Trifluoperazine (TFP) blocks spreading and migration of cultured mammalian cells. These are calcium-dependent and microfilament-mediated processes. Calmodulin, a regulator of many calcium-dependent processes in cells, is selectively inhibited by TFP. Cell spreading on a plastic- or collagen-coated substratum was reversibly inhibited by 10 micro M TFP. The drug blocks cell spreading even in the presence of 1 mM cAMP. TFP is as effective as cytochalasin B (CB), in inhibitor of microfilament function, in blocking cell spreading. All cell lines tested, whether "normal" or virally transformed, failed to spread to TFP. The drug, at a concentration sufficient to inhibit spreading, does not interfere with the initial attachment of a cell to a plastic surface. Cells plated in the presence of 10 micro M TFP attach at a rate and to an extent equal to untreated controls. TFP added to already spread cells results in a reversible cell rounding. Detection of fibronectin by indirect immunofluorescence suggests TFP-induced cell rounding is not due to shedding of fibronectin from the cell surface. TFP reversibly blocks cell migration into a would edge almost as effectively as CB. We suggest that TFP interferes with these microfilament-mediated functions by direct action on the microfilaments or indirect action by inactivating calmodulin.

Cells of the established preadipose line, 3T3-L1, appear to be undifferentiated fibroblasts during exponential growth. When cells become quiescent, a small percentage of them accumulate triglyceride and become morphologically indistinguishable from mature adipocytes. When insulin is added to quiescent cultures, up to 50% of the cells differentiate into adipocytes. The distribution of lipid-containing cells which appear in clusters of varying sizes was analyzed to determine whether commitment to differentiation occurred after quiescence or during exponential growth and whether insulin was required as an inducer of commitment. The spatial arrangement of 3T3-L1 cells at quiescence on some culture dishes was destroyed by replating. This resulted in random distribution of these cells. The distribution of adipocytes among replated and nonreplated cells in these experiments was compared to a computer generated random distribution of differentiated among undifferentiated cells. Dispersal of cells at confluence resulted in a distribution of fat among nonfat cells not significantly different from the computer generated random distribution. In undisturbed cultures, the distribution of fat cells is not random and is consistent with a commitment event in single cells at any cell division during exponential growth followed by divisions of both committed and uncommitted cells. Since insulin affected the number of mature adipocytes only when added after cessation of exponential growth, insulin is not the inducer of commitment but merely enhances lipid production in previously committed cells.

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Some cells of the established preadipose cell line, 3T3-L1, synthesize triglyceride after becoming confluent and quiescent. An analysis of the distribution of clusters of lipid-containing cells was consistent with a commitment event during exponential growth followed by clonal growth of committed cells. Experiments were designed to determine if the final clonal pattern of fat among nonfat cells could be described by a probability model. Undifferentiated cells (fibroblastic cells with no detectable accumulation of triglycerides) were plated at various cell numbers so that the total number of cell divisions to confluence could be controlled. Cells were passaged by trypsinization and replating, or trypsinization followed by passage through a narrow-bore needle before replating. Passing cells through a 22G needle seems to eliminate already committed cells from the population. We determined the percentage of fat cells and the range of clone sizes in cultures in which clone sizes depended upon the number of allowed cell divisions. Patterns of clone sizes in experimental cultures were compared to expected patterns obtained by computer simulations of several programmed and stochastic commitment models. Both the observed range of clone sizes and pattern of clones can be approximated by a simple stochastic model, suggesting that commitment to fat production in 3T3-LI cells is a random process occurring with a fixed probability in single cells in exponential growth, followed by division of both committed and uncommitted cells. The probability of commitment was essentially constant at each cell division. The number of cells committed during each passage i s just large enough to replace "terminally differentiated" lipidcontaining cells that have been lost, thereby maintaining a constant percentage of fat cells in any given culture of 3T3-Ll.Differentiation i s the process by which cells acquire the ability to express a particular phenotype. Differentiated cells possess specialized properties or functions such as t h e ability t o produce substances l i k e melanin, hemoglobin, crystallin, or fat (Wigley, 1975). At a point in t h e i r life history some change in t h e organization of t h e genome or the expression of certain genes occurs and such cells eventually acquire a differentiated phenotype. This change, assumed for t h e present t o be irreversible, i s referred t o as the commitm e n t point. Commitment and different i a t i o n have been studied in several established cultured cell lines, including F r i e n d erythroleukemia cells (FEL), keratinocytes, muscle cells, and adipocytes (Friend e t al., 1965; F r i e n d e t al., 1971; Dienstman and Holtzer, 1975;Green, 1977;Nadal-Ginard, 1978;Levenson et al., 1979;Pragnell et al., 1980). Many hypotheses based u p o n genetic or epigenetic causes have been offered t o explain how commitment occurs, but a satisfactory explanation has remained elusive.