Vacuolar cytoplasmic phase separation in cultured mammalian cells involves the microfilament network and reduces motional properties of intracellular water

Hep‐2, human epithelial carcinoma cells, and human foreskin fibroblasts (FF9 and FF13) were exposed to either an ultrafiltrate (< 50 kD) of human sera or the weak base, procaine hydrochloride, to induce reversible cytoplasmic vacuolization. The formation of vacuoles was shown not to be due to imbibition of medium. Ultrastructural details obtained from various stages of vacuole formation were compared. In both cases of induction vacuoles were irregular and often appeared membraneless, with little in the way of electron‐dense content. They started to form in the perinuclear cytoplasm and progressed towards the periphery. Osmotic stress was not involved since mitochondria remained normal throughout a vacuolization episode. Vacuoles were often seen in close contact with filamentous structures, and this association remained detectable at late stages of the phenomenon. Fluorescent visualization of F‐actin confirmed that the vacuoles were frequently bordered by microfilaments. No major metabolic impairment was apparent in vacuolized cells as judged by protein synthesis measurements, but nuclear fluorescence (DNA content) and forward light scatter (nuclear volume) by flow cytometric analysis suggested late S phase and G2 retardation. 1 H‐nmr relaxation measurements indicated intracellular water restricted in motional characteristics in vacuolized cells. The possibility of a restricted cytoplasmic phase separation as part of a transient adaptation response is raised, and a hypothesis to explain the findings is discussed.