Effects of cytochalasin B on the fine structure of organized endodermal cells of the early chick embryo

Fresh pullet eggs (White Leghorn) were incubated for 36 to 48 hours. The blastoderms were exposed to cytochalasin B (CB), 10 or 40 microgram/ml, for 2, 5, and 15 minutes prior to fixation by immersion in buffered chick Ringers solution containing CB, previously dissolved in dimethysulfoxide (DMSO), or by sub-blastodermic injection. Controls fixed in ovo possess relatively flat surfaces with bulges due to uptake of yolk. Numerous microappendages (blebs, microvilli and ruffles) are present, especially at cell margins. DMSO-controls present a similar cell surface except that small blebs are more prominent. The plasmalemmas of CB-treated endodermal cells possess numerous large blebs (2-10 micron in diameter), smaller blebs (0.2 micron) and microvilli. Cell dissociation occurs in selected areas resulting in rounded cells, devoid of microappendages, with peripheral processes. Transmission electron microscopic preparations of tissues similar to those used for scanning electron microscopy reveal that large blebs are filled with membranous material. Microfilaments are present but lack their normal subplasmalemmal arrangement. Microtubules and other cell organelles are apparently unaffected by CB. Evidence in this study supports the concept that cytochalasin B exerts its influence through alteration of the plasmalemma.     

Cytochalasin D and cationized ferritin as probes for the morphological investigation of blebbing in two human cell lines

Summary The potent fungal metabolite cytochalasin D (CD) and cationized ferritin (CF) are used in combination to test for negative charge distribution on blebs (knobs). Two established human epithelial cell lines, WISH and HeLa, that display blebs in various phases of the cell cycle or under certain culture conditions (37,46) are investigated. CD alone, applied at a low concentration (1.0 μg/ml) and for a short time period (3 min), causes blebs to appear as the prevalent surface feature. These are filled mainly with free ribosomes. Additionally, feltlike mats, presumed to be disorganized, compacted microfilaments, are formed directly beneath the cell membrane. These are especially evident in the cortical cytoplasm below the blebs or bleb clusters. CF (0.345 mg/ml), applied for a 5-min period after CD administration (1.0 μg/ml) for 3 min, appears along the surface of microvilli, at the base of blebs, and in vesicles beneath the bleb clusters. In some cases, microfilaments (6 nm in diameter) are closely related to the vesicles. CF does not preferentially bind to the apical cell membrane of blebs. Above areas of the subplasmalemmal microfilaments, CF membrane binding is apparent, even under circumstances where the filaments are disorganized by cytochalasin treatment. These results seem to show the following: (a) bleb membranes are different from the remainder of the cell and do exhibit a loss of negative charge and (b) surface charge may be dependent on the presence or structural integrity of membrane-related 6-nm microfilaments. The support of this research by a grant from the Baylor College of Dentistry and The Oklahoma College of Osteopathic Medicine and Surgery is gratefully acknowledged. The assistance of Dr. J. H. Martin, Department of Pathology, Baylor University Medical Center, is also greatly appreciated.     

Apically administered cytochalasin B and D decreases sensitivity of electroreceptor organs in the North-American catfish,Ictalurus nebulosus

The receptor cells of the ampullary electroreceptor organs of Ictalurus nebulosus bear microvilli on the apical membrane. Whereas microvilli in mechanoreceptive hair cells and in chemoreceptor cells have a transduction function, the function of these membrane specializations in electroreceptor cells is not fully understood. To study the role of the microvilli of the electroreceptor cells, the ampullary electroreceptor organs were apically exposed to the microfilament-disrupting agents cytochalasin B and D. Electrophysiological measurements showed that cytochalasin caused a high decrease in sensitivity and a slight decrease in spontaneous activity. Exposure to cytochalasin B resulted in a striking disorganization of the microvilli on the apical membrane of the electroreceptor cells. The most plausible explanation for the results is that treatment with cytochalasin mainly affects the actin filaments of the microvilli causing an increase of the resistance of the apical membrane. A high apical resistance results in a decrease of the voltage over the basal membrane, which in turn reduces the sensitivity. The conclusion is that intact apical microvilli are necessary for proper functioning of ampullary electroreceptor organs. Alterations in microvillar properties, like surface area and ion channel conductancy might play a considerable role in the regulation of the sensitivity.     

Effects of cytoskeletal perturbant drugs on ecto 5''-nucleotidase, a concanavalin A receptor

Differences in cell morphology, concanavalin A-induced receptor redistributions, and the cooperativity of the inhibition of 5'-nucleotidase (AMPase) by concanavalin A (Con A) have been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma cells treated with microfilament- and microtubule-perturbing drugs. By scanning electron microscopy MAT-C1 cells exhibit a highly irregular surface, covered with microvilli extending as branched structures from the cell body. MAT-A, MAT-B, and MAT-B1 cells have a more normal appearance, with unbranched microvilli, ruffles, ridges, and blebs associated closely with the cell body. MAT-C cells have an intermediate morphology. Treatment of MAT-A, MAT-B, or MAT-B1 cells with Con A causes rapid redistribution of Con A receptors. Both cytochalasins and colchicine cause alternations in the receptor redistributions. Receptors on MAT-C1 cells are highly resistant to redistribution, even in the presence of cytoskeletal perturbant drugs. The cooperativity of the inhibition of AMPase by Con A was investigated in MAT-A and MAT-C1 cells. Untreated cells exhibit no cooperativity. If either subline is treated with colchicine, cytochalasin B or D, or dibucaine, cooperativity is observed. Lumicolchicine has no effect. Theophylline or dibutyryl cyclic AMP prevents the effects of either colchicine or cytochalasin. The concentration required for half-maximal induction of cooperativity is 0.3--0.4 microM for both colchicine and cytochalasin D, which is in the appropriate range for specific microtubule and microfilament disruptions. The effectiveness of the cytochalasins (E greater than D greater than B) is consistent with their known effects on microfilaments. No direct correlation was observed between the induction of cooperativity and drug-induced changes in Con A receptor redistribution or cell morphology. The morphology of MAT-A cells is grossly altered by cytochalasins or dibucaine and somewhat less by colchicine. MAT-C1 cells exhibit more minor alterations in morphology as a result of these drug treatments. The results of this study indicate that the inhibition of AMPase, which is a Con A receptor, is a different process from the redistribution of the bulk of the Con A receptors, possibly short range membrane interactions rather than global effects on the cell.     

Site-specific maturation of enveloped viruses in L cells treated with cytochalasin B

Treatment of infected L cells with 10 micrograms/ml cytochalasin B (CB) was found to promote a rapid relocalization of viral glycoproteins on the cell surface. Whereas the vesicular stomatitis virus G protein and the influenza virus hemagglutinin were uniformly distributed on the surface of untreated cells, in CB-treated cells, they were strikingly concentrated at cell extremities in the regions of clustered blebs. Glycoprotein concentration at cell extremities was accompanied by preferential maturation of virus particles from the same sites; both vesicular stomatitis and influenza viruses budded predominantly from the vicinity of clustered blebs. This effect of CB was completely reversible. Removal of CB from the cell growth medium resulted in a return of viral glycoproteins to the uniform distribution characteristic of untreated cells and to uniform virus budding. The results of this study are interpreted in terms of a model that suggests that preferential budding of viruses from the regions of bleb clusters is due to the concentration of viral glycoproteins at these sites.     

Response of murine erythroleukemia cells to cis-and trans-diamminedichloro-platinum (II)

Summary Cis-diamminedichloroplatinum II (cis-DDP), an antitumor drug and the inactive trans-isomer were studied to evaluate their effects on cell multiplication, DNA synthesis, and surface morphology of the murine erythroleukemia cells (clone 6A11A). Short-term treatment of cells (1h) with 5 and 10μg/ml of cis-DDP resulted in a significant inhibition of cell multiplication. Continuous treatment with cis-DDP (up to 144 h) significantly arrested cell growth at 1,5, and 10μg/ml. The cells exposed to 10 μg/ml trans-DDP exhibited a slight decrease in cell multiplication; however, the 25-μg/ml treatments showed a modest inhibition of cell growth. Continuous treatment with cis-DDP resulted in a concentrationdependent decrease in DNA synthesis, although low-dose treatment (0.05 and 0.1 μg/ml), with a few exceptions, had no relative inhibitory effect. Likewise, trans-DDP treatments decreased tritiated thymidine incorporation; however, this inhibitory effect was not as drastic as with corresponding concentrations of cis-DDP. Scanning electron microscope studies revealed the formation of many giant cells and blebs at all short-term treatment concentrations of cis-DDP past the 48 h interval. Continuous treatment of cis-DDP at 1 μg/ml concentration produced giant cells with minute holes, whereas the 5 and 10 μg/ml exposure resulted in the formation of blebs and large holes and reduction of microvilli past the 48-h treatment period. At higher concentrations the continuous treatment of cis-DDP completely destroyed the cells. The surface morphology of trans-isomer treated cells, in most instances, resembled the corresponding untreated control cells.     

Bleb formation in granulosa cells of rat pre-ovulatory follicles in vivo and in vitro

To determine if hormone-induced events leading to ovulation an granulosa cell luteinization might be associated with changes in the surface configuration of granulosa cells we have studied the morphology of granulosa cells from the preovulatory follicles both in vivo and in vitro. In vivo, granulosa cells in follicles from rats primed with estradiol and FSH developed bulbous protrusions termed blebs in response to injected hCG. The blebs were restricted to the adluminal granulosa cells which possess the least number of receptors for hCG. When granulosa cells from follicles of rats primed with estradiol and FSH were cultured in vitro, in the absence of serum, approximately 10% of the cells formed blebs. In the presence of 10% rat or fetal calf serum, nearly 90% of the cells formed blebs by 18 hr. Serum-induced bleb formation was prevented by 1 mM dibutyryl cycle-AMP plus 0.5 mM methyl isobutyl xanthine and by cytochalasin B (25 mug/ml), while 0.1 muM colchicine had no effect. Fibronectin at 25 mug/ml increased bleb formation three-fold over control values in serum-free medium. When hCG was included in serum containing medium, the majority of the cells remained smooth without any blebs. Thus, in contrast to its action in vivo, hCG inhibited the formation of blebs in vitro. When the cells incubated in the presence of dbcAMP plus methyl isobutyl xanthine in serum-containing medium, none of the cells formed blebs. One explanation for the seemingly opposite actions of hCG in vivo and in vitro is that hCG might act to alter the permeability of the pre-ovulatory follicles, and thereby allow the admission of serum. The admitted serum component(s) could then induce the formation of blebs on receptor-deficient adluminal cells that did not have elevated cAMP concentrations. The results suggest that fibronectin and/or other serum components, act to induce microfilament-dependent, cAMP-inhibited bleb formation on granulosa cells in vivo and in vitro.     

Cytoskeletal control of the apical surface transformation of rat uterine epithelium

Summary— During early pregnancy, in the lead up to blastocyst implantation, the apical cell surface of luminal epithelial cells of the rat uterus undergo a dramatic shape transformation. This study aims to investigate the role of the cytoskeleton in this apical transformation by considering the effects of the drugs cytochalasin D and colchicine on the uterine luminal cell surface. The results are determined using transmission and scanning electron microscopy. In vivo exposure to cytochalasin D during oestrus, as well as on day 1 of pregnancy, did not affect the long, regular surface microvilli. This drug, however, did disrupt the terminal web within the apical cytoplasm of these cells. Disruption of microfilament (MF) polymerization by cytochalasin D on day 4 of pregnancy induced a cell surface transformation, resulting in the appearance of numerous irregular projections normally present during blastocyst implantation on day 6 of pregnancy. Colchicine did not alter the uterine microvilli of oestrus or day 1 pregnant tissue. Unlike the effect of cytochalasin D, colchicine-induced microtubule (MT) disruption on day 4 of pregnancy did not increase irregular projections and hence this treatment did not result in the cell surface appearance associated with blastocyst implantation. These results indicate that the disruption of MF, rather than MT, contributes to the transformation of the uterine luminal cell surface during the lead up to blastocyst attachment.     

Cytochalasins distinguish by their action resting human T lymphocytes from activated T-cell blasts

In the majority of resting human peripheral T lymphocytes obtained from separate individuals cytochalasin B (CB) and D (CD) cause a disappearance of microvilli and induce a rapid formation of prominent sac and bleb-like projections with a length of 1–10 μm randomly distributed over the cell surface. During mitogen stimulation the cells lose the tendency to develop such projections when subsequently exposed to CB and CD. By contrast, in activated T lymphocytes the cytochalasins provoke an asymmetric localization of microvilli including cell surface antigens and actin to a prominent protuberance often separated from the cell body by a constriction. This protuberance is distinct from conventional spontaneous uropods formed by conA-stimulated lymphocytes in relation to contact with other cells and with non-cellular surfaces. The cytochalasins therefore in their action distinguish resting small lymphocytes from activated T-cell blasts.     

Modulation of agglutinability by alteration of the surface topography in mouse ascites tumor cells

Factors involved in controlling agglutinability of cells with plant lectins include number, distribution, availability and mobility of cell surface lectin receptor sites. We have examined the concanavalin A (ConA)-mediated agglutination of mouse sarcoma 180 ascites tumor cells in the presence or absence of cytochalasin B (CB) using a quantitative electronic particle counter assay. These cells become substantially more agglutinable after brief treatment with low concentrations of CB. Scanning and transmission electron microscopy indicate that CB causes formation of large, broad, cell surface ruffles and loss of narrow projections that appear to be microvilli. Studies using fluorescent ConA suggest that lectin receptor sites concentrate on these ruffles and that the ruffles seem to directly mediate increased agglutinability in this system. Electron spin resonance studies suggest that CB does not alter lipid “fluidity” in these cells. The results indicate that the gross cell surface topography favoring high agglutinability is one displaying broad ruffles, not numerous narrow projections.     

Inhibition of virus-induced fusion of Ehrlich ascites tumor cells by cytochalasin B and D.

Cytochalasin B and D were found to inhibit HVJ (Sendai virus)-induced fusion of Ehrlich ascites tumor cells. Nearly complete inhibition was attained by 4 uM (2 μg/ml) cytochalasin D, whereas cytochalasin B was a less effective inhibitor. The inhibition was largely reversible. Since the transport of 2-deoxy-glucose into the tumor cells was not affected by cytochalasin D (though inhibited by cytochalasin B), the observed inhibition was not related to the effect of the drugs on sugar transport. Instead, it was suggested that the inhibition was due to the action of the drugs on microfilaments. The requirement of ATP for the cell fusion could be explained at least partly by the involvement of microfilaments in the cell fusion process.     

The mechanism of matrix vesicle formation. Studies on the composition of chondrocyte microvilli and on the effects of microfilament-perturbing agents on cellular vesiculation

The mechanism of matrix vesicle (MV) formation by growth plate chondrocytes in primary cell culture was assessed both by using drugs which interfere with assembly or disassembly of microfilaments and microtubules, as well as by comparison of the composition of chondrocyte microvilli with MV. Cytochalasin D, which is known to inhibit assembly of actin microfilaments, was found to stimulate the release of alkaline phosphatase-rich MV. This stimulatory effect was confirmed by studies with [3H]palmitate- and 32P-prelabeled cells which showed that cytochalasin D enhanced the release of labeled MV. In contrast, phalloidin, which blocks disassembly of microfilaments, suppressed release of cellular alkaline phosphatase into MV. The phospholipid composition of vesicles released by cells treated with cytochalasin D and phalloidin was virtually identical with that of the controls. In contrast, colchicine, which interferes with the assembly of microtubules, was found to cause fragmentation of the cells, producing large vesicles significantly different in lipid composition from MV. Microscopic studies revealed that cytochalasin D caused marked rounding and retraction of the cells, with evidence of actin withdrawal from the cell periphery. This led to cell surface blebbing and formation of small zeiotic bodies at the tips of cell processes. In contrast, phalloidin enhanced and stabilized the actin network within the cells. Chemical analysis of microvilli prepared from isolated chondrocytes revealed high levels of alkaline phosphatase and a phospholipid composition almost identical to MV. Electrophoretic profiles of microvillar proteins were again like that of MV, except for the presence of high levels of actin. This cytoskeletal protein was nondetectable in MV. Taken together with the effects of the drugs, the data indicate that cell surface microvilli are the precursors of MV and that retraction of the supporting microfilament network is essential for the release of these structures.     

Role of the microfibrillar system in knob action of transformed cells

Transformed cells often display knobs (or blebs) distributed over their surface throughout most of interphase. Scanning electron microscopy (SEM) and time-lapse cinematography on CHO-K1 cells reveal roughly spherical knobs of 0.5–4 μm in diameter distributed densely around the cell periphery but sparsely over the central, nuclear hillock and oscillating in and out of the membrane with a period of 15–60 sec. Cyclic AMP derivatives cause the phenomenon of reverse transformation, in which the cell is converted to a fibroblastic morphology with disappearance of the knobs. A model was proposed attributing knob formation to the disorganization of the jointly operating microtubular and microfilamentous structure of the normal fibroblast. Evidence for this model includes the following: (1) Either colcemid or cytochalasin B (CB) prevents the knob disappearance normally produced by cAMP, and can elicit similar knobs from smooth-surfaced cells; (2) knob removal by cAMP is specific, with little effect on microvilli and lamellipodia; (3) immunofluorescence with antiactin sera reveals condensed, amorphous masses directly beneath the membrane of CB-treated cells instead of smooth, parallel fibrous patterns of reversetransformed cells or normal fibroblasts; (4) transmission electron microscopy (TEM) of sections show dense, elongated microfilament bundles and microtubules parallel to the long axis of the reverse-transformed CHO cell, but sparse, random microtubules throughout the transformed cell and an apparent disordered network of 6-nm microfilaments beneath the knobs; (5) cell membranes at the end of telophase, when the spindle disappears and cleavage is complete, display typical knob activity as expected by this picture.     

Studies on cell surface conformation following injury. II. Scanning and transmission electron microscopy of cell surface changes following anoxic injury in Ehrlich ascites tumor cells.

Exposure of Ehrlich ascites tumor cells to anoxia resulted in rapid and characteristic conformational changes of cell surface topography. Combined scanning and transmission E/M studies revealed rapid alterations including simplification of the cell surface configuration with disappearance of microvilli which were replaced with formation of blebs and recesses at the cell periphery. These surface changes were accompanied by characteristic organelle alterations inside the cells which in this and other cellular systems have been shown to be reversible. Later, the cell surface topography became smoother and monotonic with small blebs and cribriform invaginations in addition to larger eruptions of the cell periphery. Combined transmission E/M studies revealed fragmentation of cellular membrane systems and lysis of organelles indicating the irreversible phase of anoxic injury. The rapid conformational surface changes encountered in Ehrlich ascites tumor cells following anoxia suggest the important role of the plasma membrane and its unfolding as a virtually instantaneous response of the cells to this injury.     

Mitotic cells and their microappendages in the primitive streak of the chick embryo.

Fresh pullet eggs (White Leghorn Strain) were incubated to the primitive streak stage of development. Blastoderms were fixed in situ with isotonic aldehyde fixatives and prepared for scanning electron miscropy by means of post-osmication, critical point drying and gold-palladium coating. Cells judged to be in various stages of mitosis by their surface contours were numerous on the ventral surface of the chick blastoderm. Cells which were in the late preparatory stages for mitosis had rounded up from their surroundings. Microvilli dominated the surface. The degree of separation and number of microvilli increased until late metaphase or anaphase. Mitotic cells did not completely separate themselves from adjacent cells. Ruffles and blebs were not prominent during mitotis and long filopodia were absent. A definite localization of microappendages (microvilli, blebs, ruffles) to the area of cytokinesis was evident in early telophase and persisted through daughter cell formation.     

Cell cycle-dependent surface changes in Chinese hamster cells grown in suspension culture

The cell surface appears to play an important part in the control of cell replication. It has been demonstrated that the cell membrane undergoes cyclic changes in appearance which bear a relation to the cell cycle phase, irrespective of close intercellular contact. The surface of Chinese hamster (CHO) cells was investigated using the scanning electron microscope (SEM). The cells were synchronized in suspension culture and were sampled at frequent intervals during the cell cycle. During mitosis, the cells showed microvilli and few blebs. In early G 1 phase, profuse microvilli were seen. In late G 1 phase, blebs appeared and persisted in great numbers. During the synthesis of DNA in the S phase, blebs were observed in the early stages and then declined in number; in G 2 phase, the blebs appeared to be larger (1–2 μm) and more sparsely distributed than in late S phase. Some of these blebs were pedunculated and, in some instances, the diameter of the pedicles approximated the diameter of microvilli. Since the reasons for these changes are not understood, our long-range goal is to correlate the observed surface changes with internal biochemical events during the cell cycle.     

The corpus luteum of the pig. Scanning electron microscopic study of surface features at different times of incubation

The surface ultrastructure of porcine early corpus luteum cells (days 1-3 of the luteal phase) was studied in SEM and correlated with progesterone secretion. Luteal cells were divided into 2 groups: small cells (10-20 microns) and large cells (20-30 microns) and their surface features were observed after 1, 3, and 5 h of incubation in the control medium and in a medium supplemented with prolactin (PRL). The surface morphology of control cells was characterized by numerous smooth blebs and the presence or absence of thin microvilli. Small and large cells showed a tendency to adhere to the glass during the experiment, but on the large cells the number of thin adhesive filopodia was greater. After the 1st and 3rd h of incubation with PRL the number of microvilli and numerous filopodia on the small cells increased substantially. Nodular blebs were scattered and appeared to protrude from the cell surface. Many small cells adhered to the glass by thick, layered and thin thread-like cytoplasmic processes. After the 5th h distinct smoothing of the surface of the small cells was seen. The number of microvilli seen on the PRL stimulated surface of the large cells was smaller and in some cases even entirely absent. After the 1st and 3rd h of the experiment the large cell surface was ruffled with minute folds. Numerous nodular blebs protruded from the cell surface. The number of adhesive filopodia attaching the cells to the glass decreased or vanished during the experiment. After the 5 h of incubation most of the cells had smooth surface with smooth blebs. Progesterone secretion was measured by radioimmunoassay. The cells in the medium without exogenous hormone (control) secreted relatively low levels of progesterone throughout 1-5 h of the incubation period. After addition of PRL to the medium the amount of secreted progesterone increased.     

The cytoskeleton and rat granulosa cell steroidogenesis: possible involvement of microtubules and microfilaments

The participation of both microtubules and microfilaments in granulosa cell steroidogenesis was assessed by monitoring the effects of colchicine (0-250 microM) and/or cytochalasin B (0-10 micrograms/ml) or dihydrocytochalasin B (0-2.0 micrograms/ml) on cellular morphology and production of progestins during 24 h of culture. Both colchicine and the cytochalasins increased granulosa cell production of progesterone and of 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-progesterone) in a dose-dependent manner. The largest increase in steroidogenesis (about 2- to 3-fold) was observed at 4-250 microM colchicine and at 2-10 micrograms/ml cytochalasin. Those concentrations of the inhibitors of microtubule or microfilament polymerization that stimulated basal progestin production also markedly influenced cell spreading. Whereas cells cultured for 24 h in medium alone became very flattened with numerous cytoplasmic extensions, those cultured with colchicine (0.2-250 microM) or cytochalasin (0.4-2 micrograms/ml) were much less spread and progressively became more rounded and regular in outline. These changes in cell morphology were reflected by decreases in the mean area occupied by the cells on the culture surface of up to 60-65% and reductions in mean contour index values from 5.7 +/- 0.1 (control) to 3.9 +/- 0.1 (250 microM colchicine), 4.2 +/- 0.1 (2 micrograms/ml cytochalasin B), or 4.1 +/- 0.1 (2 micrograms/ml dihydrocytochalasin B). Cultures containing both colchicine and cytochalasin B exhibited a greater steroidogenic response than that elicited by either inhibitor alone. For example, granulosa cell progesterone production was stimulated almost 2-fold by 4 microM colchicine or 2 microM/ml cytochalasin B, but 5.5-fold by 4 microM colchicine plus 2 micrograms/ml cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cytochalasin D on the adhesion of a neuroblastoma x glioma cell line (NG108-15) to laminin and plastic substrates

Summary Adhesion of the neuronal cell surface to its underlying substrate plays an important role in neurite outgrowth in vitro. I have investigated the adhesive basis for neurite outgrowth in the presence of cytochalasin D, a disruptor of actin-containing microfilaments, and in the presence of vinblastine, a depolymerizer of microtubules. Scanning electron microscopy shows that cytochalasin D does not alter the branching configuration of filopodia on a laminin substrate, although processes are shorter and tapered distally in the presence of the drug. Using a standard attachment assay for the neuroblastoma x glioma cell line (NG108-15) I show that vinblastine does not influence attachment of NG108-15 cells to either plastic or laminin. Cytochalasin D-treated cells normally attach to high concentrations of a laminin substrate (20 g/ml). However, when cell are seeded on a laminin substrate at lower concentrations (0.001–10 g/ml), or on YIGSR, a fragment of laminin, cytochalasin D increases cell attachment. Cytochalasin D increases attachment in a dose-dependent manner when cells are seeded on plain polystyrene plastic, so that the number of cells attached to plastic in 1 M cytochalasin D is similar to the number attached to laminin (20 g/ml). Combining low concentrations of cytochalasin D and laminin results in greater attachment than with either agent alone. Mild trypsinization of the cell surface reduces the CD-enhanced attachment to plastic, indicating that a protein on the cell surface may be involved. The effect of cytochalasin D appears to be cell specific since cytochalasin D does not affect the attachment of a fibroblast cell line (NIH 3T3) to laminin and plastic. I hypothesize that a molecular mechanism is involved in which cytochalasin D promotes attachment by interacting with the cell surface via the actin microfilament system.     

Cell surface protein decreases microvilli and ruffles on transformed mouse and chick cells.

Transformation of cultured fibroblasts usually results in a decrease in a high molecular weight cell surface glycoprotein (LETS protein) and often in increased numbers of surface microvilli and ruffles. We have isolated such a major cell surface glycoprotein from chick embryo fibroblasts; this protein, CSP, is decreased after transformation. Treatment of a mouse tumor cell line (SV1), L929 cells, and transformed chick fibroblasts with CSP results in a decrease in the number of microvilli and marginal ruffles, accompanied by restoration of a more normal morphology.