Proc. Nati. Acad. Sci. USA Vol. 91, pp. 1510-1514, February 1994 Cell Biology

Dynamic actin structures stabilized by profilin (actin cytoskeleton/overexpression/nudeotide exchange) TOREN FINKEL*t, JULIE A. THERIOT*§, KIRK R. DISE*, GORDON F. TOMASELLI*, AND PASCAL J. GOLDSCHMIDT-CLERMONT*¶ Departments of *Medicine, Cardiology Division, and 1Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and tDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94143 Communicated by Thomas D. Pollard, October 5, 1993

ABSTRACT We describe the production and analysis of MATERIALS AND METHODS clonal cell lines in which we have overexpressed human pro- to Production of Stable Clonal Lines Overexpressing Profilin. filin, a small ubiquitous actin monomer binding protein, two (i) LK- on actin function in vivo. The We constructed profilin expression plasmids: assess the role of profilin HPS. The human profilin cDNA (19), a gift from D. J. is increased in cells with concentration of filamentous actin Kwiatkowski (Harvard University), was inserted into the Sal higher profilin levels, and actin filament half-life measured in I-BamHI site of the eukaryotic expression vector LK444 these cells is directly proportional to the steady-state profrin (20), which contains the 3-actin promoter and encodes con- concentration. The distribution of actin flaments is altered by stitutive resistance to G418 (GIBCO/BRL). (ii) pCMV- profihin overexpression. While parallel actin bundles crossing profilin. The human profilin cDNA was inserted into the the cells are virtually absent in cells overexpressing profflin, the unique EcoRV-BamHI sites ofthe pCMV expression vector, submembranous actin network of these cells is denser than in which uses the promoter from the immediate early region of control cells. These results suggest that in vivo profilin regulates human cytomegalovirus (a gift from E. R. Fearon, Yale the stability, and thereby distribution, ofspecific dynamic actin University). structures. For the first transfection, CHO cells grown in a-MEM (GIBCO) with 10% fetal calf serum supplemented with pen- In nonmuscle cells, actin functions as an adenine nucleotide icillin (100 units/ml) and streptomycin (100 ,ug/ml) (complete triphosphatase, cycling between monomeric and filamentous medium) were transfected at 50o confluence with Lipofectin conformations. The actin cytoskeleton of nonmuscle cells (GIBCO/BRL) and either 2 Mg of LK-HPS or 2 jug of the can undergo dramatic reorganization in response to extra- expression vector alone. On day 3, selection of clones was cellular agonists (1, 2). This restructuring of the actin cy- initiated with G418 (maintenance dose, 0.25 mg/ml), and toskeleton is controlled by actin-binding proteins (3). Profi- clonal lines were expanded from microtiter wells. For the lins are ubiquitous actin-, polyproline-, and inositol phos- second transfection, selected overexpressing clonal lines pholipid-binding proteins (4-7). The interaction of profilin were retransfected with pCMV-profiin (10 pg) together with with actin monomers has been characterized in vitro: profilin the hygromycin-resistance vector pG5Elb-Hygro (1 ,ug) (21), inhibits the nucleation of new filaments, sequesters actin using Lipofectin. Control cells were transfected with pCMV monomers, shuttles actin subunits toward the high-affinity (without profilin cDNA; 10 tag) and the hygromycin- end of filaments, and increases exchange and reduces hy- resistance vector (1 pug). Clonal lines were selected in com- drolysis of the nucleotide bound to monomeric actin (8-14). plete medium supplemented with neomycin (0.25 mg/ml) and At high actin-to-profilin ratios, the dominant effect ofprofilin hygromycin (0.25 mg/ml) and then expanded. is to increase by two to three orders of magnitude the off rate Protein Quantitation. Washed confluent cells (8 x 107 cells of the nucleotide bound to actin (12, 13). for Coomassie blue-stained gels, 107 cells for Western blots) Based on these observations, under physiological condi- were extracted with melting ice-cold lysis buffer [15 mM tions in nonmuscle cells where the actin-to-profilin ratio is Hepes (pH 7.0), 145 mM NaCl, 0.1 mM MgCl2, 10 mM high (-5:1), filament turnover is rapid (15-17), and ATP is in EGTA, 0.5% Triton X-100, 1 mM 4-(2-aminoethyl)benzene- large excess over ADP, profilin may play a key role in actin sulfonyl fluoride, and protease inhibitors (chymostatin, leu- assembly by recharging with ATP the ADP-actin monomers peptin, antipain, and pepstatin each at 20 pg/ml)], transferred produced by depolymerization of actin filaments (13). The to polycarbonate tubes (Beckman, 1 ml), and sonicated (10 affinity of thymosin (4 (the most abundant actin monomer sec, Branson Sonifier 450, energy level 1-2). Protein con- binding protein in many cells) for ATP-actin monomers is centration in each extract was determined by Bradford assay, much greater than for ADP-actin monomers (18). Thus, and extracts were normalized for total protein content (12). thymosin ,/4 selective interaction with ATP-actin monomers Profiin Quantitation. To quantitate profilin on Coomassie the ADP-actin subunits blue-stained gels, extracts were centrifuged (100,000 x g for should further target profilin towards 30 min, 4°C in 1-ml tubes) in a Beckman TLA-100.2 rotor, and produced by filament turnover. profilin in the supernatants (which contain -99% of cellular This study examines the role of profilin on actin dynamics profilin) (12) was concentrated on poly(L-proline) beads, in vivo. We have obtained stable overexpression of human boiled in SDS sample buffer (12), and analyzed by SDS/ profilin in Chinese hamster ovary (CHO) cells and have acrylamide gel electrophoresis (4-20% gradient), together examined the effect of a steady-state increase in profilin with purified profilin standards. The gels were stained with concentration on actin dynamics. tPresent address: Cardiology Branch, National Institutes of Health, The publication costs of this article were defrayed in part by page charge Bethesda, MD 20892. payment. This article must therefore be hereby marked "advertisement" §Present address: Whitehead Institute for Biomedical Research, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Cambridge, MA 02142-1479. 1510 Downloaded by guest on September 26, 2021 Cell Biology: Finkel et al. Proc. Natl. Acad. Sci. USA 91 (1994) 1511 Coomassie blue, and the intensity of the profilin bands was muscle actin monomers covalently labeled with a caged measured by densitometry and compared to standards. fluorochrome (caged resorufin) were microinjected into cells Profilin was also quantitated on Western blots. Profilin where they are incorporated into filaments. Activation ofthe from poly(L-proline)-concentrated samples or whole-cell fluorochrome with a beam of 360-nm ultraviolet light marked extracts was probed with affinity-purified rabbit polyclonal an area of the filament network, and observation of the fate IgGs against human profilin (dilution, 1:1000). Where indi- of this area by fluorescence videomicroscopy allowed the cated, two additional profilin antibody preparations were measurement of filament turnover and/or movement. used at the same dilution: (i) a rabbit antibody against the Clonal cell lines grown on acid-washed glass coverslips human profilin peptide LVGKDRSSFY, which was cross- were transferred to an aluminum chamber held at 370C by a linked to an octabranched matrix core (Research Genetics, circulating water bath. Cells were studied in F-12 medium Huntsville, AL) (22); (ii) a rabbit antibody raised against with 5% fetal calf serum, 20 mM Hepes, penicillin (100 whole recombinant human profilin (a gift from D. A. Kaiser units/ml), and streptomycin (100 1ig/ml), and the medium and T. D. Pollard, Johns Hopkins University). The blots was covered with a layer of silicon oil to minimize evapora- were developed on chemiluminograms (ECL; Amersham), tion. Cells were injected with caged resorufm-actin at 4 using horseradish peroxidase-labeled IgGs (HyClone), at a mg/ml; 5-10% of the cell volume was injected. The dilution of 1:500. The profilin bands were measured by injected densitometry, using profilin standards on the same cells were incubated for 30-60 min, and then photoactivation chemiluminograms. and imaging of resorufm were performed as described (16). Actin Quantitation. Total actin was measured on Western Fluorescence images of activated cells were averaged for blots, using an actin-specific monoclonal antibody (clone C4; eight frames every 30 sec and recorded on optical disc 1:1000 dilution; ICN) (23) and the ECL method. The fraction (Panasonic). Total fluorescence intensity in each activated of polymerized actin was measured by extracting 107 cells in area was measured using Image-1 (Universal Imaging, Me- lysis buffer (500 pl) supplemented with a saturating concen- dia, PA). Average filament half-lives were determined by tration of rhodamine phalloidin (1.0 pM; Molecular Probes), fitting an exponential decay curve to the plot of fluorescence which has been shown to prevent depolymerization of fila- intensity in the activated area versus time for each cell. ments occurring after detergent extraction (24). The extracts were centrifuged (Beckman TLA 100.2 rotor, 1-ml tubes, RESULTS 200,000 x g for 30 min at 4°C), and the pellets were rinsed (but Protein Characterization of Clonal Lines Overexpressing not disrupted) once with lysis buffer without rhodamine Profilin. Six CHO cell lines transfected with an expression phalloidin and then resuspended in 200 Au of methanol. After plasmid containing human profilin cDNA (19) regulated by 2 hr, methanol extracts were transferred to 96-well plates the 1-actin promoter expressed profilin concentrations 1.5- (Corning; flat bottom, low protein binding), and fluorescence 4.1 times control levels (Fig. 1). The profilin concentration for each sample was measured in CytoFluor 2300 (Millipore; within the three cell lines transfected with the expression with excitation and emission wavelengths of 530 nm and 590 vector lacking the cDNA insert was similar to control cells. nm, respectively), together with a standard curve of known Out of the original nine clonal lines, we selected three rhodamine phalloidin concentrations to calculate the F-actin representative clones for detailed studies. Clonal line 2-II concentration in each sample. (control) was transfected with the expression vector alone, Fluorescence Microscopy. To study F-actin, cells were whereas clonal lines 1-II and 1-V (overexpressing) were seeded in six-well plates with sterile coverslips (22 mm2), transfected with the profilin cDNA construct. grown for 48 hr, fixed for 30-60 min [3.7% formaldehyde in Profilin concentration was found to be stable in these phosphate-buffered saline (PBS)], and then permeabilized clonal lines for 24 months (data not shown). Densitometric with 0.2% Triton X-100 in PBS for 5 min (25). Actin filaments analysis of the bands corresponding to profilin reveals that were labeled for 21 min in the dark by inverting the coverslips overexpressing clones 1-II and 1-V contained 17.7 ,M and on top of a 200-4p droplet of rhodamine phalloidin (0.2 ,uM) 34.4 ,uM profilin, respectively, whereas 8.4 ,uM profilin was in PBS, which was enough to saturate all phalloidin binding present in control clone 2-II. To generate clonal lines with sites. The unbound rhodamine phalloidin was washed with even higher profilin levels, we performed a second transfec- PBS (2 ml for 5 min followed by 6 ml for 12-28 hr). The tion of clonal line 1-V with another vector-pCMV-profilin coverslips were mounted using 13 ,ul ofVectashield mounting (Fig. 2). Control line 2-II was transfected with pCMV ex- medium (Vector Laboratories) and then kept in the dark at pression vector without profilin cDNA. Although CHO cells 4°C until microscopic analysis (Zeiss) and photography (Ko- grew slightly slower in the presence of hygromycin (used for dak Ektachrome 400X films). selection after second transfection), in no instance was the To detect profilin, formaldehyde-fixed cells were perme- rate of growth of clonal lines noticeably affected by profilin abilized using either Triton X-100 in PBS (0.2%) or a quick dip overexpression (data not shown). (7 sec) in -20°C acetone. After incubation in PBS containing Effect of Profifin on Actin. The total actin concentration 2% (wt/vol) bovine serum albumin (BSA; Sigma) for 30 min, was not affected by profilin overexpression in the clonal cell the coverslips were then placed on top of a droplet (200 ,ul) lines (Fig. 1). The molar ratio of total actin to total profilin of anti-profilin antibody (1:200 dilution). Three separate concentration ranged from 2:1 to 18:1, although the exact profilin antibody preparations were used for these experi- amount of each protein free to interact with the other is ments (see quantitation of profilin). After 1 hr, the coverslips unknown. Unexpectedly, stable profilin overexpression in- were washed in PBS/BSA (30 min) and then placed on top of creased the F-actin concentration in CHO cells (Fig. 3A). An a droplet of fluorescein isothiocyanate-labeled goat anti- 8.7-fold increase in profilin concentration caused the fraction rabbit immunoglobulins (Sigma) for 1 hr (1:50 dilution). of polymerized actin to nearly double. Coverslips were rinsed twice in PBS/BSA, then washed Moreover, the stability of actin filaments was directly overnight in PBS/BSA at 4°C, and rinsed again prior to proportional to profilin cellular concentration. Using caged mounting on slides with Vectashield and microscopy. Con- resorufin-actin (16), the rate of fluorescence decay in cells trols for specificity of the staining were obtained by omitting overexpressing profilin was slower than in control cells, the first antibody or using corresponding preimmune serum consistent with a global increase in actin filament stability in in place of the first antibody (at 1:200 dilution). cells overexpressing profilin. When average actin filament "Caged" ResorufLn-Actin Assay. We used the fluorescence half-life values were plotted as a function of the profilin photoactivation technique to test the stability of actin fila- concentration in each clone, a linear relationship was ob- ments within the various clonal cell lines. Purified rabbit tained (Fig. 3B). Downloaded by guest on September 26, 2021 1512 Cell Biology: Finkel et al. Proc. Natl. Acad. Sci. USA 91 (1994)

a ::_205 116 Clones =106 80 ~ - ~ - 49 205 32 106 - 27 80 __ _- _ 18 -_ 49 - :32 b 27 ---~ -Profilin

c _ 205 ~106 FIG. 2. Profilin content in clonal lines from second transfection. Profilin concentrations in selected clonal lines from second trans- fection (pCMV-profihin vector) were measured by Western blotting. Normalized cell extracts (20 pg of protein) were analyzed together w_iif32 with profilin standards. The blot was developed using an antibody - 27 against recombinant human profilin and the ECL method. Symbols -----18'Ip - at the top of each lane refer to Fig. 3. d specific accumulation ofprofilin in this area (27), a possibility - - -~~~~~Actin supported by confocal microscopy studies (28). In cells from control clone 2-II stained for both profilin and FIG. 1. Profilin and actin concentrations within clonal lines from actin, we observed that actin filaments were organized in first transfection. (a and b) Purified profilin standards (lanes 1-3) and parallel bundles crossing the central region at the bottom of poly(L-proline) bead precipitates from clonal line extracts (lanes the cells, while peripheral actin filaments formed a dense 4-6). (a) Coomassie blue-stained gel. Lanes 1-3, 2 jig, 1 ug, and 0.5 network with some patchy structures (Fig. 5). When cells ,gg of purified human platelet profilin standards, respectively. Lanes expressing various levels of human profilin were grown side 4-6, profilin content in clonal lines 2-II (control) and 1-II and 1-V we observed that concentrations were (profilin overexpressors). The 42.5-kDa band, which coprecipitated by side, high profilin with profilin and corresponded to actin, indicated that overexpressed profilin was functional in its ability to bind actin and poly(L-proline). tJULnJ . I . I I. (b) Western blot of profilin. Lanes 1-3, 0.80, 0.40, and 0.20 ug of .A purified profilin standards, respectively. Lanes 4-6, profilin content in clonal lines. (c and d) Purified actin standards (lanes 1-3) and C.) U) normalized clonal line extracts (lanes 4-6). (c) Coomassie blue- 4 en stained gel. Lane 4, clone 2-II; lane 5, clone 1-IT; lane 6, clone 1-V 2 (20 of protein loaded per Most bands did not seem 0) pg lane). protein 'r,310 a) affected by profilin overexpression; however, multiple small changes C.) in protein concentrations could have been missed in this assay. The Ico profilin band was not detectable on this gel (<60 ng of profilin per 40 a)E lane). (d) Western blot ofactin. Lanes 1-3 correspond to 63, 31, and co_1E, IL 16 ng of skeletal muscle actin standards, respectively. Lanes 4-6 1 correspond to clonal lines 2-TI, 1-II, and 1-V, respectively; 3 pg of protein was loaded per lane. Total actin concentration was not affected by profilin overexpression (148.1 ± 11.1 p;M). 0 20 40 60 80 0 10 20 30 40 Profilin, /M Profilin, AuM Effect of Profilin on Cell Morphology and Actin Subcellular Distribution. Overexpressing clones 1-V and 1-II achieved a FIG. 3. Effect of profilin overexpression on actin filaments. (A) higher confluent density than control cells. Thus upon reach- Filamentous actin concentration. F-actin was quantitated by rhoda- ing confluence, cells from clone 1-V appeared to be more mine phalloidin fluorescence microtiter well assay. The mean F-actin retracted (1071 ± 55 um2 per cell; mean ± SD, n = 6) than concentration is shown for each clonal line (±SEM, vertical bar) and ± < was calculated from several measurements (n = number of separate cells from control clone 2-lI (1501 105 ,um2 per cell; P determinations): control clone 2-TI (A; n = 3) and overexpressing 0.001, unpaired two-tailed t test), a morphological difference clones 1-V (o; n = 3), 1-V CMV-d (v; n = 3), clone 1-V CMV-f (c; that was not always evident upon examining individual cells n = 6), clone 1-V CMV-106d (U; n = 6), and clone 1-V CMV-106a (v; by fluorescence microscopy (Fig. 4). n = 6). The P value for profilin concentration effect on F-actin Profilin in control CHO cells was present in the cytoplasm concentration is <0.0001. Thus, profilin overexpression was asso- (Fig. 5), with marked immunostaining of the perinuclear ciated with a significant increase in F-actin concentration. (B) region, which corresponded to the thickest area of the cells. Stability of actin filaments. Filament half-life was measured in cells a microinjected with caged resorufin-actin. Mean filament half-life is Profilin displayed speckled distribution, characterized by shown for three clonal lines (±SEM, vertical bar): control clone 2-IT multiple profilin "spots" of various intensities. At the pe- (A; n = 13) and overexpressing clones 1-TI (C; n = 10) and 1-V (o; riphery, where membrane ruffling is pronounced in CHO n = 12). The line corresponded to the best fit to the points (r = 0.99). cells, the staining for profilin seemed increased, which could The differences in half-life between clone 1-V and clones 2-IT and 1-IT be due to increased thickness ofthe edge ofthe cells or to the were significant (P = 0.002 and 0.049, respectively). Downloaded by guest on September 26, 2021 Cell Biology: Finkel et al. Proc. Natl. Acad. Sci. USA 91 (1994) 1513

FIG. 4. Effect ofprofilin overexpression on actin superstructure. Two CHO cells grown side by side (from clonal line 1-V CMV-f), one expressing control levels of profilin and the other overexpressing profilin, were stained for profilin using the antibody against recom- binant human profilin (a). A micrograph corresponding to a short exposure is shown to demonstrate the subcellular distribution of profilin in overexpressing cells; hence, profilin staining in the cell expressing control levels was barely above background. The same cells were stained for F-actin with rhodamine phalloidin (b). The distributions of actin and profilin shown on this figure were typical for control and overexpressing cells. Evenly spread cells were selected to ease the comparison of F-actin organization correspond- ing to each profilin condition. (Bar = 10 pan.) associated with reduced density of parallel actin bundles, a finding consistent with those obtained with profilin microin- jection experiments (29). However, in overexpressing cells, F-actin FIG. 5. Fluorescence microscopy for F-actin and profilin in bright staining was seen at the periphery of the cells, control cells. (a) Profilin in control clone 2-1I was stained by indirect in areas that may correspond to membrane ruffling (Fig. 4). immunofluorescence, using a rabbit antibody (IgGs) against recom- Thus, the observed stabilizing effect of profilin on F-actin binant human profilin. Profilin localization was similar when de- seemed to be selective for specific actin superstructures. tected with two other anti-profilin antibodies (Materials and Meth- Moreover, large fractions of the profilin in overexpressing ods) and unaffected by the method of permeabilization; thus, it is cells appeared concentrated just behind the submembranous unlikely to be artifactual (26). (b) Controls for the specificity of the actin areas where F-actin was almost excluded. staining included omitting the first antibody (data not shown) or using network, in corresponding preimmune serum in place ofthe profilin antibody. (c) It was therefore unlikely that the effect of profilin on F-actin Actin filaments in control cell II-2 were stained with rhodamine stability and density resulted mainly from the direct interac- phalloidin. (Bar = 10 pam.) tion of profilin with actin filaments. Microinjection ofpurified profilin-actin complexes in nor- DISCUSSION mal rat kidney cells provided additional evidence for the Previous genetic studies in Drosophila and Saccharomyces facilitating effect ofprofilin on the generation ofspecific actin cerevisiae have shown that reducing the profilin concentration structures (29). The rapid and marked increase in F-actin can markedly alter the actin superstructure normally present following the introduction in the cytoplasm of actin mono- in these cells. Yeast cells null for profilin display disorganized mers complexed to profilin is consistent with these com- actin superstructure (30), and Drosophila nurse cells contain- plexes representing a readily available source of actin sub- ing reduced concentrations of profilin are unable to generate units, poised for efficient association into filaments. This the actin cables that abruptly develop at the stage of oocyte result contrasted with the effect of microinjection of large maturation when nurse cells actively transport the content of concentrations of profilin alone, which was associated with their cytoplasm into the oocyte (31). These phenotypes dis- the selective disruption of actin stress fibers. played by cells deficient forprofilin are consistent with profilin We used stable overexpression techniques to increase being necessary to stabilize dynamic actin structures. profilin concentration in CHO cells and characterize its effect Downloaded by guest on September 26, 2021 1514 Cell Biology: Finkel et al. Proc. Natl. Acad. Sci. USA 91 (1994)

on the actin cytoskeleton. Three properties of actin struc- 3. Hartwig, J. H. & Kwiatkowski, D. J. (1991) Curr. Opin. Cell tures were altered by profilin overexpression: (i) actin fila- Biol. 3, 87-97. ment stability was increased in proportion to profilin con- 4. Carlsson, L., Nystrom, L.-E., Sundkvist, I., Markey, F. & Lindberg, U. (1977) J. Mol. Biol. 115, 465-483. centration, (ii) profilin overexpression altered the distribu- 5. Lassing, I. & Lindberg, U. (1985) Nature (London) 318, 472- tion of F-actin, and (iii) the concentration of F-actin was 474. proportional to the profilin concentration. Taken together, 6. Goldschmidt-Clermont, P. J., Machesky, L. M., Baldassare, our results confirm that profilin regulates actin dynamics in J. J. & Pollard, T. D. (1990) Science 247, 1575-1578. vivo and provide evidence that the distribution of actin 7. Goldschmidt-Clermont, P. J. & Janmey, P. A. (1991) Cell 66, dynamic structures in cells might be regulated by profilin, as 419-421. a toward actin 8. Mockrin, S. C. & Korn, E. D. (1980) Biochemistry 19, 5359- result of the stabilizing effect of profilin 5362. filaments. These findings are consistent with studies on the 9. Tilney, L. G., Bonder, E. M., Coluccio, L. M. & Mooseker, actin-based motility ofListeria monocytogenes in cells and in M. S. (1983) J. Cell Biol. 97, 112-124. reconstituted cell extracts, where profilin concentration is 10. Pollard, T. D. & Cooper, J. A. (1984) Biochemistry 23, 6631- limiting for the process of "actin-tail" formation and bacte- 6641. rial locomotion (32). 11. Nishida, E. (1985) Biochemistry 24, 1160-1164. It is likely that adaptation of cells to higher profilin con- 12. Goldschmidt-Clermont, P. J., Machesky, L. M., Doberstein, centrations occurs with stable overexpression, with possible S. K. & Pollard, T. D. (1991) J. 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