Cytologia 39: 709-727 , 1974

The Cellular Response to Cytochalasin B: A Critical Overview

Michelle Copeland

Children'sCancer Research Foundation , 35 BinneyStreet, Boston, Massachusetts02115 , U. S. A. ReceivedApril 3, 1973

The cytochalasins comprise a novel class of fungal metabolites which have attracted a great deal of recent interest as a result of the numerous apparently di versecytological effects which are elicited by their presence, both in vivo and in vitro. Six cytochalasins have been isolated, to date, from the cultures of Helminthospor ium dematioidem, Metarrhizium anisopliae, and Rosellina necatrix, although some what similar biological activity has been associated with the fermentation products of several additional fungi (Aldridge et al. 1967). The six currently isolable cyto chalasins are designated A, B, C, D, E, and F. In terms of their biological activity, i nsofar as this is known at present, these six cytochalasins appear qualitatively simi lar to one another, although the potencies of the isomeric cytochalasins C and D

Aldridge et al., 1967 Chem. Comm. 1: 26

Fig. 1. The chemical structure of cytochalasin B. exceedthose of cytochalasins A and B by a factor of approximately ten (Carter 1967). Themost readily isolated of these is the B variant , which has been shown to be iden tical with the antibiotic "phomin" (Aldrige and Turner 1968) (Fig. 1). The addi tional features of appropriate potency , essential nontoxicity at low concentrations and reversibility of action have made cytochalasin B the subject of most laboratory studies of the biological phenomena produced by the cytochalasins. Preliminary investigators employed the term"cytochalasin" (from the Greek cytos: cell;+chalasis: relaxation) to denote the cytological changes induced by these metabolic end-products . These substances appeared to "relax" the cell 710 Michelle Copeland Cytologia 39

through a mechanism which may have involved an inhibition of the normal process of cell motility or a structural variation of the cytoskeleton. A more thorough consideration of this phenomenon will be presented later in this discussion. A reasonable approach to the problem of formulating a realistic mechanism for the action of cytochalasin B is to attempt to establish a functional relationship between the apparently diverse cytological effects which it has been demonstrated to elicit. Among the more prominent of these effects are several which are chara acterized as morphological in nature, such as cytoskeletal variation, multinucleation,

Fig. 2. Large quantities of granular endoplasmic reticulum and annulate lamellae are seen in the cytoplasm of this multinucleated cell; this fibroblast cell was exposed to cytochalasin B (1ƒÊg/ml) for 6-7 days. Scale line is 1um. Courtesy of Dr. Awtar Krishan. diffuse organelle alteration, nuclear extrusion, and surface phenomena of diminished membrane ruffling; other effects are described as functional or physiological, and include inhibition of cell motility, of mitotic reproduction and of phagocytosis and pinocytosis, as well as alteration of surface adhesion properties and of micro filaments and depression of synthesis or uptake of membrane components. One rather convincing element which is consistent with some of these processes is an alteration in the properties of the cell plasma membrane and it is not without evi dence that this avenue may be viewed as central to the activity of cytochalasin B. 1974 Cytochalasin B 711

Alteration of cell morphology

The diverse cellular effects of cytochalasin B were first systematically noted by

Carter (1967) in experiments with fibroblasts in culture . His initial observation of these cells treated with cytochalasin B included diminished levels of both normal motile activity and characteristic membrane ruffling . At concentrations of 1 .0 ƒÊ g/ml of cytochalasin B these fibroblasts were also noted to assume a flattened

configuration, and to occupy a larger surface area on glass after treatment . Kro shan (1971) corroborated this early evidence for membrane involvement and sup

plemented it with the observation that fibroblasts exposed to similar concentra tions of cytochalasin B transformed from the normal spindle into flat , epithelioid shapes through contraction of elongated pseudopodia. These discoid fibroblasts became multinucleated and accumulated Golgi membranes, rough endoplasmic

reticulum and annulate lamellae such that after fifteen days of culture in medium

Fig. 3. A large cytochalasin-induced multinucleated fibroblast cell with 9 nuclei . Individual variation in the nuclear size is evident. Exposed to cytochalasin B (1ƒÊg/ml) for 6-7 days. Scale

line is 1ƒÊm. Courtesy of Dr. Awtar Krishan.

containing cytochalasin B, the cells attain diameters of approximately 300 microns and possessed eight or nine nuclei each (Figs. 2 and 3). The possibility that these alterations in cell structure might be due to an augmentation by cytochalasin B of the synthesis of cellular elements has been effectively eliminated by the demonstra tion that the rates of DNA, RNA and protein syntheses are unaffected by cyto chalasin B (Estensen 1971). Krishan's data are consonant with a process in which the drug influences merely the plasma membrane such that the maintenance of even normal metabolic activities results in large-scaled cytoeconomics. That is, quite simply, that cells behave metabolically as if they had been separated from one another following normal mitosis, and that each member "cell" of the cytochalasin B-induced conglomerate contributed to the mass that quantity of synthetic pro ducts which it would have formed in the independent state. This consideration points out an important aspect of the analysis of the activities of cytochalasin B; 712 Michelle Copeland Cytologia 39

that is, the drug may have a single direct action on a component of, say, the plasma membrane and yet produce in vitro variations which suggest a diversity of roles. An additional example of the application of this principal has been provided by Krishan in an explanation of the conspicuously large number of RNA virus-like particles found within the giant fibroblasts produced by cytochalasin B. Krishan has realistically suggested that the larger fibroblast cells were preferentially suited to the proliferation of endogenous L-cell virus-like particles as well as exogenously introduced virus-like reo and Herpes viruses in that the hosts contained large cyto plasmic masses, unusually large nuclei, relatively hypertrophied endoplasmic reti culum, and numerous other cell organelles in high numbers.

Arborization and cellular adhesion

Insight into the processes resulting in the observations of Carter and Krishan have been provided by Sanger and Holtzer (1972), who have validated an alteration

by cytochalasin B of the cytoskeleton. Exposure of myoblasts, fibroblasts and stellate chondroblasts for 48 hours to 5ƒÊg/m1 of cytochalasin B caused the cells to

assume a branched gross morphology and induced a virtual immotility. Reversal of this "arborization" could be achieved by withdrawal of cytochalasin B from the

medium. It was noted by these authors that the resumption of normalized cell morphology and considerable recovery of cellular motility did not entail retraction of the extruded processes, the overall dimensions of which remain unchanged.

The cells apparently lose their arborized morphology by gradually filling the area between the processes with outflowing lobopodia, the forms of which are indis tinguishable from the advancing tip of amoebae, and no membrane "ruffling" pheno mena were associated with the lobopodial protrusion. Additional experiments were conducted by these researchers in which specific metabolic inhibitors were

employed to assess the role of cytochalasin B in the arborization process. Ar borized cells incubated with cycloheximide (an inhibitor of protein synthesis) and 5ƒÊg/ml of cytochalasin B for 30 minutes and then transferred to a medium con

taining only cycloheximide underwent normal reversal of shape. From this datum it can be concluded that the motile lobopodial activity responsible for normaliza tion of shape is not dependent upon protein synthesis. On the other hand, during

exposure of arborized cells to sodium cyanide, an inhibitor of oxidative phos

phorylation, the branched conformation is maintained until this inhibitor is with drawn. It is apparent, therefore, that protrusion of lobopodia is dependent upon

energy production and independent of protein synthetic activity. In addition, it should be mentioned that not all of the cell types treated with cytochalasin B ex hibited arborization under the conditions employed. Replicating chick embryo

erthrocytes undergoing nuclear division show neither cytokinesis nor arborization; whereas presumptive myoblasts arborize upon treatment with cytochalasin B, their fusion products, myotubes, do not; furthermore, the nonfunctional chondrocyte, lacking a maturation capsule, does exhibit arborization, while the functional chon droblast encapsulated in chondroitin sulfate does not branch. It has been sug gested that these differing responses to cytochalasin B may reflect fundamental differences in cell surfaces. Myoblasts, for example, are thought to undergo con siderable surface alteration following fusion into multinucleate myotubes, and 1974 Cytochalasin B 713

this change may effect variation in the cellular response to cytochalasin B . Simi larly, while the nonfunctional chondrocyte is freely exposed to the drug , the en capsulated chondroblast may not provide a site on the surface of the membrane which is sensitive to the action of cytochalasin B. These considerations supplement other evidence which suggest that cytochalasin B acts directly upon some compo nent at the cell surface and that it influences the cytoskeleton indirectly through a surface phenomenon.

As discussed above, fibroblasts exposed to 1ƒÊg/ml of cytochalasin B underwent a gradual transformation characterized by a relative contraction of pseudopodia

and the assumption of an epitheloid conformation (Krishan 1971); higher doses of cytochalasin B (5ƒÊg/ml) appear, however, to elicit the entirely different response

morphology of arborization, as just indicated. The apparent contradiction in herent in these responses can be resolved through the following mechanistic pro

posal: cytochalasin B at a concentration of 1ƒÊg/ml causes a weakening of ceveral regions of the plasma membrane and thereby permits the cytoplasm to bulge out ward in the area of weakness, altering the shape of the cell; the "rounded" appea

rance of the cell results from the fact that pseudopodia are less pronounced as cyto

plasmic protrusions in the presence of a generally "bulged-out" morphology; at higher doses of cytochalasin B (i.e., 5ƒÊg/ml) the plasma membrane is affected more

severely and cytoplasmic protrusion is more pronounced; the result, at these drug levels is the formation of a branched morphology and the exhibition of an arborized

state. Alternatively, if viewed in light of Carter's postulation (1967) that cyto

chalasin B causes an increase in cell-to-substrate adhesion , cytoplasmic protrusions result from the increased adhesion of portions of the plasma membrane to a sub strate and, therefore, protrusions would be visible at points of lower cell surface-to substrate adhesion. It is intriguing to note that the amoeboid movement which characterizes amoebae, blood leukocytes and numerous other cells is dependent

upon the formation of pseudopodia which involves the extension of membrane bound processes. Pseudopod formation occurs at the cell periphery and may be

a surface-related phenomenon; alteration of the membrane with regard to pseudopod formation would be anticipated to inhibit amoeboid motility. Indeed, in several cell types, a change in the configuration of cytoplasmic protrusions occurs with

concommitant inhibition of normal cell motility in the presence of cytochalasin B. Similar mechanisms can be employed to explain the influence of this drug in in hibiting phagocytosis , a phenomenon considered later in this dicussion. Sanger and Holtzer (1972) have demonstrated other cytological effects of cytochalasin B which may be more directly related to the functioning of the surface membrane. In experiments conducted with three-day-old chick embryos, dis sociated two-day-old embryonal eyes and mixtures of myogenic and chondrogenic cells, these workers detected marked differences in cell-to-cell adhesion properties betweencells treated with cytochalasin B and those grown in a normal medium. Embryonal tissues cultured in a normal medium could be dislodged from the growth chamber as a cohesive pellet of cells, while those cultured in media containing cyto chalasinB dispersed into separate cells when dislodged from the wall of the chamber. Thesecells could be caused to aggregate into the normal cohesive pellet following 714 Michelle Copeland Cytologia 39 continued incubation after withdrawal of cytochalasin B from the growth medium. Under similar conditions, the embryonal eyes cultured in a normal medium formed rosettes characteristic of neural epithelium and cohesive masses of lens cells, while those treated with cytochalasin B failed to dissociate in these definitive structures. Moreover, mixtures of chondrogenic and myogenic cells exhibited distinct func tional variation upon treatment with cytochalasin B; histological sections from normal-control cell masses revealed regions of metachromatic chondrification and swirls of multinucleate myotubes, while those from cytochalasin B-treated cultures exhibited neither chondrocyte aggregation nor myotube formation. It appears

Fig. 4. a, cells from hybrid culture before exposure to cytochalasin B. L-cell-chick erthrocyte hybrid is shown on the left and contains two erythrocyte nuclei. b and c, in contrast, note size of nucleus in intact erythrocyte. Within several hours of exposure to cytochalasin B, nuclei are rando domly extruded. Nuclei become condensed and appear pyknotic. d, as extrusion progresses, nucleus is isolated from the cytoplasm and is transiently connected to it by a very thin cytoplasmic thread. Magnifications about x 1150. Courtesy of Dr. Roger L. Ladda and Dr. Richard D. Estensen. from these data that cytochalasin B alters cellular adhesion interactions-proper ties generally ascribed to the surface membrane. This evidence provides a func tional demonstration of the direct action of the drug at the cell surface. The ubi quity of this phenomenon has recently been challenged by Armstrong and Parenti (1972). Their demonstration that aggregation of neural and pigmented retinal cells was not altered by 5 to 10ƒÊg/ml of cytochalasin B suggests the possibility of a more specific locus of action for the drug.

Nuclear extrusion As alluded to above, the phenomena of nuclear extrusion and inhibition of 1974 Cytochalasin B 715

phagocytotic activity observed in the presence of cytochalasin B may also be mem brane-related. Carter (1967) has described the sequence of events seen during cyto chalasin B-induced nuclear extrusions in fibroblasts, at drug dosages of 10ƒÊg/ml. The process is initiated by migration of the nucleus toward the plasma membrane with the formation of a bulge in the membrane in this region. Shortly, the nucleus appears external to the cell mass, although its connection to the cell is maintained by a thread-like cytoplasmic bridge (Fig. 4). It is intriguing not only that rather high doses of cytochalasin B were required for the demonstration of this effect, but also that the structural alteration could be reversed simply by restoring fresh medium to the culture chamber (that is, by removal of cytochalasin B) without apparent alteration in nuclear function as a result of the episode. If, however, the cell and nucleus in the extruded state are maintained in medium containing cytochalasin B for several hours, the extrusion becomes irreversible and the nucleus becomes pyk notic. Although other researchers (e.g., Ridler and Smith 1968, Ladda and Esten

sen 1970, Prescott et al. 1971) have observed these phenomena, little critical examin ation has been conducted and the formulation of a mechanism for this unique re sponse is not feasible at present. It seems reasonable, nevertheless, that a surface

influence may once again be involved, insofar as, it may be speculated, high doses of cytochalasin B may alter the adhesion properties of the plasma membrane; if the membrane adhered more strongly to a substrate and consequently remained fixed,

the moving nucleus would eventually approach the surface to exert a pressure on the membrane until it produced a bulge. Moreover, the membrane may be coated with a negatively charged mucopolysaccaride compound such as that neces

sary for particle-binding prior to phagocytosis. Alterations of this mucoid com

ponent by cytochalasin B might sufficiently change the surface electrical properties of the membrane such that nuclear extrusion would be precipitated. A role for

cytochalasin B in this regard has already been intimated by Sanger and Holtzer

(1972), Zigmond and Hirsch (1972) and Estensen and Plagemann (1972) who have demonstrated an inhibitory effect of the drug upon mucopolysaccharide biosyn

thesis in vitro. Extrusion of the nucleus would, of course, be facilitated by the weak ening of the plasma membrane under the influence of cytochalasin B as postulated earlier, and the membrane would therefore pose relatively little physical resistance

to nuclear transport. One must, however, not discount the additional possibility that the enucleation phenomenon is a manifestation of, say, a toxic reaction to cytochalasin B.

Alterations in cell physiology

Phagocytosis The process of phagocytosis may be viewed as a functional inversion of the occurrence of nuclear extrusion in that the former involves the engulfing and in corporation of a foreign granule by the cell while the latter consists of the expulsion of an organelle native to the cell. Indeed nuclear extrusion may be compared with the process of emeiocytosis, in which a membrane-lined vacuole fuses with the plasma membrane, and its contents are excreted into the extracellular region. 716 Michelle Copeland Cytologia 39

Malawista (1971) reported that human blood leukocytes exposed to 2-5ƒÊg/ml of cytochalasin B showed a subnormal degree of bacterial incorporation; the effect increased with time and was dose-dependent. In addition to the numerical sup pression of bacterial incorporation by the cells, the burst of carbon dioxide produc tion which usually accompanies phagocytosis by leukocytes was markedly depressed in those cells treated with cytochalasin B; once again, a good dose/response rela tionship was established. Phagocytosis in these cells involves the invagination of the membrane and the subsequent inclusion of extrinsic materials by association within membrane-limited vesicles, followed by the transport of the material into the deeper interior of the cell. In view of this morphology, functional impairment of phagocytosis could be manifested by agents which prevent either membrane invagination or the surface fusion which forms the phagocytotic vacuole. Fusion, incidently, seems logically dependent upon effective synthesis of new membrane elements, and it has been suggested that cells in which phagocytosis is inhibited display an endocytotic activity-maximum which may correspond to the limitation of precursor materials available for synthesis of new membrane (Zigmond and Hirsch 1972). The depression of the rate of phagocytosis-associated CO2 release produced by cytochalasin B indicates that certain metabolic processes may be in fluenced by the drug. These speculations are dealt with more systematically below.

Cell division

The action of cytochalasin B upon mitotic processes has been among the most controversial areas of research with the compound. Carter reported in 1967 that fibroblasts exposed to 0.5-1.0ƒÊg/ml of cytochalasin B exhibited morphologically normal mitotic nuclear division in the absence of effective cytokinetic completion, and that the cells became binucleated during 24 hours of incubation with the drug.

These fibroblasts underwent a mitotic process in what began as a normal manner in the presence of cytochalasin B; nuclear division was morphologically normal and a deep cytoplasmic cleavage furrow developed. After the formation of this cleavage furrow, however, the presumptive daughter cells failed to separate from one another; instead, the progeny reunited, forming large binucleated cells. The repetition of this process of incomplete mitosis yielded, after several days, more severe multi nucleation-several cells containing as many as seven or eight nuclei. It is apparent that, even at a decreased rate of mitotic initiation, several consecutive nuclear divi sions had occurred without complete cell cleavage. It should be noted, furthermore, that Carter's data suggest a more complex role for cytochalasin B in the production of multinucleation phenomena than merely an inhibition of the cytoplasmic cleav age furrow. Indeed, a mechanism involving solely cytokinetic influence is inappli cable in light of the fact that the size of the nuclear compartments did not increase geometrically; it appears, rather, that only one additional nucleus appeared follow ing each cycle of aborted mitosis. As a response to the apparent discrepancy be tween this observation of nuclear number and clear morphological data which sug gested that all of the nuclei of the multinucleated cell enter a "mitotic" cycle simul taneously, Carter postulated a process of differential nuclear division as a means of explaining these phenomena. It was suggested that, of those nuclei within a multinucleate fibroblast, only one underwent a relatively normal mitotic process 1974 Cytochalasin B 717

resulting in the formation of the "daughter" nuclei corresponding to the presump tive, albeit unformed, progeny. Each other nucleus within the cell begins what Carter terms a "pseudomitosis" in a manner morphologically indistinguishable from that of the single nucleus undergoing a productive or 'true" mitosis; the mito tic process initially proceeds simultaneously and synchronously in all of the nuclei within a given cell. At approximately the end of prophase , after the chromatin has become condensed, the chromosomes have begun to form , and the nuclear envelope has disintegrated, the pseudomitotic nuclei reformulate themselves in their original numbers, and display no morphological changes as a result of their aborted mitosis . Such a mechanism was not empirically validated through Carter's work , but does serve as a conceptual framework within which to consider the fact that mitotic activity in multinucleated fibroblasts results in the addition of one nucleus to the cellular compartment during each cycle.

Fig. 5. Radioautographs of 3H thymidine-labelled, cytochalasin-induced multinucleate cells.

Two multinucleate cells with heavy and lightly labelled nuclei. From cultures exposed to cyto chalasin B (1ƒÊg/ml) for 72-120 hr. Magnification marker on figure is 10 µm long. Courtesy of

Dr. Awtar Krishan.

Although Carter's investigation provided a phenomenological basis for further research with cytochalasin B, it did little to elucidate the mechanism by which the compound induces the cytological changes observed. In an attempt to reveal

more completely the effects of cytochalasin B upon the course of nuclear duplica tion, experiments were conducted by Krishan and Ray-Chaudhuri (1969) in which cultured normal fibroblasts exposed to 1ƒÊg/ml of cytochalasin B were studied after

arresting the cell in various stages of mitosis through the action of vinblastine sul fate. Evaluation of the resulting mitotic figures with particular regard to the de gree of chromatin condensation yielded data which tend to corroborate Carter's earlier suggestion of dysynchrony within the nuclear compartment of the cell (Fig. 5). It was observed by Krishan and Ray-Chaudhuri, however, that during the first seventy-six hours of exposure to cytochalasin B, the number of nuclei in the multinucleate fibroblast cells increased geometrically (i.e., from one to two and 718 Michelle Copeland Cytologia 39 then to four nuclei per cell); only after more prolonged action were odd numbers of nuclei encountered. In addition, it was noted that cells with odd numbers of nuclei displayed a variety of mitotic phases; that is, no single mitotic phase was found to represent a large number of nuclei, as would be anticipated of Carter's postulated uniformly "pseudomitotic" nucleus. It was proposed therefore, that although upon initial exposure to cytochalasin B all of the nuclei in a given multi nucleated cell were characterized by mitotic synchrony and exhibited a geometric pattern of regeneration, continued exposure to the compound evoked karyokinetic dysynchrony in that nuclei were observed to enter mitosis at different times and thereby to undergo asynchronous and relatively independent mitotic cycles. Such a mechanism seems less demanding of cytochalasin B then is Carter's proposal,

Fig. 6. Time-lapse cine prints of an L-cell culture exposed to cytochalasin B (1 ƒÊg/ml) for 16 hr. 1-3, show mitosis with deep cleavage furrow dividing the two daughter cells which are seen entering a cell division after 18hr. A deep cleavage furrow and midbody are seen in frame 6. This cell subsequently reunited to form a large multinucleate cell. All figures are approximately •~250.

Courtesy of Dr. Awtar Krishan. and it requires merely that the drug effects, say, the formation of a cytoplasmic cleavage furrow in a manner which prevents the ultimate separation of post-mitotic daughter cells. The process, however, presumes that increased karyokinetic asyn chrony is simply the result of cumulative differences in the internal or external milieux of the individual nuclei. More recently, Krishan (1972) has compiled time-lapse cinematographic data which have enabled him to provide a somewhat more detailed elaboration of cytochalasin B-induced mitotic abnormalities . It can now be pre sumed that treated cells display a normal mitosis through telophase and reveal chromosomal aggregation and polar migration, division and retraction of the nu clear masses, cleavage furrow formation and the initial separation of daughter 1974 Cytochalasin B 719

cells. These progeny are connected by a thin cytoplasmic bridge prior to the ap

parent reversal of cytokineses which results in the aggregation of the would-be daughter cells into a single binucleate mass (Fig. 6). The first indication of mitotic abnormality is presented subsequent to the formation of a cytoplasmic bridge be tween the daughter cells, the nature of which has been described by Fawcett (1966)

in association with the normal mitotic process. He states that "after the two sets of chromosomes have moved to the poles the advancing constriction furrow that ultimately separates the two daughter cells closes down upon the interzonal portion

of the spindle and its progress is temporarily arrested. The daughter cells thus remain connected for a short time by a slender isthmus variously called a spindle

bridge ... intermediate body" or mid-body. It is feasible , therefore, that once the dividing cell has attained this "intermediate" stage, cytochalasin B acts either to

interfere with a signal to resume division, or to inhibit the synthesis of compounds necessary for completion of mitosis. Moreover, DuPraw (1968) has indicated that

cytokinesis includes a specialized mechanism necessary for completion of cell divi sion after contraction of the division furrow, and that this involves the synthesis

of new cell membrane; this new membrane growth during cytokinesis appears to occur at the poles (Dan et al. 1937) or at the equator (Buck and Krishan 1964) of the dividing cell. More recently, Bluemink (1971) has examined the alterations of cleavage furrow morphology and the process of cytokinesis in Xenopus egg cells treated with 7.5ƒÊg/ml of cytochalasin B and has reported that the formation of

normal intercellular junctions, necessary for new surface membrane ingrowth , is prevented; although a cleavage furrow forms initially, the drug acts to derange cell-cell junctions and membrane ingrowth and finally to induce regression of the

cleavage furrow.

Microfilament involvement

A period of active research with cytochalasin B followed the observation of cytokinetic inhibition which entailed considerable investigation of the influence of

the drug upon cell motility in general. Schroeder (1969), for instance, has studied division in sea urchin cells and in cells of human cervical carcinoma (HeLa cells) in the presence of 1ƒÊg/ml of cytochalasin B; in both of these instances, cytokinesis was spontaneously aborted prior to completion. The morphology of this response may be summarized, briefly: mitosis appeared to progress normally through prophase and metaphase; movement revealed no discernible variation from the pattern displayed by untreated cells; at the onset of telophase, however, exposed cells evidence neither the gross morphological presence of a cytoplasmic cleavage furrow, nor the concomitant ultrastructural counterparts of the furrow, namely the filaments of the contractile ring apparently requisite for effective cytokinesis. These results in sea urchin and HeLa cells conflict somewhat with those obtained by other researchers, such as Krishan (1971) and Bluemink (1971) which suggest that cleavage furrow formation is not completely inhibited, but rather that such furrowing is not followed by movement of daughter cells away from each other. Upon consider ation of his observations with regard to a structural variation, Schroeder proposed that the cytokinetic effects of cytochalasin B are principally the result of alteration in the microfilament component of the telophase contractile ring. 720 Michelle Copeland Cytologia 39

The influence of cytochalasin B upon the fine structure of microfilaments and the implication of this influence for the formulation of a cytokinetic mechanism has been the subject of conflicting subsequent research, some of which is reviewed by Wessells et al. (1971). These authors cite studies of cytochalasin B in a variety of cell types, including chick oviductal tubules, salivary gland epithelium, embryo nic neurons, fibroblasts, smooth and cardiac muscle cells, blood platelets, tadpole cells and several algae, in which the drug has been implicated as a factor effecting structural alteration of the contractile filaments involved in various motile and developmental processes. The researchers have concluded that cytochalasin B appears not only to influence microfilament structure directly, but also to demon strate a "clear correlation between the integrity of microfilament systems and vari ous biological phenomena."

In experiments with epidermal and dermal melanocytes of Rana pipiens exposed to 10ƒÊg/ml of cytochalasin B, McGuire and Moellman (1972) (and, in similar ex periments by Malawista 1971) observed that the drug prevented the dispersion of pigment granules by melanocyte stimulating hormone (MSH); those pigment gra nules treated with MSH prior to exposure to cytochalasin B aggregated under the influence of the drug. Similarly, Wagner, Haupt and Laux (1972) showed that at concentrations of 5 to 25ƒÊg/ml cytochalasin B reversibly inhibited chloroplast migration in the green alga Mougirotia.

Thoa et al. (1972) compared the effects of colchicine, vinblastine and cytochala sin B on the release of stored hormones and found that all act to depress release of dopamine-ƒÀ-hydroxylase and norepinephrine in guinea pig vas deferens. There fore, since it is known that colchicine and vinblastine alter the neurotubular pro teins necessary for hormone release, these authors speculated that cytochalasin B also depresses hormone release by a similar mechanism. Disruption of micro filament-like structures has led Orci et al. (1972) to speculate that cytochalasin B affects glucose-induced secretion of insulin in isolated islets of Langerhans. These researchers incubated pancreatic islet cells in 10ƒÊg/ml of cytochalasin B and ob served that treated cells lacked filamentous components located beneath the plasma membrane (i.e., the 'cell web" which, when intact, may serve as a barrier to emeio cytosis). Since incubated cells also showed increased insulin release it was suggested that cytochalasin B violated the integrity of the cell web and potentiated the secre tion of insulin upon stimulation with glucose. In studies exploiting the birefringence of intact myofibrils under polarized light, Manesak et al. (1972) have recently demonstrated that cytochalasin B dis turbs preformed embryonal myofibrils and inhibits further myofibril proliferation. Indeed, exposure of embryos to 0.1mM of cytochalasin B for 45 minutes prevented the development of intact myofibrils, a dose-dependent effect that was inversely related to embryonic age. In spite of the extensive body of data which form the basis for the conclusions drawn by these researchers, Goldman (1972) has presented recent evidence which suggests that cytochalasin B does not necessarily alter the structure of microfila ments. In experiments upon baby hamster kidney cells, he demonstrated differing responses of individual cells to the drug. In particular, Goldman observed that 1974 Cytochalasin B 721

in a significant number of the cells in which motility had been depressed by the action

of cytochalasin B morphologically normal bundles of microfilaments persisted after exposure for twenty-four hours to 5ƒÊg/ml of the drug, while other cells paradoxi

cally displayed abnormal microfilament configuration. Although these data are in no manner conclusive with respect to the relationship between microfilament

structure and cytochalasin B, they are illustrative of the difficulties encountered in any attempt to ascribe a specific functional significance to the morphological data obtained in this area.

The skepticism imposed by Goldman's work is particularly helpful in assessing

the mass of conflicting data which has accumulated with respect to structure-func tion relationships in microfilaments treated with cytochalasin B. Accordingly, Hammer, Sheridan and Estensen (1971) applied cytochalasin B in concentrations

of from 1 to 10ƒÊg/ml to eggs of Xenopus laevis and have observed that although

multinucleation phenomena do occur, cleavage furrowing appears completely normal; their data provide no evidence for the alteration of microfilaments during the treatment process. These findings have been substantiated by Bluemink

(1971) who also observed intact 100 A filaments present along the walls of the cleav age furrow during its regression in Xenopus eggs exposed to 7.5ƒÊg/ml of cytochala sin B. At present, therefore, mechanistic proposals involving an influence of cyto

chalasin B upon contractile microfilaments must be considered entirely speculative. Attempts to resolve the question through in vitro investigations have resulted

mainly in the persistence of confusion and controversy. Spudich and Lin (1971)

have exposed filaments of actin and myosin extracted from striated muscle to con centrations of cytochalasin B up to 0.4 millimoles, upon the assumption that cyto

plasmic contractile ring filaments include actin-like filaments capable of binding heavy meromyosin. These workers noted that cytochalasin B diminishes the vis cosity of actin to the extent that it becomes incapable of interacting with myosin.

It was suggested that the system undergoes competitive inhibition of cytochalasin B for binding of myosin to actin. From these data the possibility was extrapolated

that cytochalasin B might also induce functional changes in the structurally similar contractile ring filaments. Forer, Emmersen and Behnke (1972) on the other hand, conducted experiments in which filaments of F-actin obtained from skeletal muscle

were exposed to 5ƒÊg/ml of cytochalasin B and found that such treatment resulted in neither the structural alteration of the actin filaments nor the inhibition of bind ing of heavy meromyosin to those filaments. It is clear from the inconsistent

data collected to date that this aspect of cytochalasin investigation is in need of further systematic examination in order that the functional influence of the drug

upon the cytoskeleton can be approached from a meaningful standpoint.

Biochemical aspects

In an attempt to assess further the nature of the variables involved in cyto chalasin B-induced cytokinetic failure, Estensen et al.(1971) has investigated the rates of nuclear division, cytoplasmic growth and uptake of various precursors of DNA, RNA, proteins and membrane (i.e., choline) in eggs of Xenopus laevis culture 722 Michelle Copeland Cytologia 39

ed with 1ƒÊg/ml of cytochalasin B. It was reported that during the first twelve hours of incubation in this medium, nuclear division and cytoplasmic growth are

relatively undisturbed, and that incorporation of precursors of DNA, RNA, pro teins and membranes was only slightly altered. It has been concluded, therefore, that cytochalasin B does not inhibit major metabolic pathways in these cells to a

significant degree, a viewpoint consistent with the datum that RNA and protein

synthesis, at least, are unnecessary for completion of mitosis after prophase. Some what more speculative are further suggestions by these workers that only the later cytokinetic stages-that is, those steps independent of RNA and protein syn thesis-appear to be interrupted by cytochalasin B, the action of which likely in cludes derangement of the process of membrane fusion. Such fusion would com prise the joining of two parts of a cell membrane followed by the formation of dis crete daughter cells, most probably along the edges of a contractile ring or cleavage furrow. They suggest, moreover, that "a primary effect on macromolecular syn thesis seems unlikely ... [since] ... cleavage will still occur even when RNA and

DNA synthesis is blocked and doses of cytochalasin B which inhibit cytokinesis completely in mammalian cells have little effect on protein or membrane synthesis."

It has recently been demonstrated that the synthesis of several membrane compo nents are inhibited by 0.5ƒÊg/ml and 1ƒÊg/ml, respectively, of cytochalasin B in amnion and HeLa cells (Sanger and Holtzer 1972). These later evaluations in volved determination of the extent of inhibition of mucopolysaccharide synthesis through measurement of 14C-labelled glucosamine incorporation. In a mucopoly saccharide fraction of cells incubated whith 14C-glucosamine and 0.5ƒÊg/ml of cyto chalasin B, glucosamine uptake was decreased by 80 percent in amnion cells, by 55 percent in HeLa cells, and by 50 percent in skeletal myogenic cells, when compared with similar cells not exposed to cytochalasin B. The inhibitory effect was reversed when cells were removed from medium containing cytochalasin B, washed and cultured in normal medium for as little as two hours. The association by Esten sen's group of the progress of membrane biosynthesis and the consumption of a single presursor substance, choline, is challenged by the data offerred by Sanger and Holtzer. This deviation may be partially resolved through the consideration that although certain structures associated with the viable plasma membrane may consist of glycerophosphatides (such as phosphatidyl serine, phosphatidyl ethanol amine and phosphatidyl choline), sphingolipids and cholesterol, as presupposed by Estensen et al., the effectiveness of surface activity may be largely dependent upon the integrity of a mucopolysaccharide coating, as might be disrupted or in hibited by cytochalasin B. This supposition is quite suitably amalgamated with the finding related by DuPraw (1968) that the bulk of synthesis of substances as sociated with new membrane occurs at a locus considerably removed from the cyto plasmic cleavage furrow. It seems more reasonable, therefore, to consider the action of cytochalasin B as an inhibitor of the biosynthesis of a mucopolysaccha ride membrane constituent in the elaboration of the factors responsible for its cyto kinetic interference than to presume an influence upon a membrane fusion process for which empirical foundation is lacking. 1974 Chtochalasin B 723

Conclusion

The variety of data which have been discussed herein indicate an essential re

lationship between the action of the fungal metabolite, cytochalasin B, and common

cellular processes involving the surface membrane. The functional nature of this relationship as it pertains to cell motility, cytokinesis, surface adhesion and phago

cytotic activities will require considerable further investigation for adequate ela boration. The elucidation of the locus of influence of the drug is an issue of para mount concern in that it is presently unknown whether cytochalasin B enters the

cell or is surface-bound to an enzyme or other molecular complex located at the

plasma membrane. The latter possibility may be suggested in the observation that the effects of cytochalasin B are generally reversed shortly after withdrawal

of the compound, an occurrence intuitively consistent with a surface-limited con figuration of drug action. A fertile and promising field of investigation conceivably

would involve radioactive labelling of the cytochalasin B molecule and the auto radiographic surveillance of the drug during incubation with susceptible cells. Such

studies of mobility of labelled cytochalasin B might also demonstrate the associa tion of specific transport mechanisms with which the substance might interact. While the work of Sanger and Holtzer in demonstrating the inhibitory role of cyto chalasin B upon the synthesis of compounds intimately related to the formation of

a viable cell surface stands as a milestone in research with the compound, the physi cal means by which this interference is effected remains unestablished. Their data,

while indicative of the diminution of incorporation of the labelled mucopolysc charide precursor, 14C-glucosamine, by cytochalasin B, leave inestimable the avenue of this attenuation. That is, it is indeterminate whether cytochalasin B blocks the uptake if glucosamine by inhibition of transport into the cell, or interferes more directly with its utilization in the biosynthesis of mucopolysaccharides. A parallel

confusion is encountered in the experiments of Skowsky et al. (1972), in which the assimilation of 14C-labelled glucose was evaluated in human blood leukocytes treated

with 10ƒÊg/ml of cytochalasin B by measurement of CO2 production. Once again, although CO2 effusion was diminished, the use of this measurement as an index of

glucose oxidation provides no direct differentiation between the drug's action as an inhibitor of transport or of mucopolysaccharide synthesis. Either viewpoint could be substantiated through comparison of the experiments with glucose and gluco samine, because the structural similarity of these compounds would be consistent

with a reasonably specific action of cytochalasin B. Indeed, the recent work of Zigmond and Hirsch (1972) clearly demonstrates that the incorporation into leuko

cyte and fibroblast cells of deoxyglucose, a nonmetabolic analog of glucose, is de

pressed by approximately 80 percent when these cells are exposed to 2 and 10ƒÊg/ml of cytochalasin B. It was further determined that the effect of cytochalasin B on

glycolysis was due to the blockage of glucose transport since leukocytes incubated in medium containing no glucose produced similar amounts of lactate, irrespective of exposure to cytochalasin B, and since leukocyte homogenates were able to meta bolize glucose as well in the presence of cytochalasin B as in its absence. Similar findings were reported by Estensen and Plageman (1972), who demonstrated that cytochalasin B competitively inhibits deoxyglucose transport in Novikoff rat hepa 724 Michelle Copeland Cytologia 39

toma cells. Warner and Perdue (1972) ascertained that inhibition of glucose trans

port by cytochalasin B does not appear to result in a significant decrease in the energy levels of the treated cells in that fibroblasts treated with cytochalasin B (10ƒÊg/ml)

displayed no significant decrease in ATP content during inhibition of glucose trans port. The apparent role of cytochalasin B as an agent which disturbs the cell surface makes this antibiotic potentially useful as an investigative instrument in the de scription of membrane-oriented aspects of cell physiology. The proposal that cytochalasin B alters the synthesis of mucopolysaccharide components of the plasma membrane through inhibition of precursor incorporation has functional implica tions for the cell-cell interactions of adhesion and for membrane ruffling, mitotic cytokinesis, phagocytosis and induction of nuclear extrusion. Moreover, Du Praw (1968) has pointed out that the solute-binding reaction intimately related to incorporation processes such as phagocytosis is closely associated with a mucous layer which coats the extracellular surface of the cell membrane in a variety of cell types. Insofar as this material carries a negative electrical charge, "the binding of positively-charged inducer molecules very likely takes place by electrostatic attraction and formation of salt linkages with the mucus coat." This phenomenon may be taken as illustrative of the electrostatic level at which the surface-active mucopolysaccharide layer might act in numerous cellular processes. Alteration of the membrane coat by cytochalasin B represents a prototypical role in the elabora tion of the action of this unusual compound. More significantly, perhaps, it sig nals the enormous opportunity which is presented in cytochalasin B for the even tual clarification of the dynamics of membrane physiology so essential to improved understanding of life processes at the cellular level.

Acknowledgements

The encouragement and guidance of Dr. Don W. Fawcett, Chairman of the Department of Anatomy at Harvard Medical School, and Dr. Awtar Krishan of the Children's Cancer Research Foundation and of the Department of Pathology, Harvard Medical School, is gratefully acknowledged.

References

Aldridge, D. C., Armstrong, J. J., Speake, R. N. and Turner, W. B. 1967. The cytochalasins, a new class of biologically active mould metabolites. Chem. Comm. 1: 26-27. - , -, and 1967. The structure of cytochalasin A and B. J. Chem. Soc. 17: 1667-1676. - and Turner, W. B. 1969. The identity of zygosporin A and cytochalasin D. J. Antibiotics 22: -170 - and - 1969. Structures of cytochalasins C and D. J. Chem. Soc. 22: 923-928. Armstrong, P. B. and Parenti, D. 1972. Cell sorting in the presence of cytochalasin B. J. Cell Biol. 55: 542-553. Arnold, J. M. and Williams, L. D. 1970. Effects of cytochalasin B on cytoplasmic movement, cleavage and subsequent development of squid embryo, Loligo-Pealer. Biol. Bull. 139: 413. Behnke, O., Forer, A. and Emmersen, J. 1971. Actin in sperm tails and meiotic spindles. Na 1974 Cytochalasin B 725

ture 234: 408-409. -, -, and - 1972. Cytochalasin B: does it affect actin-like filaments? Science 175: 774-775. Bluemink, J. G. 1971. Effects of cytochalasin Bon surface contractility and cell junction formation during cleavage in Xenopus laevis. Cytobiologie 3: 176. - 1971. Cytokinesis and cytochalasin-induced furrow regression in the first-cleavage zygote of Xenopus-laevis. Z. Zellforsch. 121: 102-126. Buck, R. C. and Krishan, A. 1964. Site of membrane growth during cleavage of amphibian epi thelial cells. Exptl. Cell Res. 38: 426. Burnside, B. 1972. Cytochalasin B: problems in interpretating its effect on cells. Devel. Biol. 27: 443-444. Carter, S. B. 1967. Effects of cytochalasins on mammalian cells. Nature 213: 261-264.- 1972. The cytochalasins as research tools in cytology. Endeavour 31: 77. - 1967. Haptotactic islands-A method of confining single cells to study individual cell reaction and clone formation. Exp. Cell Res. 48: 189-193. - 1967. Haptotaxis and mechanism of cell motility. Nature 213: 256-260. Culliton, B. 1972. Cell membranes: Anew look at how they work. Science 175: 1348-1350. Dan, K., Yanagita, T., and Sugiyama, M. 1937. Behavior of the cell surface during cleavage. Protoplasma 28: 66. Davis, A. T., Estensen, R. and Quie, P. G. 1971. Cytochalasin B-III, inhibition of human poly morphonuclear leukocyte phagocytosis. P. Soc. Exp. Biol. Med. 137: 161. DuPraw, E. 1968. Cell and Molecular Biology. New York, Academic Press. Estensen, R. D. 1971. Cytochalasin B-I. Effect of cytokinesis of Novikoff Hepatoma cells Proc. Soc. Exp. Biol. Med. 136: 1256-1260. - and Plageman, P. G. W 1972 Cytochalasin B: Inhibition of glucose and glucosamine trans port. Proc. Nat. Acad. Sci. (U. S.) 69: 1430. -, Rosenberg, M. and Sheridan, J. D. 1971. Cytochalasin B: Microfilaments and "contractile" processes. Science 173: 356-357. Fawcett, D. 1966. The Cell: An Atlas of Fine Structure. Philadelphia, Saunders, Co., 244: 390-401. Fine, R. E. and Bray, D. 1971. Actin in growing nerve cells. Nature 234: 115-118. Gail, M. H. and Boone, C. W. 1971. Cytochalasin effects on BALB/3T3 fibroblasts: Dose dependent, reversible alteration of motility and cytoplasmic cleavage. Exp. Cell Res. 68: 226-228. Gawadi, N. 1971. Actin in the mitotic spindle. Nature 234: 410. Goldman, R. D. 1972. The effects of cytochalasin B on the microfilaments of baby hamster kidney (BHK-21) cells. J. Cell Biol. 52: 246-254. Hammer, M., Sheridan, J. D. and Estensen, R. D. 1971. Cytochalasin B-II. Selective inhibition of cytokinesis in Xenopus laevis eggs. Proc. Soc. Exp. Biol. Med. 136: 1158-1162. Harper, M. J. K. and McGaughe, R. W. 1970. Effects of cytochalasin B on development of rabbit eggs. Anat. Rec. 166: 315. Hoehn, H., Sprague, C., and Martin, G. 1972. Induction of polyploidy in human diploid cell lines by cytochalasin B. Fed. Proc. 31: 2213 (Abs.). Holtzer, H. and Sanger, J. W. 1972. Cytochalasin B: Microfilaments, cell movement and what else? Devel. Biol. 27: 444-445. Katagiri, K. and Matsuura, S. 1971. Antitumor activity of cytochalasin D. J. Antibiotics 24: 722-723. Krishan, A. 1972. Cytochalasin B: Time-lapse cinematographic studies on its effects on cy tokinesis. J. Cell Biol. 54: 657. - 1971. Fine structure of cytochalasin-induced multinucleated cells. J. Ultrastruct. Res. 36: 191-204. - 1970. Ribosome-granular material complexes in human leukemic lymphoblasts exposed to vinblastine sulfate. J. Ultrastruct. Res. 31: 272-281. - 1968. Time-lapse and ultrastructure studies on the reversal of mitotic arrest induced by vin 726 Michelle Copeland Cytologia 39

blastine sulfate in Earle's L Cells. J. Nat'l Cancer Inst. 41: 581-595. - and Hsu, D. 1971. Binding of colchicine-3H to vinblastine and vincristine induced crystals in mammalian tissue culture cells. J. Cell Biol. 48: 407-410.- and - 1971. Vinblastine-induced ribosomal complexes-effect of some metabolic inhibitors on their formation and structure. J. Cell Biol. 49: 927. - and Raychaud, R. 1969. Asynchrony of nuclear development in cytochalasin-induced multi nucleate cells. J. Cell Biol. 43: 618-621. Ladda, R. L. and Estensen, R. D. 1970. Introduction of a heterologous nucleus into enucleated cytoplasms of cultured mouse L-cells. Proc. Nat'l Acad. Sci. (U. S.) 67: 1528. Malawista, S. E. 1971. Cytochalasin B reversibly inhibits melanin granule movement in melano cytes. Nature 234: 254-255. - 1971. Cytochalasin B reversibly inhibits phagocytosis by human blood leukocytes. Prog. Immun. 187-192. Manasek, F. J., Burnside, B., and Struman, J. 1972. The sensitivity of developing cardiac myo fibrils to cytochalasin B. Pric. Nat'l Acad. Sci. (U. S.) 69: 308-312. McGuire, J. and Moellman, G. 1971. Cytochalasin B: Effects on microfilaments and move ment of melanin granules within melanocytes. Science 175: 642-644. Orci, L., Gabba, K. H., and Malaise, W. J. 1971. Pancreatic beta-cell web: Its possible role in insulin secretion. Science 175: 1128. Plagemann, P. G. and Estensen, R. D. 1972. Cytochalasin B. VI. Competitive inhibition of nucleoside trasport by cultured Novikoff rat hepatoma cells. J. Cell Biol. 55: 179 Prescott, D. M., Myerson, D. and Wallace, J. 1972. Enucleation of mammalian cells with cyto chalasin B. Cell Res. 71: 480-485. Ridler, M. and Smith, G. E. 1968. Response of human cultured lymphocytes to cytochalasin B. J. Cell Sci. 3: 595. Rothweiler, W. and Tamm, C. 1966. Isolation and structure of Phomin. Experientia 22: 750-752. Sanger, J. W. and Holtzer, H. 1972. Cytochalasin B: Effects on cell morphology, cell adhesion, and mucopolysaccharide synthesis. Proc. Nat. Acad. Sci. (U. S.) 69: 442-446. - and -. 1970. Nuclear completation in 5 bromodeoxyuridine and cytochalasin B treated myogenic cells. J. Cell Biol. 47: 1784. Schofield, J. G. 1971. Cytochalasin B and release of growth hormone. Nature 234: 215-216. Schroeder, T. E. 1970. Contractile ring-fine structure of dividing mammalian (HeLa) cells and effects of cytochalasin B. Z. Zellforsch 109: 431-449. - 1969. The role of "contractile ring" filaments in dividing Arbacia eggs. Biol. Bull. 137: 413 - 414. Shepro, D., Belmari, F. A., Robbler, L. and Chao, R. C. 1970. Antimotility effect of cytochalasin B observed in mammalian clot retraction. J. Cell Biol. 47: 544. Skosey, J. L., Damguard, E., and Chow, D. 1972. Effect of cytochalasin B upon glucose oxida tion by human peripheral blood leukocytes. Fed. Proc. 31: 3315 (Abs.). Smith, G. F., O'Hara, P. and Ridler, M. 1970. Production of multinucleated lymphocytes by cytochalasin B-an electron microscopic study. Ped. Res. 4: 441A. - , Ridler, M. and Faunch, J. A. 1967. Action of cytochalasin B on cultured human lymphocytes. Nature 216: 1134-1135. Spooner, B. S. 1970. Effects of cytochalasin B upon microfilaments involved in morphogenesis of salivalary epithelium. Proc. Nat. Acad. Sci. (U. S.) 66: 360. -, Yamada, K. M., and Wessells, N. K. 1971. Microfilaments and cell locomotion. J. Cell Biol. 49: 595. Spudich, J. A. and Shin, L. 1972. Cytochalasin B, its interaction with actin and actomyosin from muscle. Proc. Nat. Acad. Sci. (U. S.) 69: 442. Thoa, N. B., Wooten, G. F., Axelrod, J., and Kopin, I. J. 1972. Inhibition of dopamine p-hydra xylase and norepinephrine release from sympathetic nerves by colchicine, vinblastine, and cytochalasin B and its enhancement by dibutryl cyclic-AMP. Fed. Proc. 31: 1984 (Abs.). 1974 Cytochalasin B 727

Wagner, G., Haupt, W., and Laux, A. 1972. Reversible inhibition of chloroplast movement by cytochalasin B in the green alga mougeotia. Science 176: 808. Wagner, R., Rosenberg, M., and Estensen, R. D. 1971. Endocytosis in Chang liver cells, quanti tation by sucrose-3H and inhibition by cytochalasin. B. J. Cell Biol. 50: 804. Warner, D. A. and Perdue, J. F. 1972. Cytochalasin B and the adenosine triphosphate content of treated fibroblasts. J. Cell Biol. 55: 242. Wessells, N. K. 1971. Structure and function of cytoplasmic microfilaments. Fed. Proc. 30: 1039 (Abs.). -, Spooner, B. S., Ash, J. F., Bradly, M. 0., Luduena, M. A. , Taylor, B. L., Wrenn, J. T., and Yamada, K. M. 1971. Microfilaments in cellular and developmental processes. Sci ence 171: 135. -, -, -, -, and Wrenn, J. T. 1971. Cytochalasin B-microfilaments and contractile processes . Science 173: 358. Wrenn, J. T. and Wessells, N. K. 1970. Cytochalasin B. Effects upon microfilaments involved in morphogenesis of estrogen-induced glands of oviduct. Proc. Nat. Aced. Sci. (U. S.) 66: 904. Yamada, K. M., Spooner, B. S., and Wessells, N. K. 1970. Axon growth: Roles of microfila ments and microtubules. Proc. Nat. Acad. Sci. (U. S.) 66: 1206. - , -, and - 1971. Ultrastructure and function of growth cones of cultured nerve cells. J. Cell Biol. 49: 614. - , and Wessells, N. K. 1971. Axon elongation effect of nerve growth factor on microtubule pro tein. Exp. Cell Res. 66: 346. Yoshinaga, M., Waksman, B., and Malawista, S. 1972. Cytochalasin B inhibits lymphotoxin pro duction by antigen-stimulated lymphocytes. Science 176: 1147. Zigmond, S. H. and Hirsch, J. G. 1972. Cytochalasin B: Inhibition of D-2 deoxyglucose trans port into leukocytes and fibroblasts. Science 176: 1432. -, and -. 1972. Effects of cytochalasin B on polymorphonuclear leukocyte locomotion phago cytosis and glycolysis. Exp. Cell Res. 73: 383.

Address inquiries to: Michelle Copeland Harvard School of Dental Medicine 188 Longwood Avenue Boston, Massachusetts 02115, U. S. A.