Proc. Natl. Acad. Sci. USA Vol. 89, pp. 4678-4682, May 1992 Cell Biology Thymosin 134 (Fx peptide) is a potent regulator of actin polymerization in living cells (cytoskeleton/assembbly) MITCHELL C. SANDERS*, ALLAN L. GOLDSTEINt, AND Yu-Li WANG*t *Cell Biology Group, Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, MA 01545; and tDepartment of Biochemistry and Molecular Biology, The George Washington University School of Medicine and Health Services, Washington, DC 20037 Communicated by Paul C. Zamecnik, February 3, 1992 ABSTRACT Thymosin .84 (f84) is a 5-Ala polypeptide bind a significant portion of monomeric actin and inhibit originally identified in calf thymus. Although numerous activ- salt-induced polymerization of actin filaments in vitro (22, ities have been attributed to .34, its physiological role remains 23). Since (4 appears to be present at a higher level than most elusive. Recently, (34 was found to bind actin in human platelet other actin sequestration factors in platelets and cultured extracts and to inhibit actin polymerization in vitro, raising the cells (20-23), it is possible that (4 may represent the primary possibility that it may be a physiological regulator of actin factor responsible for the regulation of actin polymerization assembly. To eamine this potential function, we have in- (24). creased the cellular A concentration by mlcroilJecting syn- One approach to test this putative physiological role of(4 thetic (34 into living epitheial cells and fibroblasts. The injec- is to induce the "overexpression" in living cells and to tion induced a diminution ofstress fibers and a dose-dependent examine the subsequent effects on specific structures or depolymerization ofactin filaments as indicated by quantitative functions. In the present study, we have taken this approach image analysis of phaLlodin binding. Our results show that (34 by microinjecting excess synthetic (4 into living epithelial is a potent regulator of actin assembly in living cells. cells and fibroblasts. The effects on actin were then deter- mined by measuring the amount of binding of fluorescent Actin is involved in many important nonmuscle cellular phalloidin with quantitative image analysis. Our results in- processes such as cell locomotion, chemotaxis, phagocyto- dicate that (34 is indeed capable of inducing extensive de- sis, and cytokinesis. Previous studies indicate that the reg- polymerization of actin filaments in living cells. ulation ofthese processes involves the dynamic assembly and disassembly of actin filaments and high-order structures, most likely through the interplay ofa number ofactin-binding MATERIALS AND METHODS factors (for reviews, see refs. 1-3). However, important Preparation of Thymosins. (3 and thymosin a, (a,), pro- questions remain concerning the actual mechanism for the vided by Alpha 1 Biomedicals (Washington), were synthe- regulation of actin assembly in living cells. sized as described (25, 26). The concentration of 4 was One important mechanism for the regulation of actin as- determined by the bicinchoninic acid assay (BCA; Sigma) sembly involves the inhibition of spontaneous polymeriza- with bovine serum albumin as the reference and was cali- tion of the high concentration of actin subunits in nonmuscle brated with amino acid analysis of (4. cells. Previously we have demonstrated that such inhibition Actin Polymerization Assay. The activity of synthetic P4 in is probably achieved through the sequestration of actin vitro was determined by the pyrene-conjugated actin assay. monomers from addition onto active sites of assembly (4). Pyrene-actin was prepared as described by Kouyama and However, although a number of monomer sequestering pro- Mihashi (27) with minor modifications (28). The pyrene-actin teins, such as profilin, actin-depolymerizing factor/destrin, conjugate had a dye-to-protein molar ratio of 1.3. Labeled depactin, actobindin, and cofilin, have been identified (5-10), and unlabeled actin monomers were dialyzed for 2-3 days none of these factors appears to be present at a high enough with three exchanges of G-buffer (0.5 mM ATP/0.2 mM concentration to account for the levels of unpolymerized CaCl2/0.5 mM dithiothreitol/2 mM Tris HCl, pH 7.0) and actin in nonmuscle cells. In addition, despite their ability in then centrifuged at 35,000 rpm in a 42.2 Ti rotor at 40C for 2 vitro to inhibit the polymerization of purified actin subunits, hr. Pyrene-actin was mixed 1:20 (wt/wt) with unlabeled actin the activity in vivo remains in question, since a number of at a final actin concentration of 4.2 puM and incubated with factors are capable of stabilizing actin filaments or overcom- various concentrations of(3 for 10 min. At t=0, the solution ing the sequestration effect (11). was mixed with 0.5 volume of 3x polymerization buffer (6 The newest member to this family of potential actin regu- mM MgCl2/0.3 M KCI/3 mM ATP/75 mM imidazole, pH lators is thymosin (4 ((4), an =5-kDa polypeptide first 7.0), and the fluorescence intensity was measured in a isolated from an extract of calf thymus (12-14). (34 was spectrofluorimeter (LS-3; Perkin-Elmer) at an excitation originally proposed solely as a thymic hormone, based on its wavelength of365 nm and an emission wavelength of407 nm. ability to stimulate the T-lymphocyte terminal deoxynucle- For steady-state readings, the samples were allowed to otidyltransferase after intraperitoneal injection into mice (13, polymerize for 15 hr. 15) and to inhibit macrophage migration at a dosage of 5-500 Cell Culture and Mcroinjection. Normal rat kidney (NRK) pM (16). However, later studies indicated that 84 is present epithelial cells (NRK-52E; American Type Culture Collec- at a relatively high abundance in several tissues (17-19) and tion) were cultured in F12K medium (JRH Biosciences, cell lines (20, 21), raising the possibility that it may also serve Lenexa, KS) supplemented with 10%6 (vol/vol) fetal calf a more ubiquitous role. Recently, (4 was found to be identical serum (JRH Biosciences), 50 pg of streptomycin per ml, and to a small acidic peptide (Fx) in platelet extracts, which can 50 units of penicillin per ml and were maintained at 370C under 5% C02/95% air. Swiss 3T3 cells were cultured as The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: (34 and a,, thymosins (4 and a,. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 4678 Downloaded by guest on September 27, 2021 Cell Biology: Sanders et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4679 described (4). The cells were plated on microinjection dishes a (29) 36-40 hr prior to microinjection. For most of the experiments, thymosin peptides were dissolved in 5 mM Tris acetate (pH 6.95) containing 3 mg of .ta~%0.3- fixable fluorescein-conjugated dextran (Molecular Probes) cn per ml at a concentration of 0.48-1.63 mM. High concentra- CU) tions (=20 mg/ml) of either a, or 84 were found to be cytotoxic for =30% of the cells (inducing an apparent disin- 5r 0.2- tegration of the cell as observed under phase optics) and 0 therefore were not included in the analysis. Pressure micro- -W injection was performed as described (4, 30). The average 0 volume of microinjection was estimated to be 2-3% ofthe cell .5 volume by the method of Lee (31) with minor modifications (32). Lamellipodia extension was induced by scraping a conflu- ent cultured monolayer with a Pasteur pipet. Cells along the edge were microinjected following a recovery for 15 min. Fluorescent Staining. Cells were incubated for 1 hr at 370C Time (min) after microinjection, then fixed with 4% formaldehyde, per- b meabilized with acetone, and stained with rhodamine- conjugated phalloidin (Sigma) as described by Sanders and Wang (4). Lamellipodia were fixed and extracted with a 1> 0.5- C special protocol that preserves the highly labile structure (see 6) Fig. 3; ref. 33). Immunofluorescence staining with polyclonal C antibodies against actin (Biomedical Technologies, Stough- et0.4- ton, MA) or myosin II (provided by Keigi Fujiwara, National C0 0 Cardiology Research Center, Osaka) was performed essen- 0 tially as described by Amato et al. (34). Rhodamine- 0.3n conjugated secondary antibodies were obtained from Tago. Microscopy and Image Processing. Fluorescence images were recorded with a Zeiss Axiovert 10 microscope (Zeiss) 0 coupled to a cooled CCD camera (Star I; Photometrics, U- Tucson, AZ) using a 100-W quartz-halogen lamp and either a 25x/NA 0.8 Plan-Neofluar or a 10Ox/NA 1.3 Neofluar objective (Zeiss). Images recorded with the illumination lamp 4 offwere subtracted from integrated fluorescence images with a digital image processor. The boundary of injected and #4 (AsM) uninjected sister cell pairs was defined interactively by using a graphics tablet and computer software developed in this FIG. 1. Inhibition ofactin polymerization by synthetic (4 in vitro. Actin polymerization was measured by the increase of fluorescence laboratory. The intensities ofpixels within the boundary were intensity ofpyrene-labeled actin. The assay mixture contains 4.2 AM then integrated with a computer. The percentage of actin total actin and 5% pyrene-actin. The lag period for nucleation was depolymerization was calculated by dividing the integrated extended from 2 to 3 min for the control (o) to >5 min in the presence rhodamine-phalloidin fluorescence of 84-injected cells with of4 AM P4 (0); the initial rate of filament elongation in the presence that of uninjected sister cells. of 4 AM (e) and 6 MM (A) 84 was also significantly reduced (a). In addition, the level of steady-state polymerization was reduced with a linear dependence on the concentration of (34 (b).