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