actin filament severing by cofilin

doi:10.1016/j.jmb.2006.10.102 J. Mol. Biol. (2007) 365, 1350–1358

Actin Filament Severing by Cofilin

Dmitry Pavlov1⁎, Andras Muhlrad2, John Cooper3, Martin Wear3

and Emil Reisler1,4

1Department of Chemistry andBiochemistry, University ofCalifornia, Los Angeles,CA 90095, USA2Department of Oral Biology,Hebrew University HadassahSchool of Dental Medicine,Jerusalem, Israel 911203Department of Cell Biologyand Physiology, WashingtonUniversity School of Medicine,St. Louis, MO 63110, USA4Molecular Biology Institute,University of California,Los Angeles, CA 90095, USA

Abbreviations used: TRC, tetramecadaverine; HMM, heavy meromyodepolymerizing factor.E-mail address of the correspondi

[email protected]

0022-2836/$ - see front matter © 2006 E

Cofilin is essential for cell viability and for actin-based motility. Cofilinsevers actin filaments, which enhances the dynamics of filament assembly.We investigated the mechanism of filament severing by cofilin with directfluorescence microscopy observation of single actin filaments in real time. Incells, actin filaments are likely to be attached at multiple points along theirlength, and we found that attaching filaments in such a manner greatlyincreased the efficiency of filament severing by cofilin. Cofilin severingincreased and then decreased with increasing concentration of cofilin.Together, these results indicate that cofilin severs the actin filament by amechanism of allosteric and cooperative destabilization. Severing is moreefficient when relaxation of this cofilin-induced instability of the actinfilament is inhibited by restricting the flexibility of the filament. Theseconclusions have particular relevance to cofilin function during actin-basedmotility in cells and in synthetic systems.

© 2006 Elsevier Ltd. All rights reserved.

*Corresponding author
Keywords: actin; cofilin; gelsolin; severing; flexibility
Introduction

Many cellular processes involve rapid remodelingof the actin cytoskeleton and changes in the pools ofassembled and monomeric actin.1 Not surprisingly,rapid cycles of actin polymerization/depolymeriza-tion that occur at the leading edge of motile cellsdepend on factors accelerating both the polymeriza-tion and depolymerization of actin filaments.2 Thecofilin/ actin depolymerizing factor (ADF) family ofproteins appears to be a major factor contributing toactin depolymerization in cells, which is essential forrecycling actin subunits to support the growth ofnew filaments. Cofilin can sever actin filaments,creating free barbed and pointed ends available forpolymerization or depolymerization, depending onthe local concentration of actin monomer.3–5 Cofilinalso appears to increase the rate of loss of actinsubunits from the pointed end of the actin filament.6

In vivo, actin filaments are likely to be capped at one

thylrhodaminesin; ADF, actin

ng author:

lsevier Ltd. All rights reserve

or both ends over time, suggesting that severingmay be crucial for disassembly of actin filamentsand turnover of the actin filament network.5 Indeed,cofilin is essential for viability in a number of cellsystems, based on its function in actin filamentdynamics and actin-based motility.7,8

When actin-based motility was reconstituted frompure proteins, cofilin was a necessary component,along with Arp2/3 complex and capping protein.Increasing concentrations of cofilin first increasedand then decreased the rate of actin-based motility,9

showing that the balance of filament assembly anddisassembly is important. Correspondingly, themotility of glioblastoma tumor cells was reportedrecently to increase and then decrease with increas-ing over-expression of cofilin.10 These observationscorrelate with the bell-shaped dependence of therate of actin filament treadmilling on the concentra-tion of cofilin.6,11

Cofilin binding to the side of an actin filamentresults in conformational changes that have beenproposed to account for increased severing andpointed-end dissociation, the two mechanisms ofcofilin-induced actin depolymerization. These con-formational changes in the actin filament include ashift in the mean twist of actin filaments,12,13 andsubstantial changes in longitudinal and lateral inter-protomer contacts,13 which are predicted to lead

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http://dx.doi.org/10.1016/j.jmb.2006.10.102

1351Actin Filament Severing by Cofilin

to filament destabilization. Accordingly, filamentflexibility has been suggested as an importantvariable for the frequency of severing events alongthe filament, be those spontaneous14 or mediated bycofilin.6 On the other hand, recent differentialscanning calorimetry studies suggest a dual role forcofilin in actin turnover, revealing stabilization ordestabilization of the filament, depending on themolar ratio of cofilin to actin.15,16 Such F-actin stab-ilization should occur locally, at the filament sitesdirectly in contact with cofilin.17

Cofilin binds cooperatively to the actin filament,and cooperative effects of cofilin on the structure of thefilament have been revealed in measurements of thetwist and torsional flexibility of the filament,13,18,19

and through the presence of “tilted” filamentstructure in segments free of cofilin.20 Gelsolin alsoaffects the actin filament conformation coopera-tively,21,22 causing severing of the filament basedon breaks in longitudinal actin–actin contacts com-bined with distortions of lateral, across-strandsbonds.23

In this work, we asked how filaments are severedby cofilin and gelsolin. We performed directobservations of individual actin filaments withreal-time fluorescence microscopy, which allowedus to visualize the severing events. More important,this approach allowed us to vary the flexibility of thefilaments by changing their attachment to thesubstrate. Our results show that filament flexibilityand degree of saturation with cofilin and gelsolinhave important and different impacts on how thetwo proteins sever actin filaments.

Figure 1. TRC∼F-actin severing by cofilin. Snapshot imagsurface were taken before addition of cofilin and during the inmagnified images of the degradation of a single filament tacontained 2.0 mM K+-EGTA, 20 mM KCl, 2.0 mMMgCl2, 10 mglucose, 9×10003 units of catalase/ml, and 240 units of glucosHMM at a concentration of 5 μg/ml. White arrowheads indica

Results

The goal of this study was to investigate therelationship between cofilin interactions with actinfilaments and the severing of these filaments. Wehave focused on the effects of filament flexibility andfilament saturation by cofilin on the severingprocess, using gelsolin for comparison. We usedtetramethylrhodamine cadaverine (TRC)-labeledactin for fluorescence imaging of actin filaments inlieu of the commonly employed rhodamine phalloi-din, because phalloidin competes with cofilin forbinding to F-actin.24 Conveniently, the addition ofTRC label at Gln41 of actin (as well as other probesat this site)25,26 lowers the critical concentration foractin polymerization, stabilizes filaments, andreports on cofilin binding without impairing thatprocess.27 Thus, TRC∼actin filaments could beviewed easily in a fluorescence microscope atnanomolar concentration (5.0–20 nM) without sig-nificant depolymerization.28 For the observation ofsevering, TRC∼actin filaments were attached to aglass coverslip surface through binding to HMM, α-actinin, or CapZ adsorbed to that surface. Additionof yeast cofilin in assay buffer (pH 6.0–8.0) led to theappearance of filament fragments due to breakpoints introduced along the entire filament withinthe first 60 s to 120 s (Figure 1). Over time, thesefragments underwent additional severing and short-ening until almost complete disappearance (thedetached filament debris diffused away from thefocal plane). Severing activity was measured only onthe filaments that could be identified in fluorescence

es of TRC∼F-actin filaments bound to the HMM-coveredcubation for 2 min with yeast cofilin. The top panel showsken every 30 s after addition of cofilin. The assay bufferM DTT, 25 mMMops (total ionic strength 50 mM), 14 mMe oxidase/ml at pH 8.0. The glass surface was coated withte sites of severing. Identical data were obtained at pH 6.0.

1352 Actin Filament Severing by Cofilin

images taken before and after the addition of cofilin.Although the progress of severing with time wasqualitatively visible at all early time-points, fullstatistical analysis was carried out for filamentsexposed to cofilin for 2 min, at which point theirfragmentation could be measured best.Importantly, TRC-labeling of actin did not impede

or change the severing of filaments by cofilinsignificantly. This conclusion is supported by mea-suring the severing of 25% TRC-labeled filaments(copolymers of fully labeled and unlabeled actin),which resulted in similar, albeit somewhat smaller

Figure 2. Severing of actin filaments tethered (via CapZsnapshots of TRC∼F-actin being held by HMM (2) and CapZafter 2 mins of incubation with cofilin at 25 °C and pH 8.0 (forSnapshot images of TRC∼F-actin filaments were enhanced aprogram. The length of at least 150 filaments was measuredcontaining slide. Length distributions are plotted in lower pansmaller when the severed filaments were attached to the surfacCapZ (3). Length distributions are shown for TRC-F-actin filamand 75% unlabeled G-actin before the addition of 20 nM cofilinat 25 °C and pH 8.0 (5).

changes, in filament length distribution to thoseobserved for fully labeled actin (Figure 2).The ability to detect the severed filament frag-

ments in our system rests on the diffusional motionof fragment ends, which generates observable gapsbetween them. As stated above, and documented ina prior study showing pH-independence of filamentsevering by yeast cofilin,28 depolymerization wasunlikely to shorten the TRC-actin filaments signifi-cantly in our short-time-scale experiments (up to2 min). Nevertheless, we did not measure the kineticrates of actin severing by cofilin, since that would

) and attached (via HMM) to the glass surface. Several(3) were taken before the addition of 50 nM cofilin (1) andfilaments held to the surface by HMM (2) and CapZ (3)).

nd processed using the Sigma Scan Pro 5 image analysisin each case after the screen was calibrated using a grid-els 1–3. The mean filament length was two to three timese by HMM (2) rather than tethered via their barbed ends toents co-polymerized from 25% fully labeled TRC–G–actin(4) and after their incubation with 20 nM cofilin for 2 min


require more accurate accounting of all factors thatmay affect the data. Instead, we chose a singleoptimal time-point for a data-based, qualitative ana-lysis of the effects of actin flexibility and concentra-tion of cofilin on actin severing.

F-actin flexibility and the severing by cofilin andgelsolin

To investigate the role of filament flexibility in thesevering process, we varied the concentration ofHMM or α -actinin adsorbed to the coverslip as thetethering protein. In most of our experiments, weused the smallest amount of HMM (∼5 μg/ml) thatcould hold filaments at several points on theworkingsurface. Using the results of previous studies,29 weestimated that the approximate density of HMMmolecules attached to the working surface in ourexperiments was: ∼111(±33) μm−2.Using the assumption that actin filaments are

6 nm in diameter,30 and that the only crossbridgesthat interact with actin filament are those that arewithin 10 nm (i.e. within a 26 nmwide “band” couldreach the filament),29 we calculated the number ofmyosin heads that were able to interact with 1 μmlength of actin filament to be ∼6(±2) μm−1. Highconcentrations of HMM led to tethering at multiplesites along the filament, thus decreasing filamentflexibility and preventing reliable observation ofseparate filament fragments during its severing bycofilin. At a constant concentration of cofilin,increasing concentrations of HMM (up to 300 μg/ml) led to easily observed increases in the rate ofactin filament severing. However, because at higherconcentrations of HMM the diffusional motion offilament fragments (which creates the gaps betweenthem) is reduced, these observations probablyunderestimate the actual severing increase and,thus, are not analyzed in quantitative terms. Inaddition, we noted that those free actin filaments inthe solution that had not been attached or washedaway, were severed at a much slower rate than werethe surface-attached filaments. This suggests thatthe mode of filament attachment and the resultingvariation in filament flexibility may play an impor-tant role in severing by cofilin.To provide maximum flexibility of actin filaments

while tethering them to the surface, we employedCapZ, which was able to hold actin filaments ontothe surface through their barbed end. To reduce theBrownian motion of the filaments and thereby keepthem in focus, methylcellulose was added (up to0.2%, w/v) to the assay solution (methylcellulosehad no effect on the severing of HMM-tethered F-actin). Upon addition of yeast cofilin, we observedshortening of the tethered filaments (Figure 2). Somesevered filament fragments diffused away, so wecould not image all severing events. Indeed, thedebris of short filament fragments became verynoticeable above the surface plane as a result offilament severing. Severing of CapZ-tethered fila-ments was decreased compared to HMM-boundfilaments, as revealed by analysis of filament length

distributions after 2 min incubation with 50 nMcofilin (Figure 2). Qualitatively and quantitatively,the filaments tethered by one end, with CapZ, weresevered less efficiently than those tethered by HMMby multiple side attachments.Cofilins can promote depolymerization by

increasing the off-rate of actin protomers from thepointed end of the filament. In our experimentalapproach, this potential complication was mini-mized by the use of TRC-labeled actin, which isdepolymerized by cofilin much less than unlabeledF-actin.28 In previous work with TRC-actin, wefound that the decrease in filament length caused bycofilin corresponded closely with the increase in thenumber of filaments, implying little, if any, effect offilament depolymerization.28 In addition, in thatstudy, TRC-labeling of actin did not appear tochange its severing by cofilin, as actin labeled at adifferent amino acid residue with a differentfluorophore behaved in a similar manner. Thisconclusion is supported by showing similar frag-mentation of fully and partially labeled filaments bycofilin (Figure 2). Also, to exclude the possibility thatdepolymerization contributed to the filament short-ening observed above, we repeated the experimentsin the presence of the pointed-end capper tropomo-dulin. With both CapZ and tropomodulin present,filaments on the coverslip surface shortened, andshort fragments of filament were observed above thesurface, revealing severing activity.We tested the effect of filament flexibility on

severing with capped filaments. Actin filamentswere prepared in solution in the presence of CapZand tropomodulin, to cap both ends, and thenapplied to an HMM-covered surface, as above, torestrict flexibility. Again, we observed that severinginduced by cofilin was much faster than whenfilaments were tethered to the surface only by oneend via CapZ (same as in Figure 2). This resultconfirms that restriction of filament flexibilitypromotes the process of severing by cofilin.The preceding experiments were performed with

budding yeast cofilin. We repeated the key experi-ments with cofilin from nematodes (Caenorhabditiselegans UNC-60B) and from mice. Again, thesevering was greater when the actin filamentswere bound to the surface via multiple sideattachments with HMM, as opposed to single-endattachments with CapZ (data not shown). Thus, theeffect of filament flexibility on severing appears tobe a property of cofilins in general.To determine whether severing of actin filaments

increases with decreased filament flexibility ingeneral, we tested the action of gelsolin in similarexperiments. Increasing concentrations of HMMapplied to the glass surface decreased severing bygelsolin, unlike by cofilin (Figure 3). In contrast,when CapZwas used for tethering actin filaments tothe glass surface (equivalent to 0 μg/mL of HMM inFigure 3), the gelsolin-caused severing was fast andcomplete. These results are the opposite of what weobserved with cofilin, pointing to different mechan-isms of severing by cofilin and gelsolin.

Figure 4. Change of the mean length of actin filamentsafter incubation for 2 min with cofilin from differentsources. Several snapshots of TRC∼F-actin filamentsbeing held by HMM or CapZ were taken before theaddition of cofilin from different sources, and afterincubation for 2 min with cofilin at 25 °C. Images wereenhanced and processed as described for Figure 2. Thelength of at least 150 filaments was measured in each case.The change in the filament mean length distributions wasplotted versus concentration of cofilin added to theimmobilized actin filaments. (1) Yeast cofilin was addedto actin filaments attached to the glass surface by HMM;(2) yeast cofilin was added to actin filaments attached tothe glass surface by CapZ; (3) nematode cofilin was addedto actin filaments attached to the glass surface by CapZ.The data obtained for S3C mouse cofilin are not shown.

Figure 3. Changes in the mean actin filaments lengthversus HMM concentration (applied to the glass surface)after 2 min incubation with 100 pM gelsolin. Severalsnapshots of TRC∼F-actin filaments tethered by CapZ(0 μg/ml of HMM) or attached to HMMwere taken beforethe addition of 100 pM gelsolin, and after 2 min incubationwith gelsolin at 25 °C. Images were enhanced andprocessed as described for Figure 2. The length of atleast 150 filaments was measured in each case. The changein the filament mean length was plotted versus theconcentration of HMM applied to the glass surface beforethe addition of actin. Maximum filament length changewas observed when the severed filaments were tetheredvia their barbed ends to CapZ.


Complex dependence of F-actin severing on theconcentration of cofilin

To gain further insight into the mechanism ofsevering action of cofilin on F-actin, we used fluo-rescence microscopy to test the functional implica-tions of previous calorimetry results showing actinfilament destabilization and stabilization, depend-ing on filament saturation by cofilin.15,16 To this end,we varied the concentration of various cofilins (from20 nM to 1 μM) added to the actin filamentsimmobilized at a constant density of HMM. Initialincreases in cofilin concentration, from zero, causeda sharp rise in the severing activity (Figure 4), butfurther increases caused a gradual decrease insevering activity. We repeated these experimentsusing CapZ instead of HMM to tether actin filamentsvia their barbed ends to the glass surface, in order toavoid any competition between cofilin andHMM forbinding sites on the actin filament, and to retainmaximal torsional flexibility of actin filaments. Thesevering activity of cofilin was lower with CapZtethering, at any given concentration, compared tothat with HMM tethering (Figure 4). However, theshape of the profile of severing activity versus theconcentration of cofilin was similar in the two cases.Mouse (data not shown) and nematode (Figure 4)cofilins had levels of severing activities differentfrom that of yeast cofilin, but they all showed asimilar concentration-dependence with severingactivity, reaching a maximum at some optimal

concentration and decreasing at higher concentra-tions of cofilin (Figure 4). Thus, high concentrationsof cofilin stabilized actin filaments.15,16

We also tested filament severing in solution, wherethe actin:cofilin stoichiometry is known accurately.In these experiments, TRC∼F-actin (2 μM) wasmixed gently with cofilin (0.25–5.0 μM), and after30 s and 180 s, samples of the mixture were diluted(200-fold) with buffer containing 1 μM phalloidin tostop the severing process. The filaments were thenapplied to HMM-coated coverslips in order toobserve the filament lengths. Increasing concentra-tions of cofilin increased the filament severing rate,reaching a maximum at an approximately 1:2 molarration of cofilin:actin, and then decreasing graduallywith further increases in the cofilin:actin ratio,consistent with previous reports on the cofilin effecton actin treadmilling and depolymerization.6,11 Themagnitude of the decrease in the filament length wasless than in the case of “surface” experiments, per-haps because of incomplete stopping of the severingreaction by phalloidin, but the overall effect wassimilar. The results of these functional assayssupport the conclusions of the calorimetric studiesinwhich actin filamentswere stabilizedwhen satura-ted by cofilin and destabilized at lower concentra-tions of cofilin.15,16

We next tested the effect of gelsolin concentrationon filament severing activity in solution. In contrast


to cofilin, the gelsolin severing rate increased withincreasing gelsolin concentration (50 pM–100 nM).

Discussion

Filament flexibility and severing

The most important result of this study is theexperimental confirmation of a prior suggestion thatactin filament flexibility affects the ability of cofilinto sever the filaments.6 It appears that when theactin filament is tethered to a glass surface only viaits barbed ends, the filament can dissipate thestructural strain caused by the binding of cofilin.We found that filaments tethered at one end weresevered much more slowly than filaments attachedat multiple points along their sides, which did notallow dissipation of the induced strain. Twodifferent filament side-binding proteins, HMM andα-actinin, showed this effect, with the severing rateincreasing when the density of attachments wasincreased. Similar results were obtained for cofilinsfrom different sources. For the myosin complex withF-actin, several solution studies reported a dec-reased flexibility of filaments,31–33 although anotherstudy did not detect such myosin-induced changesin actin.34 Clearly, in our case, with filamentstethered to the glass slide at multiple points theresulting decrease in filament flexibility would begreater than in the solution.Our interpretation of these results, in line with the

speculation by Carlier et al.,6 is that multipleattachments of actin filaments restrict the relaxationof the cofilin-induced torsional strain, accounting formore efficient severing. This mechanism may beimportant in cells, where cofilin is essential for actinfilament dynamics and cytoskeleton reorganization.Tethering or cross-linking of actin filaments is acommon feature at the leading edge of cells, whereactin assembly is prominent.35 Nevertheless, asdemonstrated here, filament severing by cofilinoccurs also in filaments that are unconstrained byattachments.In contrast to cofilin, gelsolin severed uncon-

strained filaments much faster and more efficientlythan those held by multipoint attachments to theglass surface. This basic mechanistic differencebetween filament severing by gelsolin and cofilinmay be related to their different tasks, perhaps withone of the proteins participating in actin recycling instatic cells, and the other at the leading edge ofmotile cells.36

Filament saturation by cofilin and severing

Another important experimental finding of thisstudy is that cofilin severs actin filaments mostefficiently at subsaturating concentrations. Similaroptimal effects of substoichiometric ratios of co-filin to actin on filament treadmilling rate and the invitro motility of a reconstituted system have been

reported,6,9,11 but these effects were not connecteddirectly to the severing by cofilin or the mechanismof its action. Preferential filament severing atsubstoichiometric cofilin:actin ratios was proposedrecently,16,37 but without the direct observationprovided here. Our observation is consistent withthe recent discovery of dual effects of cofilin on actinfilaments in calorimetry studies.15,16 Those studiesrevealed allosteric and cooperative destabilization ofinterprotomer contacts in segments of actin filamentfree of cofilin, coupled with steric stabilization at thesites of cofilin binding via an interprotomer actin-cofilin-actin bridge between subdomain 2 andsubdomain 1 on adjacent protomers. The calorimetryresults showed that actin filaments saturated withcofilin were stabilized against thermal denaturation.On the other hand, subsaturating concentrations ofcofilin yielded two populations of actin filaments:one in which the filaments were stabilized, as in thecase of filaments saturated with cofilin, and anotherin which the filaments were destabilized coopera-tively. A critical prediction of these calorimetryresults is that filament severing should achieve amaximum rate at subsaturating concentrations ofcofilin and decline with further increases in theconcentration of cofilin. Our results here, with directobservation of filament severing, confirm this pre-diction (Figure 4). The same conclusion was reachedfrom examining the severing of actin filaments insolution, which was maximal at an approximately1:2 cofilin to actin molar ratio, suggesting that thesevering occurs preferentially at a site neighboringthe cofilin-bound actin. This result is important,because the binding ratios of cofilin to actin are welldefined in solution, but potentially less certain forfilaments on glass surfaces. Again, cofilin fromdifferent sources yielded similar results in all cases.In addition, gelsolin did not show this dependence offilament severing on concentration, confirming thatcofilin and gelsolin have different mechanisms forsevering.

A model for the mechanism of actin filamentsevering

Several distinctive features of the interaction ofcofilin with actin filaments must be included whenone considers models for the mechanism of filamentsevering:

(a) the high stability of severed and short cofilin-actin filaments;11

(b) the cooperative disruption of interprotomercontacts and the cooperative increase intorsional motion of filaments upon cofilinbinding;13,16,18,38

(c) the facilitation of filament assembly frompolymerization-impaired actin by cofilin,based on the bridging of adjacent actinprotomers;17,19

(d) the altered filament conformations in thepresence of cofilin (tilted state, torsionalstrain);18,19


(e) the allosteric destabilization of unoccupiedfilament segments at low densities ofcofilin.15,16,18,19

To account for these observations, we haveconfirmed that the actin filaments break preferen-tially at sites unoccupied by cofilin, but neighboringor close to the cofilin-bound actin.16,24,37 At thesesites, the structural perturbation (or strain) propa-gated from the cofilin-bound protomers is maximal,not yet dissipated over the length of the filaments.At these weak spots, energy from Brownian motionmay suffice to sever the filaments. The results of thepresent study are consistent with this strain-basedsevering model, since flexible filaments, tetheredonly by one end, should dissipate the cofilin-induced structural strain far more effectively thando less flexible filaments attached at multiple pointsalong their length.More importantly, the severing activity of several

cofilins showed a biphasic concentration depen-dence, first increasing, and then decreasing with theconcentration of cofilin. This result provides sup-port for the model of preferential filament severingat the unoccupied sites, assuming that the cumu-lative torsional strain increases up to a certain levelof cofilin saturation,18 while the probability ofconformational fluctuations that can sever filamentsat the sites occupied by cofilin is reduced. We notethat these results and our model may account forthe recently reported increase and then decrease ofthe motility of glioblastoma tumor cells withincreased overexpression of cofilin.10 The strongsevering inhibition seen at high concentrations ofcofilin is most likely a “by-product” of filamentstabilization by TRC attached to Gln41. Clearly,cofilin severs also fully saturated filaments, asdocumented before and in our solution tests.11

This severing may arise from similar, strained twiststates of F-actin, especially when the stabilizingcofilin bridge between two actin protomers istransiently broken, leaving cofilin bound to onlyone protomer.In striking contrast to cofilin, but not surprisingly,

gelsolin does not show such a complex severingbehavior. Filament flexibility may have little effecton gelsolin action, and the apparent inhibition ofsevering with increasing density of F-actin attach-ments to HMM (Figure 3) may result from decreaseddiffusional motion of filament fragments, therebyimpeding detection of severing events. Our resultsare consistent with the prevailing model in whichthe initial binding of gelsolin segment 2 (GS2) to F-actin, and the subsequent structural changes ingelsolin, culminate in the intercalation of gelsolinsegment 1 (GS1) between two actin protomers andthus, filament severing.23

Addendum

While this manuscript was under review, a studymaking similar observations was reported by

Andrianantroandro and Pollard.39 Their results areconsistent with our data on a complex mechanism ofactin filaments severing by cofilin, showing moreefficient filament severing at low concentrations ofcofilin and less efficient at high concentrations. Thisstudy showed increased probability of filamentsevering with their increased length, but did notconsider the effects of filament flexibility on thesevering.

Materials and Methods

Proteins

Skeletal myosin, actin, and heavy meromyosin (HMM)were prepared as described.40 α-Actinin was purchasedfrom Cytoskeleton Inc. (Denver, CO). Gelsolin waspurchased from Sigma Chemical Co. (St. Louis, MO) orpurified from bovine plasma.41

The labeling of Gln41 on skeletal actin with TRC wasperformed using Ca2+-independent bacterial transgluta-minase (TGase) (a generous gift from Dr K. Seguro;Ajinomoto Co, Kawasaki, Japan) as described.27 Theextent of labeling (∼100%) was determined using a TRCextinction coefficient of E544 nm=78 mM−1 cm−1. Gelsolin-capped filaments were obtained by polymerizing actin inthe presence of gelsolin (1:3000–1:200 molar ratios ofgelsolin to actin) with 1.0 mM CaCl2, 50 mM KCl and2.0 mM MgCl2.WT yeast cofilin was expressed and purified as

described.42 Nematode cofilin from Caenorhabditis elegans(UNC-60B) was a generous gift from Dr Shoichiro Ono(Emory University). S3C mouse (Mus musculus) cofilinwas a generous gift from Dr Gerard Marriott (Universityof Wisconsin). The concentrations of cofilin and unlabeledα-actin were determined spectrophotometrically, usingextinction coefficients E280

1% =9.2 cm−1 and E2901% =11.5 cm−1,

respectively.Tropomodulin was a generous gift from Dr Velia M.

Fowler (The Scripps Research Institute). Mouse actincapping protein (CapZ), (α1 and β2 subunits), wasexpressed and purified as described.43

In vitro severing assays

Fluorescence microscopy and actin covalently labeled atGln41 with TRC were used for direct observation offilament severing. In vitro severing assays were similar tothe previously described in vitromotility experiments.40 Aglass slide with spacers of double-sided Scotch tape and anitrocellulose-coated coverslip formed a ∼70 μl chamberopen on two sides. HMM (1–300 μg/ml), α-actinin(1.0 μM), CapZ (0.6–5.0 μM), or tropomodulin (0.6 μM)were applied to the chamber; then bovine serum albumin(2.0 mg/ml) was added to block the glass surface. TRC-labeled F-actin (TRC-FA) (20 nM) was then applied in anassay buffer (25 mM Mops (pH 7.4), 2.0 mM K+-EGTA,20 mM KCl, 2.0 mM MgCl2, 10 mM DTT; total ionicstrength 50 mM) containing an oxygen-scavenging systemat 25 °C.44 The unbound filaments were washed off withthe assay buffer. The slide was placed on a temperature-controlled stage (25 °C) of a Leica fluorescence microscopeand fixed with Scotch tape. The TRC-labeled actinfilaments were visualized using epifluorescence illumina-tion (100 W mercury arc lamp and a 100× numeric


aperture=1.3 objective), were imaged on a VE 1000 SITcamera (DAGE-MTI, Michigan City, IN), and wererecorded without enhancement on a Panasonic VHSVCR. Severing was initiated by adding cofilin or gelsolinin an assay buffer at the desired pH (6.0–8.0), atconcentrations ranging from 20 nM to 1.0 μM for cofilin,and from 50 pM to 100 nM for gelsolin. Assay buffercontaining cofilin or gelsolin was applied carefully to anopen side of the working chamber, and the excess liquidwas removed from the other side, while the field of viewremained unchanged and the filaments on the surfaceremained in focus. During image recording by VCR, thefilaments were exposed to light for short periods of time(10–15 s, every 30–60 s) to avoid photobleaching. Severalsnapshots were taken before the addition of cofilin, andover the first 2–5 min after its addition, using the ATI TV-tuner. The recorded images were averaged, enhanced,and analyzed using the SigmaScan Pro 5 program (SPSSInc.).

Experimental approach limitations

The goal of real-time observation of the effects of cofilinconcentration and actin filament flexibility on its severingwas achieved by using TRC-labeled actin in fluorescencemicroscopy experiments. The severed filament fragmentswere detected and measured because of the gapsgenerated between them through filament severing. Asconcluded before, for TRC-F-actin these gaps stem mainlyfrom diffusional motion of the ends of tethered fragments,rather than from the depolymerization of these fragments(within our short observation times).28 Although theevaluation of the effect of the concentration of cofilin onfilament severing was carried out at an empiricallydetermined optimal (and constant) tethering HMM con-centration, to allow for diffusional motion of filamentfragment ends, our data are interpreted mainly in terms ofqualitative trends and the overall behavior of the actin–cofilin system. This approach is dictated by experimentallimitations; i.e. the inability to measure very short filamentfragments either because of their diffusion from theobservation field or a size that is smaller than the limitof detection (∼250 nm). Similarly, in experiments invol-ving changes in the concentration of HMM (or α-actinin),the effect of such changes on the detection accuracy offilament severing measurements is difficult to calibrate,hence the results are presented either in qualitative terms(for cofilin) or are considered in terms of measurementlimitations (for gelsolin).

Acknowledgements

We thank Professor Earl Homsher for his help indata acquisition andMai Phan for excellent technicalassistance. *This work was supported by grantsfrom the USPHS to E. R. (GM-077190) and J. C.(GM38542) and the NSF to E. R. (MCB 0316269).

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Edited by J. Karn

(Received 25 August 2006; received in revised form 26 October 2006; accepted 26 October 2006)Available online 3 November 2006

actin filament severing by cofilin

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