Changes in the levels of human tropomyosins IEF 52,55, and 56 do not correlate with the loss of actin cables observed in SV 40 transformed MRC-5 fibroblasts

Summary A mouse monoclonal antibody (mAb 1D122G9) raised against human tropomyosin IEF 52 (HeLa protein catalogue number, Mr=35 kd) has been characterized both in terms of specificity and patterns of immunofluorescence staining in Triton extracted cultured cells. As determined by two dimensional gel immunoblotting of HeLa cell proteins the antibody recognized IEF 52 and two other acidic proteins (IEF 55, Mr=31.8 kd; IEF 56, Mr=31 kd) previously identified as putative tropomyosin-like proteins. Immunofluorescence staining of Triton extracted cultured cells revealed the striated or interrupted pattern on the actin cables characteristic of tropomyosin staining. Quantitation of the three tropomyosins in Triton cytoskeletons from normal and SV 40 transformed human MRC-5 fibroblasts showed that the latter contained significantly less of tropomyosin IEF's 52 (52%) and 56 (72%) as compared to their normal counterparts. The ratios of these two tropomyosins to actin however was very similar for both types of cytoskeletons. This was not the case for tropomyosin IEF 55, which was present in nearly twice the amount in the cytoskeletons from the SV 40 transformed cells. The ratio of actin to total tropomyosin for whole cells was found to be unchanged on transformation. This ratio however was 31% lower in the cytoskeletons from the transformed cells. These and other results presented here suggest that changes in the levels of these three tropomyosins are not enough to account for the magnitude of the loss of actin cables observed in the transformed cells.Abbreviations IEF isoelectric focusing - mAb monoclonal antibody - NEPHGE non equilibrium pH gradient electrophoresis     

Interaction of fluorescently-labeled contractile proteins with the cytoskeleton in cell models

To determine if a living cell is necessary for the incorporation of actin, alpha-actinin, and tropomyosin into the cytoskeleton, we have exposed cell models to fluorescently labeled contractile proteins. In this in vitro system, lissamine rhodamine-labeled actin bound to attachment plaques, ruffles, cleavage furrows and stress fibers, and the binding could not be blocked by prior exposure to unlabeled actin. Fluorescently labeled alpha-actinin also bound to ruffles, attachment plaques, cleavage furrows, and stress fibers. The periodicity of fluorescent alpha-actinin along stress fibers was wider in gerbil fibroma cells than it was in PtK2 cells. The fluorescent alpha-actinin binding in cell models could not be blocked by the prior addition of unlabeled alpha-actinin suggesting that alpha-actinin was binding to itself. While there was only slight binding of fluorescent tropomyosin to the cytoskeleton of interphase cells, there was stronger binding in furrow regions of models of dividing cells. The binding of fluorescently labeled tropomyosin could be blocked by prior exposure of the cell models to unlabeled tropomyosin. If unlabeled actin was permitted to polymerize in the stress fibers in cell models, fluorescently labeled tropomyosin stained the fibers. In contrast to the labeled contractile proteins, fluorescently labeled ovalbumin and BSA did not stain any elements of the cytoskeleton. Our results are discussed in terms of the structure and assembly of stress fibers and cleavage furrows.     

Motility-dependence of the heterogenous staining of culture cells by a monoclonal anti-tropomyosin antibody

A monoclonal antibody (CG1) which recognizes tropomyosin isoforms 1 and 3 of chicken embryo fibroblasts was used to detect what is a motility-dependent change in the availability of the antigenic determinant in tropomyosin molecules along microfilaments. Immunofluorescence microscopy with this antibody revealed a heterogenous staining pattern among chicken embryo fibroblasts cells such that a population (17%) of cells showed only background staining. Stress fibers in about half the population of the cells stained weakly with this antibody, while the stress fibers in another population of cells (35%) showed very strong staining. After glycerination or cytochalasin B treatment, all of the cells became positive in reaction to CG1 antibody, suggesting that the antigenic determinant was present in every cell. On the other hand, all of the cells after brief nonionic detergent treatment became negative to CG1 antibody. The CG1 staining pattern was not significantly changed in cells at different stages after release from colcemid blockage, nor was a brief treatment of cells with buffer containing 2 M urea, mild trypsin, chymotrypsin, or V.8 protease effective in changing the reactivity. However, most of the cells with a morphology typical of movement, and all of the contracted, glycerinated cells were strongly positive to CG1 antibody. These results suggest that the unmasking of the CG1 determinant may be motility-dependent. Immunoblot analysis showed that forced modification on the cysteine residue of tropomyosin molecules, caused either by performic acid oxidation or by disulfide cross-linking with the chemical 5,5'-dithiobis (2-nitrobenzoate), results in drastic changes in the reactivity of the different isoforms to CG1 antibody. These results indicate that the cysteine residue is involved in the CG1 determinant. The motility-dependent unmasking of this determinant may suggest an important role for nonmuscle tropomyosin in regulating cell motility.     

Gelsolin sensitivity of microfilaments as a marker for muscle differentiation

The ability of porcine smooth muscle gelsolin to sever actin filaments was used to study alterations in the organization of F-actin containing structures during skeletal myogenesis. In permeabilized fibroblasts and unfused myoblasts, gelsolin induced complete degradation of the actin cytoskeleton. After fusion of myoblasts to multinucleated myotubes, gelsolin removed a substantial amount of actin, revealing fibers with a sarcomere-like arrangement of gelsolin-insensitive actin. These fibrils were much thinner and had shorter sarcomeres than fully differentiated myofibrils. The proportion of gelsolin-resistant fibrils increased during differentiation, resulting in almost complete inertness of mature myofibrils. Fibrils isolated from adult muscle were also found nearly resistant to gelsolin. Extraction of tropomyosin and myosin in buffer of high ionic strength prior to gelsolin treatment reestablished the susceptibility to the severing protein, both in myotubes and isolated myofibrils. Only small remnants of phalloidin-stainable material were retained. We therefore conclude that during myotube differentiation either an increased interaction of actin with actin-binding proteins (e.g., myosin and tropomyosin), or the assembly of muscle-specific isoforms of these proteins protect the filaments against degradation by actin severing proteins.     

Tropomyosin from smooth and skeletal muscles initiates various conformational changes in skeletal F-actin

Changes in F-actin conformation in myosin-free single ghost fibers of rabbit skeletal muscle induced by the binding of skeletal and gizzard tropomyosin to F-actin were studied by measuring intrinsic tryptophan-polarized fluorescence of F-actin. It was found that skeletal and gizzard tropomyosin binding to F-actin initiate different conformational changes in actin filaments. Skeletal tropomyosin inhibits, while gizzard tropomyosin activates the Mg2+-ATPase activity of skeletal actomyosin. It is supposed that in muscle fibers tropomyosin modulates the ATPase activity of actomyosin via conformational changes in F-actin.     

Modulation of actin-bundling activity of 55-kDa protein by multiple isoforms of tropomyosin

Cultured rat cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J.J.-C. (1983) J. Biol. Chem. 258, 6636-6644). To explore the roles of the multiple tropomyosin isoforms in the microfilament organization of cultured cells, we have examined effects of tropomyosins on the bundling activity of the 55-kDa protein recently purified from HeLa cells (Yamashiro-Matsumura, S., and Matsumura, F. (1985) J. Biol. Chem. 260, 5087-5097). Maximum bundling of F-actin was observed at a molar ratio of 55-kDa protein to actin higher than 1:8. None of the isoforms of cultured rat cell tropomyosin significantly altered the F-actin-bundling activity of 55-kDa protein at this ratio, whereas skeletal muscle tropomyosin inhibited the bundling activity to about 50%. Also, cultured cell tropomyosins did not inhibit binding of 55-kDa protein to actin, whereas skeletal muscle tropomyosin inhibited it by 50%. The effect of 55-kDa protein on the binding of tropomyosin to actin varied with the isoform type of tropomyosin. Most (80%) of the tropomyosins with low Mr values (Mr 32,400 or 32,000) were caused to dissociate from actin by 55-kDa protein, but only 20% of tropomyosins with high Mr values (Mr 40,000 or 36,500) was dissociated from actin in these conditions. Immunofluorescence has shown that, while tropomyosin was localized in stress fibers, 55-kDa protein was found in microspikes as well as stress fibers, both of which are known to contain bundles of microfilaments. Therefore, we suggest that 55-kDa protein together with the multiple tropomyosin isoforms may regulate the formation of two types of actin-filament bundles, bundles containing tropomyosin and those without tropomyosin.     

Caldesmon restricts the movement of both C- and N-termini of tropomyosin on F-actin in ghost fibers during the actomyosin ATPase cycle

New data on the movements of tropomyosin singly labeled at alpha- or beta-chain during the ATP hydrolysis cycle in reconstituted ghost fibers have been obtained by using the polarized fluorescence technique which allowed us following the azimuthal movements of tropomyosin on actin filaments. Pronounced structural changes in tropomyosin evoked by myosin heads suggested the "rolling" of the tropomyosin molecule on F-actin surface during the ATP hydrolysis cycle. The movements of actin-bound tropomyosin correlated to the strength of S1 to actin binding. Weak binding of myosin to actin led to an increase in the affinity of the tropomyosin N-terminus to actin with simultaneous decrease in the affinity of the C-terminus. On the contrary, strong binding of myosin to actin resulted in the opposite changes of the affinity to actin of both ends of the tropomyosin molecule. Caldesmon inhibited the "rolling" of tropomyosin on the surface of the thin filament during the ATP hydrolysis cycle, drastically decreased the affinity of the whole tropomyosin molecule to actin, and "freezed" tropomyosin in the position characteristic of the weak binding of myosin to actin.     

A role for gelsolin in stress fiber-dependent cell contraction.

Gelsolin is an abundant actin binding protein that mediates the rapid remodeling of cortical actin filaments through severing, capping, and nucleating activities. Most of the attention on the intracellular function of gelsolin has focused on the remodeling of the cortical actin meshwork but the localization of gelsolin to other regions of the cell suggests that it may have other important functions elsewhere. In cultured fibroblasts, gelsolin is heavily enriched in stress fibers, where its function in these contractile organelles is unknown. To study gelsolin function during stress fiber formation and cell contraction, we first assessed gelsolin levels in stress fiber preparations from lysophosphatidic acid (LPA)-treated human fibroblasts. LPA induced a large, time-dependent, calcium-independent increase of actin, gelsolin, alpha-actinin, and tropomyosin in stress fiber preparations. A microinjected gelsolin antibody that inhibits severing by gelsolin reduced stress fibers. Anti-sense-transfected gelsolin-depleted 3T3 cell lines treated with LPA after serum starvation required approximately 6 h to form stress fibers and focal adhesions, in contrast to control lines transfected with vector only, which formed stress fibers 15 min after addition of LPA. In cells microinjected with the gelsolin antibody that inhibits severing, Mg-ATP-induced cell contraction was greatly reduced in approximately 90% of injected cells compared to cells injected with an irrelevant antibody. Gelsolin-depleted cells were incapable of collagen gel contraction and showed no apparent Mg-ATP-induced cell contraction compared to cell lines transfected with vector only. The involvement of gelsolin in cell contraction and remodeling of collagen gels suggests a novel role for gelsolin in stress fiber-dependent cell function.     

Lack of tropomyosin correlates with the absence of stress fibers in transformed rat kidney cells

We have utilized epithelial rat kidney cells and their Kirsten viral transformant (442) to examine the role of actin-binding proteins in cellular morphogenesis. Normal rat kidney cells are well spread while the transformed cells are more spherical, poorly adherent, and lack actin stress fibers (Rubin, R.W., Warren, R.H., Lukeman, D.S. and Clements, E. (1978) J. Cell Biol. 78, 28-35). By immunofluorescence, antitropomyosin prominently stains normal rat kidney cell stress fibers while only a weak, nonspecific fluorescence is observed in 442 cells. Using two-dimensional gel electrophoresis, tropomyosin can be detected in normal rat kidney cells homogenates. The tropomyosin subunits are enriched in Triton-extracted filamentous normal rat kidney cell models, and in extracts of normal rat kidney cell homogenate produced by using a rapid myosin affinity technique to isolate actin and actin-associated proteins. The identity of the tropomyosin subunits has been confirmed by electrophoretic mobility, lack of proline, and the peptide map generated by limited proteolysis. None of these techniques have detected tropomyosin in the corresponding 442 preparations. Our results suggest that the transformation of normal rat kidney cells has led to an overall reduction in tropomyosin content. This may be related to the inability of 442 cells to organize filamentous actin stress fibers.     

The effect of caldesmon on actin-myosin interaction in skeletal muscle fibers

The effects of caldesmon on structural and dynamic properties of phalloidin-rhodamine-labeled F-actin in single skeletal muscle fibers were investigated by polarized microphotometry. The binding of caldesmon to F-actin in glycerinated fibers reduced the alterations of thin filaments structure and dynamics that occur upon the transition of the fibers from rigor to relaxing conditions. In fibers devoid of myosin and regulatory proteins (ghost fibers) the binding of caldesmon to F-actin precluded structural changes in actin filaments induced by skeletal muscle myosin subfragment 1 and smooth muscle tropomyosin. These results suggest that the restraint for the alteration of actin structure and dynamics upon binding of myosin heads and/or tropomyosin evoked by caldesmon can be related to its inhibitory effect on actin-myosin interaction.     

Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy.

Past attempts to detect tropomyosin in electron micrograph images of frozen-hydrated troponin-regulated thin filaments under relaxing conditions have not been successful. This raised the possibility that tropomyosin may be disordered on filaments in the off-state, a possibility at odds with the steric blocking model of muscle regulation. By using cryoelectron microscopy and helical image reconstruction we have now resolved the location of tropomyosin in both relaxing and activating conditions. In the off-state, tropomyosin adopts a position on the outer domain of actin with a binding site virtually identical to that determined previously by negative staining, although at a radius of 3.8 nm, slightly higher than found in stained filaments. Molecular fitting to the atomic model of F-actin shows that tropomyosin is localized over sites on actin subdomain 1 required for myosin binding. Restricting access to these sites would inhibit the myosin-cross-bridge cycle, and hence contraction. Under high Ca(2+) activating conditions, tropomyosin moved azimuthally, away from its blocking position to the same site on the inner domain of actin previously determined by negative staining, also at 3.8 nm radius. These results provide strong support for operation of the steric mechanism of muscle regulation under near-native solution conditions and also validate the use of negative staining in investigations of muscle thin filament structure.     

Comparison of Ca2+-dependent effects of caldesmon-tropomyosin-calmodulin and troponin-tropomyosin complexes on the structure of F-actin in ghost fibers and its interaction with myosin heads

Comparison of two types of Ca2+-regulated thin filament, reconstructed in ghost fibers by incorporating either caldesmon-gizzard tropomyosin-calmodulin or skeletal muscle troponin-tropomyosin complex, was performed by polarized microphotometry. The changes in actin structure under the influence of these regulatory complexes, as well as those upon the binding of the myosin heads, were followed by measurements of F-actin intrinsic tryptophan fluorescence and the fluorescence of phalloidin-rhodamine complex attached to F-actin. The results show that in the presence of smooth muscle tropomyosin and calmodulin, caldesmon causes Ca2+-dependent alterations of actin conformation and flexibility similar to those induced by skeletal muscle troponin-tropomyosin complex. In both cases, transferring of the fiber from '-Ca2+' to '+Ca2+' solution increases the number of turned-on actin monomers. However, whereas troponin in the absence of Ca2+ potentiates the effect of skeletal muscle tropomyosin, caldesmon-calmodulin complex inhibits the effect of smooth muscle tropomyosin. This difference seems to be due to the qualitatively different alterations in the structure and flexibility of F-actin in ghost fibers evoked by smooth and skeletal muscle tropomyosins. Troponin can bind to F-actin-smooth muscle tropomyosin-caldesmon complex and, in the presence of Ca2+, release the restraint by caldesmon for S-1-induced alterations of conformation, and reduce that for flexibility of actin in ghost fibers. This effect seems to be related to the abolishment by troponin of the potentiating effect of tropomyosin on caldesmon-induced inhibition of actomyosin ATPase activity.     

Distribution of actin and the actin-associated proteins myosin, tropomyosin, alpha-actinin, vinculin, and villin in rat and bovine exocrine glands

Actin, myosin, and the actin-associated proteins tropomyosin, alpha-actinin, vinculin, and villin were localized in acinar cells of rat and bovine pancreas, parotid, and prostate glands by means of immunofluorescent staining of both frozen tissue sections and semithin sections of quick-frozen, freeze-dried, and plastic-embedded tissues. Antibodies to actin, myosin, tropomyosin, alpha-actinin, and villin reacted strongly with a narrow cytoplasmic band extending beneath the luminal border of acinar cells. The presence of villin, which has so far been demonstrated only in intestinal and kidney brush border, was further confirmed by antibody staining of blotted electrophoresis gels of whole acinar cell extracts. Fluorescently labelled phalloidin, which reacts specifically with F-actin, gave similar staining, within the cell apex to that obtained with antibodies to actin, myosin, tropomyosin, alpha-actinin, and villin. In contrast, immunostaining with antibodies to vinculin was restricted to the area of the junctional complex. Ultrastructurally, the apical immunoreactive band corresponded to a dense web composed of interwoven microfilaments, which could be decorated with heavy meromyosin. Outside this apical terminal web, antibodies to myosin and tropomyosin gave only a weak immunostaining (confined to the lateral cell borders) whereas antibodies to actin and alpha-actinin led to a rather strong bead-like staining along the lateral and basal cell membrane most probably marking microfilament-associated desmosomes. Anti-villin immunofluorescence was confined to the apical terminal web. It is suggested that the apical terminal web is important for the control of transport and access of secretory granules to the luminal plasma membrane and that villin, which is known to bundle or sever actin filaments in a Ca(++)-dependent manner, might participate in the regulation of actin polymerization within this strategically located network of contractile proteins.     

Studies on cardiac myofibrillogenesis with antibodies to titin, actin, tropomyosin, and myosin

Cardiac myofibrillogenesis was examined in cultured chick cardiac cells by immunofluorescence using antibodies against titin, actin, tropomyosin, and myosin. Primitive cardiomyocytes initially contained stress fiber-like structures (SFLS) that stained positively for alpha actin and/or muscle tropomyosin. In some cases the staining for muscle tropomyosin and alpha actin was disproportionate; this suggests that the synthesis and/or assembly of these two isoforms into the SFLS may not be stoichiometric. The alpha actin containing SFLS in these myocytes could be classified as either central or peripheral; central SFLS showed developing sarcomeric titin while peripheral SFLS had weak titin fluorescence and a more uniform stain distribution. Sarcomeric patterns of titin and myosin were present at multiple sites on these structures. A pair of titin staining bands was clearly associated with each developing A band even at the two or three sarcomere stage, although occasional examples of a titin band being associated with a half sarcomere were noted. The appearance of sarcomeric titin patterns coincided or preceded sarcomere periodicity of either alpha actin or muscle tropomyosin. The early appearance of titin in myofibrillogenesis suggests it may have a role in filament alignment during sarcomere assembly.     

Organization of stress fibers in cultured fibroblasts after extraction of actin with bovine brain gelsolin-like protein

Mouse and quail embryo fibroblasts were extracted with Triton X-100 and the resulting cytoskeletons were treated with gelsolin-like actin-capping protein (the 90-kDa protein-actin complex isolated from bovine brain). Staining of cells with rhodamine-conjugated phalloin or an antibody to actin did not reveal any actin-containing structures after treatment with the 90-kDa protein-actin complex. Extraction of actin was confirmed by SDS-gel electrophoresis. Immunofluorescence microscopy showed that vinculin and α-actinin were released from the cytoskeletons together with actin. However, myosin remained associated with the cytoskeleton after treatment with the 90-kDa protein-actin complex. The distribution of myosin in treated cells showed no significant difference from that in control cells: in both cases myosin was localized mainly in the stress fibers. Double-fluorescence staining showed the absence of actin in myosin-containing stress fibers of treated cells. The ultrastructural organization of actin-depleted stress fibers was studied by transmission electron microscopy of platinum replicas. On electron micrographs these fibers appeared as bundles of filaments containing clusters of globular material. It is concluded that myosin localization in stress fibers does not depend on actin.     

Distribution of fluorescently labeled actin and tropomyosin after microinjection in living tissue culture cells as observed with TV image intensification

Skeletal muscle F-actin and smooth muscle tropomyosin separately labeled with the fluorescent reporter group 5-iodoacetamidofluorescein (5-IAF) were further purified to yield G-actin fully competent to polymerize and tropomyosin able to bind specifically to F-actin. The two fluorescent proteins (dye content of 0.4–0.5 moles/mole of protein) were microinjected into tissue culture cells and their intracellular distribution was followed by TV image intensification. Fluorescent actin is found in the stress fibers and in the lamellopodia and ruffling edges of the cells. In addition a general cytoplasmic fluorescence is observed as well as fluorescent patches, which could be actin paracrystals. In contrast tropomyosin is not incorporated into ruffles although it is clearly seen along the stress fibers and gives rise to general cytoplasmic fluorescence. Control experiments using fluorescent serum albumin show no specific visualization of either stress fibers or ruffles. The specificity of the incorporation of the fluorescently labeled contractile proteins into the microfilament structures is further documented by the preparation of detergent resistant cytoskeletons which retain actin and tropomyosin in the appropriate structures but are devoid of fluorescent serum albumin. In addition the distribution of the contractile proteins in the living cells is affected by the microfilament specific drugs phalloidin and cytochalasin B (CB). The results obtained on live cells are in excellent agreement with conclusions drawn from immunofluorescence microscopical observations on fixed cells. In addition they directly prove the rather obvious point that contractile proteins are constantly rearranged in tissue culture cells.     

Tropomyosin antibody: the specific localization of tropomyosin in nonmuscle cells

An antibody against purified chicken skeletal muscle tropomyosin is used in indirect immunofluorescence to visualize the localization of tropomyosin in a variety of nonmuscle cells. The antibody produces a fluorescent pattern which is very similar to that obtained with an actin-specific antibody. This pattern is composed of fluorescent fibers which are shown to be coincident with the fibers seen with phase- contrast optics. High resolution epifluorescent microscopy reveals that fibers stained with the actin antibody show a continuous fluorescence, while fibers reacted with the tropomyosin antibody show a periodic fluorescence. Measurements indicate that the lengths of the fluorescent segments are variable with an average of 1.2 mum while the spacing between segments is approximately 0.4 mum.     

Preservation of cellular polarity in isolated hepatocytes

Summary The distribution of actin, myosin and tropomyosin in freshly isolated and short-term cultured rat hepatocytes was investigated by use of both rhodaminyl-phalloidin staining and immunofluorescence techniques. The cytoskeletal proteins were mainly located in distinct areas of the hepatocyte membrane, corresponding to their accumulation in the bile-canalicular region of liver tissue. In freshly prepared cells, these sections resembled sharp, angled or branched bands, similar to the pattern of hemicanaliculi. During incubation in a monolayer culture, these bands were transformed to circular formations. Simultaneously, enclosed bile-canalicular spaces between undissociated hepatocytes were visualized by staining of actin, myosin, and tropomyosin. The preservation of canalicular cytoskeletal structures in isolated hepatocytes is an indication of cellular polarity. Our findings suggest a uniform association of membrane-bound F-actin with myosin and tropomyosin.     

Regulation of tropomyosin expression in the maturing ovary and in primary granulosa cell cultures

Granulosa cell differentiation in vitro in response to gonadotropins is characterized by major changes in cell shape, cell aggregation, and the organization of microfilaments. These changes are associated with enhanced steroidogenesis in maturing granulosa-lutein cells. Since nonmuscle tropomyosin isoforms were implicated in stabilizing actin filaments, we studied the organization and expression of tropomyosin in differentiating primary cultures of rat granulosa cells and during ovarian folliculogenesis and luteinization. In unstimulated primary granulosa cell cultures tropomyosin was found mainly along stress fibers. In differentiating cells tropomyosin staining was diffuse with sometimes a subcortical organization. The changes in tropomyosin organization were accompanied by a pronounced decrease in the synthesis, translation in vitro, and mRNA levels of all the rat nonmuscle tropomyosin isoforms, with a greater reduction in the higher molecular weight isoforms than in the smaller isoforms. Similar results were obtained whether cells were stimulated to differentiate with gonadotropins, with cAMP, by culturing cells on an extracellular matrix, or by treatment with cytochalasin B. The effect of cytochalasin B was reversible; upon removal of the drug tropomyosin synthesis increased to near control levels, while that of proteins associated with luteinization decreased drastically. RNA isolated from ovaries with follicles at the preantral, preovulatory stage and from corpora lutea contained decreased tropomyosin mRNA levels during ovarian luteinization when the level of RNA for a key steroidogenic enzyme, cytochrome P-450 cholesterol side chain cleavage (P-450 scc), increased. The results suggest a physiological relevance for the low level of tropomyosin expression in the mechanisms which bring about the morphological and biochemical development and maturation of granulosa cells.