In our opinion, all of the phenomena that are inhibited by cytochalasin can be thought of as resulting from contractile activity of cellular organelles. Smooth muscle contraction, clot retraction, beat of heart cells, and shortening of the tadpole tail are all cases in which no argument of substance for alternative causes can be offered. The morphogenetic processes in epithelia, contractile ring function during cytokinesis, migration of cells on a substratum, and streaming in plant cells can be explained most simply on the basis of contractility being the causal event in each process. The many similarities between the latter cases and the former ones in which contraction is certain argue for that conclusion. For instance, platelets probably contract, possess a microfilament network, and behave like undulating membrane organelles. Migrating cells possess undulating membranes and contain a similar network. It is very likely, therefore, that their network is also contractile. In all of the cases that have been examined so far, microfilaments of some type are observed in the cells; furthermore, those filaments are at points where contractility could cause the respective phenomenon. The correlations from the cytochalasin experiments greatly strengthen the case; microfilaments are present in control and "recovered" cells and respective biological phenomena take place in such cells; microfilaments are absent or altered in treated cells and the phenomena do not occur. The evidence seems overwhelming that microfilaments are the contractile machinery of nonmuscle cells. The argument is further strengthened if we reconsider the list of processes insensitive to cytochalasin (Table 2). Microtubules and their sidearms, plasma membrane, or synthetic machinery of cells are presumed to be responsible for such processes, and colchicine, membrane-active drugs, or inhibitors of protein synthesis are effective at inhibiting the respective phenomena. These chemical agents would not necessarily be expected to affect contractile apparatuses over short periods of time, they either do not or only secondarily interfere with the processes sensitive to cytochalasin (Table 1). It is particularly noteworthy in this context that microtubules are classed as being insensitive to cytochalasin and so are not considered as members of the "contractile microfilament" family. The overall conclusion is that a broad spectrum of cellular and developmental processes are caused by contractile apparatuses that have at least the common feature of being sensitive to cytochalasin. Schroeder's important insight (3) has, then, led to the use of cytochalasin as a diagnostic tool for such contracile activity: the prediction is that sensitivity to the drug implies presence of some type of contractile microfilament system. Only further work will define the limits of confidence to be placed upon such diagnoses. The basis of contraction in microfilament systems is still hypothetical. Contraction of glycerol-extracted cells in response to adenosine triphosphate (53), ex...
Administration of estrogen (E) to immature chicks triggers the cytodifferentiation of tubular gland cells in the magnum portion of the oviduct epithelium ; these cells synthesize the major egg-white protein, ovalbumin . Electron microscopy and immunoprecipitation of ovalbumin from oviduct explants labeled with radioactive amino acids in tissue culture were used to follow and measure the degree of tubular gland cell cytodifferentiation . Ovalbumin is undetectable in the unstimulated chick oviduct and in oviducts of chicks treated with progesterone (P) for up to 5 days . Ovalbumin synthesis is first detected 24 hr after E administration, and by 5 days it accounts for 35% of the soluble protein being synthesized . Tubular gland cells begin to synthesize ovalbumin before gland formation which commences after 36 hr of E treatment . When E + P are administered together there is initially a synergistic effect on ovalbumin synthesis, however, after 2 days ovalbumin synthesis slows and by 5 days there is only 1 /20th as much ovalbumin per magnum as in the E-treated controls. Whereas the magnum wet weight doubles about every 21 hr with E alone, growth stops after 3 days of E + P treatment . Histological and ultrastructural observations show that the partially differentiated tubular gland cells resulting from E + P treatment never invade the stroma and form definitive glands, as they would with E alone . Instead, these cells appear to transform into other cell types-some with cilia and some with unusual flocculent granules . We present a model of tubular gland cell cytodifferentiation and suggest that a distinct protodifferentiated stage exists . P appears to interfere with the normal transition from the protodifferentiated state to the mature tubular gland cell .
Abstract. The administration of estrogen to immature chicks induces formation. of tubular glands and differentiation of cells in the oviduct. As glands begin to form, organized bundles of 40-50 A filaments appear at the luminal end of the cells. These structures are not present in uninduced oviducts. Cytochalasin treatment of oviducts early in gland formation results in the disappearanice of young glands already present and the inhibition of new gland formation. Furthermore, organized microfilaments are no longer present. When the oviducts are washed free of cytochalasin, however, organized bundles of microfilaments reappear.The correlations between the presence of filaments and formation of glands suggest that filaments are important agents in morphogenesis, presumably because of contractile properties which generate changes in cell shape and, consequently, tissue shape.The roles of intracellular structures in complex biological processes such as organ morphogenesis are difficult to define. The use of the drug colchicine has provided a tool for investigating the functions of cytoplasmic microtubules in developing cells.1 In the presence of colchicine, microtubules disappear and developmental processes are interrupted. Until recently, no analogous tool has been available to probe the function of microfilaments, structures that are also involved in organ morphogenesis.2The contractile ring of cleaving marine eggs is composed of microfilaments, 40-50 A in diameter.3 When Schroeder4 treated cleaving eggs with cytochalasin,5 cytokinesis stopped and the filaments of the contractile ring were no longer seen. Thus, there is a positive correlation between cytokinesis and the presence of filaments.Filaments of similar size are present in chick oviduct cells that have been induced by estrogen to form tubular glands and to differentiate. Such tissues have been treated with cytochalasin to see if their filaments have the same sensitivity to the drug as the contractile ring filaments and also to establish whether a positive correlation exists between gland formation and presence of filaments.Methods. Female white leghorn chicks, 7-10 days old, were injected at 0 and 24 hr with 1 mg estradiol 17 3-benzoate.6 At 36 hr, the chicks were sacrificed and their oviducts removed. Oviducts were also obtained front uninjected chicks of the same age.The uninduced oviducts and some portions of estrogen-induced oviducts were fixed immediately and prepared for electron microscopy.7 904
Changes in the distribution and organizational state of actin in the cortex of echinoderm eggs are believed to be important events following fertilization. To examine the initial distribution and form of actin in unfertilized eggs, we have adapted immunogold-labeling procedures for use with eggs of Strongylocentrotus purpuratus. Using these procedures, as well as fluorescence microscopy, we have revealed a discrete 1-micron-thick concentrated shell of actin in the unfertilized egg cortex. This actin is located in the short surface projections of unfertilized eggs and around the cortical granules in a manner that suggests it is associated with the cortical granule surface. The actin in the short surface projections appears to be organized into filaments. However, most if not all of the actin surrounding the cortical granules is organized in a form that does not bind phalloidin, even though it is accessible to actin antibody. The lack of phalloidin binding is consistent with either the presence of nonfilamentous actin associated with the cortical granules or the masking of actin-filament phalloidin-binding sites by some cellular actin-binding component. In addition to the concentrated shell of actin found in the cortex, actin was also found to be concentrated in the nuclei of unfertilized eggs.
Early lenses of mouse embryos were studied with the electron microscope to determine whether their cells contain organelles that can be causally related to the invagination of the lens placode. The cells of the placode and early lens cup are joined at their apical ends by junctional complexes. At the level of the zonula adhaerens, favorabIe sections show cytoplasmic filaments, 35 A to 50 A wide, arranged about the apex of a cell. Groups of filaments appear to originate on or near the plasma membrane and then to extend across the cytoplasm, parallel to the apical cell surface, to terminate on or near the plasma membrane some distance away. They are nearly always straight; in contrast, nearby lateral and apical cell membranes are twisted into irregular folds and projections.We present a model to explain lens invagination in which the filaments are considered to be contractile. On the basis of their location in the cells and their presumed function, these organelles are proposed to be a major component in the mechanism that leads to early morphogenesis of the lens.Morphogenetic movements of large groups of cells are known to occur frequently in animal development (for review, see Trinkaus, '65). Often a curved structure is formed from a flat sheet as the cells within that sheet appear to move to new locations. Several examples are the lung, pancreas, thyroid gland, salivary glands, olfactory organ, and otic vesicle. The mammalian lens is also formed by such a movement as the sheet of cells comprising the flat lens placode invaginates to form the lens vesicle.The cause of lens invagination is unknown. One possibility is that structures within the individual cells generate forces which are responsible for the movement of the cells as a group. We have begun to investigate this possibility by ascertaining what cell organelles are present and how they are distributed within the cells of the invaginating lens. If the cellular ultrastructure is suggestive of a method to control cell shape and movement, then this morphological investigation can provide a basis for further studies of this type of cell behavior. MATERIALS AND METHODSDeveloping eyes were obtained from mouse embryos resulting from crosses of BALB/c female with C3H male mice. The stage of each embryo was determined by somite count. Those used in the current studies ranged from 31 to 36 somites (all on the tenth day after vaginal plug discovery).Pregnant females were killed by cervical dislocation. Immediately afterward, the uteri were removed and placed in 90% Waymouth's MB 752/1 with 10% horse serum at 21°C. Within 10 minutes from the time of death of the females, the embryos were placed in the primary fixative, and the eyes were dissected out. Part of the optic nerve and side of the head were taken with the eye for purposes of orientation.For most tissues, primary fixation for 60 to 90 minutes took place in 2.5% glutaraldehyde with paraformaldehyde in 0.14 M Sorenson's buffer at pH 7.4, following a modified method of Karnovsky ('65). In order to preser...
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