Actin Filament Bundling by Fimbrin Is Important forEndocytosis, Cytokinesis, and Polarization in Fission Yeast*□S

Received for publication, March 10, 2011, and in revised form, May 12, 2011 Published, JBC Papers in Press, June 3, 2011, DOI 10.1074/jbc.M111.239004

Colleen T. Skau‡, David S. Courson§, Andrew J. Bestul‡, Jonathan D. Winkelman‡, Ronald S. Rock§,Vladimir Sirotkin¶, and David R. Kovar‡§1

From the Departments of ‡Molecular Genetics and Cell Biology and §Biochemistry and Molecular Biology, University of Chicago,Chicago Illinois 60637 and the ¶Department of Cell and Developmental Biology, SUNY Upstate Medical University,Syracuse, New York 13210

Through the coordinated action of diverse actin-binding pro-teins, cells simultaneously assemble actin filaments with dis-tinct architectures and dynamics to drive different processes.Actin filament cross-linking proteins organize filaments intohigher order networks, although the requirement of cross-link-ing activity in cells has largely been assumed rather than directlytested. Fission yeast Schizosaccharomyces pombe assemblesactin into three discrete structures: endocytic actin patches,polarizing actin cables, and the cytokinetic contractile ring. Thefission yeast filament cross-linker fimbrin Fim1 primarily local-izes to Arp2/3 complex-nucleated branched filaments of theactin patch and by a lesser amount to bundles of linear antipar-allel filaments in the contractile ring. It is unclear whether Fim1associates with bundles of parallel filaments in actin cables. Wepreviously discovered that a principal role of Fim1 is to controllocalization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized thatthe bundling ability of Fim1 is dispensable for actin patches butis important for the contractile ring andpossibly actin cables. Bydirectly visualizing actin filament assembly using total internalreflection fluorescence microscopy, we determined that Fim1bundles filaments in both parallel and antiparallel orientationsand efficiently bundles Arp2/3 complex-branched filaments inthe absence but not the presence of actin capping protein.Examination of cells exclusively expressing a truncated versionof Fim1 that can bind but not bundle actin filaments revealedthat bundling activity of Fim1 is in fact important for all threeactin structures. Therefore, fimbrin Fim1 has diverse roles asboth a filament “gatekeeper” and as a filament cross-linker.

The fission yeast Schizosaccharomyces pombe assemblesmultiple actin structures with distinct architecture and dynam-ics, including parallel bundles of filaments for polarization,antiparallel bundles for cytokinesis, and short branched net-works for endocytosis (1–4). Amajor challenge for the cell is to

simultaneously coordinate the self-assembly of diverse actinstructures from a single pool of monomers within a crowdedcytoplasm. Specification of actin organization may be initiallyestablished by different actin nucleating factors, includingformins and Arp2/3 complex (5–8). Subsequently, partiallyoverlapping sets of actin-binding proteins associate with andare required for generation,maintenance, and turnover of actinfilaments within all three structures (4). Furthermore, actin-binding proteins have distinct roles for different processes. Forinstance, coronin promotes branching by recruiting the Arp2/3complex to new, ATP-loaded, actin filaments but also syner-gizes with cofilin to turnover older ADP-rich filaments (9–11).Similarly, cofilin severs filaments and debranches Arp2/3 net-works but can also stimulate filament nucleation (12–14).Endocytic actin patches in yeast are short-lived, highly

mobile structures nucleated by the Arp2/3 complex (3, 15–18).A diverse set of actin-binding proteins that includes actin cap-ping protein, type I myosin motor, bundling proteins, and sev-ering proteins associates with actin patches and contributes torapid filament assembly and turnover (4, 19). Endocytic scaffoldproteins and actin-binding proteins accumulate to defined lev-els at actin patches in a tightly controlled sequence (3, 20–22).However, the precise biochemical and organizational contribu-tion of many of these proteins is unclear.We previously discovered that the actin-bundling protein

fimbrin Fim1 prevents access of the filament stabilizing proteintropomyosin Cdc8 to actin patches (23), which has importantfunctional consequences. First, inhibition of Cdc8 permits sev-ering by cofilin Adf1, which facilitates rapid filament turnoverin patches (23). Second, Fim1 prevents Cdc8 from inhibitingthe type-I motor Myo1, ensuring maximal Myo1 activity inactin patches (24). Antagonization of Cdc8 does not depend onthe bundling activity of Fim1 in vitro (23). Based on these find-ings and because patches are thought to be composed primarilyof branched filaments (25), we hypothesized that the bundlingactivity of Fim1 is dispensable for its role at actin patches infavor of its ability to regulate the localization of Cdc8 (23, 24).We, therefore, utilized a combination of in vitro and in vivo

approaches to examine the ability of Fim1 to bundle actin fila-ments, the polarity of filaments in Fim1-generated bundles, therole of Fim1 in Arp2/3 complex-mediated actin polymeriza-tion, and the necessity of bundling by Fim1 for diverse actinstructures. We found that Fim1 creates both parallel andantiparallel bundles, and Fim1 efficiently bundles Arp2/3 com-plex-mediated branched filament networks, which appear to

* This work was supported, in whole or in part, by National Institutes of HealthGrant RO1GM079265 (to D. R. K.) and RO1GM078450 (to R. S. R.) and Molec-ular and Cellular Biology Training Grant T32 GM007183 (to C. T. S.). Thiswork was also supported by a William Rainey Harper Fellowship (to C. T. S.)and an American Heart Association Predoctoral Fellowship (to D. S. C.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1, Figs. S1–S3, and Movies S1–S13.

1 To whom correspondence should be addressed: The University of Chicago,920 E. 58th St., Suite 915E, Chicago IL 60637. Tel.: 773-834-2810; Fax: 773-702-3172; E-mail: drkovar@uchicago.edu.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 30, pp. 26964 –26977, July 29, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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inhibit Arp2/3 complex filament branching. Capping proteinrelieves Fim1 inhibition of Arp2/3 complex in vitro. In disagree-ment with our original hypothesis, we found that the bundlingactivityofFim1 isnecessary forproperactinpatchdynamicsandatthe contractile ring. We also discovered a novel role for bundlingby Fim1 in actin cables. Together, these results indicate that, likeother actin-binding proteins, Fim1 has multiple roles at differentactin structures and is part of a complicated network of interac-tions between diverse actin-binding proteins.

EXPERIMENTAL PROCEDURES

Strains, Growth Conditions, Transformation, and CellularMethods—We used standard growth media (YE5S completemedium and EMM5Sminimal medium). Expression under thenmt1 promoter was regulated by the presence or absence of10.0 �g/ml thiamine (Sigma). Supplemental Table 1 lists theS. pombe strains used in this study. For tetrad dissection, cellswere sporulated on standard mating (SPA5S) plates for 36 h,then spread on complete medium (YE5S) plates, and tetradswere picked with a dissection scope. Plates were incubated at25 °C for 10 h or until tetrads germinated, at which point indi-vidual spores were separated.FimA2 Strain Construction—An S. pombe strain exclu-

sively expressing FimA2-mCherry was constructed as fol-lows. FimABD2-mCherry with a Nat resistance cassette wasamplified from genomic DNA prepared from the wild typeFim1-mCherry strain (VS888–3) using a forward primerhomologous to the Fim1 promoter and FimABD2 (5�-ACT-GCAAGCCACCCAAAGCACACATCGTGTGGTTTCGT-TTACTATACATTTTTTGGTCAAATTTTACTTTTA-AAGAAATGTTAAATGAAGAGGAAAAGCC-3�) and areverse primer homologous to the Fim1 sequence 68–150nucleotides downstream of the stop codon (5�-TATCTAT-AATATGACATACCACTAATACACTAGCGAGTCAAA-AGCGACGTACTCACTTCCGAAGTTGTTCGCAATAT-AAG-3�) using an Expand High Fidelity Plus PCR system(Roche Applied Science). Amplified product was insertedinto the fim1� strain (JW142) by homologous recombinationafter lithium acetate transformation. Recovered colonies wereassessed for 1) resistance to Nat, 2) red fluorescence, andfinally 3) PCR amplification of ABD2-mCherry-Nat fromgenomic DNA.Cell Microscopy—Cells were observed by differential inter-

ference contrast and epifluorescence microscopy with anOrca-ER camera (Hamamatsu, Bridgewater, NJ) on an IX-81microscope (Olympus, Tokyo, Japan) fitted with a 60� 1.4numerical aperture Plan-apo objective. Nuclei and septa werevisualized simultaneously using 4,6-diamidino-2-phenylindole(nuclei) and calcofluor (septa) as described (26). Actin fila-ments were visualized using the general actin marker GFP-CHD(rng2)2 (27).

To quantify endocytic actin patch dynamics and behavior ofMyo52–3�YFP particles, cells grown in exponential phase in

Edinburgh minimal (EMM) liquid media were spotted ontoEMM pads containing 25% gelatin on glass slides and sealed(Vasaline, Lanoline and Parafin wax). Images were acquiredwith a 100� 403/1.4 NA objective on a Zeiss Axiovert 200Mequipped with a Yokogawa CSU-10 spinning-disk unit(McBain) illuminated with a 50-milliwatt 473-nm DPSS laser(Cobolt). Time-lapse acquisitions were captured at 75-msintervals on a Cascade 512B EM-CCD camera (Photometrics)with MetaMorph (Molecular Devices). Analysis of patchdynamics was quantified for 50 randomly chosen patches foreach strain using ImageJ software (rsbweb.nih.gov) as previ-ously described (28). Only patches that originated and disap-peared during the course of the movie were quantified.For experiments reported in Figs. 6, C and D and 7A,

mCherry-tagged Fim1 and FimA2 patches and cells expressingGFP-CHD were imaged by spinning disk confocal microscopyusing an Ultraview VoX system (PerkinElmer Life Sciences)installed on a Nikon TiE microscope equipped with 100�/1.4NA lens. Time-lapse series of images through the middle of thecell were acquired at the rate of 1 frame/s, and Z-series span-ning the entire cell depth were acquired at 0.6-�m intervals.

Fluorescence intensity and position of mCherry-labeledpatches over time were tracked in single sections through themiddle of the cell using an eight-pixel-wide circular selectiontool. Intensity values were subtracted for cytoplasmic back-ground; all data were aligned to the start of patch movement ortime of patch appearance (for non-moving patches) and aver-aged at each time point. Total cell fluorescence intensities weremeasured from sections through the middle of the cells andsubtracted for background intensity. Images were processedusing ImageJ (rsbweb.nih.gov).Plasmid Constructs—Bacterial expression constructs for

fimbrin truncation mutants were prepared by PCR amplifi-cation (iProof; Bio-Rad) and restriction digest cloning into apET-21a vector (pET-21a-Fim Truncation-His). Insertswere sequenced to confirm fidelity of PCR amplification.The fission yeast tropomyosin Cdc8 expression plasmidpREP4x-cdc8 has been described (29). The bacterial expres-sion plasmid for cofilin Adf1 (pMW-SpCofilin) and GST-Wsp1 (pGEX2-(SpWsp1p)VCA) have been described (3, 12).The bacterial expression plasmid for fission yeast-capping

protein with a His6 affinity tag was prepared as follows. Acp1was amplified from pET3a-SpAcp1/2 (30), with a His6 affinitytag added to the forward primer (5�-(GACTCATATGCATC-ACCATCACCATCACGAAAAGGAGGCAATTTACAAAC-3�) and cloned into pET3a, then confirmedby sequencing.Acp2was cut from pET3a-SpAcp2 (30), and ends were blunted withKlenow (New England Biolabs, Ipswich, MA) and inserted intopET3a-HIS-Acp1 using EcoRV (New England Biolabs). Orien-tation was confirmed by restriction digest; the constructselected contained Acp1 and Acp2 ORF sequences oriented inthe same direction each expressed under the T7 promoter.Protein Purification—Tropomyosin (Cdc8) was purified

from fission yeast cells expressing pREP4X-cdc8 by successivesteps of boiling, ammonium sulfate precipitation, and ion-ex-change chromatography as described (29). Full-length recom-binant fission yeast fimbrin Fim1 andGST-Wsp1 were purifiedas previously described (3, 23).Native fission yeastArp2/3 com-

2 The abbreviations used are: CHD, calponin homology (CH) domain; TIRF,total internal reflection fluorescence; WASP, Wiskott-Aldrich syndromeprotein; CP, capping protein; a.u., arbitrary units; TMR, tetramethylrhod-amine-6-maleimide.

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plex was purified from a protease-deficient strain (TP150) bybinding to GST-N-WASP-VCA as previously described (3).Human fascin was purified as previously described (35).Recombinant fission yeast cofilin Adf1 was expressed frompMW-SpCofilin in Escherichia coli BL21-Codon Plus(DE3)-RPand purified by ammonium sulfate precipitation, S-200 gel fil-tration, and ion-exchange chromatography as described (12,31). Frozen cofilin slowly lost activity, soAdf1was usedwithoutfreezing.Tetramethylrhodamine-6-maleimide (TMR)-Fim1 was pre-

pared as described for OG488-AtFim1 (32), with a few modifi-cations. First, TMR (Sigma) was used in place of OG488-iodo-acetamide. Second, Talon Metal Affinity Resin (Clontech,Mountain View, CA) was used instead of glutathione-Sephar-ose. Third, because theHis6 tagwas not cleaved fromFim1 afterthe coupling reaction was stopped with DTT, the column wasresettled and washed with Talon extraction buffer (50 mM

NaH2PO4, pH 8.0, 500 mM NaCl, 10% glycerol, 10 mM imidaz-ole, 10 mM �-mercaptoethanol), and protein was eluted fromthe columnwithTalon elution buffer (50mMNaH2PO4, pH8.0,500 mM NaCl, 10% glycerol, 250 mM imidazole, 10 mM �-mer-captoethanol). Eluted protein was dialyzed overnight versusSource Q Buffer A (20 mM Tris-HCl, pH 8.5, 50 mM NaCl, 5%glycerol, 0.01%NaN3, 1mMDTT). Dialyzed protein was loadedonto a 5.0-mlMonoQ column (GEHealthcare) and elutedwitha linear gradient from 0 to 500 mM NaCl. Pure protein wasdialyzed into fimbrin storage buffer (20 mM HEPES, 1 mM

EDTA, pH 8.0, 200 mM KCl, 0.01% NaN3, 10% glycerol, 1 mM

DTT), flash-frozen in liquid nitrogen, and stored at �80 °C.Because TMR does not absorb at A280, protein concentrationwas checked as previously described (23). Percent labeled wasdetermined using a correction factor (A280 TMR/Amax TMR);yield was �90% labeled.Truncated versions of Fim1 and recombinant His6-Acp1/2

were purified from E. coli BL21-Codon Plus(DE3)-RP (Strat-agene) by expressing with 0.5 mM isopropyl �-D-thiogalactopy-ranoside (Sigma) for 16 h at 16 °C. Cells were harvested by sed-imentation, washed with phosphate-buffered saline, and storedat�80 °C. Pelletswere resuspended in extraction buffer (50mM

NaH2PO4, pH 8.0, 500 mM NaCl, 10% glycerol, 10 mM imidaz-ole, 10 mM �-mercaptoethanol) supplemented with 0.5 mM

phenylmethylsulfonyl fluoride and protease inhibitors andhomogenized in an Emulsiflex-C3 (Avestin, Ottawa, ON, Can-ada). The homogenate was clarified at 30,000 � g and 50,000 �g for 20min each, incubatedwithTalonMetal Affinity Resin for1 h at 4 °C, and loaded onto a disposable column. After a 50-mlwash with extraction buffer, protein was eluted with Talon elu-tion buffer (50 mM NaH2PO4, pH 8.0, 500 mM NaCl, 10% glyc-erol, 250 mM imidazole, 10 mM �-mercaptoethanol) and dia-lyzed overnight versus Source Q Buffer A (20 mM Tris-HCl, pH8.5, 50 mM NaCl, 5% glycerol, 0.01% NaN3, 1 mM DTT). Dia-lyzed protein was loaded onto a 5.0 ml Source Q column (GEHealthcare) and eluted with a linear gradient from 0 to 500 mM

NaCl. Pure protein was dialyzed into storage buffer (20 mM

HEPES, 1 mM EDTA, pH 8.0, 200 mM KCl, 0.01% NaN3, 10%glycerol, 1mMDTT), flash-frozen in liquid nitrogen, and storedat �80 °C.

Ca-ATP actin was purified from chicken skeletal muscle asdescribed (33). Gel-filtered actin was labeled on Cys-374 withpyrenyl iodoacetamide or Oregon Green iodoacetamide (Invit-rogen) (26). Immediately before each experiment, Ca-ATPactin was converted to Mg-ATP actin by the addition of 0.1volume of 2 mM EGTA and 0.5 mM MgCl2 for 2 min at 25 °C.

Extinction coefficients for Cdc8 (A280 � 2980 M�1 cm�1),Fim1 (A280 � 55140 M�1 cm�1), FimA12 (A280 � 50670 M�1

cm�1), FimA2 (A280 � 27960M�1 cm�1), FimA1 (A280 � 29700M�1 cm�1), FimEFA1 (A280 � 34170 M�1 cm�1), Arp2/3 com-plex (A290� 138570M�1 cm�1), GST-Wsp1 (A280� 80260M�1

cm�1), capping protein (A280 � 72000 M�1 cm�1), and Adf1(A280 � 13075M�1 cm�1) were estimatedwith ProtParam fromthe amino acid composition.Actin Filament Sedimentation—Actin filaments preas-

sembled for 2 h at 25 °C from 15 �MMg-ATP-actin monomerswere incubated with a range of concentrations of Fim1 for 20min at 25 °C and then spun at 100,000 � g (high speed) or10,000 � g (low speed) for 20 min at 25 °C. Equal volumes oftotal (before centrifugation), supernatant, and pellet were sep-arated by 12.5% SDS-polyacrylamide gel electrophoresis,stainedwith Coomassie Blue for 30min, and destained for 16 h.Gels were analyzed by densitometry on an Odyssey InfraredImager (LI-COR Biosciences, Lincoln, NE) as previouslydescribed (29). The amount of protein was quantified as deple-tion from the supernatant, converted to micromolar bound.Plots of the dependence of the concentration of bound Fim1 onthe concentration of actin were fit with a quadratic function.To determine the effect of nucleotide state on the affinity of

Fim1 for actin filaments, ADP-Pi actin filaments were preparedby assembling ADP-actin in the presence of 25 mM potassiumphosphate, pH 7.0 (15.37 mM H2KPO4 and 9.63 mM HK2PO4)with 19.9 mM KCl. Mg-ATP actin and Mg-ADP actin wereassembled in the presence of 25 mM potassium sulfate with 3.5mM KCl to keep the conductivity consistent (34).Fluorescence Spectroscopy—Actin assembly was measured

from the fluorescence of pyrene-actin with SpectramaxGeminiXPS (Molecular Devices) and Safire2 (Tecan) fluorescent platereaders. Spontaneous assembly has been described in detailpreviously (33). Briefly, assembly of a mixture of unlabeled andpyrene-labeled Mg-ATP-actin monomers was initiated by theaddition of 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM

imidazole, pH 7.0, and other proteins to be assayed (Fim1,Arp2/3 complex, Wsp1, etc.). Final protein concentrations andpercent pyrene-labeled actin are indicated in the figure legends.Calculation of spontaneous polymerization rates has beendescribed (33).Total Internal Reflection Fluorescence (TIRF) Microscopy—

TIRF images of a mixture of either 2.0 �M Mg-ATP-actin sup-plemented with 1.0 �M Oregon Green-labeled actin (Fig. 1) or1.0 �M Mg-ATP-actin supplemented with 0.5 �M OregonGreen-labeled Mg-ATP-actin (see Figs. 3–5), excited by eva-nescent wave fluorescence, were acquired every 10 s on anIX-71 microscope (Olympus) fit with through-the-objectiveTIRF illumination and an iXon EMCCD camera (Andor Tech-nology, SouthWindsor, CT) as described (33). Branching rateswere measured as total branches that appeared over a period of10 min and normalized to total amount of polymer.

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FIGURE 1. Fimbrin produces both parallel and antiparallel bundles. TIRF microscopy observation of filament bundling is shown. 2 �M Mg-ATP-actin with 1.0�M Mg-ATP-actin labeled with Oregon Green was assembled on slides coated with N-ethylmaleimide-myosin II. Corresponding supplemental Movies S1–S8are available online. A, quantification of Fim1 bundling as measured by skewness of pixel intensity (supplemental Movie S1) is shown. Left, shown arehistograms of the distribution of pixel intensities in 50 frames of a TIRF movie from 1000 to 1500 s after polymerization initiation in the absence or presence of250 nM Fim1. The red line indicates the mode of the data. Right, Fim1 increases pixel skewness, indicating a shift toward brighter pixels due to bundling. Valuesare the mean � S.D. B-H, time-lapse micrographs with time in seconds indicated in the upper right. Pink, blue, and yellow arrowheads indicate filament barbedends. Yellow brackets indicate bundled filaments. Bars, 5 �m. B, actin filaments growing in the absence of Fim1 (supplemental Movie S2). C, two filamentsbundled in a parallel fashion by 250 nM Fim1; 40.2% of two-filament bundles are parallel (supplemental Movie S3). D, two filaments bundled in anantiparallel fashion by 250 nM Fim1; 59.6% of two-filament bundles are antiparallel (supplemental Movie S4). E, a bundle containing both antiparallel (pinkand blue arrowheads) and parallel (yellow and pink arrowheads) filaments (supplemental Movie S5). F, a parallel bundle containing at least four filaments in thepresence of 250 nM fascin (supplemental Movie S6). G and H, bundles severed by the addition of 150 nM cofilin Adf1 (pink arrowhead). Severing events in G aremarked by pink dots (supplemental Movie S7). Severed filament barbed ends rapidly grow upon the addition of new 1.0 �M of 33% Oregon Green-labeled G-actin(green arrowhead) in H (supplemental Movie S8).

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Measuring Off-rates by TIRF—The method was modifiedfrom Courson and Rock (35). Bundles were prepared by incu-bating 10 �M actin filaments with 3 �M TMR-Fimbrin at 4 °Cfor 2 h in assay buffer (AB: 25mM imidazole, pH 7.5, 25mMKCl,1 mM EGTA, 4 mM MgCl2, and 10 mM DTT). Flow chamberswere sequentially loaded with N-ethylmaleimide-myosin, 1mg/ml BSA, actin (0.5�Mbundles or 0.1�M filaments), AB, andfinally observation buffer (AB plus 0.86 mg/ml glucose oxidase,0.14 mg/ml catalase, 9 mg/ml glucose). All proteins added dur-ing the experiment were diluted in observation buffer.

RESULTS

Fission Yeast Fimbrin Bundles Actin Filaments with MixedPolarities—To better understand the roles of fission yeast fim-brin Fim1 for various cellular processes, we biochemically char-acterized the actin binding and bundling properties of Fim1. Inagreement with previous reports (36, 37), high and low speedcosedimentation assays revealed that Fim1binds filamentswithsubmicromolar affinity irrespective of nucleotide state (supple-mental Fig. 1, A–C) and that Fim1 efficiently cross-links fila-ments into dense bundles composed ofmultiple filaments (sup-plemental Fig. 1, D–F).To determine the orientation of the filaments in these bun-

dles, we observed individual filaments growing in real time viaTIRFmicroscopy (Fig. 1, supplementalMovie 1).Wequantifiedthe amount of bundling in two ways. First, we measured thedistribution, or skewness, of pixel brightness in each sample.Samples containing actin alone have a Gaussian distribution ofpixel intensity, whereas the intensity of pixels in bundled sam-ples is shifted toward the right (skewed) (38, 39). In the presenceof 250 nM Fim1, the histogram of pixel intensity skews towardbrighter pixels as compared with actin alone (Fig. 1A, supple-mental Movie 1). Second, we quantified the percentage of fila-ments that coalign with at least one other filament over at least10 �m for at least 10 frames. In the absence of Fim1, filamentsrarely align (�3%) for more than two frames (Fig. 1B, supple-mental Movie 2). However, in the presence of 250 nM Fim1,�80% of the observed filaments coalign with other filaments(Fig. 1, C–E).Many actin cross-linking proteins including filamin, �-ac-

tinin, fascin, and other fimbrins have a strong selectivity for onefilament orientation over the other (35, 40–46). Following thegrowing barbed ends of individual filaments revealed the orien-tation of filaments within bundles (Fig. 1, C–F, supplementalMovies 3–6). Fim1 creates bundles containing filaments inboth parallel and antiparallel orientations without strong selec-tivity (Fig. 1,C andD, supplementalMovies 3 and 4).Of bundlescontaining only two filaments, 40.2% are parallel, whereas59.6% are antiparallel (the remaining 0.2% could not be deter-mined). Bundles with more than two filaments contain fila-ments in both orientations (Fig. 1E, supplemental Movie 5).Conversely, fascin creates bundles composed exclusively ofparallel filaments (Fig. 1F, supplemental Movie 6) (35, 43).Because Fim1 binds to structures composed of both paralleland antiparallel filaments in vivo, this may be an essential fea-ture of Fim1.Fim1 plays roles in the contractile ring and in actin cables

(23) (see below), where filaments are bundled (1, 2). Given that

filament severing by cofilin Adf1 is critical for turnover of actinfilaments (12, 23, 47, 48), we examined the ability of Adf1 tosever Fim1 bundles by TIRF microscopy. Adf1 is able to severindividual filaments in bundles (Fig. 1G, supplemental Movie7). The addition of new actin monomers to severed bundlesresults in the rapid thickening of bundles as severed barbedends elongate and new filaments are added to the bundle (Fig.1H, supplemental Movie 8). Therefore, Adf1-mediated sever-ing can stimulate either rapid filament turnover or bundleexpansion depending upon the availability of monomeric actin.Fim1 Is Stable in Bundles—We used a TIRFmicroscope cou-

pled to a flow chamber to assess the dissociation of Fim1 fromsingle filaments and filament bundles (Fig. 2). First, weobserved binding of labeled TMR-Fim1 to individual unlabeledactin filaments that were immobilized on a coverslip. TMR-Fim1 was washed over the filaments, and fluorescent intensityon those filaments was recorded (Fig. 2A, black curve). A singleexponential curve fit revealed a dissociation constant of0.043 � 0.001 s�1. Then we washed TMR-Fim1 off of the samefilaments with buffer (Fig. 2A, red curve). TMR-Fim1 rapidlydissociates from filaments with an identical off rate of 0.043 �0.001 s�1.We examined the stability of Fim1 in bundles by immobilizing

bundles assembledwithTMR-Fim1 on a coverslip andwashing in

FIGURE 2. Fimbrin is stably associated with actin filament bundles. Deter-mination of the fimbrin off-rate from single filaments and bundles is shown.A, a solution of 0.1 �M TMR-fimbrin was washed over single actin filaments(black). A single exponential curve fit is in black. The dissociation constantmeasured from this experiment was 0.043 � 0.001 s�1. Single filamentscoated with TMR-fimbrin were washed with observation buffer (red). Fimbrindissociated with an off-rate of 0.043 � 0.001 s�1. B, TMR-Fimbrin bundleswere washed with different concentrations of unlabeled fimbrin: 1 �M (blue,koff � 0.023 � 0.003 s�1), 5 �M (green, koff � 0.025 � 0.001 s�1), 10 �M (orange,koff � 0.025 � 0.003 s�1), 20 �M Fim1 (magenta, koff � 0.028 � 0.001 s�1), andno significant TMR-fimbrin dissociation with buffer only (red, koff � 0.0028 �0.002 s�1).

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0–20�MunlabeledFim1 (Fig. 2B).Withbufferonly,TMR-Fim1 ishighly stable in bundles (koff � 0.0028 � 0.002 s�1). TMR-Fim1dissociates frombundles in the presence of a range of unlabeledFim1 concentrationswith koff� 0.023–0.028 s�1 (Fig. 2B). Thisindicates that increasing concentrations of free fimbrin do notincrease the rate of bundle turnover but instead increase theamplitude of turnover.Fim1 Inhibits Polymerization by the Arp2/3 Complex—Fim1

is abundant at endocytic actin patches, which are thought to becomposed of Arp2/3 complex-branched filament networksrather than bundles (3, 25, 36, 49).We, therefore, examined theability of Fim1 to bundle Arp2/3 complex-generated branchednetworks. TIRF microscopy assays revealed Fim1 efficientlybundles branched filament arrays (Fig. 3A, supplementalMovie9). Fim1 produces both parallel and antiparallel bundlesbetween adjacent branched networks. Fim1 also efficientlybundles filaments within a single branched “tree,” where allfilaments can be traced back to an original mother filament.Branches bundle to each other (Fig. 3A, arrowhead) as well asback on to the mother filament (Fig. 3A, arrow). Because fila-ments within an actin tree are oriented in the same direction,the percentage of parallel bundles created by Fim1 increases

from 59.6% with actin alone to 73% in the presence of Arp2/3complex.Because Arp2/3 complex nucleates new filaments by bind-

ing to the sides of existing filaments (5, 8), we hypothesizedthat bundling might inhibit Arp2/3 complex by decreasingavailable binding sites. In the presence of the activator GST-Sp(Wsp1)VCA, hereafter referred to as Wsp1, Arp2/3 com-plex strongly promotes actin assembly (Fig. 3B) (3, 50). How-ever, increasing concentrations of Fim1 inhibit stimulationof actin polymerization by Arp2/3 complex andWsp1, even-tually reducing the rate to that of Arp2/3 complex in theabsence of Wsp1 (Fig. 3B). Quantification of the initial slopeof the polymerization reactions revealed a concentration-dependent inhibition of polymerization by Fim1 (Fig. 3C).Conversely, Fim1 does not affect spontaneous polymeriza-tion of actin monomers alone (see Fig. 5C). As expected,Arp2/3 complex is also strongly inhibited by tropomyosinCdc8 (Fig. 3C) (51).As predicted, TIRF microscopy assays revealed that Fim1

significantly decreases the overall branching rate of Arp2/3complex (Fig. 3, D and E, supplemental Movie 10). Conversely,Fim1 does not affect the rate of debranching (data not shown).

FIGURE 3. Fimbrin inhibits Arp2/3 complex-mediated actin filament assembly. A, shown is a TIRF microscopy observation of a branched actin filamentnetwork (25 nM Arp2/3 complex 100 nM GST-SpWsp1VCA) bundled by 250 nM Fim1. The arrow indicates a branch bundling onto the mother filament. Thearrowhead indicates neighboring parallel branches bundling onto each other. Wedges indicate antiparallel bundling between branches. Seconds are indicatedat the upper left. Bar, 5 �m. Corresponding supplemental Movie S9 is available online. B and C, spontaneous assembly of 20% pyrene-labeled 2.5 �M Mg-ATP-actin monomers. B, assembly of actin monomers with 25 nM Arp2/3 complex alone (thick curve) or Arp2/3 complex and 100 nM Wsp1 in the absence (F) orpresence of 50 nM (�), 1 �M (�), or 5 �M Fim1 (‚). C, dependence of the initial assembly rate on concentration of Fim1 or tropomyosin Cdc8. D and E, TIRFmicroscopy observation of Arp2/3 complex-mediated branching. D, a plot of the branching rate (branches �M

�1 min�1) by Arp2/3 complex and Wsp1 in theabsence or presence of 250 nM Fim1. Values are the mean � S.D. E, time-lapse micrographs of actin filament branching by 25 nM Arp2/3 complex and Wsp1alone (top) or in the presence of 250 nM Fim1 (bottom). Time in seconds is indicated at the top left. Bar, 5 �m. Corresponding supplemental Movie S10 is availableonline.

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Capping Protein and Fimbrin Are Antagonistic in Vitro—Al-though fimbrin inhibits branching by Arp2/3 complex in vitro,Arp2/3 complex efficiently stimulates actin assembly at endo-cytic actin patches in cells (3, 22). We reasoned that filamentsbranched at a 70° angle must reach a critical length before theycan be bundled. Actin capping protein (CP) is a major compo-nent of actin patches (30, 36), which might prevent bundling ofbranched filaments by restricting their length.Initially, low speed sedimentation assays were used to assess

whether the bundling efficiency of Fim1 is generally affected byfilament length. CP significantly reduces Fim1 bundling effi-ciency by decreasing filament length, although CP does notcompletely prevent bundling (Fig. 4A). High concentrations ofFim1 bundle short-capped filaments to essentially the sameextent as low concentrations of Fim1 bundle longer filaments(Fig. 4A).We utilized TIRFmicroscopy to examine the effects of CP on

Arp2/3 complex-mediated branched actin filament assembly inthe presence of Fim1 (Fig. 4, B and C, supplemental Movie 11).

Although CP reduces filament length by inhibiting barbed endelongation, as observed previously, CP increases branching byArp2/3 complex and Wsp1 (52–54). Likewise, the addition ofCP restores branching in the presence of Fim1 to similar levelsobserved in the absence of Fim1 (Fig. 4, B and C, supplementalMovie 11). We hypothesize that CP restores Arp2/3 complex-mediated filament branching by reducing filament length and,therefore, bundling by Fim1.The Two Actin Binding Domains of Fim1 Are Not Identical—

Arp2/3 complex-generated branched actin filaments in actinpatches are not thought to form dense bundles (25), which is inpart due to prevention of Fim1-mediated bundling by CP (Fig.4). Furthermore, amajor role of Fim1 is to exclude tropomyosinCdc8 from endocytic patches (23, 24). Because the bundlingactivity of Fim1 is not required to prevent tropomyosin frombinding actin filaments in vitro, we hypothesized that the bun-dling activity of Fim1 is dispensable in vivo (23). We, therefore,replaced fim1 at its endogenous genomic locuswith a version offimbrin that contains only one of two actin binding domains

FIGURE 4. Capping protein restores rapid Arp2/3 complex-mediated actin polymerization in the presence of Fim1. A, CP reduces Fim1-mediatedbundling efficiency. Dependence of actin in the low speed sedimentation supernatant (non-cross-linked actin) on the concentration of Fim1 in the absence (F)or presence (�) of 100 nM CP is shown. Inset, plot with a magnified x axis. B, TIRF microscopy time-lapse micrographs of actin filament branching by 25 nM

Arp2/3 complex and 100 nM Wsp1 in the presence of 100 nM CP (left), 250 nM Fim1 (center), or CP and Fim1 (right). Time in seconds is indicated at the upper right.Bar, 5 �m. Corresponding supplemental Movie S11 is available online. C, plot of branching rate (branches �M

�1 min�1) in the presence of 25 nM Arp2/3 complexwith 100 nM Wsp1 and the indicated combinations of 100 nM CP and 250 nM Fim1. Values are the mean � S.D.

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(see Figs. 6 and 7). However, we first biochemically character-ized truncated constructs of Fim1 to determine the in vitroproperties of each isolated actin binding domain (Fig. 5).Fim1 bundles actin filaments as amonomer. Fimbrin is com-

posed of anN-terminal headpiece followedby two�250-aminoacid actin binding domains, each composed of two tandem cal-ponin-homology (CH) domains (Fig. 5A) (36, 49). We clonedand purified a series of constructs that either lack the N-termi-nal headpiece (FimA12) or contain a single actin bindingdomain (FimA1 and FimA2) or the N-terminal headpiece withthe N-terminal actin binding domain (FimEFA1) (Fig. 5A)(36, 49).We tested the ability of each construct to bind and bundle

actin filaments utilizing in vitro cosedimentation assays (Fig.5A, supplemental Fig. 2,A andB).We determined the affinity ofeach construct for F-actin by incubating a single concentrationof fimbrin with a range of concentrations of F-actin followed byhigh speed centrifugation. Full-length Fim1, FimA12, FimA2,and FimEFA1 all bind to and sediment with F-actin, althoughwith different affinities. The affinity of full-length Fim1 foractin filaments is �650 nM. FimA12 binds to actin approxi-mately twice as tightly as full-length fimbrin (Kd �390 nM),whereas FimA2 has approximately the same affinity for actin asfull-length fimbrin (Kd �650 nM). FimA1, as has been previ-ously reported (36), does not bind F-actin strongly (Kd � 3�M).Interestingly, FimEFA1 binds to actin a bit better than full-length Fim1 (Kd � 220 nM).As expected, both full-length fimbrin and FimA12 (contain-

ing two actin binding domains) cross-link F-actin with similarefficiency in low speed cosedimentation assays (Fig. 5A, supple-mental Fig. 2, B and C). Neither FimA1 nor FimA2 (each con-taining an isolated actin binding domain) strongly cross-links

actin filaments. Surprisingly, FimEFA1 (containing the N-ter-minal headpiece and the isolated N-terminal actin bindingdomain) cross-links actin filaments.We next examined the effects of fimbrin truncations on the

spontaneous assembly of actin monomers (Fig. 5, B and C).Full-length Fim1, FimA12, and FimEFA1 do not affect thespontaneous polymerization of actin monomer. FimA1, whichdoes not bind filaments well, slightly inhibits polymerization.Conversely, FimA2 significantly increases the actin polymeri-zation rate. To examine the mechanism by which FimA2increases actin assembly, we visualized actin filament assemblyby TIRF microscopy (Fig. 5D). As compared with actin alone,FimA2 significantly increases the total number of filaments,suggesting that FimA2 increases nucleation (Fig. 5D). However,FimA2 does not entirely eliminate the initial lag step of bulkassembly assays (Fig. 5B), suggesting that FimA2 likely stabi-lizes the actin trimer rather than actin dimer. Neither Fim1 norFimA2 affect the filament elongation rate (data not shown).A Fimbrin Truncation Only Partially Substitutes for Full-

length Fim1 in Vivo—In general, the importance of filamentcross-linking has been assumed rather than rigorously tested.In fission yeast, cross-linked filaments are found at the con-tractile ring and cables (1, 2), but endocytic actin patches arenot thought to be composed of bundles (25). Given the abun-dance of Fim1 in actin patches, which apparently lack bundledfilaments (25), we hypothesized that the primary role of Fim1 inpatches is to prevent ectopic localization of tropomyosin Cdc8(23). Conversely, the bundling activity of Fim1 is likely to beimportant for the contractile ring and possibly cables. Todetermine whether the bundling activity of Fim1 is requiredfor any of the actin structures, we directly compared threestrains: 1) a strain exclusively expressing truncated FimA2

FIGURE 5. The two actin binding domains of Fim1 are not identical. A, left, domain organization of constructs used in this study. Numbers under constructsindicate amino acids. Right, affinity for filaments (Kd) is based on two separate high speed sedimentation assays, and the general ability to cross-link filamentsis based on low speed sedimentation assays (see supplemental Fig. 2). B and C, spontaneous assembly of 20% pyrene-labeled 2.5 �M Mg-ATP-actin monomers.B, polymerization of actin alone (thick curve) or in the presence of 2.5 �M FimA12 (�), 2.5 �M FimA2 (f), or 2.5 �M FimA1 (F). C, initial polymerization rates inthe presence of the indicated constructs. Values are the mean � S.D. D, TIRF microscopy time-lapse micrographs of increased filament nucleation in thepresence of FimA2 as compared with actin alone. Time in seconds is indicated at the top right. Bar, 5 �m.

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tagged with mCherry (fimA2) integrated at the endogenouslocus, 2) a wild type strain carrying full-length Fim1 taggedwith mCherry, and 3) a strain deleted of fim1 (fim1�) (Fig.6A). In general we found that although FimA2 is able toprevent ectopic localization of Cdc8 to endocytic actinpatches, the fimA2 strain otherwise phenocopies the fim1�strain, indicating that bundling is necessary for actinpatches, the contractile ring, and actin cables.Similar to full-length Fim1-mCherry (36, 49), FimA2-

mCherry localizes primarily to endocytic actin patches (Fig. 6A,top). FimA2-mCherry also localizes weakly to cable-likestructures.We next observed tropomyosin GFP-Cdc8 in live cells to

examine the ability of FimA2 to inhibit the binding of Cdc8 toendocytic actin patches (Fig. 6A, bottom). In wild type Fim1-mCherry cells, GFP-Cdc8 associates with the contractile ringbut not with endocytic actin patches (23, 55). However, in theabsence of Fim1 (fim1�), GFP-Cdc8 localizes to the contractilering as well as ectopically to endocytic actin patches (23). Con-versely, as expected from in vitro assays (23), GFP-Cdc8 ismostly restricted to the contractile ring in fimA2 cells (Fig. 6A,bottom).To determine whether FimA2 is capable of fully replacing

full-length Fim1 at endocytic actin patches, we tracked the life-time and dynamics of individual patches using the general actinmarker GFP-CHD(rng2) (Fig. 6B, supplemental Movie 12). Inwild type cells, patches have a lifetime of about 9.5 s. Wild typepatches originate near the cell cortex, where they remain forabout 60% of their lifetime before propelling into the cell inte-rior (3, 23). Conversely, patches in fim1� cells have a lifetime of16 s, and only about 20% of patches internalize (23). Patches infimA2 cells exist for 17 s, almost twice as long as in wild typecells. Furthermore, only 25% of patches in fimA2 internalizecompared with �90% of wild type patches. Therefore, despitethe absence of significant Cdc8 on actin patches in fimA2 cells,their behavior strongly resembles fim1� patches, indicatingthat fimbrin-mediated cross-linking is important for actinpatch function.We then compared the total intracellular concentration and

dynamics of full-length Fim1-mCherry and FimA2-mCherry inactin patches using quantitative live cell imaging (Fig. 6, C andD) (22). Even though FimA2-mCherry is expressed under thecontrol of the native promoter, total intracellular concentrationof FimA2-mCherry is �3-fold lower compared with Fim1-mCherry (Fig. 6C), resulting in the accumulation of �4-foldless FimA2 in actin patches than Fim1 (Fig. 6D). The dynamicsof FimA2-labeled patches is quite different than Fim1-labeledpatches. FimA2 patches persist for �2-fold longer than Fim1patches, and FimA2 patches have a pronounced internalizationdefect (Fig. 6D).Fimbrin-mediated Filament Bundling Is Important for Cell

Division—The othermajor fission yeast actin-bundling protein�-actinin Ain1 localizes to the contractile ring (49). AlthoughAin1 is not essential for life, deletion of both ain1 (ain1�) andfim1 (fim1�) is synthetically lethal due to cytokinesis defectspredicted to result from the loss of bundling activity in thecontractile ring (49). To examinewhether FimA2 can substitutefor full-length fimbrin in ain1� cells, we crossed ain1� with

full-length (Fim1-mCherry), fim1�, or fimA2 (FimA2-mCherry) (supplemental Fig. 3). We recovered Fim1-mCherryain1� strains as expected, but all predicted double mutantfim1� ain1� cells are inviable. Similarly, all predicted double

FIGURE 6. The cross-linking activity of fimbrin is important for endocyticactin patches. A, top, fluorescence micrographs of mCherry-tagged full-length Fim1 or fimbrin fragment FimA2. Arrows indicate actin cable-like asso-ciation of FimA2. Bottom, tropomyosin GFP-Cdc8 in cells expressing full-length Fim1-mCherry or fimbrin fragment FimA2-mCherry or fim1� cells.Bars, 5 �m. B, actin patch dynamics of cells expressing the general actinmarker GFP-CHD(rng2). Corresponding supplemental Movie S12 is availableonline. Top, plot of actin patch lifetime for full-length fim1, fim1�, and fimA2cells (n � 30 patches for each strain). Bottom, the position over time (0.075 sbetween positions) for representative full-length fim1, fim1�, and fimA2 actinpatches. Dashed lines indicate cell cortex. C and D, comparison of the dynam-ics of Fim1-mCherry and FimA2-mCherry in actin patches. C, fluorescentmicrographs of cells expressing Fim1-mCherry (top) and FimA2-mCherry(bottom) and the corresponding montages of a time series of a single actinpatch (white boxes) at 1 frame/s. Numbers to the right are mean total cell fluo-rescence intensities (�S.D.). Arrowheads mark patch appearance, initiation ofmovement, and disappearance. Bars, 1 �m. D, individual patch (thin dashedlines) and average patch (thick lines) time courses of fluorescence intensity(top) and distance moved (bottom) for 12 Fim1-mCherry (left) and 14 FimA2-mCherry (right) actin patches.

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mutant cells from 24 tetrads of fimA2 ain1� are also inviable,indicating that FimA2 is not sufficient for life in the absence of�-actinin ain1. Therefore, the redundant bundling activities ofAin1 and Fim1 appear to be important for cytokinesis.Fimbrin Is Involved in the Maintenance of Actin Cables—Al-

though fimbrin Fim1 has not been detected in cables, fim1�cells have mild polarity defects, and FimA2-mCherry weaklylocalizes to cables (Fig. 6A) (36, 49). We did not observe grossactin cable defects in fim1� and fimA2 cells expressing the gen-eral actin marker GFP-CHD(rng2) (Fig. 7A) (23). However,whereas actin cables are primarily oriented parallel to the longaxis of wild type cells, actin cables in fim1� and fimA2 cellsappear qualitatively more randomly oriented (Fig. 7A).

To quantify the integrity of actin cables, we examined motil-ity of the type VmyosinmotorMyo52 (Myo52–3�YFP), whichwalks exclusively along actin cables and has a role in cell polar-ity (Fig. 7, B–G, supplemental Movie 13) (56–58). In wild typecells with full-length Fim1 (Fim1-mCherry), Myo52–3�YFPparticles are localized primarily to the cell poles or division siteand move linearly along the length of the cell, with an averagelifetime of �3.5 s (Fig. 7, B–G). In both fim1� and fimA2 cells,Myo52–3�YFPparticles are dispersed throughout the cell (Fig.7, B and C). Although more than 30% of Myo52–3�YFP parti-cles remain stationary in fim1� and fimA2 cells (Fig. 7D), thosethat do move are frequently perpendicular to the long axis andoften change direction (Fig. 7, E and F). Furthermore, the life-

FIGURE 7. Actin cable integrity is likely impaired in cells lacking full-length fimbrin. A, comparison of actin cables in full-length (FL) Fim1-mCherry, fim1�,and fimA2 cells. Maximum intensity projections of a Z-series through cells expressing the general actin filament marker GFP-CHD(rng2). Bar, 3.5 �m. B, left,maximum intensity Z projections of Myo52–3�YFP distribution in full-length Fim1-mCherry, fim1�, or fimA2 cells. Right, time-lapse micrographs of Myo52–3�YFP particles in full-length Fim1-mCherry, fim1�, or fimA2. Seconds are indicated at the upper left. Bar, 1 �m. Corresponding supplemental Movie S13 isavailable online. C, Myo52–3�YFP fluorescence value distribution across the length of interphase cells (n � 25 for each strain). Bar, 5 �m. D, the fraction ofMyo52–3�YFP particles that remain stationary throughout their lifetimes in full-length Fim1-mCherry, fim1�, or fimA2 cells (n � 25 particles for each strain).Values are the mean � S.D. E, the position over time (0.8 s between marks) for representative Myo52–3�YFP particles. Patch start position is indicated in red,and end position is indicated by blue/violet. F, the fraction of Myo52–3�YFP moving particles that change direction in full-length Fim1-mCherry, fim1�, orfimA2 cells (n � 25 for each strain). Values are the mean � S.D. G, the average duration of Myo52–3�YFP particle movement in full-length Fim1-mCherry, fim1�,or fimA2 cells (n � 25 for each strain). Values are the mean � S.D.

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time of Myo52–3�YFP particles in fim1� and fimA2 cells ismore than double that of wild type cells (Fig. 7G). These dataare consistentwith Fim1bundling activity playing a role in actincable organization.

DISCUSSION

Filament cross-linking proteins are fundamental compo-nents of the actin cytoskeleton. In fission yeast, fimbrin Fim1plays a role in endocytic actin patches and the contractile ring,although there is little evidence about a possible involvement inpolarizing actin cables. Previously we discovered that a majorrole of Fim1 is to regulate the localization of tropomyosin Cdc8(23), which allows cofilin-mediated filament severing andensures type-I myosin motorMyo1 activation (23, 24). Becauseinhibition of Cdc8 does not require that Fim1 cross-links fila-ments (23), we hypothesized that the cross-linking activity ofFim1 might not be important for actin patches.Through a combination of in vitro and in vivo approaches, we

determined that Fim1 bundles filaments in both parallel andantiparallel orientations, and the ability of Fim1 to cross-linkfilaments is in fact important for patches, rings, and cables.Furthermore, in vitro Fim1 bundles Arp2/3 complex-mediatedbranched networks and inhibits Arp2/3 complex-mediatedactin assembly. However, actin CP prevents Fim1 from effi-ciently bundling branched filaments and inhibiting Arp2/3complex. We, therefore, postulate that CP contributes to rapidpolymerization by Arp2/3 complex in the presence of Fim1 invivo. The diverse roles of Fim1 in regulating tropomyosin local-ization and cross-linking filaments as well as affecting Arp2/3complex-mediated polymerization highlight the importance ofcoordinating the balance of actin-binding proteins for diversecellular roles (23, 24).The Bundling Properties of Fimbrin Are Likely Tailored for Its

Cellular Role—In general, the selectivity of particular filamentorientations by bundling proteins in vitromatches their in vivolocalization. For example, chicken and mammalian fimbrinsstrongly favor parallel bundles in vitro (42, 44, 45) and localizeto parallel bundled filaments in the inner core of intestinalmicrovilli (59). Fascin also preferentiallymakes parallel bundlesin vitro (Fig. 1F) (35, 41, 43) and localizes to cellular structurescomposed of parallel bundles including filopodia (60, 61). Fis-sion yeast Fim1 localizes to endocytic patches composed ofbranched filaments with barbed ends thought to be orientedtoward the membrane as well as the contractile ring composedof antiparallel bundles of filaments and likely actin cables com-posed of parallel filaments (Figs. 6A and 7) (2, 25, 36, 49). Wefind that in vitro Fim1non-selectively bundles filaments in bothorientations (Fig. 1).It is possible that either the bundling protein defines filament

orientation of a structure or that the pre-existing filamentarchitecture selectively recruits particular actin-bundling pro-teins. These possibilities are notmutually exclusive. Nucleationfactors establish the initial filament orientation that recruitsparticular bundling proteins, which would in turn strengthenand maintain the existing architecture. Many bundling pro-teins, which do not select a particular orientation, are alsorecruited to specific cellular structures. We, therefore, proposethat bundling proteins are initially recruited to new filaments

due to intrinsic properties conferred by the nucleation factorsuch as filament twist, flexibility, or nucleotide state (62). Onceat a structure, actin-bundling proteins reinforce existing fila-ment orientation, thus organizing the architecture of actinstructures as they assemble rather than being passivelyrecruited to a completed structure.Role of Fimbrin in Arp2/3 Complex-nucleated Structures—

Yeast endocytic actin patches are composed primarily of short-branched filaments nucleated by the Arp2/3 complex, similarto the leading edge of motile cells (4, 20, 63). Short-branchedfilaments at endocytic sites are thought to help promote vesicleinternalization (20, 63, 64). Unlike lamellipodium of motilecells (65), fimbrin concentrates in yeast endocytic actin patches(3, 36, 49, 66, 67). In both budding and fission yeast, deletion offimbrin severely disrupts actin patch dynamics (23, 68, 69).Because Arp2/3 complex nucleates new filaments from the

side of pre-existing mother filaments (70, 71), bundling couldinhibit the Arp2/3 complex by masking mother filament bind-ing sites. We found that Fim1 inhibits Arp2/3 complex in vitro(Fig. 3) and by a greater extent when pre-existing filaments arebundled by Fim1 before the addition of Arp2/3 complex (datanot shown). However, we cannot rule out the possibility thatFim1 competes directly with Arp2/3 complex for binding tofilament sides. Interestingly, heat shock protein HSP90 canbind to N-WASP and bundle branched filaments but does notinhibit polymerization by Arp2/3 complex and N-WASP (72).Capping Protein Reduces Bundling by Fim1andRelieves Inhi-

bition of Arp2/3 Complex—Given that themajority of filamentsin actin patches are probably not bundled (25) and that bun-dling inhibits Arp2/3 complex, mechanismsmust be in place toreduce bundling in vivo. Branched filaments nucleated at a 70°angle must reach a critical length before they can bend backonto the mother filament or “zipper-up” with neighboringbranches. Actin CP is a major actin patch component thatkeeps filaments short by tightly binding the barbed end of fila-ments (30, 73). The average length of filaments in an actin patchis estimated to be only 50–100 nm (20–40 actin monomers)(21, 22, 25).We found that CP significantly reduces bundling byFim1 in vitro and restores rapid branching by Arp2/3 complex.According to our model, deletion of CP should produce

patches containing longer, bundled filaments. Additional actindoes accumulate in patches of fission yeast cells lackingCP (30).Conversely, electronmicroscopy indicates that patches in bud-ding yeast cells lacking a single subunit of CP appear normal(25). We would also predict that deletion of fim1 should rescuedefects in cells lacking CP. However, fim1� and acp2� are syn-thetically lethal (30), and fim1� acp1� strains show grossdefects in cell morphology and division (73). Interpretation ofthese interactions is complicated because Fim1 also has impor-tant roles at the contractile ring (23, 24) and actin cables (Fig. 7).Because deletion of CP (acp2�) partially suppresses contractilering nucleation factor formin cdc12 mutants (30), defects infim1� acp1� could be explained by their roles in cytokinesis aswell as general perturbation of the actin cytoskeleton. It is alsopossible that other actin patch components help alleviate Fim1-mediated bundling and inhibition of Arp2/3 complex.

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The Two Actin Binding Domains of Fim1 Play Different Rolesin Filament Binding—The cross-linking core of fimbrin forms ahorseshoe shape of two ABDs, ABD1 and ABD2 (Fig. 5A) (74–76). Sequence homology is generally higher between a singleABD of different fimbrin family members (60–80% homology)than between the two ABDs of a single fimbrin (20–30%homology) (77). Identical mutations in the two ABDs of bud-ding yeast fimbrin Sac6 do not have the same effect in vivo,suggesting that the twoABDs are functionally distinct (78–80).Structural and biochemical studies also indicate that ABD1 andABD2 do not bind filaments equivalently (Fig. 5A, supplemen-tal Fig. 2, A and B) (76, 81, 82). In general, isolated ABD1s bindfilamentsmuchmoreweakly thanABD2s (81–83). ABD1 ismoreflexible thanABD2 and appears to cause a conformational changein subdomain 1 of actin upon binding to a filament (76, 84). Thehigher affinity ABD may function as a positioning head for theother to form a stronger contact, particularly when filaments arenot perfectly in register (77, 81, 82).As previously suggested, we found that Fim1 ABD1 does not

bind filament efficiently in vitro (Fig. 5A,Kd � 3�M) (36). How-ever, ABD2 binds with submicromolar affinity (Fig. 5A, Kd �620 nM) (36), supporting amodel by which ABD2 initially bindsto a filament, allowing ABD1 to find a nearby filament.Interestingly, we found that inclusion of the EF-hand-like

N-terminal headpiece of Fim1 significantly increases the affin-ity of ABD1 for actin filaments (Kd � 220 nM). Many fimbrinisoforms are calcium-sensitive (85), whereas Fim1 is not (36, 49,86). The EF hands might, therefore, have an additional role. Incrystal structures, the EF hands of some fimbrins face awayfrom the filament between CH domains of ABD1, possibly act-ing as a wedge to position ABD1 (75). Deletion of the EF handsof other fimbrins reduces actin binding by ABD1 (75). We pro-pose that inclusion of the EF hands increases the affinity ofFim1 ABD1 by changing the conformation of ABD1, aligningactin binding residues at positions where they can access thefilament in the proper orientation. Structural studies arerequired to further explore this possibility.The Bundling Activity of Fim1 Is Important for Actin Patches,

the Contractile Ring, and Actin Cables—Both the contractilering and actin cables are composed of bundled filaments (1, 2).However, despite high Fim1 levels (3, 22, 36, 49), endocyticactin patch filaments do not appear to be extensively bundled(25). We proposed that bundling by Fim1 is dispensable at theendocytic patches, where instead the ability of Fim1 to regulatecofilin-mediated severing by inhibiting the binding of tropomy-osin Cdc8 is critical (23). The cross-linking activity of Fim1 ispredicted to play an overlapping role with �-actinin Ain1 in thecontractile ring because small amounts of Fim1 are detected inthe ring (36, 49), and deletion of both fim1 and ain1 is synthet-ically lethal (49). Conversely, it is not known whether Fim1cross-links filaments in actin cables. We, therefore, hypothe-sized that FimA2, which can bind but not bundle actin fila-ments (Fig. 5) (23), would be fully functional for actin patchesbut not the contractile ring and possibly actin cables. However,we found that the bundling activity of Fim1 appears to berequired for all three processes (Figs. 6 and 7).Although FimA2 inhibits localization of tropomyosin Cdc8

to actin patches, patches otherwise behave like those in fim-

brin-deleted cells. Therefore, some patch filaments are proba-bly cross-linked. Similarly, FimA2 is not sufficient for life in theabsence of �-actinin Ain1 (supplemental Fig. 3), indicating thatactin cross-linking activity is essential at the contractile ring.Alternatively, it is important to note that because FimA2 is

composed of only a single actin binding domain, there are halfthe ABDs per molecule of FimA2. Furthermore, FimA2 is�4-fold less abundant in actin patches comparedwithwild typeFim1 (Fig. 6D), so it is certainly possible that defects in FimA2cells are due to less protein rather than deficient cross-linkingactivity. Conversely, FimA2 localizes to cables more stronglythan wild type Fim1. Furthermore, as we do not yet fully under-stand the function of all other actin patch components, we can-not account for their behavior in fimbrin mutant cells.A limited number of actin-binding proteins have been local-

ized to actin cables, including coronin Crn1, tropomyosinCdc8, and transgelin Stg1 (17, 87, 88). Neither of the canonicalbundling proteins, Fim1 or Ain1, has been localized to actincables (36, 49). However, both fim1� and fimA2 strains showmild polarity defects (Fig. 6A) (36, 49). We found that actincables are functionally compromised in both fim1� and fimA2cells, as they no longer provide effective tracks for the type Vmyosin motor Myo52. However, these defects can be inter-preted multiple ways. The simplest explanation is that Fim1does bundle filaments in cables but has not been detected dueto low levels. In agreement with this, overexpression of eitherfull-length Fim1or FimA2 induces extra cable-like structures ina tropomyosin-dependent manner (36). Conversely, cabledefects could be due to global defects in the actin cytoskeleton(23, 36, 49). Because turnover of actin patches is significantlyslower in fim1� and fimA2 cells (Fig. 6B) (23), less actin may beavailable for assembling cables.Conclusion—Fimbrin Fim1 has multiple important roles at

different actin structures in fission yeast.We propose that Fim1acts as a central regulator of actin structure organization boththrough its intrinsic function as an actin-bundling protein andthrough regulation of other proteins such as tropomyosin andmyosin, which can in turn affect severing by cofilin and nucle-ation by Arp2/3 complex (23, 24, 51). However, interpretationsremain clouded because the roles of many actin-binding proteinsremain poorly understood. For example, budding yeast transgelinhas vital roles working with fimbrin at actin patches (89), but thefunction of fission yeast transgelin is less clear (88). Similarly,although extensive work has detailed the mechanisms by whichthe contractile ring assembles, the function of several contractilering-specific proteins, including the F-BAR protein Cdc15 andIQGAP Rng2, are multifaceted and only partially understood (37,90–93). Because we believe that diverse actin structures self-as-semble in a crowded cytoplasm from a common pool of nuclea-tors, profilin-bound actin monomer, and actin-binding proteins,elucidating the functional interaction among actin-binding pro-teins remains a daunting but essential task.

Acknowledgments—We thank Jessica Henty and Chris Staiger (Pur-due University) for technical assistance and members of the Kovarlaboratory for strains, technical assistance, and insightful comments.

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S. Rock, Vladimir Sirotkin and David R. KovarColleen T. Skau, David S. Courson, Andrew J. Bestul, Jonathan D. Winkelman, Ronald

and Polarization in Fission YeastActin Filament Bundling by Fimbrin Is Important for Endocytosis, Cytokinesis,

doi: 10.1074/jbc.M111.239004 originally published online June 3, 20112011, 286:26964-26977.J. Biol. Chem. 

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