Microtubules, organelle transport, and steroidogenesis in cultured adrenocortical tumor cells. 1. An ultrastructural analysis of cells in which basal and ACTH-induced steroidogenesis was inhibited by taxol

In adrenocortical cells, the first step in the enzymatic processing of cholesterol to steroid end products occurs in the mitochondria. ACTH increases mitochondrial cholesterol and steroidogenesis. In cultured mouse adrenocortical tumor cells, microtubule-based organelle motility may increase the proximity of mitochondria to the SER, lipid droplets and endoscome-derived lysosomes, thereby facilitating the transfer of cholesterol from these organelles to the mitochondrial outer membrane. ACTH may increase opportunities for the transfer by promoting organelle motility and by increasing the number of lysosomes. Taxol, a microtubule polymerizer, inhibits basal and ACTH-induced steroidogenesis in these cells, presumably at the step where mitochondria obtain cholesterol. We examined the ultrastructure of taxol-treated, unstimulated and ACTH-stimulated cells, seeking alterations which conceivably could interefer with the proposed organelle transport and encounters, and thus correlate with taxol's inhibition of steroidogenesis. Primary cultured cells were incubated in serum-containing medium for 4 hr with and without ACTH (10 mU/ml), with 10 micrograms/ml and 50 micrograms/ml of taxol, and with ACTH and taxol 10 or taxol 50 simultaneously. Culture media were analyzed for the presence of secreted steroids at the end of 1, 2, and 4 hr of incubation. At the end of the fourth hour, unstimulated cells and cells treated with ACTH, taxol 50, and both agents simultaneously, were fixed and processed for EM. Taxol inhibited basal and ACTH-induced steroidogenesis in a dose-dependent fashion. In both unstimulated and ACTH-stimulated cells, taxol 50 formed numerous microtubule bundles, but did not markedly change the distribution of mitochondria and lipid droplets. SER tubules, and clusters of Golgi fragments, endosomes, and lysosomes appeared to be translocated towards the cell periphery along some of the microtubules. Taxol permitted an ACTH-induced cell rounding and microfilament rearrangement considered to facilitate organelle motility. Our data indicate that taxol disrupts the formation of lysosomes by these adrenal cells, but it seemed unlikely that taxol's ultrastructural effects could prevent organelle transport proposed to cause meetings between mitochondria and the SER or lipid droplets, or prevent ACTH-caused increases in these encounters. Taxol may instead prevent the transfer of lipid droplet or SER-contained cholesterol to adjacent mitochondria, by a means not detectable in our electron micrographs.     

The reversibility of the effects of ACTH and cytochalasin B on the ultrastructure and steroidogenic activity of adrenocortical tumor cells in vitro

We have demonstrated previously that the steroidogenic activity of ACTH on cultured adrenal tumor cells is associated with cell rounding and a rearrangement of microfilaments. Cytochalasin B (CB) also induces cell rounding, but changes the conformation of microfilaments and severely inhibits steroidogenesis. ACTH and CB may have different modes of action on the contractile machinery which are related to their opposing actions on steroidogenesis. To investigate this possibility further, we have examined the reversibility of the morphological and functional effects of these agents. Cultures were incubated for 1 hr, with and without ACTH (10 microU/ml of media), or with CB (50 micrograms/ml), or with both agents simultaneously. After a media wash, the cultures were incubated for 1 hr, with and without ACTH. The steroid production of the cells during pre- and post-washout incubations was determined, and some cultures were fixed for electron microscopy at the end of both incubation periods. The three- to ten-fold increases in steroidogenic activity of ACTH-stimulated cells declined during recovery incubations, but remained well above basal values. These cells nearly reflattened and began to regain stress fibers which had been 'pulled apart'. The 'washed out' ACTH-stimulated cells were often refractory to restimulation. Cells recovering from CB also reflattened. Masses of filamentous felt induced by the drug disappeared from the cytoplasm, lost microvilli reappeared and stress fibers reformed. The 20-50% inhibition of basal steroidogenesis by CB was completely reversed. When ex-CB-treated cells were incubated with ACTH, their morphology and steroid production were typical of acutely stimulated cells. The recovery behavior of cells incubated with ACTH and CB simultaneously reflected the observation that there were cell-specific responses to one agent or the other during initial incubations. The persistence of heightened steroidogenic activity following a washout of ACTH and the rapid reversal of the effects of CB strongly support the concept that regulated actomyosin interactions are an integral part of the steroidogenic process.     

Microtubules, organelle transport, and steroidogenesis in cultured adrenocortical tumor cells. 2. Reversibility of taxol's inhibition of basal and ACTH-induced steroidogenesis is unaccompanied by reversibility of taxol-induced changes in cell ultrastructure

Taxol inhibits the basal and ACTH-stimulated steroidogenesis of cultured mouse adrenocortical tumor cells, presumably by preventing the arrival of cholesterol in mitochondria. In these cells, taxol polymerizes and rearranges microtubules, disperses SER masses, disrupts the Golgi, and impedes the formation of cholesterol-containing lysosomes. However, taxol's alterations in ultrastructure appear likely to permit both a microtubule-based organelle transport proposed to bring mitochondria of unstimulated cells close to alternate sources of cholesterol--the SER and lipid droplets--and postulated ACTH-caused increases in these encounters. Conceivably, taxol may prevent the transfer of cholesterol from the SER and lipid droplets to mitochondria, once the meetings are achieved. To investigate this possibility, we determined the reversibility of taxol's ultrastructural effects and inhibition of steroidogenesis. Primary cultured adrenal tumor cells were incubated for 4 hr with and without ACTH (10 mU/ml). with taxol (50 micrograms/ml), and with ACTH and taxol 50 simultaneously. Some cultures from each set were washed with fresh medium and re-incubated for 1.5 hr. with and without ACTH. Media taken from cultures at the ends of pre- and post-washout incubations were analyzed for the presence of secreted steroids. Sample cultures were fixed for electron microscopy at the ends of both incubations. Data derived from pre-washout incubations confirmed previous reports of taxol's ultrastructural changes and inhibition of steroidogenesis. When cells recovered from taxol in the absence of ACTH, the inhibition of steroidogenesis was completely reversed. In the presence of ACTH, ex-taxol-treated cells demonstrated a "rounding up' and an increased steroid production that are characteristic responses to the hormone. However, in all cases, there was a persistence of taxol's alterations in organelle numbers and arrangements. Our findings establish that the ultrastructural effects of taxol which we recorded cannot prevent mitochondria of unstimulated and ACTH-stimulated adrenal tumor cells from gaining cholesterol. They strengthened the possibility that in pre-washout incubations, taxol allowed organelle motility to bring mitochondria adjacent to cholesterol-containing SER tubules and lipid droplets, but inhibited steroidogenesis by preventing the cholesterol transfer. Taxol might limit the availability of a protein required for the transfer, an effect not visible in our electron micrographs.     

Structural changes induced in capillary endothelial cells by growth factors: Altered distribution of cytoskeletal elements and organelles in cells spreading in the presence of tumor conditioned medium and hypothalamus derived growth factor

Using high-voltage and conventional electron microscopy of cell whole mounts, we have investigated the effects of tumor-conditioned medium and hypothalmus-derived growth factor on the structure of capillary endothelial cells during their attachment and spreading in tissue culture. Cells were cultured in A, Dulbecco's Modified Eagle's Medium (DMEM) and 10% calf serum; B, equal parts of A and 48 hr mouse sarcoma conditioned medium; and C, A containing 10 units of hypothalamus-derived growth factor. Cells cultured in all three media were fully spread, and to the same extent, by 4 hr after plating. While spreading, cells cultured in DMEM alone developed prominent stress fibers and contained numerous bundles of microtubules which formed radical tracts along which mitochondria and other organelles rapidly moved to the cell periphery. Stress fibers were thinner and microtubule tracts fewer in number in cells cultured in tumor-conditioned medium. In 4 hr, organelles moved only part of the distance to the cell margin. Stress fibers were rudimentary and microtubules randomly orientated in cells exposed to hypothalamus-derived growth factor. Most organelles remained near the cell nucleus. The dramatic decrease in stress fibers and microtubule tracts in cells grown in tumor-conditioned medium and hypothalamus-derived growth factor and the subsequent decreased capacity of the cells to move organelles toward their periphery could have some functional significance relative to the growth-promoting activity of these substances.     

The effects of ACTH on acid phosphatase activity in endosomes, GERL and lysosomes of cultured adrenal tumor cells

Cultured cells derived from a mouse adrenocortical tumor transplant are unspecialized in appearance, but produce basal levels of steroids and demonstrate a near-immediate steroidogenic response to ACTH. There is biochemical evidence that ACTH induces increases in the uptake of serum lipoproteins by these cells and that this material is hydrolyzed in lysosomes to free cholesterol, a precursor for steroid end products. To investigate morphologically the role of lysosomes in the steroidogenic activity of these cells, cultures were incubated for 4 h with and without ACTH, then processed for the ultrastructural localization of acid phosphatase (ACPase), a marker enzyme for lysosomes, and for GERL, the lysosome-forming subcompartment of the Golgi, and examined by TEM and HVEM. Steroid output was determined by a fluorometric technique. Unstimulated cells secreted basal levels of steroids. By TEM, large endosomes, some containing semi-compact material and ACPase reaction product, were occasionally seen at the cell periphery and in the Golgi region. The Golgi and GERL were poorly developed. Residual bodies, a few of them ACPase+, appeared in the Golgi region and in microtubule-associated clusters near the cell membrane. ACTH-stimulated cells secreted steroids at 8-10 fold basal values. In TEM records, they displayed numerous ACPase+ endosomes between the cell periphery and the Golgi. The Golgi and GERL regions appeared to be hypertrophied and many large, inclusion-containing, strongly ACPase+ residual bodies appeared here and in elongated microtubule-containing cell processes. HVEM micrographs showed more definitively that ACTH produced distinct increases in the size of GERL and in the number of ACPase+ organelles. Our results suggest that in unstimulated cells, endosomes, presumably containing media-derived material, gain lysosomal enzymes in or near GERL, are transformed to residual bodies as their contents are hydrolyzed, and are subsequently translocated by microtubules to the cell periphery for exocytosis. ACTH appears to intensify all of these effects. The "giant" lysosomes seen in stimulated cells may result from a fusion of smaller lysosomes. Their amorphous contents may reflect an inefficient hydrolysis of LDL to free cholesterol.     

The effects of colchicine,vinblastine and cytochalasins on the corticotropic responsiveness and ultrastructure of inner zone adrenocortical tissue in the Pekin duck

Tissues slices superfused with medium containing no ACTH released only traces of corticosterone. Addition of ACTH to the medium caused the rate of corticosterone release to increase to a maximum about 45 min after the addition of ACTH, after which time it either remained constant or started to wane slightly. The rate of release was affected by tissue thickness; the maximum rate of corticosterone release occurred when the tissue slices were 200 microns. Stimulated adrenocortical cells had large spherical nuclei, numerous mitochondria with tubular cristae, numerous lipid droplets, and a large amount of smooth endoplasmic reticulum. Many cells had an extensive network of microfilaments adjacent to the plasma membrane and some microtubules. Prolonged superfusion caused degenerative changes in some of the cells. Both cytochalasin B and cytochalasin D, dissolved in DMSO before addition to the superfusion medium, inhibited the corticotropic responsiveness in a dose-dependent manner. Control tissue samples superfused with medium containing DMSO, but no ACTH and no cytochalasin, released significantly more corticosterone than corresponding unstimulated samples. Few or no microfilaments were observed in adrenocortical cells after treatment with cytochalasin. Neither colchicine nor vinblastine had any discernible effect on the corticotropic responsiveness. After treatment with colchicine, adrenocortical cells had an ultrastructure characteristic of inner zone stimulated cells except that they were mainly devoid of microtubules.     

Determination of cell form in cultured fibroblasts : Role of surface components and cytokinetic elements

Functions involved in the determination of the morphology of mouse embryo fibroblasts have been studied using as a system the cell rounding and reflattening related to the presence or absence of a protein component of the cell surface [18]. Cells that had undergone rounding following the removal of the surface protein gradually reflattened if their protein synthesis was not impaired but could rapidly revert to the flattened state if a fraction containing the surface protein was returned to the cells. Studies with sodium azide and potassium cyanide showed that both the rounding and reflattening were energy-dependent. When the rounded state was induced the organised appearance of the microfilaments present in the cortical layer adjacent to the plasma membrane was lost. However, microfilament structures reappeared when rounded cells respread over the glass substrate. On the other hand no reflattening occurred if microfilament function was impaired by treatment with cytochalasin B (CB), showing a requirement of microfilament integrity for the flattened form to be manifest. Microtubules also were shown to be involved in shape determination as their integrity was necessary for cells to progress from the flattened form to the rounded state, but they did not appear to be primarily involved in the process of reflattening. The distribution of macromolecular components of the plasma membrane was also shown to affect changes of cell shape as cross-linking of surface receptors by concanavalin A (ConA) or specific anti-cell immunoglobulins prevented cells from acquiring the rounded form and treatment of cells with media of low pH, which causes aggregation of intra-membrane particles, also prevented rounding. A comparison between quiescent cells and cells in S phase showed the changes of morphology to be similar in nature, but more prompt and marked in the S population. The indication of the studies presented is that determination of cell form requires the cooperative interaction between the protein component of the cell surface, integral constituents of the plasma membrane and cytokinetic organelles.     

Cytoskeletal changes accompanying ACTH-induced steroidogenesis in cultured embryonic adrenal gland cells from the Pekin duck

Summary Cells derived from the adrenal glands of duck embryos immediately prior to hatching were grown in culture and used to study the morphological and cytoskeletal changes and steroidogenic responses induced by 1–24 ACTH. Changes in the cytoskeletal components were observed by rhodamine-phalloidin staining for actin and by staining the tubulin immunoreactive components with FITC. The cultures were comprised of a small population of chromaffin cells and a larger population of steroidogenic cells. The chromaffin cells were distinguished by their tyrosine hydroxylase immunoreactivity. The steroidogenic cells were characterized by the presence of sudanophilic lipid droplets, numerous mitochondria, abundant smooth endoplasmic reticulum, microtubules distributed as a fairly even network throughout the cytoplasm, and microfilaments that formed an extensive and elaborate system of stress fibers with many parallel arrays. The cells readily responded to stimulation with ACTH by releasing corticosterone, aldosterone and deoxycorticosterone. Stimulation with ACTH also induced changes in both the cell morphology and the cytoskeleton. Exposure of the cells to Krebs-Henseleit buffer containing 1–24 ACTH caused them to form numerous fine filopodia, to lose their stress fibers, and to form a thick ring of actin at the periphery of the cell. In addition, many cells became extremely arborized with many long branched dendritic processes. The morphological changes appeared to be related to a redistribution of the actin components, and may be explained only in part by the rounding up or retraction of the cytoplasm. The results strongly suggest an involvement of the actin components of the cytoskeleton in the steroidogenic response to corticotropic stimulation.     

Cinemicrographic observations of cultured adrenocortical tumor cells. Dynamic responses to ACTH and cytochalasin B

ACTH increases the basal steroidogenic activity of cultured adrenocortical tumor cells, whereas moderate-high doses of cytochalasin B (CB) inhibit both basal and ACTH-induced steroidogenesis. Previous ultrastructural studies have revealed that ACTH rearranges microfilaments in these adrenal cells, whereas CB causes microfilaments to aggregate into felt-like masses. It has been postulated that the ACTH effects may facilitate organelle motility and increase organelle interactions that are required for steroid biosynthesis, and that the CB-created "foci" may impede or prevent the organelle meetings. To shed light on these possibilities, we have employed 16 mm cinemicrography of unstimulated adrenal tumor cells and cells incubated for 1-2 h with ACTH (10 mU/ml), or low (10 micrograms/ml), or high (50 micrograms/ml) doses of CB. ACTH caused initial increases in membrane ruffling and a "flurry" of particle (organelle) activity above that seen in unstimulated cells. The stimulated cells then retracted from each other and began their characteristic "rounding up" in response to the hormone. Particles appeared to move towards the nucleus, and in fully-rounded cells were extremely congested. Steroid production rose several fold above basal levels. CB10 produced slight-marked cell convexities, nearly stopped particle motility and inhibited steroid production moderately. CB50 produced an asymmetrical, spidery cell form, stopped membrane ruffling and particle motility and abolished steroidogenesis. After a washout of CB50, particle motility resumed nearly immediately. Our CB data indicate that associations between particles, presumably between mitochondria and various sources of cholesterol, are prerequisite for basal steroidogenesis in the adrenal tumor cells. In ACTH-stimulated cells, increases in steroid output correspond with increased opportunities for particle associations. These opportunities appear to arise directly or indirectly from ACTH effects on microfilaments. The responses of microfilaments to the hormone may be particularly intense in tumorous forms. By these means, the cells may express their differentiated function, although their cytoplasm has a distinctly unspecialized appearance.     

The roles of microfilaments and intermediate filaments in the regulation of steroid synthesis

Much of the cholesterol used in steroid synthesis is stored in lipid droplets in the cytoplasm of steroid-forming cells. The cholesterol ester in these droplets is transported to the inner mitochondrial membrane where it enters the pathway to steroid hormones as free cholesterol—the substrate for the first enzyme, namely P450_scc. It has been shown that this transport process governs the rate of steroid synthesis and is specifically stimulated by ACTH and its second messenger. The stimulating influence of ACTH on cholesterol transport is inhibited by cytochalasins, by monospecific anti-actin and by DNase I demonstrating that the steroidogenic cell must possess a pool of monomeric actin available for polymerization to F actin if it is to respond to ACTH and cyclic AMP. It has been shown that the two structures involved in cholesterol transport (droplets and mitochondria) are both bound to vimentin intermediate filaments in adrenal and Leydig cells. In addition these filaments are closely associated with the circumferential actomyosin ring in which they are crosslinked by actin microfilaments. In permeabilized adrenal cells Ca²⁺/calmodulin phosphorylates vimentin and this change is known to disrupt intermediate filaments and to cause contraction of actomyosin by phosphorylating myosin light chain kinase. Ca²⁺/calmodulin stimulated cholesterol transport and steroid synthesis and causes rounding of the responding cells by contraction of the actomyosin, if ATP is also added at the same time. Other agents that disrupt intermediate filaments include anti-vimentin plus ATP in permeabilized cells which also results in rounding of the cell. Acrylamide exerts a similar effect in intact adrenal cells and in addition causes rounding of the cells and increase in steroid synthesis without increase in cyclic AMP. It is also known that if adrenal cells are grown on surfaces treated with poly(HEMA), the cells grow in rounded form and steroid synthesis is increased in proportion to the degree of rounding (r = 0.92). This response does not involve increase in cellular levels of cylic AMP. It is proposed that in vivo where the cell is always round and cannot show more than strictly limited change in shape, ACTH activates Ca²⁺/calmodulin possibly by redistributing cellular Ca²⁺. Ca²⁺/calmodulin in turn promotes phosphorylation of vimentin and myosin light chain. The first of these phosphorylations shortens intermediate filaments and the second promotes contraction of the actomoyosin ring with internal shortening and approximation of lipid droplets and mitochondria. Details of the earlier events (activation of Ca²⁺/calmodulin) and later changes (transfer of cholesterol to the inner membrane) remain to be elucidated. It is clear however that the action of ACTH requires increase in cellular cyclic AMP. These experimental responses bypass this step in the response to ACTH.     

Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique

We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end-on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles.     

Changes in plasma iodohormone concentrations during the day before hatching in Gallus domesticus

Plasma concentrations of thyroxine (T4) and triiodothyronine (T3) were measured at the onset of breathing, clicking and hatching in unstimulated chick embryos. There was a progressive increase in plasma T3 concentration from the onset of breathing (482 hr of incubation) to hatching (496 hr). Plasma T4 concentration did not change significantly between the start of breathing and clicking (494 hr) but decreased between the onset of clicking and hatching. Embryos stimulated with artificial clicks hatched 19 hr before their unstimulated counterparts but their plasma concentrations of T3 and T4 did not differ at hatching from those of unstimulated embryos; however, the plasma T3 concentration, but not T4, at hatching was higher than in unstimulated embryos incubated for a similar length of time, i.e. to the onset of breathing.     

Transmembrane interactions and the mechanisms of transport of proteins across membranes

We have made observations, by double fluorescence staining of the same cell, of the distributions of surface receptors, and of intracellular actin and myosin, on cultured normal fibroblasts and other flat cells, and on lymphocytes and other rounded cells. The binding of multivalent ligands (a lectin or specific antibodies) to a cell surface receptor on flat cells clusters the cell receptors into small patches, which line up directly over the actin- and myosin-containing stress fibers inside the cell. Similar ligands binding to rounded cells can cause their surface receptors to be collected into caps on the surface, and these caps are invariably found to be associated with concentrations of actin and myosin under the capped membrane. Although these ligand-induced surface phenomena appear to be different on flat and rounded cells, we propose that in both cases clusters of receptors become linked across the membrane to actin- and myosin-containing structures. In flat cells these structures are very long stress fibers; therefore, when clusters of receptors become linked to these fibers, the clusters are immobilized. In round cells, membrane-associated actin- and myosin-containing structures are apparently much less extensive than in flat cells; therefore, clusters of receptors linked to these structures are still mobile in the plane of the membrane. We suggest that in this case the clusters are then actively collected into a cap by an analogue of the muscle sliding filament mechanism. To explain the transmembrane linkage, we propose that actin is associated with the plasma membrane as a peripheral protein which is directly or indirectly bound to an integral protein (or proteins) X of the membrane. Individual molecules of any receptor are not bound to X, but after they are specifically clustered into patches, a patch of receptors then becomes bound to S and hence to actin/myosin. Patching and capping of specific receptors on rounded cells is often accompanied by a specific endocytosis of the ligand-receptor complexes. This represents one common transport mechanism of a protein (the ligand) across the plasma membrane. The possibility is discussed that this type of endocytosis is mediated by a transmembrane linkage of the clustered receptor to actin/myosin. Another mechanism of endocytosis involves the “coated pit” structures that are observed by electron microscopy of plasma membranes. The possible relationships between an actin/myosin and a coated pit mechanism of endocytosis are explored.     

High hydrostatic pressure effects in vivo: changes in cell morphology, microtubule assembly, and actin organization

We present the first study of the changes in the assembly and organization of actin filaments and microtubules that occur in epithelial cells subjected to the hydrostatic pressures of the deep sea. Interphase BSC-1 epithelial cells were pressurized at physiological temperature and fixed while under pressure. Changes in cell morphology and cytoskeletal organization were followed over a range of pressures from 1 to 610 atm. At atmospheric pressure, cells were flat and well attached. Exposure of cells to pressures of 290 atm or greater caused cell rounding and retraction from the substrate. This response became more pronounced with increased pressure, but the degree of response varied within the cell population in the pressure range of 290-400 atm. Microtubule assembly was not noticeably affected by pressures up to 290 atm, but by 320 atm, few microtubules remained. Most actin stress fibers completely disappeared by 290 atm. High pressure did not simply induce the overall depolymerization of actin filaments for, concurrent with cell rounding, the number of visible microvilli present on the cell surface increased dramatically. These effects of high pressure were reversible. Cells re-established their typical morphology, microtubule arrays appeared normal, and stress fibers reformed after approximately 1 hour at atmospheric pressure. High pressure may disrupt the normal assembly of microtubules and actin filaments by affecting the cellular regulatory mechanisms that control cytological changes during the transition from interphase into mitosis.     

Colchicine-induced tubular,vesicular and cisternal organelle aggregates in absorptive cells of the small intestine of the rat. I. Morphology and phosphatase cytochemistry

Treatment of rats with colchicine administered intraperitoneally at a dosage of 0.5 mg per 100 g of body weight for 6 hr induces extensive accumulations of tubular-vesicular and cisternal organelles in the absorptive cells of the small intestine. The formation of these organelle aggregates coincides with a reduction of microtubules and massive changes in the cellular organization including alterations of the Golgi apparatus and the plasma membrane. In most cases the accumulated tubules and vesicles contain a homogeneous electron-dense matrix, the cisternae often having the character of rigid lamellae. The organelle aggregates mainly occupy apical cell portions subjacent to the terminal web as well as basal cellular regions close to the basolateral plasma membrane. Tubular-vesicular as well as cisternal organelles react strongly for thiamine pyrophosphatase (TPPase), inosine diphosphatase (IDPase), acid phosphatase (AcPase) and trimetaphosphatase (TMPase). The staining pattern of TMPase differs from that of the other phosphatases in that the reaction is restricted to the colchicine-induced tubular-vesicular and cisternal aggregates, whereas TPPase, IDPase, and AcPase, respectively, also appear over Golgi stacks, multivesiculated bodies and plasma membrane. This phosphatase reactivity indicates the lysosomal character of the organelle aggregates.     

Role of microtubule assembly in lysosomal enzyme secretion from human polymorphonuclear leukocytes. A reevaluation

The dose-related inhibition by colchicine of both lysosomal enzyme release and microtubule assembly was studied in human polymorphonuclear leukocytes (PMN) exposed to the nonphagocytic stimulus, zymosan-treated serum (ZTS). Cells were pretreated with colchicine (60 min, 37 degrees C) with or without cytochalasin B (5 microng/ml, 10 min) and then stimulated with ZTS (10%). Microtubule numbers in both cytochalasin B- treated and untreated PMN were increased by stimulation and depressed below resting levels in a dose-response fashion by colchicine concentrations above 10(-7) M. These concentrations also inhibited enzyme release in a dose-response fashion although the inhibition of microtubule assembly was proportionately greater than the inhibition of enzyme release. Other aspects of PMN morphology were affected by colchicine. Cytochalasin B-treated PMN were rounded, and in thin sections the retracted plasma membrane appeared as invaginations oriented toward centrally located centrioles. Membrane invaginations were restricted to the cell periphery in cells treated with inhibitory concentrations of colchicine, and the centrioles and Golgi apparatus were displaced from their usual position. After stimulation and subsequent degranulation, the size and number of membrane invaginations greatly increased. They remained peripheral in cells pretreated with greater than 10(-7) M colchicine but were numerous in the pericentriolar region in cells treated with less than 10(-7) M. Similarly, untreated PMN that were permitted to phagocytose immune precipitates had many phagosomes adjacent to the centriole. After colchicine treatment, phagosomes were distributed randomly, without any preferential association with the centrioles. These data suggest that microtubules are involved in maintaining the internal organization of cells and the topologic relationships between organelles and the plasma membrane.     

Effects of cytochalasin D on the distribution of actin and ACTH-induced steroidogenesis in cultured embryonic adrenal gland cells from the Pekin duck (Anas platyrhynchos)

Cultured steroidogenic cells derived from the adrenal glands of duck embryos were used to study changes in the distribution of actin associated with the corticotropic responsiveness. Actin-containing components were identified by rhodamine-phalloidin staining. The actin in most of the unstimulated cells occurred as stress fibers that either ran parallel throughout the cell or were present as domains of parallel fibers at angles to one another. When incubated in Krebs-Henseleit buffer containing 1–24 ACTH, the cells released approximately equal amounts of corticosterone and aldosterone. Incubation of the cells in buffer containing cytochalasin D caused the cells to lose their stress fibers, and the actin became distributed at the periphery in what appeared to be fragments of stress fibers and clumps of fibrous material in the central cytoplasm. Although cytochalasin D did not affect the basal output of corticosterone and aldosterone, the 1–24 ACTH-induced rates of both hormones were suppressed significantly. After the cells had been washed in unadulterated buffer, the normal distribution of actin stress fibers was restored and the cells responded normally when incubated in buffer containing 1–24 ACTH. These results suggest that the actin components of the cytoskeleton are important determinants of corticotropin-induced steroidogenic responsiveness.     

Modulation of endothelial cell shape by SPARC does not involve chelation of extracellular Ca2+ and Mg2+.

SPARC (secreted protein, acidic and rich in cysteine) is an extracellular, Ca(2+)-binding protein that inhibits the spreading of newly plated cells and elicits a rounded morphology in spread cells. In this study, I investigated whether the rounding effect of SPARC depends on the ability of the protein to chelate Ca2+ at the cell surface. Bovine aortic endothelial cells were plated in the presence of different concentrations of SPARC and Ca2+; control experiments were performed with 1 mM EGTA and with Mg2+. Quantitative estimates of cell rounding were calculated according to a rounding index. SPARC, at concentrations between 0.15 and 0.58 microM, elicited rounding (or prevented spreading) of cells cultured for 16-38 h in 0.5-2.0 mM Ca2+. Addition of 0.5-2.0 mM Mg2+ to cells previously rounded in the presence of SPARC did not abrogate the effect of SPARC. When the levels of extracellular Ca2+ were adjusted with 1 mM EGTA to maximum values ranging from 7.1 to 320 microM, cells displayed a rounded morphology in the presence of exogenous SPARC. Although the rounding induced by 1 mM EGTA was essentially reversed by the inclusion of 2 mM Ca2+, cultures containing these reagents together with SPARC maintained the rounded phenotype. These results do not support a mechanism that involves the abstraction of Ca2+ from proteins at the cell surface or the provision of Ca2+ from native extracellular SPARC to cells. Therefore, SPARC does not appear to act as a local chelator of extracellular Ca2+ and Mg2+ and presumably exerts its function as a modulator of cell shape via a different pathway.     

Enhanced uptake of calcium by transforming lymphocytes

Phytohemagglutinin caused a rapid increase in calcium accumulation by lymphocytes. The enhanced uptake was observed within 1 hr of initiation of transformation in both human lymphocyte and mouse spleen cell cultures. Increased uptake was also found in mixed lymphocyte cultures although not until late in the response. The rate of calcium uptake increased with time after stimulation and depended upon the PHA concentration. The lowtemperature coefficient (Q₁₀) for calcium permeability in unstimulated cells was indicative of a passive diffusion process, but the Q₁₀ was slightly greater for PHA-stimulated cells. Various chemical agents which alter membrane properties and/or cellular metabolism inhibited uptake to a greater extent in stimulated cultures than in control cultures. Ouabain did not affect the calcium permeability of controls or stimulated cells within 1 hr after PHA addition, but it partially inhibited calcium uptake 12 hr after PHA treatment. Cyclic AMP, dibutyryl cyclic AMP, and theophylline also altered calcium transport providing evidence for an effect of cyclic AMP on an early event in the transformation process.     

Effects of local anesthetics on cell morphology and membrane-associated cytoskeletal organization in BALB/3T3 cells

Tertiary amine local anesthetics (dibucaine, tetracaine, procaine) reversibly affect the morphology of untransformed BALB/3T3 cells and the organization of membrane-associated cytoskeletal elements. In the presence of these drugs cells contract and become rounded in shape with the appearance of numerous surface "blebs." Electron microscope examination of anesthetic-treated cells revealed significant reductions in plasma membrane-associated microtubules and microfilaments and/or their plasma membrane attachment. The relationship of the findings on local anesthetic-induced changes in cellular cytoskeletal systems is discussed in relation to previous proposals on plasma membrane organization and control of cell surface receptor topography and mobility.