Quantitative Fluorescent Speckle Microscopy (QFSM) to Measure Actin Dynamics

Michelle C. Mendoza1, Sebastien Besson1, Gaudenz Danuser1

1 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 2.18
DOI:  10.1002/0471142956.cy0218s62
Online Posting Date:  October, 2012
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Quantitative fluorescent speckle microscopy (QFSM) is a live‐cell imaging method to analyze the dynamics of macromolecular assemblies with high spatial and temporal resolution. Its greatest successes were in the analysis of actin filament and adhesion dynamics in the context of cell migration and microtubule dynamics in interphase and the meiotic/mitotic spindle. Here, focus is on the former application to illustrate the procedures of FSM imaging and the computational image processing that extracts quantitative information from these experiments. QFSM is advantageous over other methods because it measures the movement and turnover kinetics of the actin filament (F‐actin) network in living cells across the entire field of view. Experiments begin with the microinjection of fluorophore‐labeled actin into cells, which generate a low ratio of fluorescently labeled to endogenously unlabeled actin monomers. Spinning disk confocal or wide‐field imaging then visualizes fluorophore clusters (two to eight actin monomers) within the assembled F‐actin network as speckles. QFSM software identifies and computationally tracks and utilizes the location, appearance, and disappearance of speckles to derive network flows and maps of the rate of filament assembly and disassembly. Curr. Protoc. Cytom. 62:2.18.1‐2.18.26. © 2012 by John Wiley & Sons, Inc.

Keywords: live‐cell imaging; microinjection; image processing; actin; speckles

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Table of Contents

  • Introduction
  • Basic Protocol 1: Microinject Fluorescent Actin into Cell
  • Basic Protocol 2: Confocal Imaging of Actin Dynamics
  • Basic Protocol 3: Quantify Actin Dynamics with QFSM Software
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Microinject Fluorescent Actin into Cell

  • 1 N hydrochloric acid (HCl; store for 1 year at room temperature)
  • Phosphate buffered saline (PBS, see recipe), prewarm to 37°C just before use
  • 70% (v/v) ethanol in water (store for 1 year at room temperature)
  • PtK1 cells (ATCC, http://www.atcc.org)
  • PtK1 growth medium (see recipe), prewarm to 37°C just before use
  • 100 mM adenosine tri‐phosphate dissolved in water (ATP; store for 1 year at −20°C)
  • 100 mM dithiothreitol dissolved in water (DTT; store for 1 year at −20°C)
  • G‐buffer stock (see recipe)
  • Alexa568‐conjugated actin (Molecular Probes, http://www.invitrogen.com/site/us/en/home/brands/Molecular‐Probes.html)
  • Liquid nitrogen
  • Double‐distilled water (filter through a 0.2‐µm filter to remove particulates and store at 4°C)
  • 35‐mm glass‐bottom culture dishes (MatTek, http://www.glass‐bottom‐dishes.com)
  • 37°C, 5% CO 2 incubator
  • 0.2‐ml and 1.5‐ml opaque microcentrifuge tubes
  • Ultramicrocentrifuge tubes
  • Refrigerated ultramicrocentrifuge (75,000 × g)
  • P‐1000 micropipet puller with a 2.5 × 4.5‐mm box filament (Sutter Instrument Company, http://www.sutter.com)
  • 10‐cm length borosilicate glass capillaries with filaments (1.0‐mm o.d., 0.78‐mm i.d.; Sutter Instrument Company)
  • Microsyringe or 2‐ to 10‐µl pipet
  • Microloader pipet tips
  • Inverted fluorescent microscope with a microinjector systems (phase ring, 40× phase contrast objective, fluorescence light source, 568‐compatible excitation and emission filters, transjector capable of controlled backpressure, micromanipulator for injection of single cells)

Basic Protocol 2: Confocal Imaging of Actin Dynamics

  • Cells microinjected with fluorescent actin (see protocol 1)
  • L‐15 imaging medium (see recipe)
  • EC‐oxyrase or oxyFluor (store in 100‐µl aliquots for 1 year at −80°C; Oxyrase, http://www.oxyrase.com)
  • Mineral oil
  • Ethanol
  • Glass cleaner
  • Immersion oil for objective lens
  • Inverted spinning disc confocal microscope (transmitted light source, fluorescence lamp, appropriate excitation laser, appropriate excitation and emission filters, motorized shutters, 0.4‐NA, 100× oil‐immersion Plan‐Apochromatic objective, cooled CCD camera with pixels <7 µm, vibration table, imaging software)
  • Lens paper
  • Tissues

Basic Protocol 3: Quantify Actin Dynamics with QFSM Software

  • Computer with 64‐bit processing capabilities and 4‐GB RAM
  • Matlab (Mathworks, version R2010b and above) with the following toolboxes: Image Processing, Curve Fitting, and Statistics
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Literature Cited

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   Delorme, V., Machacek, M., DerMardirossian, C., Anderson, K.L., Wittmann, T., Hanein, D., Waterman‐Storer, C., Danuser, G., and Bokoch, G.M. 2007. Cofilin activity downstream of Pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks. Devel. Cell 13:646‐662.
   Gupton, S.L. and Waterman‐Storer, C.M. 2006. Spatiotemporal feedback between actomyosin and focal‐adhesion systems optimizes rapid cell migration. Cell 125:1361‐1374.
   Hebert, B.S., Costantino, S., and Wiseman, P.W. 2005. Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells. Biophys. J. 88:3601‐3614.
   Hu, K., Ji, L., Applegate, K.T., Danuser, G., and Waterman‐Storer, C.M. 2007. Differential transmission of actin motion within focal adhesions. Science 315:111‐115.
   Jaqaman, K., Loerke, D., Mettlen, M., Kuwata, H., Grinstein, S., Schmid, S.L., and Danuser, G. 2008. Robust single‐particle tracking in live‐cell time‐lapse sequences. Nat. Methods 5:695‐702.
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   Lim, J. and Danuser, G. 2009. Live cell imaging of F‐actin dynamics via fluorescent speckle microscopy (FSM). J. Vis. Exp. (30) e1325.
   Lim, J.I., Sabouri‐Ghomi, M., Machacek, M., Waterman, C.M., and Danuser, G. 2010. Protrusion and actin assembly are coupled to the organization of lamellar contractile structures. Exp. Cell Res. 316:2027‐2041.
   Otsu, N. 1979. Threshold selection method from gray‐level histograms. IEEE Trans. Syst. Man Cybernet. 9:62‐66.
   Ponti, A., Vallotton, P., Salmon, W.C., Waterman‐Storer, C.M., and Danuser, G. 2003. Computational analysis of F‐actin turnover in cortical actin meshworks using fluorescent speckle microscopy. Biophys. J. 84:3336‐3352.
   Ponti, A., Machacek, M., Gupton, S.L., Waterman‐Storer, C.M., and Danuser, G. 2004. Two distinct actin networks drive the protrusion of migrating cells. Science 305:1782‐1786.
   Ponti, A., Matov, A., Adams, M., Gupton, S., Waterman‐Storer, C.M., and Danuser, G. 2005. Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithelial cells analyzed by quantitative fluorescent speckle microscopy. Biophys. J. 89:3456‐3469.
   Rosin, P.L. 2001. Unimodal thresholding. Pattern Recogn. 34:2083‐2096.
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   Thomann, D., Rines, D.R., Sorger, P.K., and Danuser, G. 2002. Automatic fluorescent tag detection in 3D with super‐resolution: Application to the analysis of chromosome movement. J. Microsc. 208:49‐64.
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   Waterman‐Storer, C.M., Desai, A., Bulinski, J.C., and Salmon, E.D. 1998. Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells. Curr. Biol. 8:1227‐1230.
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Internet Resources
  Web site for downloading the most recent version of the QFSM analysis software.
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