4332 Research Article

IntroductionCell migration is essential for life; it is required throughoutembryo development, and for tissue repair and immunity inboth the embryo and the adult. It also contributes to severalimportant diseases, including vascular disease, inflammatorydiseases and the spread of cancer.

To reach new territory, a cell must polarize, which meansthat it must form a cell front and rear. For clarity of terms, inthis work we define ‘initiation of cell polarization’ as the firstvisual sign of a break in cell symmetry, ‘elaboration’ as furtherelaboration of this first break, ‘fully polarized’ whenmorphologically both the cell front and cell rear have fullyformed, and ‘cell polarization’ as the entire process. The cellmigrates once it is fully polarized. Cell protrusion at the frontof the cell is roughly diametrically opposed to cell retractionat the rear, and cell migration occurs approximately along thisaxis in the direction of protrusion.

To polarize, there must be coordination between formationof the cell front and cell rear in both time and space. In somemigrating cell types, such as neutrophils and amoeba cells,pathways based on phosphoinositide 3-kinases and membersof Rho family proteins control formation of the cell front andrear (reviewed in Dormann and Weijer, 2006; Kimmel andFirtel, 2004; Niggli, 2003; Ridley et al., 2003). In these celltypes, feedback loops between pathways ensure that the cellfront and rear form coordinately (Charest and Firtel, 2006;

Schneider and Haugh, 2006; Van Keymeulen et al., 2006;Wong et al., 2006; Xu et al., 2003). However, in othermigrating cell types, such as fibroblasts, mathematical modelsdo not invoke feedback loops (Schneider and Haugh, 2005)and coordination is poorly understood in these cells.Furthermore, in all migrating cell types, the very early eventsin the cell that establish spatial and temporal differencesneeded in cellular processes for cell polarization are unclear.

Intuitively the cell front might be expected to form first inresponse to a localized source of chemoattractant. However, allmigrating cell types tested have the capacity to polarize in theabsence of an externally applied gradient of chemoattractant.This infers that a significant contribution to establishing the cellfront and rear must come from self-organization and self-amplification of important molecules within the cell (Bernsteinand Bamburg, 2004; Onsum et al., 2006; Verkhovsky et al.,1999b; Wong et al., 2006; Xu et al., 2003). In this regard, it isnot obvious whether the cell front or rear should be expected toform first, which has implications for overall mechanism. Insome types of already polarized cells, cell retraction precedescell protrusion (Chen, 1981; Dunn, 1980; Dunn and Zicha,1995; Small and Resch, 2005). However, whether this alsooccurs to polarize a cell has not yet been explicitly investigated.

In most cell types, correctly organized actin networks arerequired for morphological cell polarity as well as providing thedriving force needed to move the cell (reviewed by Ridley et

How formation of the front and rear of a cell arecoordinated during cell polarization in migrating cells isnot well understood. Time-lapse microscopy of liveprimary chick embryo heart fibroblasts expressing GFP-actin show that, prior to cell polarization, polymerizedactin in the cell body reorganizes to form oriented actin-filament bundles spanning the entire cell body. Within anaverage of 5 minutes of oriented actin bundles forming,localized cell-edge retraction initiates at either the side orat one end of the newly formed bundles and then elaboratesaround the nearest end of the bundles to form the cell rear,the first visual break in cell symmetry. Localized netprotrusion occurs at the opposing end of the bundles toform the cell front and lags formation of the rear of the cell.Consequently, cells acquire full polarity and start tomigrate in the direction of the long axis of the bundles, as

previously documented for already migrating cells. WhenADF/cofilin family protein activity or actin-filamentdisassembly is specifically blocked during cell polarization,reorganization of polymerized actin to form oriented actin-filament bundles in the cell body fails, and formation of thecell rear and front is inhibited. We conclude that formationof oriented actin-filament bundles in the cell body requiresADF/cofilin family proteins, and is an early event neededto coordinate the spatial location of the cell rear and frontduring fibroblast polarization.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/120/24/4332/DC1

Key words: ADF/cofilin, Actin bundles, Cell motility, Cellpolarization

Summary

ADF/cofilin family proteins control formation oforiented actin-filament bundles in the cell body totrigger fibroblast polarizationTayamika Mseka1, Jim R. Bamburg2 and Louise P. Cramer1,3,*1MRC-Laboratory Molecular Cell Biology, UCL, London, WC1E 6BT, UK2Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA3Department of Biology, UCL, London, WC1E 6BT, UK*Author for correspondence (e-mail: l.cramer@ucl.ac.uk)

Accepted 24 September 2007Journal of Cell Science 120, 4332-4344 Published by The Company of Biologists 2007doi:10.1242/jcs.017640

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al., 2003; Small and Resch, 2005; Wittmann and Waterman-Storer, 2001). Other cytoskeletal proteins, such as microtubules(depending on the cell type) and myosin II, can also play animportant role (reviewed by Ridley et al., 2003; Rodriguez etal., 2003; Small et al., 2002; Small and Kaverina, 2003;Wittmann and Waterman-Storer, 2001). Although there is someinformation in cells (Cassimeris and Zigmond, 1990; Kaverinaet al., 2000) and cytoplasmic fragments (Verkhovsky et al.,1999b), how reorganization of the actin cytoskeletoncontributes to early events needed to drive local cellulardifferences to form the front and back of the cell has not beeninvestigated widely experimentally. Particularly unclear is therole of actin within the cell body; previous studies have, for themost part, focused on actin in the leading cell edge. Althoughactin organization within the cell body has been studied in a fewmigrating cell types, such as keratocytes (Svitkina et al., 1997),fibroblasts (Cramer et al., 1997) and myoblasts (Swailes et al.,2004), in these studies the cells were already polarized andmoving, and thus cell polarization and associated actinreorganization in the cell body were not studied.

To address these issues, here we have studied cellpolarization in primary chick heart fibroblasts, a well-established migrating cell system with well-characterized cellpolarity (Abercrombie and Heaysman, 1966; Couchman andRees, 1979; Cramer, 1999b; Cramer et al., 1997). In alreadypolarized and moving fibroblasts, actin comprises an actin-richlamellipodium at the front of the cell, and oriented actin-filament bundles (graded polarity actin-filament bundles)spanning the cell body and oriented in the direction ofmigration (Cramer et al., 1997).

Determining regulation of actin organization is important inthe identification of actin mechanisms driving cell polarization.It is becoming clear that the ADF/cofilin (AC) family ofproteins, which catalyze actin-filament severing anddepolymerization in cells (reviewed by Moon and Drubin,1995; Sarmiere and Bamburg, 2003; Zigmond, 2004), isnecessary for cell migration (reviewed by Bamburg andWiggan, 2002). AC proteins are also known regulators of cellpolarity, both in yeast (Drees et al., 2001; Okada et al., 2006)and also in migrating cell types, both to sustain alreadyestablished polarity (Dawe et al., 2003) and to trigger cellpolarization (Helen R. Dawe. The role of ADF/cofilin familyproteins in the acquisition and maintenance of cell polarityduring fibroblast migration. PhD thesis, University CollegeLondon, 2003) (Ghosh et al., 2004; Mouneimne et al., 2006;Nishita et al., 2005). Despite the clear role of AC in controllingcell polarity, studies of AC in the context of actin organizationduring cell polarization are limited; this process is either not thefocus of the work (Ghosh et al., 2004; Mouneimne et al., 2006;Nishita et al., 2005) or the studies are preliminary (seePhDcit_addRef.doc). Furthermore, most studies focusing on thefunction of AC during motility have been limited to actin in theleading cell edge or lamellipodium (Chan et al., 2000; Dawe etal., 2003; Ghosh et al., 2004; Nishita et al., 2005; Zebda et al.,2000). By contrast, any role for AC in regulating actinorganization within the cell body of migrating cells during cellpolarization has yet to be explored.

ACs are regulated in cells by several mechanisms (reviewedby Moon and Drubin, 1995; Sarmiere and Bamburg, 2003;Zigmond, 2004). In chick fibroblasts, AC is at least regulated byphosphorylation on serine 3 by LIM kinase (Dawe et al., 2003),

which inactivates AC. Here, we employ mutant AC proteins,including an AC mutant that acts as a dominant negative, andconstitutively active LIM kinase 1 to explore the function of ACduring cell polarization in chick heart fibroblasts.

We found that, during cell polarization in chick fibroblasts,AC activity and actin-filament disassembly are required for theformation of oriented actin-filament bundles within the cell bodyof these cells. This is an early event that consequently triggerscoordinate formation of the cell front and rear around opposingends of the bundles. We conclude that formation of orientedactin-filament bundles within the cell body requires AC and isa required, early step in triggering fibroblast polarization.

ResultsCell-polarization assayWe developed three types of cell-polarization assays, all inprimary fibroblasts obtained from chick heart explants from 7-to 8-day-old embryos. These assays were: latrunculin A (LatA) washout of pre-treated cells growing out of explant tissue,plated for 24-36 hours; cell plating of individual, untreated,dissociated cells prepared directly from tissue explants; andspontaneous cell polarization of untreated cells growing out ofexplant tissue, plated for 24-36 hours. Because we obtainedsimilar data in each different assay, we think that the processwe describe here is a fundamental property of fibroblasts andthe mechanism we uncover important for cell polarization. Ineach assay, cells spontaneously polarized in cell culturemedium, indicating that polarization was driven by self-organization of the actin cytoskeleton with no externallyapplied chemoattractant gradient. The fact that thecytoskeleton can self-organize to propagate cell polarizationhas been recognized for some time and might be a basalproperty of migrating cells (Verkhovsky et al., 1999b).

Because the Lat-A-washout cell-polarization assay was themost robust and experimentally manipulable, we routinelyused this method (presented throughout the paper). Motilityand polarity in chick fibroblasts require an organized actincytoskeleton (Cramer et al., 1997). Pre-treating live cells withLat A, which sequesters actin monomers, causing filamentdisassembly (Ayscough et al., 1997), abolishes cell polarity.Upon subsequent washout of Lat A, we found that cellsspontaneously repolarized.

We first tested empirically and found that, by 1-hour pre-treatment with 1-5 �M Lat A (depending on different batches ofLat A from the manufacturer, see Materials and Methods),normal actin organization and cell-adhesion patterns wereabolished (supplementary material Fig. S1). Thus, aftersubsequent washout, cell repolarization occurs by formation ofnewly polymerized and newly organized actin structures ratherthan from any pre-existing organized structures. Consistent withthis, after Lat A washout, cells repolarized and had a cell frontand back apparently unrelated to their original position prior toLat A treatment (e.g. for the individual cell in Fig. 1A, compare–1:18:05 and 1:06:30). This is also represented as a schematicfor this cell (Fig. 1B) and for the entire cell population (Fig. 1C).

In the presence of Lat A, cells appeared rounded whileremaining adherent to the Matrigel substratum. At around 1-hour total pre-treatment, Lat A was thoroughly and rapidlywashed out (see Materials and Methods). By 1 hour afterwashout, approximately 70% of cells were fully repolarized(quantified in Fig. 1D) – the expected proportion that are

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polarized in a given population of untreated chick embryo heartfibroblasts – and showed the expected cell shapes (Cramer,1999b; Cramer et al., 2002; Cramer et al., 1997; Dawe et al.,2003). As soon as cells fully polarize, they begin to migrate atan average speed of 1 �m per minute, the same speed as inuntreated cells (Cramer, 1999b; Cramer et al., 1997; Dawe etal., 2003). Actin organization (see below) in migrating cells,once repolarized, appeared no different to control untreatedmigrating cells. Together, these data indicate that the Lat Acell-polarization assay faithfully reproduces the expected cellbehaviour of these cells.

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Formation of the cell front and rear during fibroblastpolarizationTo study cell polarization, we analyzed fibroblasts between 0-1 hours after washing out of Lat A, and then typically beyondthis to continue to monitor cell migration. Over this time, usingphase-contrast time-lapse microscopy of live cells, weobserved a reproducible sequence of shape-change events asfibroblasts repolarized (example of typical individual cell inFig. 1A from 0:00:00 minutes).

Immediately after washing off Lat A, cells re-spread to forma discoid or ovoid shape with the nuclear mass roughly in the

Fig. 1. Cell polarization in chick embryo fibroblasts. (A) Still images from a phase-contrast movie of fibroblast polarization. Time ishours:minutes:seconds. Lat A was added at –1:07:03 for just over 1 hour: within minutes in the drug, the cell rounded up (–1:00:44 to 0:00:00)and remains rounded and retracted in Lat A (0:00:00). Lat A was washed out at 0:00:00. The cell re-spreads (0:00:00 to 0:15:00), then a smallstable localized retraction of the cell edge occurs (by 0:35:00, black arrow). The retracted area elaborates to form the cell rear (0:58:30, blackarrows) then net protrusion and cell migration occur in the direction of the white arrow (1:06:30; net protrusion occurring for 6 minutes by thistime – see E). Further migration then occurs (1:27:02). Red and green dots (in 0:15:00 to 1:27:02) mark the position of the fully re-spread cell(at 0:15:00) at the presumptive location of where the rear initiates and front forms, respectively, determined from the movie. (B) Trace of thecell in A showing direction of polarity (arrow) before and after cell repolarization. (C) Quantification of the cell population showing directionof polarity for individual cells before and after repolarization (n=15 live-cell movies). (D) Quantification of cell polarization with time afterwashout (% total of 15 live cells). (E) Line graph showing cell retraction to form the cell rear and protrusion at the cell front of the live cell inA measured from the red (see above) and green (see above) dot, respectively, during cell polarization (the process of re-spreading is not shownon the graph). Note that net protrusion lags net retraction. (F) Histogram of time (to nearest minute) that individual cells in the populationinitiate cell polarization, as identified by initial, stable, localized cell-edge retraction (n=15 live-cell movies). (G) Delay in individual cellsbetween initial cell-edge retraction and net protrusion at the cell front (n=15 live-cell movies). Bar, 10 �m.

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cell centre and delocalized cell protrusions around the entirecell periphery (Fig. 1A, 0:15:00 minutes). In the cellpopulation, 98% of cells had re-spread by 15 minutes afterwashing out Lat A. Delocalized protrusions in re-spread cellswere dynamic – protruding and retracting (see Movie 1 in thesupplementary material). Because these protrusions/retractionsfluctuated around the entire cell periphery, they did not alteroverall cell shape and thus re-spread cells are overallsymmetrical with a non- polarized cell shape.

The first visual sign of a break in cell symmetry in these cellswas a small, discrete, localized zone of cell-edge retraction (Fig.1A, black arrow), which formed by 35 minutes after washingout Lat-A for the individual cell shown in Fig. 1A,E and onaverage by 31 minutes in the cell population (ranging from 26to 35 minutes for the majority of cells) (quantified in Fig. 1F).Within the cell population, initiation of cell polarization is thusfairly synchronous. This localized cell-edge retraction gave theappearance of a small ‘bite’ taken out from the cell edge and wasapproximately 5-15 �m across and 3-5 �m deep, taking about

5 minutes to form. In contrast to the earlier fluctuations, thisretraction was very stable and permanently broke thesymmetrical shape of the cell. This initial break in cell symmetrythen elaborated with further cell-edge retraction for aboutanother 20-30 minutes (varying with individual cells) to formthe cell rear (Fig. 1A, 0:58:30 minutes, two black arrows, Fig.1E, 35-60 minutes).

Visually, a localized cell front became apparent at theopposing cell edge (Fig. 1A, 0:35:00 and 0:58:30, green dot) asthe cell rear formed. However, although recognizable as a cellfront, net protrusion at this location only occurred towards theend of formation of the cell rear (Fig. 1A, compare red and greendots; Fig. 1E, at 60 minutes). In the cell population, there wasan average 21-minute delay between initial cell-edge retractionand net protrusion at the cell front, ranging from 4 to 37 minutesin individual cells (Fig. 1G), and, in 13/15 cells, the delay was10 minutes or more. Once both the cell rear and front had fullyformed, the cell started to migrate (Fig. 1A, compare 0:58:30and 1:27:02; Fig. 1E, from 60 minutes). Thus, during cell

Fig. 2. Two main types of actin organization are detectable in cells during cell polarization. Cells were treated with 1 �M Lat A for 1 hour (A),then washed in medium (B-E) (see Materials and Methods) to initiate cell polarization, then fixed at various time points and stained withAlexa-Fluor-594-conjugated phalloidin to visualize actin organization. (A) Rounded-up cell, after 1 hour of Lat A treatment, prior to initiationof cell polarization with actin aggregates (note that no visible actin-filament bundles are present). (B,B�) Re-spread cells early in cellpolarization: actin is organized as a non-oriented actin-filament meshwork (B) or non-oriented actin-filament bundles (B�). Red arrows (B�)indicate different directions of unaligned filament bundles. (C,C�) Re-spread cells later in cell polarization: actin is organized as oriented actin-filament bundles. Bundles are more than 70% (C) to 100% (C�) aligned with each other (red arrow, C�) ±0-30°, as in fully polarized andmoving cells (Cramer et al., 1997) (and see E). (D) The cell edge is retracted (arrow) and actin remains organized as oriented actin-filamentbundles. (E, fully polarized cell) Actin-filament bundles within the cell body are oriented in the direction of migration. (F) Quantitative analysisof actin organization in fixed and stained polarizing cells at fixed time points during the cell-polarization assay (0-60 minutes after Lat Awashout). (G) Stacked histogram illustrating actin organization in fixed cells as a function of cell shape during cell polarization. Graphs (F,G)are expressed as mean ± s.e.m. (n=600 cells per time point, triplicate experiments). Bar, 10 �m.

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Fig. 3. Formation of oriented actin-filament bundles precedes initial, localized cell-edge retraction, the first visible sign of cell polarization.Cells expressing GFP-actin were imaged live by spinning-disc confocal microscopy during cell polarization. (A) Still images from a time-lapsemovie of a single polarizing cell (time in hours:minutes:seconds, indicates time after washout of Lat A). This movie was representative of atotal of 22 live GFP-actin-expressing cells analyzed that polarized. A meshwork of actin increases in visibility (0:02:20 to 0:06:01) and, by0:29:34, oriented actin-filament bundles have formed (also see enlarged views beneath). The initial, permanent break in cell symmetry thenoccurs (by 00:36:39, arrow). Cell-edge retraction then elaborates around the bundles (ongoing at 00:49:59, arrows) forming a polarized cellwith a defined front and rear, and the cell migrates (to 1:06:13, the end of the movie-sequence taken). Red and green dots (0:29:34 to 1:06:13)mark the position of the fully re-spread cell (at 15 minutes, image not shown) at the presumptive locations of where the rear initiates and frontforms, respectively. Small fluctuations at the cell edge, which do not permanently break cell symmetry, cause slight differences in the positionof the re-spread cell at 0:15:00 (image not shown) and 0:29:34. (B) Stacked histogram illustrating actin organization in live cells expressingGFP-actin as a function of cell shape during cell polarization (n=22 live-cell movies). (C) Actin organization in cells that polarized (22/24 cells)scored 60 seconds before the initial break in cell symmetry occurred (light-grey bar), identified in each movie, and in cells in whichpolarization naturally failed (2/24 cells) during the course of observation (up to 2 hours) (dark-grey bar). Note that, in all (22/22) cases, orientedactin-filament bundles formed prior to initiation of cell polarization. (D) Histogram of the time interval between oriented actin-filament-bundleformation and initiation of cell-edge retraction in 22 individual live cells. (E) Histograms of time taken to form oriented actin-filament bundles(to 70% total actin bundles) (upper panel) and to initiate cell-edge retraction (lower panel) in 22 individual live cells. (F) Detailed analysis ofelaboration of cell-edge retraction (n=22 live cells expressing GFP-actin). Cells either: (i) initiate and elaborate cell-edge retraction (arrows)end-on to bundles; or (ii) initiate retraction in one location, off-set to bundles, then (i) elaborate further from this site; or (iii) initiate retractionin two locations, off-set to bundles, then (i) elaborate further between the two sites of initiation. Note that, irrespective of where retractioninitiates, common to all is that retraction then elaborates around one end of oriented actin-filament bundles to form the cell rear (iv).(G) Distance that the cell-edge retracted (red) and protruded (green) measured at the presumptive location of where the rear initiates (A, reddot) and front forms (A, green dot), respectively, during cell polarization from the re-spread stage (0:15:00) for the cell in A. As in Fig. 1, netretraction precedes net protrusion. Bar, 10 �m.

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polarization in this system, formation of the cell rear occurs priorto net protrusion at the cell front.

Actin organization during cell polarizationHaving identified the sequence of shape-change events thatoccur in live cells during cell polarization, we assessed actinorganization in each identified shape by two approaches (Figs2 and 3). In one approach, to obtain a large data set we fixedcells in a time-course series during cell repolarization and thenstained them with phalloidin (Fig. 2). In rounded, Lat-A-treated cells prior to repolarization, aggregates of actin wereinduced by Lat-A treatment (Fig. 2A), as expected. For cellshapes during repolarization upon Lat A washout, in all cellshapes, as expected, F-actin was enriched in protrusions at thecell periphery. For actin in the cell body, the focus of this work,in re-spread cells, two distinct actin populations formed, oneearlier after washing out Lat A (by around 15 minutes afterwashout) and one later (by around 30 minutes after washout).In the earlier population, actin in the cell body formed a non-oriented array, either an actin meshwork (Fig. 2B) or non-oriented actin-filament bundles (Fig. 2B�). However, adramatic shift in actin organization apparently occurred in re-spread cells: in the later population, actin in the cell body wasreorganized into oriented (parallel) actin-filament bundlesspanning the length of the cell (Fig. 2C,C�). This organizationof oriented actin-filament bundles was also present in cells thathad initiated cell polarization, identified as cells with a discretezone(s) of cell-edge retraction (Fig. 2D), and in fullyrepolarized cells, identified as cells that appeared similar toalready characterized polarized cells of this cell type (Cramer,1999b; Cramer et al., 2002; Cramer et al., 1997; Dawe et al.,2003) (Fig. 2E). We defined actin-filament bundles as orientedin cells when more than 70% of total visible bundles wereparallel (±30°) to each other (e.g. Fig. 2C has roughly 80%oriented actin and Fig. 2C� has 100% oriented actin).

This dramatic shift in actin organization is clearly noticeablein the cell population, as shown by the quantification of actinorganization as a function of time (Fig. 2F) and shape (Fig. 2G).Also, the timing of initiation of cell polarization in live cells fromFig. 1A,F was similar to when there was a shift towards anoriented actin-filament-bundle organization in the fixed cellpopulation (Fig. 2F). From these two analyses, we thereforehypothesize that formation of oriented actin-filament bundles inthe cell body is important for triggering cell polarization.

Formation of oriented actin-filament bundles within thecell body precedes the first visual sign of a break in cellsymmetryTo begin to directly test this hypothesis, we first investigatedwhether formation of oriented actin-filament bundles in the cellbody precedes initial cell-edge retraction, by identifying actinorganization in live cells expressing GFP-actin using spinning-disc confocal time-lapse microscopy (see Materials andMethods) during cell polarization (Fig. 3).

Live-cell analysis (Fig. 3) revealed – as in the fixed time-point analysis above (Fig. 2) – that, during cell polarization,non-oriented actin meshwork/bundles is the first actinorganization visibly formed within the cell body (Fig. 3A, see0:02:20-0:06:01 minutes). Strikingly, prior to any break in cellsymmetry, actin then appeared to form oriented actin-filamentbundles spanning the cell body (Fig. 3A, by 0:29:34), which

remained as the cell continued to establish cell polarity (Fig.3A, 0:29:34-1:00:27) and migrate (Fig. 3A, compare 1:00:27and 1:06:13) (Movie 2 in the supplementary material). Thistransition from non-oriented to oriented actin organization asa function of cell polarization occurred in all 22 cells observedin the live-cell population expressing GFP-actin that polarized(quantified in Fig. 3B), similar to data obtained in the fixedtime-point assay (Fig. 2G).

Time-history analysis of each of these 22 (22/22) individualcells that polarized showed that oriented actin-filament bundlesformed, prior to initiation of cell polarization (individual cellshown in Fig. 3A, compare 0:29:34 and 0:36:39; quantified forthe cell population in Fig. 3C, light-grey bar). A further 2 (2/24)cells failed to polarize by up to 2 hours of observation. In thesecells, actin remained disorganized (Fig. 3C, dark-grey bar).

Formation of the cell front and rear occur aroundopposing ends of oriented actin-filament bundlesspanning the cell bodyWithin an average of 5 minutes (average from 22 live cells),ranging from 0.5-9.6 minutes in individual cells (Fig. 3D), oforiented actin-filament bundles forming in the cell body, cellpolarization initiated with a small stable retraction event, asdocumented in Fig. 1 (see representative example in Fig. 3A,0:36:39, arrow). For most of these cells (19/22), the averagetiming of this initial retraction event was 36.3 minutes, rangingfrom 21-47 minutes in the cell population (Fig. 3E, lowerpanel), similar to live cells analyzed by phase-contrastmicroscopy (Fig. 1). The other minority of cells expressingGFP-actin (3/22) were delayed up to around 1 hour, due to adelay in forming oriented actin bundles (Fig. 3E, compareupper and lower panels). We suspect the delay was due to anincrease in total exposure to UV light and/or higher levels ofexpression of GFP-actin in a minority of cells.

From fixed images of cells stained with phalloidin (Fig. 2),the geometric relationship between actin-filament-bundleorientation and cell polarization was unclear because the initialbreak in cell symmetry sometimes appeared on one side of thebundles (as in Fig. 2D), yet in fully polarized cells the rear waspositioned around one end of the bundles (as in Fig. 2E). Toreveal this relationship, we therefore studied precisely howretraction elaborates to form the cell rear by assessing this stepin live cells expressing GFP-actin in detail (Fig. 3F). In 14/22cells that polarized, both initial, stable retraction of the celledge and subsequent elaboration of the retraction occurred end-on to the oriented actin bundles (Fig. 3Fi). In the remaining8/22 cells, initiation occurred, side-on to the bundles, either atone (Fig. 3Fii) or both sides (Fig. 3Fiii). In both cases,elaboration then occurred around the end of bundles (as in Fig.3Fi). Thus, in all cases, elaboration of the retracted cell areaalways occurred around one end of oriented actin-filamentbundles to form the rear of the cell (Fig. 3Fiv) (exemplified inFig. 3A, and Movie 2 in the supplementary material). Alocalized protruded area remained at the opposing end of thebundles to form the front of the cell (Fig. 3Fiv, blue band),which then underwent net protrusion after formation of the cellrear (Fig. 3G), as documented in Fig. 1E. We noticed that theexact timing of the onset of migration was either coincidentwith the onset of net protrusion (as in Fig. 1E) or slightlydelayed (as in Fig. 3G). This appears to correlate with thetiming of the end-point of forming the cell rear, implying that

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cell migration is only triggered once both the cell rear and frontare fully formed. Once the cell was migrating, migrationcontinued in the direction of the oriented actin-filamentbundles (net migration down the page in the direction of thebundles, compare last two frames in Fig. 3A), as documentedin previous work (Cramer et al., 1997).

Thus, formation of oriented actin-filament bundles within thecell body appears to coordinate the position of the front and backof the cell, a new finding. This process also thus establishes thecorrect actin geometry (long length of bundles in the directionof migration) within the cell body required (Cramer et al., 1997)for subsequent cell migration in these cells.

These data provide direct evidence that formation oforiented actin-filament bundles in the cell body precedes andis linked with triggering cell polarization.

Formation of oriented actin-filament bundlesEvidently from our inspection of time-lapse movies of GFP-actin in live cells, formation of oriented actin-filament bundlesinvolves a large number of actin dynamic processes, whichform a complete body of work in their own right and are notdescribed here in detail. These dynamic processes includeactin-filament flow, growth, disassembly, bundling andthickening of bundles (all of which are apparent by closeinspection of Movie 2 in the supplementary material), andassociation with myosin II (data not shown); together, theseprocesses culminate in the straightening and strengthening ofbundles into an oriented array spanning the cell body. Here, wefocus on testing a role for actin-filament disassembly andsevering in this process because it is becoming clear that thisis involved in controlling cell polarity (see Introduction fordetails).

Jasplakinolide blocks formation of oriented actin-filament bundles within the cell body and cellpolarizationTo further test our hypothesis that formation of oriented actin-filament bundles in the cell body is important for triggeringcell polarization, and to test whether actin-filamentdisassembly controls oriented actin-bundle formation, wetreated cells with 0.5 �M jasplakinolide during cellpolarization. At such low doses, jasplakinolide specifically andrapidly inhibits actin-filament disassembly within 1-2 minutesof its addition to fibroblasts (Cramer, 1999b). Because this isfaster than the formation of oriented actin-filament bundles(see Fig. 2F, Fig. 3A), we were able to specifically target a rolefor these bundles during cell polarization.

To specifically target these bundles, we added 0.5 �Mjasplakinolide to cells early in the cell-polarization process: LatA was washed off cells and cells were incubated in culturemedium for a total of 15 minutes, a time when we know (fromFigs 2, 3 and Fig. 4A) that actin is not yet oriented, and thenjasplakinolide was added. We also know that actin shouldorient within about the next 15 minutes. We empirically testedin these cells that treating them for up to 45 minutes with 0.5�M jasplakinolide was specific in blocking actin-filamentdisassembly, because we did not observe any actin aggregatesuntil after this time (e.g. see Fig. 4C) [aggregates are inducedin cells when the weaker activity of jasplakinolide of inducingactin-filament polymerization in cells occurs (Cramer,1999b)]. This meant that the timing and duration of treating

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Fig. 4. Actin-filament disassembly is required for formation of orientedactin-filament bundles and cell polarization. Cells infected andexpressing GFP-actin (A-C) were imaged live by spinning-disc confocaltime-lapse microscopy during normal cell polarization (after Lat Awashout; A); or Lat A was washed off, cells left in culture medium for 15minutes, then 0.5 �M jasplakinolide added and cells left in the continuedpresence of jasplakinolide (B,C); or Lat A was washed off uninfectedcells, then 0.5 �M jasplakinolide added 15 minutes later and cells left inthe continued presence of jasplakinolide then fixed in a time-course assayand stained for actin (D,E). (A-C) Time is hours:minutes:seconds andindicates total time after washout of Lat A. (A) As expected, in thecontrol, actin initially forms a meshwork (by 0:12:00) then formsoriented actin-filament bundles (by 0:17:00). Stable retraction of the celledge occurs (by 0:27:00) and the cell is fully polarized and migrating by0:58:40. (B) In the jasplakinolide-treated cell, at 0:19:00 (19 minutesafter Lat A washout), jasplakinolide has been present for the last 4(0:15:00-0:19:00) minutes and, at 0:55:30, for the last 40 minutes, 30seconds (0:15:00-0:40:30). Both formation of oriented actin-filamentbundles and cell polarization fails. Note that delocalized protrusions stilloccur in the presence of jasplakinolide, as expected and similar to non-polarized cells (Cramer, 1999b) (see text for further details). (C, 1:07:30,last 0:52:30 in jasplakinolide) Prolonged treatment with 0.5 �Mjasplakinolide causes formation of actin aggregates and disruptedbundles, as documented previously (see text). Note that this occurs wellafter the specific block in formation of oriented actin bundles, shown in Band D. (D, jasplakinolide added at 15 minutes after Lat A washout)Quantification of formation of oriented actin-filament bundles in cellsfixed and stained for actin. (E, jasplakinolide added at 15 minutes afterLat A washout) Quantification of polarized cells in cells fixed at 30 and60 minutes after washout of Lat A. Note the failure of jasplakinolide-treated cells to form oriented actin-filament bundles (D) and to polarize(E) when compared to the control cells. Graphs are expressed as mean ±s.e.m. for three independent experiments (n=600 cells counted for eachcondition). Bar, 10 �m.

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cells with 0.5 �M jasplakinolide during the polarization assaywould specifically test a role for actin-filament disassembly informing oriented actin-filament bundles.

In 8/8 live cells tested, this jasplakinolide treatment blockedboth formation of oriented actin-filament bundles within thecell body and cell polarization (Fig. 4, compare A with B).Formation of a cell front and rear was inhibited and the cellremained at the re-spread stage with transient protrusions andretractions around the entire cell periphery (Fig. 4B). The blockin polarization was specific to a block in actin depolymerizationand not due to any effects of UV light or GFP-actin expressionlevels, because all 8/8 cells were blocked; less than two wouldbe expected if the effect were non-specific. In addition, weobtained similar data in a separate assay without using GFP-actin; quantification of 600 fixed cells in a fixed time-pointassay showed that, at 30 minutes after Lat A washout, only 10%of treated (last 15 minutes in jasplakinolide) cells had orientedactin-filament bundles compared with 68% of controls (Lat Awashout only) (Fig. 4D). By 60 minutes after Lat A washout,less than 12% of treated (last 45 minutes in jasplakinolide) cellswere polarized compared with 70% of controls (Lat A washoutonly) (Fig. 4E), the expected proportion in untreated cells(Cramer, 1999b; Cramer et al., 1997; Dawe et al., 2003).

In contrast to a block in formation of oriented actin-filamentbundles, jasplakinolide did not block delocalized protrusion(see Fig. 4B). This is similar to previous data showing thatjasplakinolide does not block delocalized protrusions in non-polarized cells that naturally occur when fibroblasts are culturedlong-term (Cramer, 1999b). This suggests that delocalizedprotrusion does not require actin-filament disassemblyirrespective of whether delocalized protrusion occurs during thecell-polarization process or in non-polarized cells. We note, aspreviously described, that there was an apparent accumulationof F-actin at the rear of the lamellipodium in jasplakinolide-treated cells (Fig. 4B), which we interpret as actin filaments thathave not been depolymerized (Cramer, 1999b).

These results show that actin-filament disassembly isrequired for the formation of oriented actin-filament bundles inthe cell body and provides further evidence that oriented actin-filament-bundle formation is necessary for cell polarization.

ADF localizes to actin filaments in the cell bodyBecause AC catalyzes actin-filament severing anddisassembly in cells, we examined the distribution of AC

during cell polarization with an antibody that recognizes ADFin chicken cells (Morgan et al., 1993). On initial inspection ofcells (images not shown), ADF localized to protrusions, asexpected from previous work on ADF and cofilin (Dawe etal., 2003; Svitkina and Borisy, 1999), and dispersed fairlyhomogeneously throughout the cell body with no apparentlocalization to actin-filament bundles in this location.However, in studies of cofilin in keratocytes, cells were firstextracted live in order to more clearly visualize cofilin boundto actin filaments in the leading cell edge (Svitkina and Borisy,1999). To test the possibility that a proportion of ADFlocalized to actin-filament bundles in the cell body but wasmasked by unbound ADF, we extracted cells in cytoskeleton-stabilizing buffer (to prevent actin depolymerization) prior tofixation using a similar method to the keratocyte work(Svitkina and Borisy, 1999) (Fig. 5). We estimate that weremoved 50-70% of total ADF from cells prior to subsequentcell fixation. In this method, ADF localized to actin filamentsin the cell body both before (Fig. 5E,E�) and after (Fig. 5F,F�)formation of oriented actin-filament bundles. This is clearlyseen in the enlarged views (Fig. 5E�,F�, orange colour).Localization of ADF on the actin-filament meshwork/non-oriented bundles within the cell body prior to formation oforiented actin-filament bundles was striking (Fig. 5E�). Thespotty/granular appearance of ADF is similar to previouslypublished images of AC in cells (Aizawa et al., 1996; Chanet al., 2000; Dawe et al., 2003; Mouneimne et al., 2006;Nishita et al., 2005; Svitkina and Borisy, 1999), includingactin-colocalization studies (Aizawa et al., 1996; Chan et al.,2000; Svitkina and Borisy, 1999), and is probably indicativeof the cooperative F-actin binding of ADF (Hayden et al.,1993), which, at substoichiometric amounts, gives localregions of saturation (McGough et al., 1997). Interestingly,once oriented actin bundles had formed (Fig. 5F-F�),localization of ADF appeared more obvious on thinner/less-bundled actin bundles (Fig. 5F�) than on thicker/more-bundledfilament bundles (Fig. 5F�) within the cell body of the samecell. This might infer a difference in age of bundles becausewe noticed that bundles thicken during cell polarization.

ACs control formation of oriented actin-filament bundlesin the cell body and cell polarization in fibroblasts.To test AC function (Fig. 6), we expressed either inactivepseudo-phosphorylated Xenopus AC1 E3 (XAC1 E3 mutant),

Fig. 5. ADF colocalizes with F-actin in the cell bodyduring cell polarization. During cell polarization, cellswere extracted with detergent (see Materials andMethods) to remove soluble ADF then fixed and co-stained with Alexa-Fluor-594-conjugated phalloidin (A,B,green) and anti-chick ADF antibody (C,D, red), and thetwo respective staining patterns were merged (E,F).(A,C,E,E�) A re-spread cell with a non-oriented meshworkof F-actin. (B,D,F-F�) A cell with localized cell-edgeretraction and oriented actin-filament bundles. Boxedareas in the merged images (E,F) are enlarged in E�-F�,respectively, and show colocalization (orange/yellow) ofADF and the actin meshwork (E�) and in a proportion(F�), but not all (F�), of the oriented actin-filamentbundles. Bar, 6 �m in A for A-F; 1.3 �m in E�,F�,F�.

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which acts as a dominant negative (Dawe et al., 2003), alone(Fig. 6B), or constitutively active LIMK1 EE508 mutant alone(Fig. 6C) to increase the proportion of inactive phosphorylated-AC in cells, and assessed cells fixed and stained in a time-course assay during cell polarization. We have previouslycharacterized these mutants in live chick fibroblasts (Dawe etal., 2003) (see Materials and Methods).

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Cells expressing these mutants to block AC function (Fig.6B,C) were competent at forming actin-filament bundles in thecell body early during cell polarization (Fig. 6B,C, 15minutes), similar to control cells (Fig. 6A, 15 minutes).However, cells expressing these mutants failed to subsequentlyform oriented actin-filament bundles in the cell body (Fig.6B,C, 25, 30, 60 minutes; and quantified in Fig. 6E, red, yellow

Fig. 6. AC is required for formation of orientedactin-filament bundles and cell polarization. Cellpolarization was initiated in chick heart fibroblastsby washing out Lat A. Cells were then incubatedlive for 15, 25, 30 or 60 minutes after the washout,fixed and stained with Alexa-Fluor-380-conjugated or FITC-conjugated phalloidin.(A) Control cells expressing GFP only. (B) Cellsexpressing inactive pseudo-phosphorylated AConly (XAC1 E3 mutant). (C) Cells expressingLIMK1 EE508 only to block AC function.(D) Rescue: cells co-expressing LIMK1 EE508and active, non-phosphorylatable AC (XAC1 A3mutant). Cells expressing GFP only wereidentified by GFP expression (inset in A); LIMK-EE508-expressing cells by GFP expression off aseparate promoter (insets in C and right insets inD); and XAC1-E3- and XAC1-A3-expressingcells by anti-XAC-antibody staining (insets in Band left insets in D), which has minimal cross-reactivity with endogenous AC (Shaw et al.,2004). Note the lack of formation of orientedactin-filament bundles in cells in which ACfunction is blocked (B,C). (E) Quantification ofthe formation of oriented actin-filament bundles inthe cell population. (F) Quantification of cellpolarization in the cell population. Graphs areexpressed as mean ± s.e.m. of triplicateexperiments (n=600 cells per condition). Bar,10 �m.

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lines) compared with control cells (Fig. 6A, 25, 30, 60 minutes;and Fig. 6E, green line). They also failed to polarize; instead,multiple protrusions and retractions occurred that weredelocalized around the cell periphery (Fig. 6B,C, 60 minutes).By 60 minutes after Lat A washout 70% of control cells(expressing GFP alone) polarized; by contrast, only 10% ofcells that expressed the inactive AC (XAC E3) mutantpolarized and 15% of cells that expressed the LIMK EE508mutant (to inactivate AC) (Fig. 6F). These data are similar tothose from cells treated with jasplakinolide (Fig. 4B,D,E).

Failure to form oriented actin-filament bundles in the cellbody and to trigger cell polarization was AC-specific, becauseco-infection of cells with both LIMK EE508 and the active non-phosphorylatable Xenopus AC1 A3 (XAC1 A3) mutant detectedwith an anti-XAC1 antibody (see Materials and Methods)rescued both formation of oriented actin-filament bundles (Fig.6D, 25-60 minutes; quantified in Fig. 6E, blue line) and cellpolarization (Fig. 6F, blue bar) to expected wild-type (GFP-only,control) levels (Fig. 6E, green line and Fig. 6F, green bar). Thetime taken for oriented actin-filament bundles to form in rescuedcells was indistinguishable from control cells (Fig. 6E, comparegreen and blue lines). The active non-phosphorylatable XenopusAC1 A3 mutant used to rescue cells, when expressed alone, hadno detectable effect on cells (data not shown).

These two different approaches to block AC function showthat AC activity is required for the formation of orientedbundles in the cell body and for the triggering of cellpolarization in fibroblasts.

DiscussionWe discovered that formation of oriented actin-filamentbundles within the cell body requires the AC family of proteinsand is an early event required for subsequent cell polarization(summarized in Fig. 7). The fact that actin within the cell bodyis needed for cell polarization is consistent with the alreadyappreciated role for actin in this cellular location for cellmigration (Cramer et al., 1997; Small et al., 1998; Svitkina etal., 1997; Verkhovsky et al., 1999a) and with the known rolefor actin-filament cables in the cell body for yeast cell polarity(reviewed by Moseley and Goode, 2006). Although our workshows that oriented actin-filament bundles within the cell bodyare necessary for cell polarization, it does not address whetherthey are sufficient, an important direction for future studies.

Function of oriented actin-filament bundles in the cellbodyOne apparent function of oriented actin-filament bundles in thecell body in fibroblast polarization is coordinating the

formation of the cell rear and front around opposing ends oflong lengths of the bundles (summarized in Fig. 7C-E). Howmight this occur? It is unlikely that these bundles generatepower to drive initial shape-change events per se because,when oriented bundles were absent (Fig. 4B and Fig. 6B,C),delocalized protrusion and retraction could still occur aroundthe cell periphery (the cell is not polarized and does nottranslocate under these conditions). More likely is that orientedactin bundles actively determine at least the position of the cellrear, because this event occurred only after bundles wereformed. Positioning of the cell front then could simply be aconsequence of the cell rear forming first, and subsequent netprotrusion at the cell front could perhaps be promoted byretraction-mediated protrusion (Chen, 1981; Dunn, 1980;Dunn and Zicha, 1995; Small and Resch, 2005). Becauseprotrusion is driven by a separable actin-based mechanism,however, it is likely that additional cellular factors are alsolocally activated at the cell front to ensure that net actinpolymerization is locally maintained.

An important area for future work is to identify precisedetails of how oriented actin-filament bundles function indetermining the position of the cell rear and front in fibroblasts.Plausible ideas are that oriented actin bundles provide a trackfor actin-based nuclear movement, a movement that is knownto play a role in NIH 3T3 fibroblast polarization (Gomes et al.,2005), or that they act as a guide for polarity determinants,perhaps similar to that discussed within the leading cell edgeof already polarized cells (reviewed by Ettienne-Manneville,2004; Gundersen and Bretscher, 2003).

Any role for actin in the cell body for positioning the cellrear and front in other migrating cell types is yet to beuncovered. In myoblasts, graded polarity actin-filament sheetsspan the cell body under the plasma membrane and are alignedin the direction of migration (Swailes et al., 2004), comparableto the alignment of actin bundles that we describe in thecytoplasm here, and they might perhaps have a similar role inpolarizing these cells. During polarization of cytoplasmicfragments of keratocytes, as with fibroblasts, oriented actinbundles also form in the cell body, but, unlike fibroblasts, arealigned transversely to the direction of migration (Verkhovskyet al., 1999b), and, in migrating amoeba and neutrophils, mostpolymerized actin in the cell body is a meshwork localized inthe cell cortex. Thus, the precise mechanism of positioning thefront and back of the cell during cell polarization might varywith distinct types of actin organization in the cell body indifferent migrating cell types. Such variation is notunexpected, because the precise mechanism of cell migrationis itself probably varied among distinct cell types (Cramer,

Fig. 7. Model for forming the cell rear andfront during fibroblast polarization. Prior tocell polarization in these cells, delocalizedcell protrusions occur around the entire cellperiphery (A,B, blue band). F-actin isenriched in protrusions, as expected. Priorto cell polarization (A,B), within the cellbody, actin initially assembles to form non-oriented meshwork/bundles (A, black lines) and subsequently forms oriented actin-filament bundles(B, black lines). Oriented actin-bundle formation triggers localized cell-edge retraction, the first visual break in cell symmetry (C, thin arrow).Cell-edge retraction elaborates around one end of oriented actin-filament bundles (D, jagged edge) to form the cell rear, leaving a stableprotrusion (D, blue band) at the opposing end of the bundles. Once cell-edge retraction is elaborated (D,E), net protrusion at the front and cellmigration occurs (E). AC (A-E, red dots) localizes to polymerized actin within the cell body early in this process (A) and is appropriatelylocalized, spatially and temporally, to promote formation of oriented actin-filament bundles (B, black lines) prior to cell polarization.

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1999a; Lauffenburger and Horwitz, 1996; Mitchison andCramer, 1996; Sheetz, 1994; Small and Resch, 2005;Verkhovsky et al., 1999a). This highlights the importance ofadvancing knowledge on detailed mechanisms in all types ofmigrating cells in order to derive any global principals.

Formation of the cell rearIn this work in chick fibroblasts, formation of the cell rear duringpolarization occurred first in the absence of any externallyapplied chemoattractant gradient, implying a basic property ofself-organization in the cell. This does not appear to be restrictedto this cell type. From close inspection of the images presentedin the primary literature in polarizing cells, formation of the cellrear can also precede local net protrusion at the opposing end ofthe cell in a variety of other cell types, including mousefibroblasts, neutrophils, keratocytes and dictyostelium cells(Gomes et al., 2005; Swanson and Taylor, 1982; Verkhovsky etal., 1999b; Wong et al., 2006). This not only occurs in a uniformconcentration of applied chemoattractant (Wong et al., 2006) butalso in the presence of a localized source (Swanson and Taylor,1982) or mechanical stimulus (Verkhovsky et al., 1999b).Although this needs to be explicitly studied in other cell typesand cell polarization systems, it seems, then, that a primarycontrol of cell polarization can be governed by activation of cell-rear formation first, perhaps related to the observation in somecells that retraction occurs first in already polarized andmigrating cells (Chen, 1981; Dunn, 1980; Dunn and Zicha,1995; Small and Resch, 2005). This might explain – apparentfrom the images shown – why some cells do not initiallyprotrude a localized cell front in the direction of a suppliedlocalized source of chemoattractant (Gerisch and Keller, 1981;Mouneimne et al., 2006; Ridley et al., 1999).

Local membrane events govern protrusion at the cell front(Niggli, 2003; Ridley et al., 2003) and these are also important.Whether the cell front or rear forms first might vary withprecise cellular situation. Activating the cell front first mightbe sufficient for short-range excursions, for example. Allowingpathways to activate cell-rear formation first, by contrast,might be more important in situations in which individual cellscompletely change direction of migration, mediated by thefront and back of the cell reversing their position, as in reversechemotaxis, for example (Keizer-Gunnink et al., 2007; Mathiaset al., 2006), or in response to chemorepulsion duringembryogenesis.

Mechanism of formation of oriented actin-filamentbundles and the role of ACAC functions to sever/depolymerize actin filaments andregulates actin dynamics within cell protrusions of motile cells(Chan et al., 2000; Dawe et al., 2003; Ghosh et al., 2004;Nishita et al., 2005; Zebda et al., 2000). Here, we uncovered anew role for AC, the formation of oriented actin-filamentbundles within the cell body during cell polarization. Our workis consistent with the known requirement for AC in theformation of oriented actin cables in the cell body for polarizedgrowth in yeast (Okada et al., 2006). This might imply that ACfunctions both in cell protrusions and within the cell body ingeneral in migrating cells, although this remains to be directlytested in a single assay in a single cell type.

How, then, does AC function to form oriented actin-filamentbundles during cell polarization? The jasplakinolide data (Fig.

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4) provides an argument for actin-filament depolymerizationbeing important. With a time resolution of seconds, we did notobserve complete depolymerization of the existing non-orientedactin-filament meshwork in live cells (Fig. 7A) and thensubsequent formation of oriented actin-filament bundles. Thisargues against a simple model of complete depolymerizationof one actin organization and then temporally distinctrepolymerization to form another. Several models are plausible,then, for the function of depolymerization, and more than one isprobably required to explain formation of the oriented bundles.

Actin-filament depolymerization might directly contributeactin monomers required to make oriented bundles, perhapssimilar to the known function for depolymerization inproviding actin monomers for polymerization within theleading cell edge of migrating fibroblasts (Cramer, 1999b;Dawe et al., 2003) and other cell types (Kiuchi et al., 2007).This idea is also consistent with data on actin bundles in cellsdepleted of AC (Hotulainen et al., 2005). This scenario wouldrequire new actin polymerization to make bundles, perhaps atsites of cell adhesions observed during cell polarization(supplementary material Fig. S1), although we do not yet knowwhether adhesions actively promote actin-bundle formation. Inaddition to this function, actin depolymerization might have alesser role in removing off-axis bundles during cellpolarization, a phenomenon we have observed for a fewbundles towards the end of forming oriented actin bundles incells (data not shown).

For this theory to work, actin depolymerization of theexisting non-oriented actin meshwork must occur nearlysimultaneously with the formation of oriented bundles, whichis difficult to test. AC severing activity can also form actinbundles (Aizawa et al., 1996; Maciver et al., 1991; Moriyamaet al., 1996) and severing would generate shorter actinfilaments that could be manoeuvred into a bundledorganization by proteins such as a-actinin and myosin II. Insupport of this idea, we observed both movement of bundlesin live cells (data not shown) and association of polymerizedactin with myosin II during formation of oriented actin-filament bundles (T.M. and L.P.C., unpublished). Alternatively,or in addition to, a proportion of severed filaments could beused as a source of free-actin barbed ends for new actin-filament growth (Ichetovkin et al., 2002), although see Kiuchiet al. (Kiuchi et al., 2007).

To distinguish between these plausible AC mechanisms forforming oriented actin-filament bundles during polarization,the relative importance of depolymerization and severingactivities of AC, respectively, need to be assessed, as well asany separate functions for individual AC family members.High local concentrations of cofilin, but not of ADF, cannucleate assembly of actin filaments (Andrianantoandro andPollard, 2006; Chen et al., 2004). Such a difference in activitycould be important in polarized cell migration. Silencing ofADF, but not cofilin, in a colorectal-tumour cell line blockedtheir transwell migration through Matrigel, a measure of themetastatic potential of a cell (Estornes et al., 2007).Significantly, these cells express 83% cofilin and 17% ADF,almost identical to the cofilin and ADF percentages in chickcardiac fibroblasts (Tahtamouni and J.R.B., unpublished).

We conclude that AC control formation of oriented actin-filament bundles in the cell body, which in turn coordinates theformation of the cell rear and front during cell polarization.

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The finding that oriented actin in the cell body contributes totriggering cell polarization is new and potentially opens novelavenues in which to explore the overall mechanism and targetsfor regulating this important process in migrating cells.

Materials and MethodsInitiation of cell polarizationMigrating primary fibroblasts were freshly prepared from explants of embryonicday (E)7 or E8 chick embryo hearts for each experiment and grown in DMEM lowglucose (GIBCO) containing 10% chick serum (Sigma) and 10% FCS (CEFmedium) at 37°C and 5% CO2 for 24-36 hours after plating the explants, aspreviously described (Cramer, 1999b; Cramer et al., 1997). Coated glass coverslipswere used for immuno-cell-staining and live-cell imaging. The glass was coatedwith 1 mg/ml poly-L-lysine (Sigma) and then coated for 30 minutes with Matrigel(Becton Dickinson) on the day of plating explants. Once the explants had adheredand cells migrated out, cells were treated with 1-5 �M Lat A (Calbiochem) for 1hour then washed thoroughly with CEF medium. Lat A was thawed from a 1000�DMSO stock immediately before treatment. The final concentration of DMSO inthe drug mixture was 0.1% and this has no effect on actin organization and cellbehaviour when added to fibroblasts alone. Lat A concentration (1-5 �M) wasstandardized so that all normal organized actin in cells was abolished by 1 hour oftreatment; this required periodically titrating Lat A over 1-5 �M because strengthvaried between batches and with age of drug. To monitor polarization, cells werethen analyzed from 0 minutes to 2 hours after washing out Lat A.

Immunostaining of CEFTo stain for actin and XAC1, cells were fixed and stained at 37°C in 4% methanol-free formaldehyde (TAAB) in cytoskeleton buffer (10 mM MES pH 6.1, 3 mMMgCl2, 138 mM KCl, 2 mM EGTA) with 0.32 M sucrose for 30 minutes, aspreviously described (Cramer and Mitchison, 1993). In brief, they were thenpermeabilized in 0.5% Triton X-100 for 10 minutes. For indirectimmunofluorescence, cells were incubated with an anti-XAC1 antibody (Rosenblattet al., 1997) for 45 minutes, washed and then incubated simultaneously with a Cy-3 or Alexa-Fluor-594-conjugated anti-rabbit secondary antibody (JacksonImmunoResearch Laboratories) and 0.1 �g/ml FITC or Alexa-Fluor-350-conjugated phalloidin (Molecular Probes) for 45 minutes. Cells treated withjasplakinolide were gently fixed in –20°C methanol for 1 minute then stained withanti-actin C4 (ICN Biomedicals) for 45 minutes, then an Alexa-Fluor-594-conjugated anti-mouse secondary antibody (Jackson ImmunoResearchLaboratories) for 45 minutes.

Extraction of ADF from CEF and immunostainingCells were treated with 0.5-3.0% Triton X-100 (Sigma) in cytoskeleton, extractionbuffer (PEM; Pipes 100 mM, EGTA 1 mM, MgCl2 1 mM and 4% PEG) at 37°Cfor 1 minute, similar to previous methodology (Svitkina and Borisy, 1999). Thecells were then immediately fixed at 37°C in 4% methanol-free formaldehyde(TAAB) in cytoskeleton buffer (10 mM MES pH 6.1, 3 mM MgCl2, 138 mM KCl,2 mM EGTA) with 0.32 M sucrose, for 30 minutes. They were then permeabilizedin 0.5% Triton X-100 for 10 minutes. For indirect immunofluorescence, cells wereincubated with an anti-ADF antibody (rabbit 12977) (Shaw et al., 2004; Morgan etal., 1993) for 45 minutes, and incubated simultaneously with an Alexa-Fluor-488-conjugated anti-rabbit secondary antibody (Molecular Probes) and 0.1 �g/ml Alexa-Fluor-594-conjugated phalloidin (Molecular Probes) for 45 minutes. The anti-ADFantibody has previously been characterized in chick fibroblasts and specificallyrecognizes both chicken ADF and chicken phospho-ADF (Dawe et al., 2003).

Infection of fibroblasts with adenoviral constructsWe used the Ad Easy adenoviral system (Stratagene), consisting of adenoviruscontaining recombinant expression vectors. Replication-incompetent adenovirusescontaining the cDNA for expressing covalently fused actin-GFP (Tanner et al.,2005), LIM kinase (T508EE) with GFP on a separate promoter (Edwards et al.,1999), Xenopus AC1 (XAC1) (S3A) (Meberg and Bamburg, 2000), XAC1 (S3E)(Meberg and Bamburg, 2000) and, as a control, GFP with a lacZ promoter weremade, expanded and titred as described (Minamide et al., 2003). These were usedto infect CEF as previously described (Dawe et al., 2003). Using this method ofinfection, we found that chick embryo fibroblasts expressed exogenous proteinsefficiently. Control viral infection, containing only GFP, has no effect on eithermigration speed or cell polarity in chick fibroblasts (Dawe et al., 2003). When cellswere infected, we aimed for 60-70% of fibroblasts at the edge of the explant to beexpressing the exogenous proteins. This was achieved by using 1.0�107 to 3.0�107

virus particles per E7-E8 chick heart (approximately 0.9-1.4 million cells);variations in transfection levels occurred between different viruses and the age ofthe virus aliquot. Indirect immunofluorescence with an anti-XAC1 antibody(Rosenblatt et al., 1997) was used to distinguish cells infected by the XAC1 A3 andXAC E3 viruses from uninfected cells because this antibody has minimal crossreactivity with endogenous chick AC (Dawe et al., 2003; Shaw et al., 2004).

Matrigel-coated glass-bottomed cell culture dishes (WillCo Wells) were used forlive imaging of cells exposed to virus.

Treatment of CEF with jasplakinolideLat A was washed off cells then, 15 minutes later, 0.5 �M jasplakinolide (a giftfrom M. Sanders, Dep. Chemistry UC, Santa Cruz, CA) was added. Cells were theneither imaged live by time-lapse microscopy or fixed at various time points forimmuno-cell-staining. Jasplakinolide was diluted to the correct concentration, justbefore use, from a thawed 1000� DMSO stock.

Cell imagingHigh-resolution fluorescent images of fixed cells were obtained digitally by a 12-bit cooled CCD camera (SenSys KAF 1400, Roper Scientific) on an uprightmicroscope (Nikon eclipse e800) using a 60�1.4 NA oil objective controlled byMetamorph software (Universal Imaging).

Digitally acquired phase-contrast time-lapse movies of live cells were made usingan inverted microscope (Zeiss Axiovert S1000-TV) and a 12-bit cooled CCDcamera (MicroMax KAF 1400, Roper Scientific) with a 63�1.40 NA oil Zeissobjective controlled by Metamorph software. The cells on glass coverslips wereheated to 36.5°C by a temperature-controlled water bath, custom built into a heatingchamber.

Spinning-disc confocal microscopy (Perkin Elmer) controlled by Metamorph wasused to obtain time-lapses of actin dynamics in live cells expressing GFP-actin. Theimages were captured on an inverted microscope (Nikon TE 2000-U) by a high-performance camera (Orca II-ER, Hamamatsu) and a 60� �A1.40 oil Nikon lens.Cells on glass-bottomed dishes (WillCo Wells) were heated to 37°C in anelectronically controlled chamber (Heatwave-30, World Precision Instruments) andthe objective was also electronically heated to minimize focal drift (Bioptechs largeobjective heater, Intracell).

We are very grateful to Vania Braga (Imperial College London) forhelpful discussion and comments on the manuscript. Helen Dawe, aspart of her thesis work, made some preliminary discoveries in arelated cell system that AC blocks cell polarization. We gratefullyacknowledge grant support from the Cancer Research UK andWellcome Trust (L.P.C.) and the National Institutes of Health, grantNS40371 (J.R.B.). L.P.C. is a Royal Society University ResearchFellow.

ReferencesAbercrombie, M. and Heaysman, J. E. (1966). The directional movement of fibroblasts

emigrating from cultured explants. Ann. Med. Exp. Biol. Fenn. 44, 161-165.Aizawa, H., Sutoh, K. and Yahara, I. (1996). Overexpression of cofilin stimulates

bundling of actin filaments, membrane ruffling and cell movement in Dictyostelium.J. Cell Biol. 132, 335-344.

Andrianantoandro, E. and Pollard, T. D. (2006). Mechanism of actin filament turnoverby severing and nucleation at different concentrations of ADF/cofilin. Mol. Cell 24,13-23.

Ayscough, K. R., Stryker, J., Pokala, N., Sanders, M., Crews, P. and Drubin, D. G.(1997). High rates of actin filament turnover in budding yeast and roles for actin inestablishment and maintenance of cell polarity revaealed using the actin inhibitorLatrunculin-A. J. Cell Biol. 137, 399-416.

Bamburg, J. R. and Wiggan, O. P. (2002). ADF/cofilin and actin dynamics in disease.Acting-depolymerizing factor. Trends Cell Biol. 12, 598-605.

Bernstein, B. W. and Bamburg, J. R. (2004). A proposed mechanism for cellpolarization with no external cues. Cell Motil. Cytoskeleton 58, 96-103.

Cassimeris, L. and Zigmond, S. H. (1990). Chemoattractant stimulation ofpolymorphonuclear leucocyte locomotion. Semin. Cell Biol. 1, 125-134.

Chan, A. Y., Bailly, M., Zebda, N., Segall, J. E. and Condeelis, J. S. (2000). Role ofcofilin in epidermal growth factor-stimulated actin polymerization and lamellipodprotrusion. J. Cell Biol. 148, 531-542.

Charest, P. G. and Firtel, R. A. (2006). Feedback signaling controls leading-edgeformation during chemotaxis. Curr. Opin. Genet. Dev. 16, 339-347.

Chen, H., Bernstein, B. W., Sneider, J. M., Boyle, J. A., Minamide, L. S. andBamburg, J. R. (2004). In vitro activity differences between protiens of theADF/cofilin family define two distinct subgroups. Biochemistry 43, 7127-7142.

Chen, W.-T. (1981). Mechanism of retraction of the trailing edge during fibroblastmovement. J. Cell Biol. 90, 187-200.

Couchman, J. R. and Rees, D. A. (1979). The behaviour of fibroblasts migrating fromchick heart explants: changes in adhesion, locomotion, and growth, in the distributionof actomyosin and fibronectin. J. Cell Sci. 39, 149-165.

Cramer, L. P. (1999a). Organization and polarity of actin filament networks in cells:implications for the mechanism of myosin-based cell motility. Biochem. Soc. Symp.65, 173-205.

Cramer, L. P. (1999b). Role of actin-filament disassembly in lamellipodiumprotrusion in motile cells revealed using the drug jasplakinolide. Curr. Biol. 9, 1095-1105.

Cramer, L. and Mitchison, T. J. (1993). Moving and stationary actin filaments areinvolved in spreading of postmitotic PtK2 cells. J. Cell Biol. 122, 833-843.

Jour

nal o

f Cel

l Sci

ence

4344 Journal of Cell Science 120 (24)

Cramer, L. P., Siebert, M. and Mitchison, T. J. (1997). Identification of novel gradedpolarity actin filament bundles in locomoting heart fibroblasts: implications for thegeneration of motile force. J. Cell Biol. 136, 1287-1305.

Cramer, L. P., Briggs, L. J. and Dawe, H. R. (2002). Use of fluorescent DNase tospatially measure G-actin levels in migrating and non-migrating cells. Cell Motil.Cytoskeleton 51, 27-38.

Dawe, H. R., Minamide, L. S., Bamburg, J. R. and Cramer, L. P. (2003). ADF/cofilincontrols cell polarity during fibroblast migration. Curr. Biol. 13, 252-257.

Dormann, D. and Weijer, C. J. (2006). Chemotactic cell movement duringDictyostelium development and gastrulation. Curr. Opin. Genet. Dev. 16, 367-373.

Drees, B. L., Sundin, B., Brazeau, E., Caviston, J. P., Chen, G. C., Guo, W.,Kozminski, K. G., Lau, M. W., Moskow, J. J., Tong, A. et al. (2001). A proteininteraction map for cell polarity development. J. Cell Biol. 154, 549-571.

Dunn, G. A. (1980). Cell adhesion and motility. In Symposia / British Society for CellBiology, 3 (ed. A. S. G. Curtis and J. D. Pitts), pp. 409-423. Cambridge: CambridgeUniversity Press.

Dunn, G. A. and Zicha, D. (1995). Dynamics of fibroblast spreading. J. Cell Sci. 108,1239-1249.

Edwards, D. C., Sanders, L. C., Bokoch, G. M. and Gill, G. N. (1999). Activation ofLIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletaldynamics. Nat. Cell Biol. 1, 253-259.

Estornes, Y., Gay, F., Gevrey, J. C., Navoizat, S., Nejjari, M., Scoazec, J. Y.,Chayvialle, J. A., Saurin, J. C. and Abello, J. (2007). Differential involvement ofdestrin and cofilin-1 in the control of invasive properties of Isreco1 human colon cancercells. Int. J. Cancer 121, 2162-2171.

Ettienne-Manneville, S. (2004). Cdc42 – the centre of cell polarity. J. Cell Sci. 117, 1291-1300.

Gerisch, G. and Keller, H. U. (1981). Chemotactic reorientation of granulocytesstimulated with micropipettes containing fMet-Leu-Phe. J. Cell Sci. 52, 1-10.

Ghosh, M., Song, X., Mouneimne, G., Sidani, M., Lawrence, D. S. and Condeelis, J.S. (2004). Cofilin promotes actin polymerisation and defines the direction of cellmotility. Science 304, 743-746.

Gomes, E. R., Jani, S. and Gundersen, G. G. (2005). Nuclear movement regulated byCdc42, MRCK, myosin and actin flow establishes MTOC polarization in migratingcells. Cell 121, 451-463.

Gundersen, G. G. and Bretscher, A. (2003). Cell biology. Microtubule asymmetry.Science 300, 2040-2041.

Hayden, S. M., Miller, P. S., Brauweiler. A. and Bamburg, J.R. (1993). Analysis ofthe interactions of actin depolymerizing factor with G-and F-actin. Biochem. 32, 9994-10004.

Hotulainen, P., Paunola, E., Vartiainen, M. K. and Lappalainen, P. (2005). Actin-depolymerizing factor and Cofilin-1 play overlapping roles in promoting rapid F-Actindepolymerization in mammalian nonmuscle cells. Mol. Biol. Cell 16, 649-664.

Ichetovkin, I., Grant, W. and Condeelis, J. (2002). Cofilin produces newly polymerizedactin filaments that are preferred for dendritic nucleation by the Arp2/3 complex. Curr.Biol. 12, 79-84.

Kaverina, I., Krylyshkina, O., Gimona, M., Beningo, K., Wang, Y. L. and Small, J.V. (2000). Enforced polarisation and locomotion of fibroblasts lacking microtubules.Curr. Biol. 10, 739-742.

Keizer-Gunnink, I., Kortholt, A. and Van Haastert, P. J. M. (2007). Chemoattractantsand chemorepellents act by inducing opposite polarity in phospholipase C and PI3-kinase signaling. J. Cell Biol. 177, 579-585.

Kimmel, A. R. and Firtel, R. A. (2004). Breaking symmetries: regulation ofDictyostelium development through chemoattractant and morphogen signal-response.Curr. Opin. Genet. Dev. 14, 540-549.

Kiuchi, T., Ohashi, K., Kurita, S. and Mizuno, K. (2007). Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actinmonomers. J. Cell Biol. 177, 465-476.

Lauffenburger, D. A. and Horwitz, A. F. (1996). Cell migration: a physically integratedmolecular process. Cell 84, 359-369.

Maciver, S. K., Wachsstock, D. H., Schwarz, W. H. and Pollard, T. D. (1991). Theactin filament severing protien actophorin promotes the formation of rigid bundles ofactin filaments crosslinked with a-actinin. J. Cell Biol. 115, 1621-1628.

Mathias, J. R., Perrin, B. J., Liu, T. X., Kanki, J., Look, A. T. and Huttenlocher, A.(2006). Resolution of inflammation by retrograde chemotaxis of neutrophils intransgenic zebrafish. J. Leukoc. Biol. 80, 1281-1288.

McGough, A., Pope, B., Chiu, W. and Weeds, A. (1997). Cofilin changes the twist ofF-actin: implications for actin filament dynamics and cellular function. J. Cell Biol.138, 771-781.

Meberg, P. J. and Bamburg, J. R. (2000). Increase in neurite outgrowth mediated byoverexpression of actin depolymerizing factor. J. Neurosci. 20, 2459-2469.

Minamide, L. S., Shaw, A. E., Sarmiere, P. D., Wiggan, O., Maloney, M. T., Bernstein,B. W., Sneider, J. M., Gonzalez, J. A. and Bamburg, J. R. (2003). Production anduse of replication-deficient adenovirus for transgene expression in neurons. MethodsCell Biol. 71, 387-416.

Mitchison, T. J. and Cramer, L. P. (1996). Actin-based cell motility and cell locomotion.Cell 84, 371-379.

Moon, A. and Drubin, D. G. (1995). The ADF/cofilin proteins: stimulus-responsivemodulators of actin dynamics. Mol. Biol. Cell 6, 1423-1431.

Morgan, T. E., Lockerbie, R. O., Minamide, L. S., Browning, M. D. and Bamburg,J. R. (1993). Isolation and characterization of a regulated form of actin depolymerizingfactor. J. Cell Biol. 122, 623-633.

Moriyama, K., Iida, K. and Yahara, I. (1996). Phosphorylation of Ser-3 of cofilinregulates its essential function on actin. Genes Cells 1, 73-86.

Moseley, J. B. and Goode, B. L. (2006). The yeast actin cytoskeleton: from cellularfunction to biochemical mechanism. Microbiol. Mol. Biol. Rev. 70, 605-645.

Mouneimne, G., DesMarais, V., Sidani, M., Scemes, E., Wang, W., Song, X., Eddy,R. and Condeelis, J. (2006). Spatial and temporal control of cofilin activity is requiredfor directional sensing during chemotaxis. Curr. Biol. 16, 2193-2205.

Niggli, V. (2003). Signaling to migration in neutrophils: importance of localised pathways.Int. J. Biochem. Cell Biol. 35, 1619-1638.

Nishita, M., Tomizawa, C., Yamamoto, M. Y., Horita, Y., Ohashi, K. and Mizuno, K.(2005). Spatial and temporal regulation of cofilin activity by LIM kinase and slingshotis critical for directional cell migration. J. Cell Biol. 171, 349-359.

Okada, K., Ravi, H., Smith, E. M. and Goode, B. L. (2006). Aip1 and cofilin promoterapid turnover of yeast actin patches and cables: a coordinated mechanism for severingand capping filaments. Mol. Biol. Cell 17, 2855-2868.

Onsum, M. D., Wong, K., Herzmark, P., Bourne, H. R. and Arkin, A. P. (2006).Morphology matters in immune cell chemotaxis: membrane asymmetry affectsamplification. Phys. Biol. 3, 190-199.

Ridley, A. J., Allen, W. E., Peppelenbosch, M. and Jones, G. E. (1999). Rho familyproteins and cell migration. Biochem. Soc. Symp. 65, 111-123.

Ridley, A. J., Schwartz, M. A., Burridge, K., Firtel, R. A., Ginsberg, M. H., Borisy,G., Parsons, J. T. and Horwitz, A. R. (2003). Cell migration: Integrating signals fromfront to back. Science 302, 1704-1709.

Rodriguez, O. C., Schaefer, A. W., Mandato, C. A., Forscher, P., Bement, W. M. andWaterman-Storer, C. M. (2003). Conserved microtubule-actin interactions in cellmovement and morphogenesis. Nat. Cell Biol. 5, 599-609.

Rosenblatt, J., Agnew, B. J., Abe, H., Bamburg, J. R. and Mitchison, T. J. (1997).Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover ofactin filaments in Listeria monocytogenes tails. J. Cell Biol. 136, 1323-1332.

Sarmiere, P. D. and Bamburg, J. R. (2003). Regulation of the neuronal actincytoskeleton by ADF/ Cofilin. J. Neurobiol. 58, 103-117.

Schneider, I. C. and Haugh, J. M. (2005). Quantitative elucidation of a distinct spatialgradient-sensing mechanism in fibroblasts. J. Cell Biol. 171, 883-892.

Schneider, I. C. and Haugh, J. M. (2006). Mechanisms of gradient sensing andchemotaxis: conserved pathways, diverse regulation. Cell Cycle 5, 1130-1134.

Shaw, A. E., Minamide, L. S., Bill, C. L., Funk, J. D., Maiti, S. and Bamburg, J. R.(2004). Cross-reactivity of antibodies to actin-depolymerizing factor/ cofilin familyproteins and identification of the major epitope recognized by a mammalian actindepolymerizing factor/ cofilin antibody. Electrophoresis 25, 2611-2620.

Sheetz, M. P. (1994). Cell migration by graded attachment to substrates and contraction.Semin. Cell Biol. 5, 149-155.

Small, J. V. and Kaverina, I. (2003). Microtubules meet substrate adhesions to arrangecell polarity. Curr. Opin. Cell Biol. 15, 40-47.

Small, J. V. and Resch, G. P. (2005). The comings and goings of actin: couplingprotrusion and retraction in cell motility. Curr. Opin. Cell Biol. 17, 517-523.

Small, J. V., Rottner, K., Kaverina, I. and Anderson, K. I. (1998). Assembling an actincytoskeleton for cell attachment and movement. Biochim. Biophys. Acta 1404, 271-281.

Small, J. V., Geiger, B., Kaverina, I. and Bershadsky, A. (2002). How do microtubulesguide migrating cells? Nat. Rev. Mol. Cell Biol. 3, 957-964.

Svitkina, T. M. and Borisy, G. G. (1999). Arp2/3 complex and actin depolymerizingfactor/cofilin in dendritic organization and treadmilling of actin filament array inlamellipodia. J. Cell Biol. 145, 1009-1026.

Svitkina, T. M., Verkhovsky, A. B., McQuade, K. M. and Borisy, G. G. (1997).Analysis of the actin-myosin II system in fish epidermal keratocytes: mechanism ofcell body translocation. J. Cell Biol. 139, 397-415.

Swailes, N. T., Knight, P. J. and M. P. (2004). Actin filament organization in alignedprefusion myoblasts. J. Anat. 205, 381-391.

Swanson, J. A. and Taylor, D. L. (1982). Local and spatially coordinated movements inDictyostelium discoideum amoebae during chemotaxis. Cell 28, 225-232.

Tanner, G. A., Sandoval, R. M., Molitoris, B. A., Bamburg, J. R. and Ashworth, S.L. (2005). Micropuncture gene delivery and intravital two-photon visualization ofprotien expression in rat kidney. Am. J. Physiol. Renal Physiol. 289, F638-F643.

Van Keymeulen, A., Wong, K., Knight, Z. A., Govaerts, C., Hahn, K. M., Shorkat, K.M. and Bourne, H. R. (2006). To stabilize neutrophil polarity, PIP3 and Cdc42 augmentRhoA activity at the back as well as signals at the front. J. Cell Biol. 174, 437-445.

Verkhovsky, A. B., Svitkina, T. M. and Borisy, G. G. (1999a). Network contractionmodel for cell translocation and retrograde flow. Biochem. Soc. Symp. 65, 207-222.

Verkhovsky, A. B., Svitkina, T. M. and Borisy, G. G. (1999b). Self-polarization anddirectional motility of cytoplasm. Curr. Biol. 9, 11-20.

Wittmann, T. and Waterman-Storer, C. M. (2001). Cell motility: can Rho GTPases andmicrotubules point the way? J. Cell Sci. 114, 3795-3803.

Wong, K., Pertz, O., Hahn, K. and Bourne, H. (2006). Neutrophil polarisation:spatiotemporal dynamics of RhoA activity support a self-organizing mechanism. Proc.Natl. Acad. Sci. USA 103, 3639-3644.

Xu, J., Wang, F., Van Keymeulen, A., Herzmark, P., Straight, A., Kelly, K., Takuwa,Y., Sugimoto, N., Mitchison, T. and Bourne, H. R. (2003). Divergent signals andcytoskeletal assemblies regulate self-organizing polarity in neutrophils. Cell 114, 201-214.

Zebda, N., Bernard, O., Bailly, M., Welti, S., Lawrence, D. S. and Condeelis, J. S.(2000). Phosphorylation of ADF/Cofilin abolishes EGF-induced actin nucleation at theleading edge and subsequent lamellipod extension. J. Cell Biol. 151, 1119-1128.

Zigmond, S. H. (2004). Beginning and ending an actin filament: control at the barbedend. Curr. Top. Dev. Biol. 63, 145-188.

Jour

nal o

f Cel

l Sci

ence