Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells

Clare Waterman‐Storer1

1 The Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.10
DOI:  10.1002/0471143030.cb0410s13
Online Posting Date:  February, 2002
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Abstract

Fluorescent speckle microscopy (FSM), a combination of conventional wide‐field fluorescent light microscopy and digital imaging with a low‐noise, charge‐coupled device (CCD) camera, has been developed to allow visualization of assembly/disassembly dynamics, movement, and turnover of macromolecule assemblies in vivo and in vitro. FSM uses a low level of fluorescent subunits to avoid high background. This produces an image of speckled molecules that co‐assemble with endogenous molecules and are followed to characterize dynamic events in living cells.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Designing a Microscope System for Time‐Lapse Digital FSM
  • Basic Protocol 2: Time‐Lapse FSM Imaging of the Cytoskeleton in Living Cells
  • Basic Protocol 3: Qualitative and Quantitative Analysis of Time‐Lapse FSM Image Series
  • Support Protocol 1: Preparation of Flourescently Labeled Tubulin for FSM
  • Support Protocol 2: Preparation of Fluorescently Labeled Actin for FSM
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Designing a Microscope System for Time‐Lapse Digital FSM

  Materials
  • Upright or inverted epi‐fluorescent microscope and optics including:
  •  Epi‐illuminator
  •  High‐magnification objective lens (e.g., 60×, 63×, or 100×)
  •  Excitation filter, emission filter, and dichromatic mirror
  •  Electronically controlled shutter
  •  Cooled CCD camera
  •  Computer, digital image acquisition board, and software for control of shutter and image acquisition
  • Microscope stand

Basic Protocol 2: Time‐Lapse FSM Imaging of the Cytoskeleton in Living Cells

  Materials
  • ∼0.5 mg/ml labeled cytoskeletal protein (tubulin or actin, see protocol 4 or protocol 5, respectively)
  • Cultured tissue cells grown on 22 × 22–mm glass coverslips in small plastic petri dishes
  • Valap (unit 13.1)
  • Buffered filming medium (see recipe)
  • Oxyrase EC (Oxyrase Inc.)
  • Microultracentrifuge (Optima TL or TLX, Beckman Instruments; or Discovery M120, Sorvall)
  • 0.6‐ml microcentrifuge tubes
  • Swinging bucket rotor for microultracentrifuge with adapters for holding 0.6‐ml microcentrifuge tubes (for the Beckman TLS‐55 rotor, the standard thick‐walled 1.4‐ml polycarbonate tubes are used; and for the Sorvall, custom adapters are needed)
  • Microinjection needles (see recipe)
  • Microloader pipet tips (Eppendorf or equivalent) or narrow‐gauge syringe (narrow enough to fit in the bore of the microinjection needle, Hamilton Company)
  • Single‐cell microinjection system capable of controlled backpressure of 0.1 to 20 psi and with a precision micromanipulator for injection of single cells, mounted on an inverted microscope equipped with a long‐working‐distance phase‐contrast condenser and a 40× dry phase‐contrast objective lens with a working distance long enough to focus through the plastic petri dish and coverslip.
  • Microscope stage incubator (optional)
  • 1 × 3–in. glass microscope slides
  • Scotch double‐stick tape (3M)
  • Cotton swabs
  • FSM imaging system (see protocol 1)

Basic Protocol 3: Qualitative and Quantitative Analysis of Time‐Lapse FSM Image Series

  Materials
  • Stage micrometer (Fisher)
  • Image analysis software

Support Protocol 1: Preparation of Flourescently Labeled Tubulin for FSM

  Materials
  • 10‐ml aliquots of phosphocellulose‐purified tubulin in column buffer (CB; totaling 40 to 60 mg of tubulin; unit 13.1)
  • CB/BRB‐80 conversion buffer (see recipe)
  • 100 mM GTP (see recipe)
  • Glycerol
  • Labeling buffer (see recipe)
  • High‐pH cushion (see recipe)
  • Quench (see recipe)
  • Low‐pH cushion (see recipe)
  • Succinimidyl‐ester derivative of fluorescent probe of choice
  • Anhydrous DMSO
  • Injection buffer (IB; see recipe)
  • 1× BRB‐80 (see recipe for 10×)
  • 1 M MgCl 2 ( appendix 2A)
  • 37°C water bath
  • 100‐ and 10‐ml graduated cylinder
  • Parafilm
  • Ultracentrifuge (with a Beckman 70.1 Ti rotor; or equivalent)
  • 13.5‐ml ultracentrifuge screw‐cap polycarbonate tubes
  • Manual pipet‐pump type pipettor (VWR or equivalent)
  • 1.6‐ml microcentrifuge tubes
  • Microultracentrifuge (Beckman Optima TLX with TLA‐100.4 rotor or Sorvall RC M120 GX with S100AT4 rotor)
  • 5.1‐ml microultracentrifuge polycarbonate tubes
  • 7‐ml dounce homogenizer with “B” pestle
  • Spectrophotometer with a small‐volume quartz cuvette
  • 0.6‐ml UV‐impermeant microcentrifuge tubes

Support Protocol 2: Preparation of Fluorescently Labeled Actin for FSM

  Materials
  • Muscle (rabbit psoas or chicken breast) acetone powder
  • 1 M KCl ( appendix 2A)
  • 1 M MgCl 2 ( appendix 2A)
  • 100 mM ATP (see recipe)
  • Succinimidyl ester derivative of the fluorescent probe of choice, pre‐equilibrated to room temperature
  • Anhydrous DMSO
  • 1 M sodium bicarbonate (see recipe), freshly prepared
  • 1 M NH 4Cl (see recipe)
  • G‐buffer, 4°C (see recipe; ATP should be added just prior to use)
  • 50‐ml beaker
  • Refrigerated ultracentrifuge with rotor (e.g., Beckman 70.1 Ti rotor)
  • 13.5‐ml screw‐cap ultracentrifuge tubes
  • 50‐ml and 1‐liter graduated cylinder
  • Spectrophotometer and quartz cuvette
  • 1.6‐ml microcentrifuge tubes
  • Aluminum foil
  • Refrigerated centrifuge with rotor (e.g., Sorvall SS‐34 rotor)
  • 50‐ml open‐top polycarbonate centrifuge tubes (or equivalent)
  • 7‐ml dounce homogenizer with type “B” pestle
  • Pretreated glycerol‐free cellulose dialysis tubing (see recipe), ∼2 cm diameter, 20,000 MWCO (i.e., Spectra/Por 6, Fisher)
  • Dialysis tubing clips
  • 0.6‐ml UV‐impermeant microcentrifuge tubes
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Figures

Videos

Literature Cited

Literature Cited
   Desai, A. and Mitchison, T.J. 1997. Microtubule polymerization dynamics. Ann. Rev. Cell Dev. Biol. 13:83‐117.
   Faire, K., Waterman‐Storer, C.M., Gruber, D., Masson, D., Salmon, E.D., and Bulinski, J.C. 1999. Dynamic behavior of GFP‐labeled E‐MAP‐115 (ensconsin) in cultured cells. J. Cell Sci. 112:4243‐4255.
   Hyman, A., Drechsel, D., Kellogg, D., Salser, S., Sawin, K., Steffen, P., Wordeman, L., and Mitchison, T. 1991. Preparation of modified tubulins. Methods Enzymol. 196:478‐485.
   Inoué, S. and Spring, K. 1997. Video Microscopy: The Fundamentals (2nd Ed.) Plenum, New York.
   Mitchison, T.J., Sawin, K.E., Theriot, J.A., Gee, K., and Mallavarapu, A. 1998. Caged fluorescent probes. Methods in Enzymol. 291:63‐78.
   Pardee, J.D. and Spudich, J.A. 1982. Purification of muscle actin. Methods Enzymol. 85:164‐181.
   Prasher, D.C. 1995. Using GFP to see the light. Trends Genet. 11:320‐323.
   Turnacioglu, K.K., Sanger, J.W., and Sanger, J.M. 1998. Sites of monomeric actin incorporation in living PrK2 and REF‐52 cells. Cell Motil. Cytoskel. 40:59‐70.
   Wang, Y.L. 1989. Fluorescent analog cytochemistry: Tracing functional protein components in living cells. Methods Cell Biol. 29:1‐12.
   Wang, Y.L. and Taylor, D.L. 1980. Preparation and characterization of a new molecular cytochemical probe: 5‐Iodoacetamidofluorescein‐labeled actin. J. Histochem. Cytochem. 28:1198‐206.
   Waterman‐Storer, C.M. and Salmon, E.D. 1997. 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. J. Cell Biol. 139:417‐434.
   Waterman‐Storer, C.M. and Salmon, E.D. 1998. How microtubules get fluorescent speckles. Biophys. J. 75:2059‐2069.
   Waterman‐Storer, C.M. and Salmon, E.D. 1999. Fluorescent speckle microscopy of microtubules: How low can you go. FASEB J. 13:S225‐S230.
   Waterman‐Storer, C.M., Sanger, J.W., and Sanger, J.M. 1993. Dynamics of organelles in the mitotic spindles of living cells: Membrane and microtubule interactions. Cell Motil. Cytoskel. 26:19‐39.
   Waterman‐Storer, C.M., Desai, A., Bulinski, J.C., and Salmon, E.D. 1998. Fluorescent speckle microscopy: Visualizing the movement, assembly, and turnover of macromolecular assemblies in living cells. Curr. Biol. 8:1227‐1230.
   Waterman‐Storer, C.M., Desai, A., and Salmon, E.D. 1999. Fluorescent speckle microscopy of spindle microtubule assembly and motility in living cells. Methods Cell Biol. 61:155‐173.
   Wolf, D.E. 1989. Designing, building, and using a fluorescence recovery after photobleaching instrument. Methods Cell Biol. 30:271‐306.
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