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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...

Abstract. The motile tips of elongating axons consist of growth cones from which microspikes protrude. Cytochalasin B causes retraction of microspikes, rounding-up of growth cones, and cessation of axon elongation. Drug withdrawal is followed by resumption of growth cone and microspike activity and of axon elongation. In contrast, colchicine causes shortening and retraction of axons, but it does not initially affect the tips. Growth cones and microspikes of elongating axons contain a network of 50 A microfilaments, the pattern of which is altered by cytochalasin treatment. These experiments indicate that both structural integrity of the axon and continuing function of its motile tip are essential elements in axonal elongation.The tips of elongating axons consist of expanded regions called "growth cones" from which project long slender microspikes (filopodia). These microspikes continually wave about, extend, and retract as the growth cone moves over a substratum.1 Although it is assumed that growth cones and microspikes play a significant role in axon extension, it has not been previously possible to analyze their functions.We have investigated the effects of cytochalasin B on axon elongation since this drug halts cell movement,2 a process similar in many respects to growth cone movement. Cytochalasin B is also known to inhibit cytokinesis3 and morphogenetic movements,4 apparently by disrupting contractile 50 A microfilaments. The drug's effects upon nerve cells are compared with those of colchicine and colcemid, which disrupt microtubules' and cause retraction of elongating axons.6Methods. Nerve ganglion culture: Lumbosacral dorsal root ganglia from 8-day-old white leghorn chick embryos were cultured in Grobstein tissue culture dishes7 (Microchemical Specialties). The semisolid culture medium consisted of 0.05 ml of 1% agar in Hanks' balanced salt solution plus 0.1 ml of medium 199 containing 10% embryo extract,8 20% fetal calf serum, and antibiotics (100 units/ml penicillin, 100 Ag/ml streptomycin, and 2.5 ug/ml amphotericin B). Nerve growth factor was present as a 1: 300,000 homogenate of submandibular glands from highly inbred BALB/c adult male mice;9 this concentration of homogenate was found to be optimal for producing rapid outgrowth of axons.Cell cultures: Dorsal root ganglia and the ventral halves of spinal cords were dissociated by a modification of Scott's procedure.10 Lumbosacral dorsal root ganglia (80-120) from 8-day-old embryos were incubated at 370C in 0.25% trypsin solution (Grand Island Biological). After 1 hr, the trypsin was aspirated, culture medium containing serum was added, and the ganglia were repeatedly flushed through a narrow bore pipet. The resulting cell suspension was pelleted at 500 g for 2 min and resuspended.After two such washes, the final dilution was made to approximately 105 cells/ml in nerve 1206

The role of microfilaments in generating cell locomotion has been investigated in glial cells migrating in vitro . Such cells are found to contain two types of microfilament systems : First, a sheath of 50-70-A in diameter filaments is present in the cytoplasm at the base of the cells, just inside the plasma membrane, and in cell processes . Second, a network of 50-A in diameter filaments is found just beneath the plasma membrane at the leading edge (undulating membrane locomotory organelle) and along the sides of the cell . The drug, cytochalasin B, causes a rapid cessation of migration and a disruption of the microfilament network . Other organelles, including the microfilament sheath and microtubules, are unaltered by the drug, and protein synthesis is not inhibited . Removal of cytochalasin results in complete recovery of migratory capabilities, even in the absence of virtually all protein synthesis . Colchicine, at levels sufficient to disrupt all microtubules, has no effect on undulating membrane activity, on net cell movement, or on microfilament integrity . The microfilament network is, therefore, indispensable for locomotion .

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Mesoderm present in early developing lung is of two sorts: bronchial, which can evoke formation of bronchial buds from tracheal epithelium; and tracheal, which inhibits bronchial morphogenesis. The latter process correlates with the presence of highly ordered sheaths of collagen and mesoderm cells near the epithelial surface. In contrast, bronchial mesoderm lacks such regularly arrayed components. Initial formation of supernumerary buds on the trachea can be evoked by nonspecific mesoderms, but such buds never branch. Bronchial mesoderm is required if the latter morphogenetic process is to occur.Thymidine labeling experiments fail to provide evidence that differential mitotic activity leads to initial bud formation.

The manner in which mouse lung primordia form has been analyzed by observation of trypsin-isolated gut endoderm from nine-day embryos. The tissue interactions involved in initial formation of lung primordia and those involved in subsequent morphogenesis of the primordia have been investigated by various organ culture procedures. Mouse lungs form in a manner different from that generally described for amniote embryos. Thus, two lung buds appear first and form the primary bronchi, whereas the trachea appears secondarily.The initial lung buds form at the 25-27 somite stage. Whole-guts from embryos as young as the two-somite stage form lung buds in culture. Such buds do not undergo branching morphogenesis if the guts are taken from embryos younger than the 25-somite stage, but undergo characteristic dichotomous branching if combined with older bronchial mesoderm. If 20-25 somite-stage endoderm is isolated and cultured in direct combination with gut, older salivary or older bronchial mesoderms, lung buds form. However, such buds only branch in combinations containing bronchial mesoderm. Our results reveal two levels of mesodermal control of lung development. Interactions between nonspecific mesoderm and gut endoderm result in lung bud formation, while those between specific bronchial mesoderm and bud endoderm are required for branching morphogenesis.

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