The cytoskeleton plays several fundamental roles in the cell, including organizing the spatial arrangement of subcellular organelles, regulating cell dynamics and motility, providing a platform for interaction with neighboring cells, and ultimately defining overall cell shape. Fluorescence imaging has proved to be vital in furthering our understanding of the cytoskeleton, and is now a mainstay technique used widely by cell biologists. In this review we provide an introduction to various imaging modalities used to study focal adhesions and the actin cytoskeleton, and using specific examples we highlight a number of recent studies in animal cells that have advanced our knowledge of cytoskeletal behavior.
References
[1]
Huang, S.; Ingber, D.E. Shape-dependent control of cell growth, differentiation, and apoptosis: Switching between attractors in cell regulatory networks. Exp. Cell. Res. 2000, 261, 91–103, doi:10.1006/excr.2000.5044.
[2]
Keren, K.; Pincus, Z.; Allen, G.M.; Barnhart, E.L.; Marriott, G.; Mogilner, A.; Theriot, J.A. Mechanism of shape determination in motile cells. Nature 2008, 453, 475–480, doi:10.1038/nature06952.
Zemel, A.; Rehfeldt, F.; Brown, A.E.X.; Discher, D.E.; Safran, S.A. Cell shape, Spreading symmetry and the polarization of stress fibers in cells. J. Phys. Condens. Matter. 2010, 22, 194110, doi:10.1088/0953-8984/22/19/194110.
[5]
Fletcher, D.A.; Mullins, R.D. Cell mechanics and the cytoskeleton. Nature 2010, 463, 485–492, doi:10.1038/nature08908.
[6]
Jaffe, A.B.; Hall, A. Rho gtpases: Biochemistry and biology. Annu. Rev. Cell. Dev. Biol. 2005, 21, 247–269, doi:10.1146/annurev.cellbio.21.020604.150721.
[7]
Kavallaris, M. Microtubules and resistance to tubulin-binding agents. Nat. Rev. Cancer. 2010, 10, 194–204, doi:10.1038/nrc2803.
[8]
Wang, J.; Pelling, A.E. An approach to visualize the deformation of the intermediate filament cytoskeleton in response to locally applied forces. ISRN Cell Biol. 2012, 2012, 513546.
[9]
Schoenenberger, C.A.; Bischler, N.; Fahrenkrog, B.; Aebi, U. Actin’s propensity for dynamic filament patterning. FEBS Lett. 2002, 529, 27–33, doi:10.1016/S0014-5793(02)03267-2.
[10]
Tojkander, S.; Gateva, G.; Lappalainen, P. Actin stress fibers—Assembly, Dynamics and biological roles. J. Cell. Sci. 2012, 125, 1855–1864, doi:10.1242/jcs.098087.
Ciobanasu, C.; Faivre, B.; Le Clainche, C. Actin dynamics associated with focal adhesions. Int. J. Cell Biol. 2012, 2012, 941292.
[13]
Geiger, B.; Yamada, K.M. Molecular architecture and function of matrix adhesions. Cold Spring Harb. Perspect. Biol. 2011, 3, a005033, doi:10.1101/cshperspect.a005033.
[14]
Docheva, D.; Popov, C.; Mutschler, W.; Schieker, M. Human mesenchymal stem cells in contact with their environment: Surface characteristics and the integrin system. J. Cell Mol. Med. 2007, 11, 21–38, doi:10.1111/j.1582-4934.2007.00001.x.
[15]
Brakebusch, C.; Fassler, R. The integrin-actin connection, an eternal love affair. EMBO J. 2003, 22, 2324–2333, doi:10.1093/emboj/cdg245.
Eleniste, P.P.; Bruzzaniti, A. Focal adhesion kinases in adhesion structures and disease. J. Signal Transduction 2012, 2012, 296450.
[18]
Liu, T.; Sims, D.; Baum, B. Parallel rnai screens across different cell lines identify generic and cell type-specific regulators of actin organization and cell morphology. Genome Biol. 2009, 10, R26, doi:10.1186/gb-2009-10-3-r26.
[19]
Worth, D.C.; Parsons, M. Advances in imaging cell–matrix adhesions. J. Cell Sci. 2010, 123, 3629–3638, doi:10.1242/jcs.064485.
[20]
Severin, S.; Gaits-Iacovoni, F.; Allart, S.; Gratacap, M.P.; Payrastre, B. A confocal-based morphometric analysis shows a functional crosstalk between the actin filament system and microtubules in thrombin-stimulated platelets. J. Thromb. Haemost. 2012, 11, 183–216.
[21]
Rino, J.; Braga, J.; Henriques, R.; Carmo-Fonseca, M. Frontiers in fluorescence microscopy. Int. J. Dev. Biol. 2009, 53, 1569–1579, doi:10.1387/ijdb.072351jr.
[22]
Wang, E.; Babbey, C.M.; Dunn, K.W. Performance comparison between the high-speed yokogawa spinning disc confocal system and single-point scanning confocal systems. J. Microsc. 2005, 218, 148–159, doi:10.1111/j.1365-2818.2005.01473.x.
Le Devedec, S.E.; Geverts, B.; de Bont, H.; Yan, K.; Verbeek, F.J.; Houtsmuller, A.B.; van de Water, B. The residence time of focal adhesion kinase (fak) and paxillin at focal adhesions in renal epithelial cells is determined by adhesion size, Strength and life cycle status. J. Cell Sci. 2012, 125, 4498–4506, doi:10.1242/jcs.104273.
[25]
Pfaendtner, J.; Volkmann, N.; Hanein, D.; Dalhaimer, P.; Pollard, T.D.; Voth, G.A. Key structural features of the actin filament arp2/3 complex branch junction revealed by molecular simulation. J. Mol. Biol. 2012, 416, 148–161, doi:10.1016/j.jmb.2011.12.025.
[26]
Rouiller, I.; Xu, X.; Amann, K.J.; Egile, C.; Nickell, S.; Nicastro, D.; Li, R.; Pollard, T.D.; Volkmann, N.; Hanein, D. The structural basis of actin filament branching by the arp2/3 complex. J. Cell Biol. 2008, 180, 887–895, doi:10.1083/jcb.200709092.
[27]
Kim, M.C.; Kim, C.; Wood, L.; Neal, D.; Kamm, R.D.; Asada, H.H. Integrating focal adhesion dynamics, cytoskeleton remodeling, and actin motor activity for predicting cell migration on 3d curved surfaces of the extracellular matrix. Integr. Biol. 2012, 4, 1386–1397, doi:10.1039/c2ib20159c.
[28]
Wehrle-Haller, B. Structure and function of focal adhesions. Curr. Opin. Cell Biol. 2012, 24, 116–124, doi:10.1016/j.ceb.2011.11.001.
Petchprayoon, C.; Suwanborirux, K.; Tanaka, J.; Yan, Y.; Sakata, T.; Marriott, G. Fluorescent kabiramides: New probes to quantify actin in vitro and in vivo. Bioconjug. Chem. 2005, 16, 1382–1389, doi:10.1021/bc050006j.
[35]
M?hl, C.; Kirchgessner, N.; Sch?fer, C.; Hoffmann, B.; Erkel, R. Quantitative mapping of averaged focal adhesion dynamics in migrating cells by shape normalization. J. Cell Sci. 2012, 125, 155–165, doi:10.1242/jcs.090746.
[36]
Théry, M. Micropatterning as a tool to decipher cell morphogenesis and functions. J. Cell Sci. 2010, 123, 4201–4213, doi:10.1242/jcs.075150.
[37]
Berginski, M.E.; Vitriol, E.A.; Hahn, K.M.; Gomez, S.M. High-resolution quantification of focal adhesion spatiotemporal dynamics in living cells. PLoS One 2011, 6, e22025.
[38]
Zaidel-Bar, R.; Geiger, B. The switchable integrin adhesome. J. Cell Sci. 2010, 123, 1385–1388, doi:10.1242/jcs.066183.
[39]
Geiger, B.; Zaidel-Bar, R. Opening the floodgates: Proteomics and the integrin adhesome. Curr. Opin. Cell Biol. 2012, 24, 562–568, doi:10.1016/j.ceb.2012.05.004.
[40]
Grashoff, C.; Hoffman, B.D.; Brenner, M.D.; Zhou, R.; Parsons, M.; Yang, M.T.; McLean, M.A.; Sligar, S.G.; Chen, C.S.; Ha, T.; et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 2010, 466, 263–266, doi:10.1038/nature09198.
Carisey, A.; Tsang, R.; Greiner, A.M.; Nijenhuis, N.; Heath, N.; Nazgiewicz, A.; Kemkemer, R.; Derby, B.; Spatz, J.; Ballestrem, C. Vinculin regulates the recruitment and release of core focal adhesion proteins in a force-dependent manner. Curr. Biol. 2013, 23, 271–281, doi:10.1016/j.cub.2013.01.009.
[43]
Simpson, K.J.; Selfors, L.M.; Bui, J.; Reynolds, A.; Leake, D.; Khvorova, A.; Brugge, J.S. Identification of genes that regulate epithelial cell migration using an sirna screening approach. Nat. Cell Biol. 2008, 10, 1027–1038, doi:10.1038/ncb1762.
[44]
Winograd-Katz, S.E.; Itzkovitz, S.; Kam, Z.; Geiger, B. Multiparametric analysis of focal adhesion formation by rnai-mediated gene knockdown. J. Cell Biol. 2009, 186, 423–436, doi:10.1083/jcb.200901105.
[45]
Bai, S.W.; Herrera-Abreu, M.T.; Rohn, J.L.; Racine, V.; Tajadura, V.; Suryavanshi, N.; Bechtel, S.; Wiemann, S.; Baum, B.; Ridley, A.J. Identification and characterization of a set of conserved and new regulators of cytoskeletal organization, Cell morphology and migration. BMC Biol. 2011, 9, 54, doi:10.1186/1741-7007-9-54.
[46]
Prager-Khoutorsky, M.; Lichtenstein, A.; Krishnan, R.; Rajendran, K.; Mayo, A.; Kam, Z.; Geiger, B.; Bershadsky, A.D. Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing. Nat. Cell Biol. 2011, 13, 1457–1465, doi:10.1038/ncb2370.
[47]
Wurflinger, T.; Gamper, I.; Aach, I.; Sechi, A.S. Automated segmentation and tracking for large-scale analysis of focal adhesion dynamics. J. Microsc. 2011, 241, 37–53, doi:10.1111/j.1365-2818.2010.03404.x.
[48]
Stehbens, S.; Wittmann, T. Targeting and transport: How microtubules control focal adhesion dynamics. J. Cell Biol. 2012, 198, 481–489, doi:10.1083/jcb.201206050.
[49]
Stephens, D.J. Functional coupling of microtubules to membranes—Implications for membrane structure and dynamics. J. Cell Sci. 2012, 125, 2795–2804, doi:10.1242/jcs.097675.
[50]
Schuh, M. An actin-dependent mechanism for long-range vesicle transport. Nat. Cell Biol. 2011, 13, 1431–1436, doi:10.1038/ncb2353.
[51]
Simpson, J.C.; Joggerst, B.; Laketa, V.; Verissimo, F.; Cetin, C.; Erfle, H.; Bexiga, M.G.; Singan, V.R.; Hériché, J.K.; Neumann, B.; et al. Genome-wide rnai screening identifies human proteins with a regulatory function in the early secretory pathway. Nat. Cell Biol. 2012, 14, 764–774, doi:10.1038/ncb2510.
[52]
Chudakov, D.M.; Matz, M.V.; Lukyanov, S.; Lukyanov, K.A. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev. 2010, 90, 1103–1163, doi:10.1152/physrev.00038.2009.
[53]
Burkel, B.M.; von Dassow, G.; Bement, W.M. Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin. Cell Motil. Cytoskel. 2007, 64, 822–832, doi:10.1002/cm.20226.
[54]
Smith, A.W.; Smoligovets, A.A.; Groves, J.T. Paterned two-photon photoactivation illuminates spatial reorganization in live cells. J. Phys. Chem. A 2011, 115, 3867–3875, doi:10.1021/jp108295s.
[55]
Riedl, J.; Crevenna, A.H.; Kessenbrock, K.; Yu, J.H.; Neukirchen, D.; Bista, M.; Bradke, F.; Jenne, D.; Holak, T.A.; Werb, Z.; et al. Lifeact: A versatile marker to visualize F-actin. Nat. Methods 2008, 5, 605–607, doi:10.1038/nmeth.1220.