Computational Analysis of F-Actin Turnover in Cortical ActinMeshworks Using Fluorescent Speckle MicroscopyINTRODUCTIONMATERIALS AND METHODSALGORITHMRESULTSDISCUSSIONREFERENCES3336 Biophysical Journal Volume 84 May 2003 3336–3352Computational Analysis of F-Actin Turnover in Cortical ActinMeshworks Using Fluorescent Speckle MicroscopyA. Ponti,* P. Vallotton,* W. C. Salmon,yC. M. Waterman-Storer,yand G. Danuser**BioMicroMetrics Group, Laboratory for Biomechanics, ETH Zurich, 8952 Schlieren, Switzerland; andyDepartment of Cell Biologyand Institute for Childhood and Negl ected Diseases, The Scripps Research Institute, La Jolla, California 92037 USAABSTRACT Fluorescent speckle microscopy (FSM) is a new imaging technique with the potential for simultaneousvisualization of translocation and dynamic turnover of polymer structures. However, the use of FSM has been limited by the lackof specialized software for analysis of the positional and photometric fluctuations of hundreds of thousand speckles in an FSMtime-lapse series, and for translating this data into biologically relevant information. In this paper we present a first version ofa software for automated analysis of FSM movies. We focus on mapping the assembly and disassembly kinetics of a polymermeshwork. As a model system we have employed cortical F-actin meshworks in live newt lung epithelial cells. We lay out thealgorithm in detail and present results of our analysis. The high spatial and temporal resolution of our maps reveals a kineticcycling of F-actin, where phases of polymerization alternate with depolymerization in a spatially coordinated fashion. The cyclerates change when treating cells with a low dose of the drug latrunculin A. This shows the potential of this technique for futurequantitative screening of drugs affecting the actin cytoskeleton. Various control experiments demonstrate that the algorithm isrobust with respect to intensity variations due to noise and photobleaching and that effects of focus plane drifts can beeliminated by manual refocusing during image acquisition.INTRODUCTIONFluorescent speckle microscopy (FSM) is a recently de-veloped method to analyze the movement, assembly, anddisassembly dynamics of macr omolecular structures in vivoand in vitro (Waterman-Storer and Danuser, 2002). Origi-nally, it has been applied to the analysis of in vivo microtubulemovements in mitotic spindles and actin retrograde flow inmigrating cells (Waterman-Storer et al., 1998), and has nowbecome the tool of choice for investig ating many aspects ofcytoskeleton dynamics. Speckled images of such polymerstructures are obtained by microinjecting low amounts oflabeled monomers into cells. Random incorporation of fewlabeled and many endogenous unlabeled monomers intocellular structures yields a fluorophore distribution with highspatial density variation, which is, by light microscopic imag-ing, convolved with a diffraction-limited point spread func-tion. We define a speckle as a peak in the image signal,significantly brighter than its surroundings. Most of the spec-kle data hitherto published refer to multifluorophore specklesformed by 3–7 fluorophores clustered within a diffraction-limited region, and only recently Watanabe and Mitchison(2002) were able to acquire single-fluorophore speckles inactin structures in fibroblasts.Speckles act as local reporters throughout the polymerlattice. In time-lapse FSM, speckle appearance and disap-pearance are linked to polymerization and depolymerization,whereas speckle movem ent is associated with polymer trans-locations. Therefore, FSM allows simultaneous and localmeasurements of polymer turnover and movement in largeareas of the cell, with a time resolution equal to the frame rateof the movie. This offers a novel tool for studying nonsteady-state molecular processes in live cells.In this paper we apply FSM to the study of cortical F-actinmeshwork dynam ics in contac t-inhibited newt lung epithelialcells. Contact-inhibited cells form tight adherens junctionswith neighboring cells (Harris, 1999). In contrast to migratingcells, where F-actin meshworks and thus actin fluorescentspeckles undergo a coordinated motion known as ‘‘retrogradeflow’’ (Abercromb ie et al., 1972; Small, 1981; Wang, 1985;Forscher and Smith, 1988; Danuser and Oldenbourg, 2000;Salmon et al., 2002), in contacted cells cortical F-actinmeshworks remain spatially stationary. However, FSMmovies still show a strong photometric activity, indicatingthat F-actin is undergoing steady turnover (Waterman-Storeret al., 2000). We investigated how to extract quantitativeinformation of this turnover to shed new light on the mech-anisms of actin dynamics. Our long-term goal is to developa quantitative live-cell assay to study the effect of bioche mi-cal reagents, molecular, and genetic factors involved in the reg-ulation of actin dynamic processes.Speckles are a weak and inherently stoch astic image featurewith a low signal-to-noise ratio (SNR). For this reason, anymeasurement derived from FSM data must rely on a statisticalprocessing of large numbers of speckles. Typically, FSMmovies contain[500,000 speckles that fluctuate in intensity,move and disappear, while new speckles appear. Specklefluctuations are induced by stochastic molecular processesand their interpretation requires mathematical models of therelation between changes in speckle signal and the alterationof the polymer. Hence the demand for computational tools,where each speckle is automatically tracked in position andintensity and statistically processed according to the models.Submitted August 19, 2002, and accepted for publication December 9,2002.Address reprint requests to G. Danuser, Tel.: 141-1-633-6214; Fax: 141-1-633-1124; E-mail: [email protected].Ó 2003 by the Biophysical Society0006-3495/03/05/3336/17 $2.00In the core of this paper we propose a first version of analgorithm wor king along these lines and we demonstrate itsperformance on synthetic and experimental data. Materialsand Methods introduces the experimental protocols and asimulation package that enabled us to establish a forwardmodel of speckle formation dependent on several biochemicaland imaging parameters. Artifacts in the imaging and analysissteps are discussed in the concluding section along witha summary of the key features of our computational model.MATERIALS AND METHODSSoftware developmentAll programs for speckle analysis and graphical representation have