The ultrastructure of process formation following treatment with db-cAMP of a Chinese hamster ovary X Chinese hamster brain cell hybrid

Scanning (SEM) and transmission (TEM) electron microscopy studies were performed on a hybrid, resulting from the Sendai-virus fusion of a Chinese hamster ovary (CHO) Glycine A (Gly^-A) auxotrophic mutant cell [1,2] with a freshly-biopsied suspension of Chinese hamster cerebral cortex cells. In normal growth medium the hybrid differs from the CHO parental cell in displaying a squamous, polygonal, epithelioid appearance with sparse microvilli and ribosome-filled knobs (blebs). Slender filopodia, which sometimes reach a length of 25 μm, extend from interphase cells. Bundles of microfilaments (6 nm diameter) are observed closely associated with the cell membrane and the perinuclear region, arranged more or less in parallel to the glass substrate. The untreated hybrid has a relatively unpatterned arrangement of microtubules and reveals desmosomes at points of cell contact. When treated with N⁶,O²-dibutyryl adenosine 3′:5′-cyclic monophosphoric acid (db-cAMP) plus the Synergist testololactone, the response of the hybrid differs markedly from the fibroblastic habitus assumed by CHO [3, 4, 5]. The hybrid cells become stellate, forming processes or cytoplasmic extensions which radiate from a central, microvillus-covered, rounded, cell body. The arborizing processes number 2-8 per cell and form a contiguous network between cells of a colony. Desmosomes are seen at these points of process-to-cell body junctions. Parallel microtubules, 10 nm filaments, and 6 nm microfilaments, as well as organelles of the unstimulated cytoplasm such as free ribosomes, lipid granules, mitochondria, rough ER, and myelin figures are present in the nerve-like extensions. Knobs disappear completely following cAMP treatment. On removal of the db-cAMP, disappearance of the processes is apparent in 1-2 h so that the cell returns to its original morphology. This reversal is accompanied by ruffling activity at the cell borders. The central, rounded portion of the cell returns to the former flattened state somewhat more slowly. These studies demonstrating a cAMP-induced change in morphology and microtubule arrangement, produced in CHO X brain cell hybrids support the previous proposals that: (1) cAMP action is necessary for organization of cellular microtubules to form a pattern; (2) this pattern is a function of the cellular differentiation state and is determined by genetic or epigenetic factors.