Levels of filamentous and globular actin in Chinese hamster ovary cells throughout the cell cycle

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SHORT NOTE Levels of Filamentous and Globular Actin in Chinese Hamster Ovary Cells Throughout the Cell Cycle C. S. HEACOCK*

Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any fomt reserved 0014-4827/83$3.00

and J. R. BAMBURG

Department of Biochemistry and Graduate Program of Cellular and Molecular Colorado State University, Fort Collins, CO 80523, USA

Biology,

Summary. Synchronous Chinese hamster ovary (CHO) cells were obtained by mitotic selection and the levels of globular (G) actin, filamentous (F) actin, and cytoskeletalassociated F-actin were determined as cells progressed through the cell cycle. Total actin levels remained quite constant when expressed as a percent of the total protein. An increase in F-actin occurred upon plating the mitotic cells, but this increase was shown to be a result of attachment to the substratum, since cells which remained attached during the second mitosis failed to show these changes. No large variation in the levels of either Factin or cytoskeletal-associated F-actin occurred throughout the cell cycle. Therefore, changes in the morphology of the CHO cells which are accompanied by a reorganization of a&n-containing microtilaments during the cell cycle are not accompanied by significant changes in the size of the monomeric actin pool.

Actin, an abundant protein found in eukaryotic cells, plays an important role in cell shape and motility [ 1, 21. Actin exists in a dynamic equilibrium between its two forms: monomeric, G-actin and filamentous, F-actin. The amount and organization of the filamentous actin form has been postulated to regulate many physiological functions [3]; thus, changes in the G-/F-actin ratio as well as changes in total actin amount may be important. Chinese hamster ovary (CHO) cells which contact one another undergo changes in cellular morphology during the cell cycle [4,5]. Fluorescent antibodies [6] and fluorescent heavy meromyosin [7] have been used to localize actin filaments in the cleavage furrow at cytokinesis, in pseudopods shortly after cytokinesis, and in stress fibers during interphase. Fluorescent antibody techniques have also been used to show that the F-actin-binding proteins myosin [8, 91, a-actinin [lo], and filamin [l I] are localized near microfilaments at various times during the cell cycle. These studies provide qualitative information about the location of these proteins, but quantitative information about the amount or assembly state of actin during the cell cycle is lacking. In HeLa cells, actin synthesis, as measured by [35S]methionine incorporation, remained relatively constant throughout the cell cycle with actin accounting for 24% of the total methionine incorporation [12]. However, actin has been identitied as one of the first major proteins whose rate of synthesis increased upon stimulation of serum-arrested (GO phase) 3T3 cells [13]. Light and electron microscopy studies of 3T3 cells have shown that anchorage-dependent growth can be correlated with the presence of actin-containing sheaths and that the expression of such sheaths is independent of serum concentration [14]. Thus, it appears that the distribution of actin between the filamentous and globular state might be of more significance in the regulation of cellular growth control than the * Present address: Experimental Therapeutics Division, University of Rochester Cancer Center, Box 704, Rochester, NY 14642, USA.

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total actin levels in the cell. Total actin in cultured cells was first quantitated by SDS-gel electrophoresis of cell homogenates followed by densitometry of stained gels [15]. More recently, an actin assay based on the inhibition of bovine pancreatic DNase I was developed [16], which was capable of distinguishing between the fdamentous and monomeric actin pools in cultured cells [17]. We recently modified this procedure [18] and studied the actin pools in several cultured cell lines under various conditions [19]. This report describes the application of this assay in determining the amounts of G-, F- and total actin throughout the cell cycle of CHO cells. Materials and Methods Cell culture. CHO cells were maintained as monolayers on Falcon plastic in McCoy’s 5a medium (Gibco) supplemented with 10% calf serum (K.C. Biological Inc., Lenexa, Kans.) in an atmosphere of 5% COZ, 95 % air at 37°C. Prior to synchronization, cells were grown in magnesium acetate-treated glass roller bottles. After a 40 h growth period, synchronous cells were obtained by selectively removing mitotic cells (mitotic indices (MIS) of >95 %) from asynchronous populations by a shake-off technique [20] modified to roller bottle cultures [21]. The mitotic cells were immediately cooled to 4°C collected by centrifugation at 800 g for 15 min, pooled and held at 4°C (up to 3 h) until replating for synchronous growth. Plating densities were chosen such that cell number would reach about 50 % confluency (106 cells/dish) at the time of cell lysis. Determination of labeling and MIS. Synchronized cells were plated onto 35 mm diameter plastic culture dishes in 2 ml of medium and incubated at 37°C. Thirty minutes before each hourly time point, [3H]thymidine (25 pl of 100 &i/ml) was added to the medium to give a final concentration of 1.25 @i/ml. The cells were removed from each plate using a 0.4% trypsin solution, centrifuged, and resuspended as single cells in calcium and magnesium-free Hanks’ balanced salt solution (HBSS) (Gibco). To assure total cell recovery the initial culture medium was added back to this cell suspension. Cell number was determined from passing an aliquot of the cell suspension through a particle counter (Data Particle Counter, Elmhurst, Ill.). The labeled cells in a second aliquot were swollen in a hypotonic buffer, sedimented, fixed in methanol : acetic acid (3 : 1), sedimented, and a small aliquot fixed on glass slides [22]. The percent of cells in mitosis was determined by staining with freshly faltered 2% aceto-orcein; mitotic cells were identified as those containing condensed chromatin. The remainder of the slides were immersed in Kodak NTB nuclear track emulsion (Eastman Kodak CO., Rochester, NY), exposed for 10 days at 4”C, developed in Kodak D-19, and treated with 3 % Gurr’s Giemsa for 1 h [23]. Cells categorized as being in S phase had more than 20 grains/nuclei and were easily distinguished from non-labeled cells. Actin quantitation. Multiple plates containing identical cell numbers were harvested at each time point. The cells were lysed and actin-quantitated as previously described [18]. Briefly, the medium was removed by aspiration and the cultures rinsed with HBSS. An aliquot of myosin sufficient to give a molar ratio of myosin to actin of one was added to the plate which was placed on ice. Cells were lysed by addition of 1% Triton X-100, 10 mM Tris, 2 mM MgQ, 0.2 mM dithioerythritol, pH 7.4 (cell lysis buffer) containing 15% glycerol and kept at - 10°C. The plate was scraped with a rubber scraper and the cell lysate removed. To assure complete collection of loosely attached mitotic cells, the medium as well as the HBSS wash of the mitotic cultures (as identified by phase microscopy) were centrifuged and the collected cells were added back to the cell lysis buffer. The plate was then rinsed with cell lysis buffer and the rinse combined with the initial lysate. The solution was centrifuged at 10000 g for 1 min to pellet the F-actin. The supematant, containing G-actin, was removed and 2 mM Tris, 1 mM Naz-ATP, 0.2 mM CaClz, 0.5 mM dithioerythritol pH 8.0 was added to the pellet to depolymerize the F-actin. Actin was quantitated in each fraction using the DNase I inhibition assay [16] which was standardized with freshly purified F-actin. The sum of the amounts of G-actin and F-actin equals total actin. Protein-was determined by a modification of the method of Lowry et al. [24] using bovine serum albumin as the standard [25].

Results The degree of synchrony achieved in the initial mitosis ranged from a low of 95% to a high of 97% in the two separate experiments reported. Only the cells

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Fig. 1. Quantity of (a) total actin and (b) total protein per lo6 CHO cells throughout the cell cycle. Results from five plates for total actin, two plates for total protein and one plate for cell number were used for each data point presented. Averaging of the data over the first cell cycle gives 6.0+ 1.O pg actin/106 cells and 237k31 ug protein/106 cells which compare with the values of 6.Ok1.2 ug actin/lO’ cells and 254+34 ug protein/106 cells obtained for asynchronous cultures [18].

MITOSIS

from the group with 97% initial synchrony were carried through a second cell cycle, where the maximum mitotic index reached 18%. The labeling index gave a maximum value of 90 % in the first S phase and dropped to a maximum of 80 % in the middle of the second S phase. A total of 2x lo8 cells were harvested by the roller bottle synchrony procedure for plating in culture dishes. The total amount of actin per CHO cell decreased rapidly during mitosis and slowly increased throughout the subsequent cell cycle only to decrease again at the next mitosis (fig. 1 a). The levels of total cellular protein showed a similar pattern (fig. 1 b). The larger errors associated with the samples harvested around mitosis reflect difftculties in obtaining accurate cell numbers. Collecting and adding back the mitotic cells which detached and were in the growth medium helped correct the problem to some extent. However, significant variations in measurement of cell numbers still occurred at this point in the cell cycle. The total amount of actin as a percent of total protein did not change dramatically during the entire cell cycle (fig. 2). Although some variation in these values was evident through the first cell cycle, the same changes were not repeated in the next cell cycle. A large increase in the percent F-actin occurred following the initial mitosis of freshly plated cells (fig. 3). However, cells progressing through the second mitotic and Gl phases of the cycle did not show these significant changes in the levels of F-actin. It does not appear that these alterations in the F-actin level arose from the prolonged cold storage of the cells (about 3 h for the major synchrony experiments), since cells which were collected in the cold but rapidly replated at 37°C showed similar changes in levels of F-actin (fig. 3). Few other significant and reproducible changes in the percent F-actin occurred during the cell cycle. A small increase in the percent F-actin was observed in early to mid-S

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2. Actin expressed as a percent of protein for CHO cells throughout the cell cycle. 0, Total actin as a percent of total protein; A, G-a&in as a percent of soluble protein. Values are the average of the results from two plates with bars representing the range. Averaging the data for total actin over the first cell cycle gives a value of 2.44?;0.3% of total protein as actin, while the actin content of asynchronous CHO cells is 2.5+0.2% [18]. Fig. 3. F-actin as a percent of total CHO cell actin during the cell cycle. All cells were lysed in the presence of exogenous myosin. A, Synchrony no. 1 cells pooled and held on ice A, s3 h, n , <30 min. Synchrony no. 2 0, first, 0, second cell cycle. Values are the mean of results from three plates. Bar, SD.

Fig.

phase followed by a slight decrease in F-actin levels in mid-S phase and another slight increase in late S phase. However, all these changes in percent F-actin are relatively small compared with the changes which were observed upon initially plating the mitotic cells. Previously we showed that purified F-actin added to cell lysates totally cosedimented with exogenously added myosin; however, in the absence of added myosin virtually none of the added F-actin sedimented under the centrifugation conditions employed (10 000 g for 1 min) [ 181.Therefore, the amount of CHO cell actin in cell lysates that sedimented in the presence of exogenous myosin is considered to be F-actin. However, a large percentage of CHO cell actin also sedimented without the addition of myosin, and this actin must have been associated with other cellular structures which sedimented at a low centrifugal force. Since it has been shown morphologically that microfilamentous structures are preserved after Triton X-100 treatment [26], we have assumed that the F-actin which sedimented in the absence of added myosin must have been closely associated with the cytoskeleton. The variation in quantities of this cytoskeletonassociated F-actin throughout the CHO cell cycle is shown in fig. 4~. These changes parallel those observed for total F-actin; thus, a fairly constant difference of about 10% between the values of total F-actin and cytoskeleton-associated F-actin is observed (fig. 4b). Discussion When grown at densities such that cell-cell contact occurs, CHO cells in S phase are flat, smooth and thinly spread over the substratum, whereas cells in mitosis are round and loosely attached to the substratum [4, 51. The intervening phases have intermediate morphological forms. The cytoskeleton which is composed of several fdamentous protein systems is involved in determining the

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Fig. 4. Comparison throughout the CHO cell cycle of the percent of actin sedimented in the presence (total F-actin) and in the absence (cytoskeletal-associated F-actin) of exogenous myosin. (a) Variation of % Factin determined by lysis of cells in 0, the presence or A, absence of exogenous myosin. The average of results from three plates with bars representing the SD. A, Average of results from two plates with bars representing the range. Values of 0, averaged over the first cell cycle are 72.9+5.0% compared with 72.4+5.7% for asynchronous CHO cells [18]. (b) Difference in the cytoskeletal-associated F-actin from the total F-actin determined from the difference of the two curves in fig. 4 a. The differences shown in (b) averaged over the first cell cycle are 8.7% compared to a difference of about 8% for asynchronous CHO cells [18].

structure of the cytoplasm which, in turn, determines the overall cellular morphology. Two of the most studied lilamentous systems are the microtubule and microfilament networks. Cell cycle studies have shown that microtubule structures appear to be rather static, except during mitosis when the elaborate mitotic apparatus assembles at a time when the total amount of tubulin is constant [27]. The sudden shift to assembled microtubules may be triggered by a number of factors important in microtubule assembly which are known to change at or before mitosis (e.g., phosphorylation of tubulin, levels of Ca*+ or Ca2+-activated ATPase, and CAMP concentration) [28]. The organization of the microfilament system changes concomitantly with the morphological changes [6, 71. In S phase cells, stress fibers are evident but disappear as cells approach mitosis. However, as the stress fibers disappear other microlilament containing structures form, such as the numerous microvilli, the contractile ring, and pseudopods. The mechanism by which the reorganization of the actin microlilaments occurs has not been elucidated. Previous studies on the assembly state of microtilaments in secretory cells have shown that the proportion of actin in the filamentous form increases following stimulation of the cells with a secretogogue [29, 301. However, cells undergo a wide variety of morphological changes in response to drugs and other agents without altering either the total amount of actin-or the amount of actin in the filamentous pool [17, 191.The cell cycle provides a system in which to study the reorganization of microtilaments in response to intracellular signals regulating cell growth and division. In this report, a large decrease in the amount of F-actin was observed during the first mitosis with a gradual increase in the F-actin levels during the subsequent Gl phase. This pattern was also observed in asynchronous CHO cells

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which were plated from suspension culture [18]. When synchronized cells were allowed to progress through the next mitotic phase, no such decrease in F-actin amount was noted. Therefore, the decreased amount of F-actin which occurred during the first mitosis probably results from the plating of mitotically selected cells from suspension to monolayer; CHO cells which remain attached to the substratum during mitosis do not show this shift in the actin pools. However, given as mitotic index in the second cell cycle of under 20% and a maximum change of 20% in F-actin levels during plating of cells grown in suspension culture, a change in F-actin levels of only 4% would probably not be detected in this study. The results of this study which show that during the cell cycle very small changes occur in the levels of either total actin, F-actin or cytoskeletal-associated F-actin do not rule out the possibility that actin filaments disassemble in one region of the cell concomitant with an equivalent amount of reassembly in another region. However, data obtained from the studies on asynchronous cells suggest that changes in cellular morphology do not require changes in the F/Gactin ratio [17, 191.Thus, the changes which occur in the localization of microfilaments during the cell cycle could be due to rearrangement of F-actin without the requirement of going through a monomeric intermediate. These rearrangements may involve the activation of F-actin fragmenting and capping proteins [3 1, 321. Support in part by grants NS10429, CA18334 and a BRS grant from the National Institutes of Health, and a Grant-In-Aid from the Muscular Dystrophy Association. Taken in part from a Ph.D. thesis submitted by C. S. H. to Colorado State University, Fort Collins, CO 80523, USA.

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28. Snyder, J A & McIntosh, J R, Ann rev biochem 45 (1976) 699. 29. Swanston-Flatt, S K, Carlsson, L 8z Gylfe, E, FEBS lett 117 (1980) 299. 30. Carlsson, L, Markey, F, Blikstad, I, Persson, T & Lindberg U, Proc natl acad sci US 76 (1979) 6376. 31. Weeds, A, Nature 296 (1982) 811. 32. Craig, S W & Pollard, T D, Trends biochem sci (1982) 88. Received March 16, 1983

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