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The ability of adherent cells such as fibroblasts to enter the cell cycle and progress to S phase is strictly dependent on the extent to which individual cells can attach to and spread on a substratum. Here we have used microengineered adhesive islands of 22 and 45 m diameter surrounded by a nonadhesive substratum of polyhydroxyl methacrylate to accurately control the extent to which individual Swiss 3T3 fibroblasts may spread. The effect of cell shape on mitogen-evoked Ca 2؉ signaling events that accompany entry into the cell cycle was investigated. In unrestricted cells, the mitogens bombesin and fetal calf serum evoked a typical biphasic change in the cytoplasmic free Ca 2؉ concentration. However, when the spreading of individual cells was restricted, such that progression to S phase was substantially reduced, both bombesin and fetal calf serum caused a rapid transient rise in the cytoplasmic free Ca 2؉ concentration but failed to elicit the normal sustained influx of Ca 2؉ that follows Ca 2؉ release. As expected, restricting cell spreading led to the loss of actin stress fibers and the formation of a ring of cortical actin. Restricting cell shape did not appear to influence mitogen-receptor interactions, nor did it influence the presence of focal adhesions. Because Ca 2؉ signaling is an essential component of mitogen responses, these findings implicate Ca 2؉ influx as a necessary component of cell shape-dependent control of the cell cycle.

We describe a fluorescence assay for nitric oxide synthase activity based on a new indicator, 4,5-diaminofluorescein (DAF-2). The method offers the advantage of being safer and more convenient than the citrulline radioassay in common use. The rapid and irreversible binding of DAF-2 to oxidized nitric oxide (NO) enables NO production to be measured in real time. The protocol is applied to the measurement of nitric oxide synthase in crude extracts of skeletal muscle.