols for migration screens in C. elegans are posted via the Protein Discovery - Expression protocols page .

  Preliminary data and methodology available upon request

  BC1000 and MDA-MB-231 cDNA library screens from the Geiger laboratory are completed and submitted for publication. Data and methodology are available via the Protein Discovery - Expression activity table

• Publication Highlights

  Novel analysis method opens correlation microscopy for the production of cellular maps of concentrations, interactions, and dynamics

  Consortium investigators have extended image correlation (ICS) to probe spatial correlations in previously inaccessible temporal windows. Using standard laser confocal imaging techniques (raster-scan mode), the spatial information of image correlation spectroscopy (ICS) not be obtained on a broad range of time scales including those previously only accessible by single-point fluorescence correlation spectroscopy (FCS). In this publication the technique called RICS (raster image correlation microscopy) is used to determine spatially resolved variations in the diffusion of paxillin-EGFP stably expressed in CHOK1 cells.

  Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E. Measuring fast dynamics in solutions and cells with a laser scanning microscope. 2005 Aug;89(2):1317-27. Epub 2005 May 20. PubMed

  Migration models break into 3D and tackle signaling questions

  (1) A mechanical model has been developed for cells migrating in three-dimensions. The model confirms some events predicted by 2D models; but it also reveals others that appear to be particular to the 3D environment. (2) A finite-element model that includes cell contact area geometries demonstrates that cell morphology alone cannot account for the distribution of phosphoinositide (PI) 3-kinase PI lipids in the plasma membrane of PDGF stimulated fibroblasts. (3) An efficient Brownian dynamics algorithm has been developed to analyze biochemical signal transduction events at the cell membrane. It can readily be adapted for the stochastic simulation of more complex cell signaling systems.

  1. Zaman MH, Kamm RD, Matsudaira PT, Lauffenburger DA. Computational Model for Cell Migration in Three- Dimensional Matrices. Biophys J. 2005 Aug;89(2):1389-97. Epub 2005 May 20. PubMed
  2. Schneider IC, Parrish EM, Haugh JM. Spatial Analysis of 3' Phosphoinositide Signaling in Living Fibroblasts, III: Influence of Cell Morphology and Morphological Polarity. Biophys. J. 2005 Aug;89(2):1420-30. Epub 2005 May 27 PubMed
  3. Monine MI, Haugh JM. Reactions on cell membranes: Comparison of continuum theory and Brownian dynamics simulations. J Chem Phys. 2005 Aug 15;123(7):074908. PubMed

August 2005

• Data posting to the CMC Activity Center

  Binding partner date on key migration proteins are posted to the Proteomics pages

  Mass spectrometry has been used to identified potential binding partners for Pix, paxillin, calpain and the beta and delta isoforms of CamK-II . Go to the Protein Discovery - Proteomic activity table to view these data.

• Publication Highlights

  The physics of filopidial protrusion

  Modeling was used to examine the mechanics of filopodia. The model shows that more than 10 actin filaments need to be bundled to overcome the membrane resistance, that the filopodial length is limited by buckling for 10 - 30 filaments, and by G-actin diffusion for more than 30 filaments. There is a predicted optimal number of bundled filaments, ~ 30, at which the filopodial length can reach a few microns. The model correctly predicts the observed number of filaments in the filopodial bundle, but fails to explain emergence of long filopodia, which suggest important undiscovered mechanisms of motor transport and adhesion in filopodial maintenance.

  Mogilner A and Rubinstein B. The physics of filopodial protrusion. Biophys J. 2005 89:782-95. PubMed

• Focal adhesions as mechanosensors

  A thermodynamic model for the mechanosensitivity of focal adhesions is proposed, which accounts for their self-assemble and elongate upon application of pulling forces and dissociate when these forces are decreased.

  Shemesh T, Geiger B, Bershadsky AD, Kozlov MM. Focal adhesions as mechanosensors: a physical mechanism. Proc Natl Acad Sci U S A. 2005 Aug 30;102(35):12383-8. Epub 2005 Aug 19. PubMed

July 2005

• PhosphoArray for Migration Proteins now available

  The Consortium and BioSource International collaborate to produce the Mercator PhosphoArray

  BioSource International has produced an initial phosphoarray capable of simultaneously measuring 8 phospho-proteins, several of which are important migration-related proteins: (EGFR [pY1068], FAK [pY397], Paxillin [pY118], Akt [pS473], p38 [pTpY180/182], HSP27 [pS82] and ATF2 [pTpt69/71]). This product is available through Biosource International http://www.biosource.com/ by searching product number BHM9021 and a PDF describing it can be downloaded by clicking here

• Data posting to the CMC Activity Center

  Genetic screening data for migration proteins in Drosophila posted

  Data comparing the genes expressed in migratory and non-migratory cells of the drosophila ovary are posted via the Protein Discovery - Expression activity table

• Publication Highlights

  Chemistry facilitates signaling and imaging studies in Cell Migration

  In two publications utilize chemical approaches to address issues in signal transduction. The first describes methods for engineering membrane permeant peptides. The second uses a caged phosphopeptide from FAK that inhibits cell migration when photactivated.

  1. Carrigan CN, Imperiali B. The engineering of membrane-permeable peptides. Anal Biochem. 2005 Jun 15;341(2):290-8. PubMed
  2. Humphrey D, Rajfur Z, Vazquez ME, Scheswohl D, Schaller MD, Jacobson K, Imperiali B. In situ photoactivation of a caged phosphotyrosine peptide derived from FAK temporarily halts lamellar extension of single migrating tumor cells. J Biol Chem. 2005 Jun 10;280(23):22091-101. PubMed

June 2005

• Publication Highlights

  Two correlation microscopy and a modeling paper highlight this months publications. One paper details methods for producing maps of flow velocities in migrating cells using correlation methods (1). Another details a method for producing cellular maps of fast and slow dynamics using a confocal microscope (2). The third paper applies decision tree analysis to signaling networks.

  1. Hebert B, Costantino S, Wiseman PW. Spatiotemporal Image Correlation Spectroscopy (STICS) Theory, Verification, and Application to Protein Velocity Mapping in Living CHO Cells. Biophys J. 2005 May;88(5):3601-3614. PubMed
  2. Digman MA, Sengupta P, Wiseman PW, Brown CM, Horwitz AR, Gratton E. Fluctuation correlation spectroscopy with a laser-scanning microscope: exploring the hidden time structure. Biophys J. 2005 May;88(5):L33-L36. PubMed
  3. Hautaniemi S, Kharait S, Iwabu A, Wells A, Lauffenburger DA. Modeling of signal-response cascades using decision tree analysis. Bioinformatics. 2005 21:2027-35. PubMed

May 2005

• Data posting to the CMC Activity Center

  Phosphorylation sites on cortactin and FAK

  The utilized phosphorylation sites on cortactin and FAK have been elucidated and are posted via the Protein Discovery - Proteomics activity table.

  Migration hits from GFP-tagged cDNA library screen posted

  Data from a localization screen showing the migration related hits using a GFP-tagged HT1080 cDNA library in REF52 cells are posted via the Protein Discovery - Expression activity table

April 2005

• Publication Highlights

  Modeling, Signaling and Imaging working hand in hand

  Three publications present progress in Modeling and Signaling. Schults et al. (1) describe a new method for quantifying protein kinase activities in cell extracts. The key to the assay is a fluorescence based sensor on peptide substrates for protein kinases. One modeling study (2) develops a Baysian network learning algorithm to model signaling data, the first set of available data are for ES cell fates; the model will be applied to migration signaling data as well. The second model (3) is a multi-scale computational model for the lamellipodium.

  1. Schults MD, Janes KA, Lauffenburger DA, Imperiali B. A multiplexed homogenous fluorescence-based assay for protein kinase activity in cell lysates. Nat Methods. 2005 Mar 23;2(4):277-284. PubMed
  2. Woolf PJ, Prudhomme W, Daheron L, Daley GQ, Lauffenburger DA. Bayesian Network Analysis of Signaling Networks Governing Stem Cell Fate Decisions. Bioinformatics. 2005 Mar 21:741-53. PubMed
  3. Rubinstein B, Jacobson K, Mogilner A. Multiscale Two-Dimensional Modeling of a Motile Simple-Shaped Cell. SIAM J. MMS, 2005 3:413-439. A PDF of the manuscript is available and the movies for this model are at: http://www.math.ucdavis.edu/~mogilner/CompKerat1.mpghttp://www.math.ucdavis.edu/~mogilner/turn.mpg

Structures of migration molecules

The structure of the interaction between PIP kinase gamma and talin (1) and the integrin binding site on alpha-actinin (2) are elucidated by X-ray and tomography, respectively.

1. de Pereda JM, Wegener KL, Santelli E, Bate N, Ginsberg MH, Critchley DR, Campbell ID, Liddington RC. Structural basis for phosphatidylinositol phosphate kinase type Igamma binding to talin at focal adhesions. J Biol Chem. 2005 Mar 4;280(9):8381-6. PubMed
2. Kelly DF, Taylor KA. Identification of the beta1-integrin binding site on alpha-actinin by cryoelectron microscopy. J Struct Biol. 2005 Mar;149(3): 290-302. PubMed

March 2005

• Data posting to the CMC Activity Center

  Migration Hits from a small Chemical Library are posted

  Compounds, identified from the Chem Bridge Chemical Diversity library, that perturb migration-related structures in REF52 cells expressing YFP-paxillin are posted via the Protein Discovery - Expression activity table

• Publication Highlights

  Deconstructing the lamellipodium

  The current dogma that the lamellipodium is the leading edge of the engine of a crawling cell was challenged by demonstrating that lamellipodia can be disrupted without stopping the cell leading to a provocative hypothesis that the lamella that is the actual driving engine of the crawling cell.

  Gupton SL, Anderson KL, Kole TP, Fischer RS, Ponti A, Hitchcock-DeGregori SE, Danuser G, Fowler VM, Wirtz D, Hanein D, Waterman-Storer CM. Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin. J Cell Biol. 2005 Feb 14;168(4):619-31. PubMed

  Environment Sensitive Fluorophore 6DMN Exposed

  6-N,N-Dimethylamino-2,3-naphthalimide (6DMN) is a chromophore, whose photophysical properties are influenced by the polarity of its environment. The introduction of Fmoc-protected amino acid derivatives containing the fluorophore and the utility of these probes for biological applications is addressed.

  Vazquez ME, Blanco JB, Imperiali B. Photophysics and Biological Applications of the Environment-Sensitive Fluorophore 6-N,N-Dimethylamino-2,3-naphthalimide. J Am Chem Soc. 2005 Feb 2;127(4):1300-6. PubMed

February 2005

• Publication Highlights

  Coupling adhesion dynamics, signaling events and actin cytoskeletal reorganization

  A simple mathematical model is developed for the coupled dynamics of cell adhesions, small GTPases (Rac and Rho) and actin stress fibers in guiding a directional reorganization of the actin cytoskeleton.

  Civelekoglu-Scholey G, Orr AW, Novak I, Meister JJ, Schwartz MA, Mogilner A. Model of coupled transient changes of Rac, Rho, adhesions and stress fibers alignment in endothelial cells responding to shear stress. J Theor Biol. 2005 <<<<<<< cmcnews05.shtml Feb 232:569-85. PubMed

  Arp2 and Arp3 nucleotide binding for nucleating activity

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  The importance of Arp2 and Arp3 nucleotide binding for nucleating activity, and Arp3 nucleotide binding for the maintenance of cortical actin cytoskeleton cytoarchitecture is demonstrated using nucleotide-binding pocket (NBP) mutants in S. cerevisiae.

  Martin AC, Xu XP, Rouiller I, Kaksonen M, Sun Y, Belmont L, Volkmann N, Hanein D, Welch M, Drubin DG. Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function. J Cell Biol. 2005 Jan 17;168(2):315-28. PubMed

  A general method for preparing caged phosphoproteins

  Caged amino acids afford researchers spatial and temporal control over the effective phosphorylation state at a specific site in a target protein. This publication presentes the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense suppression methodology.

  Rothman DM, Petersson EJ, Vazquez ME, Brandt GS, Dougherty DA, Imperiali B. Caged phosphoproteins. J Am Chem Soc. 2005 Jan 26;127(3):846-7. PubMed

January 2005

• Data posting to the CMC Activity Center

  Phosphorylation sites on talin, paxillin and GIT1 are posted on the Proteomics pages

  The utilized phosphorylation sites on three key migration proteins – talin, paxillin and GIT1– are presented via the Protein Discovery - Proteomic activity table .

  Data from Breast Cancer 1000 cDNA library screen posted

  Genes identified from the Breast Cancer 1000 cDNA library that increase migratory behavior in MCF-10A cells expressing activated ErbB2 in the transwell migration assay are posted via the Protein Discovery - Expression activity table

  Data from a Natural Products Screen posted

  Compounds, identified from the Tel Aviv Natural Products Library, that perturb migration-related structures in REF52 cells expressing YFP-paxillin are posted via the Protein Discovery - Expression activity table