Changes in plasma membrane and microfilaments accompanying morphologic differentiation in Chinese hamster ovary (CHO) cells

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Experimental Cell Research 118 (1979) 191-203

CHANGES

IN PLASMA

ACCOMPANYING IN CHINESE

MEMBRANE

AND MICROFILAMENTS

MORPHOLOGIC HAMSTER

DIFFERENTIATION

OVARY

(CHO) CELLS

OMILA SAGAR KOCHHAR’ Department

of Biology,

Universiry

of Virginiu.

Charlorresville.

VA 22901, USA

SUMMARY Chinese hamster ovary cells undergo a change from epithelioid to fibroblastic morphology when cultured in the presence of 0.4 mM db-CAMP. This study was initiated to determine if the change in morphology was associated with any change in the levels of the membrane-bound enzyme, adenylate cyclase. Plasma membrane ‘ghosts’ were isolated from treated and untreated control cells; higher than normal specific activity of the enzyme was found in the treated cells. The treated cells also revealed that, during the course of plasma membrane isolation procedure, the membranous organelles were more resilient than those of the control cells. Electron microscopic study revealed a distinct increase in the number of microfilaments in the treated cells; the microfilamentous structures were seen as bundles in the cytoplasm, but more important, they were observed in association with the plasma membrane ‘ghosts’. No such association was found in the control cells. It is proposed that microfilaments generate motile force which establishes bipolar elongation of cells, producing conformational change in the molecular architecture of the plasma membrane, thereby unmasking or generating active adenylate cyclase sites.

Morphologic differentiation has been induced in several cell types by varying the intracellular levels of cyclic adenosine monophosphate (CAMP). When intracellular level of CAMP is raised Chinese hamster ovary cells (CHO) become fibroblastic [I], neuroblastoma cells generate neurites [2, 31, Dictyostelium discoideum produces stalk cells and [4] fusion occurs between myoblasts [5]. On the other hand, BHK cells become rounded and flat when intracellular level of CAMP is decreased [6]. Morphologic changes are also accompanied by changes in other characteristics of the cells [7]. For example, the growth rate slows down [8], adhesion to the substrate is increased [9], cell motility decreases [lo], agglutinability of tumor cells by lectins is reduced and the antigenic properties of the 13-781X0?

cell surface are changed [ 111. The intracellular levels of CAMP can be raised in the transformed cells by (a) the addition of CAMP and its analogs such as dibutyryl CAMP (db-CAMP); (6) by the stimulation of adenylate cyclase (AC) by hormones [12, 13, 141and by cholera toxin [15, 161,or (c) by the inhibition of cyclic nucleotide phosphodiesterase [17]. The cell shape change induced by any of these agents can be inhibited by the addition of colchicine, colcemid [l] or vinblastine [18]. Since colchicine and vinblastine inhibit microtubule assembly, it was inferred that microtubules are responsible for the production of cell shape change. The electron microscopic ’ Mailing address: Department of Anatomy, Jefferson Medical College, 1020 Locust Street, Philadelphia. PA 19107,USA.

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studies by Porter et al. [19] consequently showed an increase in number and a more orderly orientation of the microtubules. Microtubule disrupting drugs also inhibit plasma membrane-related processes and it is suggested that the mobility of macromolecules in the plasma membrane is regulated by microfilaments and microtubules [20, 211. Cell shape change can also be prevented by cytochalasin B (CB) [l] which is known to disrupt microfilaments, and by Concanavalin A (ConA) bound to the cell surface [18]. Some recent studies have documented increases in the microfilaments concomitant with change in cell morphology [22, 23, 241. Thus it appears that plasma membrane and microfilaments may play a more significant role in bringing about morphologic differentiation than is understood at present. In this report CHO cells were induced to undergo cell shape change upon treatment with db-CAMP. It was found that increases in microfilamentous structures and in levels of adenylate cyclase (AC) enzyme accompany morphological change of epithelioid CHO cells into bipolar, fibroblastic shape. A suggestion as to how these events interrelate is advanced. A preliminary report of this work has been presented [25]. MATERIALS

AND METHODS

Cell cultures CHO cells were obtained from American Type Culture Association. The cells were grown in roller bottles in F-12 medium supplemented with 10% fetal calf serum (FCS) at 3PC, 100% humidity, and 5 % CO,. The cells were first grown for 2-3 days to confluency in T-75 Falcon flasks. These cells were then harvested with 0.25 % trypsin and reseeded in glass roller bottles with IO6cells/culture bottle in 200 ml of the medium. The cell cultures become confluent in 5 days. The growth medium was changed once on the 2nd or 3rd day. For inducing differentiation, the non-confluent cell cultures were treated with 0.4 mM db-CAMP [25] on the 4th day of culture. After 24 h the majority of the treated cells change their cell shape and become elongated with fibroblastic morphology. Exp

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118 f 1979)

Fig. 1. Flow diagram of the isolation procedure.

Plasma membrane isolation procedure The control and the treated cells in the roller bottles were rinsed twice with 0.25 M Tris-HCI buffer at pH 7.3 at room temperature, then incubated further with 15 ml of 0.05 M Tris-HCI buffer at the same pH for 5 min at 37°C. The cells were then eased off the culture surface with a rubber policeman and collected in four 50 ml capacity conical centrifuge tubes. A flow diagram ihustrates (fig. 1) the various steps in the isolation of a pure plasma membrane fraction of CHO cells. The cells in each step of isolation procedure were monitored by phase contrast microscopy. To each 50 ml suspension of CHO cells, 1 ml of 60% sucrose solution was added to increase the tonicity of the suspension buffer to prevent damage to the nuclei and nuclear membrane. The cells were pelleted for 15 min at 1000 rpm in a SorvaIl centrifuge at PC. The supematant was repelleted to obtain smaller cells or cells staggering behind in the suspension. The cell pellet was resuspended in 15 ml of 0.01 M Tris buffer at pH 7.3 in a 15 ml capacity Dounce homogenizer, and incubated at ice temperature for 3-g min. This treatment enabled gradual swell-

Differentiation ing of the cells. The plasma membrane ‘ghosts’ were liberated from the intact swollen cells by gentle screwing motion of the tight-fitting pestle of the glass homogenizer. About 20-30 strokes were sufficient to liberate the majority of the plasma membrane ‘ghosts’. Sucrose solution was then added to the homogenate to achieve a final sucrose concentration of 10%. The homoeenate now in 10% sucrose in 0.05 M Tris buffer was liyered on top of a discontinuous gradient of 45 %/35 % (10 ml/3 ml) sucrose in 0.01 M Tris buffer at pH 7.3 in two conical centrifuge tubes of 50 ml capacity. The gradients were spun at 1 100 rpm for 30 min.in the Sorvall centrifuge-at 3°C in a swhrg out rotor. The top layer was removed within 5 ml of the interface between 45 and 35 %. This interface suspension as well as 5 ml of the gradient on either side of the interface was diluted with the Tris buffer to 10% with regards to sucrose. This was layered on a second 45 %/35 % sucrose eradient and centrifueed as before. The interface was again removed as in thi first gradient without the 5 ml aradient from either side. This interface was again &luted with the Tris buffer to 20% sucrose, layered on 35 % sucrose in the conical centrifuge tube and spun at 2200 rpm for 1 h to pellet the plasma membrane ‘ghosts’. The pellet was resuspended with 10 ml of the buffer in a 15 ml capacity conical plastic tube and centrifuged at the 2 200 rpm for 1 h. This washing step may be repeated. The washed plasma membrane pellet was examined under the phase contrast microscope and processed for electron microscopy and for enzyme analysis.

of CHO cells

oxide and fixed in Epon. Ultrathin sections were cut on a Sorvall MT-2 microtome, and stained with lead citrate according to Venable & Coggeshall[28]; otherwise the sections were fist stained with 3% aqueous many1 acetate before lead citrate staining. The sections were mounted on coated copper grids and examined at an operating voltage of 75 kV in a Hitachi model EU 11 electron microscope.

RESULTS Cell morphology: Control and db-CAMP treated cells

Chinese hamster ovary cells in the control cultures were primarily of epithelioid morphology (fig. 2). However, among these confluent cultures, where ever the cells were crowded, a few elongated cells were seen. The non-confluent control cell cultures after incubation with 0.4 mM dbCAMP for 24 h, became differentiated and the cells acquired fibroblastic morphology (fig. 3).

Enzyme assay

Effect of isolation procedure

The cell homogenate and the isolated plasma membrane fractions (25-100 ~1) were incubated with equal volumes of the adenvlate cvclase incubation mixture. which consisted of 20 mM Tris-HCl at pH 8.3-8.5; 0.2 mM AMP-PNP (adenylyl imidodiphosphate), 0.2 mM methyl isoxanthine, 10 mM MgSO, and 10mM NaP according to Maguire & Gilman [26]. After incubation at 30°C for 10 min, the reaction was stopped by diluting the mixture with 200 ~1 of sodium acetate buffer (75 mM) at pH 3.9. The incubation mixture was prepared fresh each time. The incubated fractions were kept frozen at -20°C. The CAMP generated from AMP-PNP during this incubation of plasma membranes was estimated according to the protein binding assay of Gilman [27].

Tris buffer treatment for harvesting

Electron microscopy The control and db-CAMP treated cells and these cells after hvootonic Tris-HCl buffer treatment were fixed in situ*& cell cultures with 2.8% glutamldehyde in 0.1 M cacodylate buffer containing 3 mM CaCl, at 3°C for 1 h. Different fractions from the isolation procedure were similarly fixed with glutaraldehyde and embedded in 1% agar. The fixed-cells and kmbedded, fixed fractions were washed with 0.1 Mcacodylate buffer. All fixed preparations were then postfixed with l-2 % osmium tetroxide (OSOJ; they were further fixed with 0.5 % many1 acetate in water or Kellenberger’s buffer for l-2 h. Dehydration was carried out in a graded series of ethanol, cleared in propanol

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cells.

Both the control and treated cell cultures when incubated with 50 mM Tris-HCl buffer at pH 7.2 at room temperature show the following results: The cells began to swell and they gradually withdrew and retracted from the adjoining cells and the substratum (figs 4,5). It took 15-20 min for the majority of control cells to finally become rounded and loosened from the substrate (figs 6, 7). This process could be accelerated by lowering the molarity of the Tris buffer and by raising the temperature. db-CAMPtreated, elongated cells took longer to retract their processes (fig. 7) and to round up under the same conditions. Thus Tris buffer treatment was chosen to harvest the cells from the roller bottles. The optimum conditions to harvest cells was to incubate cells with 0.1-0.5 M Tris buffer at 37°C for only 5 min when they were just sufficiently Exp Cell Res 118 (1979)

194 0. s. KO(.llllNt

Differentiation of CHO cells detached from the substrate and could be easily and gently liberated either by squirting or with a rubber policeman. Much of the mechanical or chemical damage to the cells was avoided by this procedure. The use of Tris buffer treatment for harvesting the cells eliminates the use of trypsin or any other treatments commonly employed for the same purpose and which are known to temporarily damage the cell surface to some extent. The cells harvested in this way were intact but slightly swollen and they frequently came off the surface as cell sheets. However if cells were treated with 0.01 M Tris HCl buffer, they rounded up and came off the surface by themselves, but some of the cells swell and lyse in the process. Morphologic characterization of various fractions. Before homogenizing, the cells (fig. 8) were treated with 0.01 M Tris buffer to separate plasma membranes from the underlying cytoplasm (fig. 9). The cells swell within 4-g min. The control cells began to lyse and clump after this period, whereas the db-CAMP-treated cells remained swol-

Figs 2-12. PM, plasma membrane; N, nucleus; C, cytoplasm. Fig. 2. Control CHO cells in culture. The cells show epithelioid morphology; jig. 3, elongated CHO cells in culture treated for 24 h with db-cAMP;fig. 4, control CHO cell culture washed with 50 mM Tris buffer for 5 min. Cells have withdrawn from each other creating intercellular spaces;j?g. 5, db-CAMP treated cells washed with Tris buffer as above. The intercellular spaces are large due to greater retraction of cells; jig. 6, control CHO cell culture incubated for 15 min with hypotonic Tris buffer. The cells have completely rounded off and are loosened from the substratum; fig. 7, db-CAMP-treated cells incubated for I5 min with hvnotonic Tris buffer, Most cells have rounded off but still show retracting processes (arrow) and firm adhesion to the substratum;Jg. 8, control CHO cells harvested with Tris buffer method are slightly swollen;fig. 9, the swollen cells obtained as above (fig. 8) treated with 0.01 M Tris buffer for 10min at 4°C. Plasma membrane is loose and cytoplasm adheres to the nucleus; fig. 10, control CHO cell homogenate. Plasma membrane ‘ghosts’ are separated from the nucleus. Cytoplasm is dispersed. Figs 2-10. Phase contrast. x 350.

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len for a much longer period and were resistent to the hypotonic condition. The treated cells produced a majority of larger ‘ghosts’ compared to control cells, and the latter probably fragmented during homogenization. The membranous organelles in the homogenate of treated cells appeared more prominent and resilient under the phase contrast microscope. After the first centrifugation of the homogenate (fig. 10) most of the finer cell debris, ribosomes, mitochondria remained in the upper 10% sucrose layer of the gradient. The interface between the lo%/35 % and 35 %/45% contained most of the plasma membrane ‘ghosts’ and some small nuclei, mitochondria, and some cell debris. The bottom 45 % layer of the gradient contained nuclei, some cells, a few larger plasma membrane ‘ghosts’ and aggregates of the cell debris. The second centrifugation removes most of the remaining cell debris, contained in the interface suspension. In the case of db-CAMP-treated cell homogenate, since the plasma membrane ‘ghosts’ were denser and larger, they tended to sediment with the nuclei during the first centrifugation. Also many nuclei remained attached to the plasma membranes obtained in the interface. Therefore, fractionation and isolation of plasma membranes from the treated cells were more tedious than from the control cells. As a result some plasma membranes were lost in the 45% layer of the first fraction. Increasing the sucrose density from 45 to 65 % did not separate the nuclei and the plasma membrane ‘ghosts’. Attempts to refractionate the 45 % fraction containing the nuclei and plasma membranes were also not successful. Therefore, some plasma membranes had to be sacrificed in order to achieve the maximum purity of the plasma membrane fraction. Exp Cd/ Re3 1I8 ( 1979)

1%

0. S. Kochhar

Figs II, 12. PM, plasma membrane; N, nucleus: C. cytoplasm. Fig. II. Final plasma membrane fraction from control cells. The cell ‘ghosts’ are mostly rolled up sheets and

fragments; fig. from db-CAMP to some extent, Phase contrast.

12, final plasma membrane fraction treated cells. The cell ‘ghosts’ retain, their original morphology. FigA I I, 12. x700.

The purity of the fiial plasma membrane fraction was judged both morphologically, under phase contrast (figs 11, 12) and electron microscopes and by the enrichment of adenylate cyclase after the enzyme assay (fig. 13). Adenylate cyclase assay

The specific activity of adenylate cyclase was measured in whole cell homogenates Figs /4-18. PM, plasma membrane; M, mitochondria; MF, microfdaments; MT, microtubules; RER, rough

endoplasmic reticulum.

1 0

Fig. 14. Control cells show rough surface with extenabed

Fig. 13. Ordinate:

pmoles/min/mg protein. The specific activity of adenylate cyclase in control and db-CAMP-treated CHO cells. (n) Control homogenate; (b) control plasma membrane fraction; (c) treated homogenate; (d) treated plasma membrane fraction. The relative increase in the treated plasma membrane fraction is approximately twice that in the control fraction.

sive blebbing;fig. 15, control cell shows microprojections at the cell surface. Well-defined microfdaments and microtubules are not present; jig. 16, db-CAMPtreated cell shows smooth cell surface;.&. 17, treated cell shows welldefined microfilament bundles and microtubules in the cytoplasm; fig. 18, treated cell showing a bundle of microfilaments in association with the plasma membrane. The plasma membrane is detached at one site (arrow) revealing its association with microfilaments. Figs 14, 16, x27000; figs 15, 17, x34500; fig. 18, x54000.

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Figs 19-23. PM. plasma membrane. Fig. IY. Control CHO cell after exposure to hypotonic Tris buffer. The cell is swollen as are its membranous organelles. Note the lack of microfilaments in the cytoplasm, particularly in the vicinity of the plasma membrane;,fig. 20, db-CAMP-treated cell after exposure to hypotonic Tris buffer as in fig. 19. The cell and its organelles are swollen as in the control cell. but note the presence of microfilaments mostly associated with the plasma membrane:,fi,q. -I/, db-CAMP-treated cell after exposure to Tris buffer as in fig. 20. Shown is a micro-

filament bundle in association with the plasma membrane. Cross connections between individual microfilaments are present (nrro,t~);fi~. 22. cross-section of rolled up plasma membrane ‘ghost’ isolated from control cells. The membranes are clean;.&. 23. crosssection of plasma membrane ‘ghost’ isolated from dbCAMP-treated cells. Several microfilaments are associated with the plasma membrane and are present within the ‘ghost’ space. Figs 19. 20. 12. 23. X39000: fig. 21. X66000.

Differentiation

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199

and the plasma membrane fractions of the microfilament formations were not seen in control and the db-CAMP-treated cells. the cytoplasm of control cells (compare There was an average of 7-fold enrichment fig. 15). The hypotonic Tris buffer treatment of of the enzyme in the plasma membrane fraction over that of the homogenate both in the cells further clarified the status regarding control and the treated fractions (fig. 13). microfilaments. In the control cells (fig. 19) The most important and striking feature of there were no microfilaments to be seen these paired experiments was that the db- either in the cytosol or below the cell surCAMP-treated cell fractions presented a face. Infrequently some amorphous material of fine density could be observed in consistent increase in the specific activity of the adenylate cyclase enzyme. As shown association with the plasma membrane. in fig. 13, there was about 1.5fold increase This suggested that either the microfilain the activity of the enzyme in both frac- ments were not present or they were untions from the treated cells. No enzyme ac- stable and could easily be removed from the tivity was detected in the pellets and super- control cells by the hypotonic Tris buffer natants from the first and the second frac- treatment. In striking contrast were the treated cells, which contained microfilationation steps. ments firmly attached to the plasma memMorphology of microfilaments and brane (fig. 20). Several microfilaments were plasma membranes loosely present in the cytosol. Some of the Thin sections of control CHO cells grown microfilamentous bundles were observed to confluency and fixed in situ showed sur- below the plasma membrane and some face blebbing (figs 14, 15). Associated with cross connections between these microfilathe blebbing some granular material was ments were noticed (fig. 21, arrow). These seen beneath the plasma membrane, but microfilamentous bundles were present towell defined fibers were not easily seen. wards the free cell surface and were not to The cytoplasm contained the usual array of be confused with the stress bundles present organelles, that is, free ribosomes, rough towards the ventral surface of the cells. The endoplasmic reticulum, Golgi membranes microfilaments were about 7 nm thick. The isolated plasma membranes from the and vesicles. Only a few microtubules were seen. The treated cells differed from control control cells were obtained as clean sheats cells in several fine structural features (fig. and, as mentioned above, did not contain 17). The cell surface in these elongated cells microfilaments (fig. 22). On the other hand, was smooth for the most part except for a isolated plasma membranes from db-CAMPfew filliform microprojections at a few treated cells enclosed several microfilaplaces. Associated with the surface projec- ments, some in association with the plasma tions were well formed microfilaments (figs membrane (fig. 23). 16, 18). Profiles of rough endoplasmic reticulum, free ribosomes, Golgi membranes, DISCUSSION and mitochondria were not affected by the treatment but it appeared that there was an Several recent studies suggest that changes increase in the Golgi membranes. The cyto- in cell morphology, growth rate and difplasm of the treated cells also contained ferentiation of cells may be regulated by bundles of microfilaments (fig. 17). Such intracellular levels of CAMP [29]. The levels 13t-781802

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of CAMP have been known to vary during the cell cycle [8, 241 accompanied by a change in the cell shape. Neoplastic and transformed cells contain lower levels of CAMP and differ from their parent cell type in morphology and other characteristics. The molecular mechanisms underlying these morphologic changes in response to varing levels of CAMP are not fully understood. In this paper I report an attempt to study the molecular changes that occur in the plasma membranes of CHO cells due to cell shape change under the influence of db-CAMP. For this purpose, plasma membranes were isolated to study differences in (1) the adenylate cyclase activity in the plasma membranes; and (2) the presence and relationship of microfilamentous structures to the cell surface in the db-CAMPtreated cells. To date most of the plasma membrane isolation procedures using tissue culture cells employ enzymatic treatments for harvesting cells. As an alternative, CHO cell cultures were treated with hypotonic TrisHCl buffer for a brief period. This treatment appeared to reduce adhesive forces between the cells and the substratum, which encouraged rounding up of the cells in quite the same morphologic fashion as seen with trypsin treatment; however, the process was slower. Tris buffer has been considered to be a mild chelator of Mg2+ ions. Among other factors, Mg2+ ions are considered to influence polymerization or appearance of microfilaments [30-321. Therefore, it is possible that the entry of this buffer into the cell periphery induced partial depolymerization or dissociation of the microfilaments. As a consequence the previously extended cell processes were retracted. Simultaneously the hypotonic buffer produced swelling and the cells became rounded. Since Tris-HCl buffer is comExp Cell Res 118 (1979)

monly used in enzyme assay mixtures. which are usually incubated at 37°C for 5-15 min, and as cells can be cultured in Tris-HCl buffer [33], these conditions were considered not to impose any experimental alterations for later analysis of the plasma membranes. At the same time this procedure prevented the danger of removing part of the cell surface which is the case with the enzyme treatments. The db-CAMPtreated cells took longer to retract, confirming the contention that the treated cells show stronger adhesions [9] possibly due to increased glycoproteins at the cell surface [34], and increased microfilaments in close proximity to the cell surface [22, 27, 301. Plasma membranes from several cell types have been isolated by Emmelot’s [35], Warren’s [36] or Kamat & Wallach’s [37] methods and by several modifications thereof. The isolation procedure outlined in this paper is a modification of Warren’s [36] and Kochhar’s [38] method. Essentially this procedure yields plasma membrane ‘ghosts’ within 68 h. The method of removing ‘ghosts’ from intact cells is gentle and no detergents or other additives were employed which could interfere with the native plasma membrane enzymes. Phase contrast and electron microscopic evaluation of the final fraction indicated minimal contamination with the cell debris. Plasma membrane ‘ghosts’ from db-CAMP-treated cells contained microfilaments which possibly strengthen and stabilize the plasma membranes. Perdue [22] also assigns the plasma membrane stabilization role to microfilamerits. The isolated plasma membranes from dbCAMP-treated CHO cells contain almost twice the specific activity of adenylate cyclase as compared with the control plasma membrane fractions under identical conditions. Even though the enzyme activity was

Differentiation of CHO cells variable from one set of experiments to the other, there was in each set a consistent increase in the enzyme activity. There are several reports where AC enzyme activity has been determined after treating the transformed cells with hormones [29]; however, there is no account which describes the levels of this enzyme when morphologic differentiation is induced by culturing cells in the presence of db-CAMP. In such a case, one would expect that the activity of adenylate cyclase would be inhibited or at least remains the same. Since exogenously introduced db-CAMP is considered to raise intracellular CAMP levels, the increase in the active adenylate cyclase sites in the plasma membranes of CHO cells appear incongruent with the expected results and needs an explanation. It has been shown that intracellular levels of CAMP decrease with transformation with an accompanied decrease of AC activity [29]. When transformed cells were treated with prostaglandin E which elevates CAMP levels, the cells revert back to fibroblastic morphology [ 13, 291. On the other hand BHK cells became rounded when treated with insulin which lowers CAMP and produces a decrease in AC activity [6]. Thus it appears that high AC activity is associated with fibroblastic morphology and the lower AC activity with rounded cells. CHO cells were originally derived by a spontaneous transformation of a fibroblastic type parent cell to an epithelioid cell type [l]. The increase in enzyme activity possibly reflects a reversal of the transformed state to parent type fibroblast. The increase in AC activity may also suggest that as the cell elongates, the restraints in the molecular architecture of the plasma membrane either become relaxed or altered and this conformational change results in the exposure of active adenylate cyclase sites. But this inference

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presupposes that the cell morphology must alter before the change in AC sites could occur, which means that those structures in the cell which generate movement and produce cell shape change must be active before this event. Microtubules and microfilaments are considered to be involved in cell movement [39,40] and in the maintenance of cell shape [22-241. The contractile proteins, actin and myosin, and actomyosin in non-musclecells are associated with those cellular functions which involve motility [31, 411; the movement may be at the cell surface such as exocytosis, endocytosis and cell surface modulations [20, 21, 39, 421 or it may involve the movement of the whole cell such as in amoeboid movement and spreading [39,43, 441. This and other studies [22, 43451 suggest that microfilaments, in association with the cell surface, are the primary structures to generate motive force that produces cell shape change whereas microtubules are secondary in this process and help to maintain the altered cell shape [19]. The experiments by Perdue [22] and Miranda et al. [46], in which cells plated and grown in the presence of 10 pg/ml colchicine spread and attached to the substratum while microtubule assembly was inhibited, tend to support this proposal. Increase in microtubule assembly in a CHO mutant M7 grown in the presence of db-CAMP, and a lack of change in morphology also support this view [47]. Thus, it appears that it is not the orientation [19] of the microtubules which is of prime importance in bringing about the bipolar morphology of cells. Instead, orientation is determined by the activity and direction of the contractile proteins. In summary, our data supports the following conclusions and suggestions. (1) The microfilaments, associated with the cell surface, generate the motive force Exp Cd Res I18 f 1979)

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for the elongation of the cells. Since high concentrations of Ca2+ and Mg”+ ions encourage, or are necessary, for the polymerization of microfilaments [30-321 whereas the polymerization of microtubules require low Ca2+ concentration [48], these two organelles seem to require opposite molecular environments for their assembly and function. As the microfilaments are being formed, they create an environment requisite for the production of microtubules. It is suggested, then, that microtubules function to maintain the altered cell morphology originally brought about by the action of microfilaments. (2) As evidenced from the adenylate cyclase studies, the molecular organization of plasma membrane is considered to change with the appearance of increased enzyme sites in response to cell elongation, we suggest that the association of microfilaments with the cell surface, which produces cell shape change, also induces conformational changes in the molecular structure of the plasma membrane. Similar proposal has come from Stossal[49] that actomyosin-like systems associated with the membrane are responsible for producing mobility of the macromolecules in the plasma membranes. The variations in intracellular levels of CAMP is responsible for the appearance of those cytoplasmic organelles which generate motive force for bringing about cell shape change. Not only do these structures change the gross morphology of the cells, they also result in changing molecular organization of the plasma membranes of these cells and thereby altered functions. This work was supported by NlH grant no. GM20523 to Dr L. I. Rebhun, to whom I am grateful for provision of all laboratorv facilities. I also thank N. Ivev, T. Schnaitman, A. Sorensen, C. Amy and J. Penner for technical assistance. A preliminary report of this work has been presented previously (J cell biol 67, 219a (1975).

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44. Taylor, D L, Condeelis, J S, Moore, P L & Allen, R D, J cell biol 59 (1973) 378. 45. Lazarides, E, J cell biol 70 (1976) 359a. 46. Miranda, A F, Godman, G C & Tanenbaum, S W, J cell biol 62 (1974) 406. 47. Borman, L S, Dumont, J N & Hsie, A W, Exp cell res 91 (1975) 422. 48. Weisenberg, R C, Science 177 (1972) 1104. 49. Stossel, T P, Seminars in hematol I2 (1975) 83. Received May 8, 1978 Revised version received August 2, 1978 Accepted August 7. 1978

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