Quantitative fluorescent speckle microscopy: where it came from and where it is going

Fluorescent speckle microscopy (FSM) is a technology for analysing the dynamics of macromolecular assemblies. Originally, the effect of random speckle formation was discovered with microtubules. Since then, the method has been expanded to other proteins of the cytoskeleton such as f‐actin and microtubule binding proteins. Newly developed, specialized software for analysing speckle movement and photometric fluctuation in the context of polymer transport and turnover has turned FSM into a powerful method for the study of cytoskeletal dynamics in cell migration, division, morphogenesis and neuronal path finding. In all these settings, FSM serves as the quantitative readout to link molecular and genetic interventions to complete maps of the cytoskeleton dynamics and thus can be used for the systematic deciphering of molecular regulation of the cytoskeleton. Fully automated FSM assays can also be applied to live‐cell screens for toxins, chemicals, drugs and genes that affect cytoskeletal dynamics. We envision that FSM has the potential to become a core tool in automated, cell‐based molecular diagnostics in cases where variations in cytoskeletal dynamics are a sensitive signal for the state of a disease, or the activity of a molecular perturbant. In this paper, we review the origins of FSM, discuss these most recent technical developments and give a glimpse to future directions and potentials of FSM. It is written as a complement to the recent review (Waterman‐Storer & Danuser, 2002, Curr. Biol., 12, R633–R640), in which we emphasized the use of FSM in cell biological applications. Here, we focus on the technical aspects of making FSM a quantitative method.

[1]  J. Small.  Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks , 1981, The Journal of cell biology.

[2]  Y. Wang,et al.  Fluorescent analog cytochemistry of contractile proteins. , 1982, Methods in cell biology.

[3]  D. Taylor,et al.  Chapter 1 luorescent Analog Cytochemistry of Contractile Proteins , 1982 .

[4]  Y. Wang,et al.  Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling , 1985, The Journal of cell biology.

[5]  Kenneth R. Spring,et al.  Video Microscopy: The Fundamentals , 1986 .

[6]  T D Pollard,et al.  Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments , 1986, The Journal of cell biology.

[7]  E. Salmon,et al.  Analysis of the treadmilling model during metaphase of mitosis using fluorescence redistribution after photobleaching , 1986, The Journal of cell biology.

[8]  D. E. Wolf,et al.  Designing, building, and using a fluorescence recovery after photobleaching instrument. , 1989, Methods in cell biology.

[9]  T. Mitchison,et al.  Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence , 1989, The Journal of cell biology.

[10]  Julie A. Theriot,et al.  Actin microfilament dynamics in locomoting cells , 1991, Nature.

[11]  C. Waterman-Storer,et al.  Dynamics of organelles in the mitotic spindles of living cells: membrane and microtubule interactions. , 1993, Cell motility and the cytoskeleton.

[12]  C. Lumsden,et al.  Cellular automaton model of the actin cytoskeleton. , 1993, Cell motility and the cytoskeleton.

[13]  A. Mikhailov,et al.  Centripetal transport of microtubules in motile cells. , 1995, Cell motility and the cytoskeleton.

[14]  D. Prasher,et al.  Using GFP to see the light. , 1995, Trends in genetics : TIG.

[15]  Gaudenz Danuser,et al.  Observing deformations of 20 nm with a low-numerical-aperture light microscope , 1996, Other Conferences.

[16]  Gaudenz Danuser.  Observing Deformations of 20 Nanometer with a Low Numerical Aperture Light Microscope , 1996 .

[17]  E. Wachman,et al.  AOTF microscope for imaging with increased speed and spectral versatility. , 1997, Biophysical journal.

[18]  Gary G. Borisy,et al.  Analysis of the Actin–Myosin II System in Fish Epidermal Keratocytes: Mechanism of Cell Body Translocation , 1997, The Journal of cell biology.

[19]  E. Salmon,et al.  Actomyosin-based Retrograde Flow of Microtubules in the Lamella of Migrating Epithelial Cells Influences Microtubule Dynamic Instability and Turnover and Is Associated with Microtubule Breakage and Treadmilling , 1997, The Journal of cell biology.

[20]  T. Mitchison,et al.  Microtubule polymerization dynamics. , 1997, Annual review of cell and developmental biology.

[21]  A. Desai,et al.  Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells , 1998, Current Biology.

[22]  M. Oshiro.  Cooled CCD versus intensified cameras for low-light video--applications and relative advantages. , 1998, Methods in cell biology.

[23]  E. Salmon,et al.  How microtubules get fluorescent speckles. , 1998, Biophysical Journal.

[24]  A K Harris.  A dozen questions about how tissue cells crawl. , 1999, Biochemical Society symposium.

[25]  E. Salmon,et al.  Fluorescent speckle microscopy of microtubules: how low can you go? , 1999, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[26]  P. Hollenbeck,et al.  Axonal microtubules stay put , 1999, Nature Cell Biology.

[27]  A. Desai,et al.  Fluorescent speckle microscopy of spindle microtubule assembly and motility in living cells. , 1999, Methods in cell biology.

[28]  T. Svitkina,et al.  Speckle microscopic evaluation of microtubule transport in growing nerve processes , 1999, Nature Cell Biology.

[29]  Gary G. Borisy,et al.  Self-polarization and directional motility of cytoplasm , 1999, Current Biology.

[30]  F. Perez,et al.  CLIP-170 Highlights Growing Microtubule Ends In Vivo , 1999, Cell.

[31]  E. Salmon,et al.  The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae , 1999, Nature Cell Biology.

[32]  K. Kalil,et al.  Reorganization and Movement of Microtubules in Axonal Growth Cones and Developing Interstitial Branches , 1999, The Journal of Neuroscience.

[33]  E. Salmon,et al.  Microtubule Dynamics from Mating through the First Zygotic Division in the Budding Yeast Saccharomyces cerevisiae , 1999, The Journal of cell biology.

[34]  E D Salmon,et al.  Feedback interactions between cell-cell adherens junctions and cytoskeletal dynamics in newt lung epithelial cells. , 2000, Molecular biology of the cell.

[35]  G Danuser,et al.  Tracking differential interference contrast diffraction line images with nanometre sensitivity , 2000, Journal of microscopy.

[36]  Targeting regulatory factors to intranuclear replication sites. , 2000, Critical reviews in eukaryotic gene expression.

[37]  E. Salmon,et al.  Microtubules Remodel Actomyosin Networks in Xenopus Egg Extracts via Two Mechanisms of F-Actin Transport , 2000, The Journal of cell biology.

[38]  T D Pollard,et al.  Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. , 2000, Annual review of biophysics and biomolecular structure.

[39]  D. Odde,et al.  Rapid dynamics of the microtubule binding of ensconsin in vivo. , 2001, Journal of cell science.

[40]  A. Periasamy,et al.  Fluorescence resonance energy transfer microscopy: a mini review. , 2001, Journal of biomedical optics.

[41]  Benjamin Geiger,et al.  TRANSMEMBRANE EXTRACELLULAR MATRIX – CYTOSKELETON CROSSTALK , 2001 .

[42]  K. Kalil,et al.  Axon Branching Requires Interactions between Dynamic Microtubules and Actin Filaments , 2001, The Journal of Neuroscience.

[43]  W. Sossin,et al.  Protein Kinase C Activation Promotes Microtubule Advance in Neuronal Growth Cones by Increasing Average Microtubule Growth Lifetimes , 2001, The Journal of cell biology.

[44]  V. Malikov,et al.  Self-organization of a radial microtubule array by dynein-dependent nucleation of microtubules , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[45]  L. Gerace,et al.  Nuclear pore complexes: dynamics in unexpected places , 2001, The Journal of cell biology.

[46]  T D Pollard,et al.  Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[47]  V. Doye,et al.  A Mechanism for Nuclear Positioning in Fission Yeast Based on Microtubule Pushing , 2001, The Journal of cell biology.

[48]  D. Toomre,et al.  Lighting up the cell surface with evanescent wave microscopy. , 2001, Trends in cell biology.

[49]  Timothy J. Mitchison,et al.  Eg5 is static in bipolar spindles relative to tubulin , 2001, The Journal of cell biology.

[50]  Torsten Wittmann,et al.  The spindle: a dynamic assembly of microtubules and motors , 2001, Nature Cell Biology.

[51]  Michael P. Lisanti,et al.  Emerging Themes in Lipid Rafts and Caveolae , 2001, Cell.

[52]  Kenneth M. Yamada,et al.  Transmembrane crosstalk between the extracellular matrix and the cytoskeleton , 2001, Nature Reviews Molecular Cell Biology.

[53]  E D Salmon,et al.  Microtubule treadmilling in vitro investigated by fluorescence speckle and confocal microscopy. , 2001, Biophysical journal.

[54]  I. Vernos,et al.  The Mitotic Spindle: A Self-Made Machine , 2001, Science.

[55]  J. M. McDonnell,et al.  Surface plasmon resonance: towards an understanding of the mechanisms of biological molecular recognition. , 2001, Current opinion in chemical biology.

[56]  J. Monypenny,et al.  Fluorescence localization after photobleaching (FLAP): a new method for studying protein dynamics in living cells. , 2002, Journal of microscopy.

[57]  C. Waterman-Storer,et al.  Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells , 2002, Current protocols in cell biology.

[58]  K. Oegema,et al.  Poleward Microtubule Flux Is a Major Component of Spindle Dynamics and Anaphase A in Mitotic Drosophila Embryos , 2002, Current Biology.

[59]  J. Scholey,et al.  Microtubule flux and sliding in mitotic spindles of Drosophila embryos. , 2002, Molecular biology of the cell.

[60]  S. Gasser,et al.  Visualizing Chromatin Dynamics in Interphase Nuclei , 2002, Science.

[61]  C. Waterman-Storer,et al.  Converging Populations of F-Actin Promote Breakage of Associated Microtubules to Spatially Regulate Microtubule Turnover in Migrating Cells , 2002, Current Biology.

[62]  Timothy J Mitchison,et al.  Single-Molecule Speckle Analysis of Actin Filament Turnover in Lamellipodia , 2002, Science.

[63]  C. Cohan,et al.  Focal loss of actin bundles causes microtubule redistribution and growth cone turning , 2002, The Journal of cell biology.

[64]  Robert Sedgewick,et al.  Algorithms in C : Part 5 : Graph Algo-rithms , 2002 .

[65]  D. Schafer,et al.  Coupling actin dynamics and membrane dynamics during endocytosis. , 2002, Current opinion in cell biology.

[66]  G. Danuser,et al.  New Directions for Fluorescent Speckle Microscopy , 2002, Current Biology.

[67]  A. W. Schaefer,et al.  Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones , 2002, The Journal of cell biology.

[68]  C. Waterman-Storer,et al.  Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells , 2002, The Journal of cell biology.

[69]  James Monypenny,et al.  Rapid Actin Transport During Cell Protrusion , 2003, Science.

[70]  P. Vallotton,et al.  Recovery, visualization, and analysis of actin and tubulin polymer flow in live cells: a fluorescent speckle microscopy study. , 2003, Biophysical journal.

[71]  P. Vallotton,et al.  Computational analysis of F-actin turnover in cortical actin meshworks using fluorescent speckle microscopy. , 2003, Biophysical journal.

[72]  D. Axelrod.  Total Internal Reflection Fluorescence Microscopy in Cell Biology , 2001, Traffic.

[73]  Torsten Wittmann,et al.  A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells. , 2003, Methods.

[74]  Timothy J. Mitchison,et al.  Direct observation of microtubule dynamics at kinetochores in Xenopus extract spindles , 2003, The Journal of cell biology.