Previous studies (Neff et al., 1982. J. Cell. Biol. 95:654-666;Decker et al., 1984. J. Cell. Biol. 99:1388-1404 have described a monoclonal antibody (CSAT Mab) directed against a complex of three integral membrane glycoproteins of 120,000-160,000 mol wt (CSAT antigen [ag]) involved in the cell matrix adhesion of myoblasts and fibroblasts. In localization studies on fibroblasts presented here, CSAT ag has a discrete, well-organized distribution pattern. It co-aligns with portions of stress fibers and is enriched at the periphery of, but not directly beneath vinculin-rich focal contacts. In this last location, it co-distributes with fibronectin, consistent with the suggestion that the CSAT ag participates in the mechanism by which fibroblasts attach to fibronectin. In prefusion myoblasts, which are rapidly detached by CSAT Mab, CSAT ag is distributed diffusely as are vinculin, laminin, and fibronectin. After fusion, myotubes become more difficult to detach with CSAT Mab. The CSAT ag and vinculin are organized in a much more discrete pattern on the myotube surface, becoming enriched at microfilament bundle termini and in lateral lamellae which appear to attach myotubes to the substratum. These results suggest that the organization of CSAT ag-adhesive complexes on the surface of myogenic cells can affect the stability of their adhesive contacts. We conclude from the sum of the studies presented that, in both myogenic and fibroblastic cells, the CSAT ag is localized in sites expected of a surface membrane mediator of cell adhesion to extracellular matrix. The results from studies that use fibroblasts in particular suggest the involvement of CSAT ag in the adhesion of these cells to fibronectin.CSAT monoclonal antibody (CSAT Mab), ~ raised against chick embryo myoblast surface membrane vesicles, rapidly rounds and detaches 48-h cultured chick myoblasts from their extracellular matrices (ECM). With additional time in culture, developing myotubes become increasingly resistant to detachment. CSAT Mab binds equally well to the skeletal fibroblasts present in the myoblast cultures, but does not detach them (24). The CSAT antigen (ag) is clearly relevant to myoblast adhesion in vivo since a population of ceils is released when I 1-d chick embryo breast muscle explants are treated with CSAT Mab. When subsequently cultured in the absence of t Abbreviations used in this paper: ag, antigen; anti-GP-3, polyclonal antiserum raised in rabbits against the third glycoprotein band of immunoaffinity-purified CSAT antigen complex; CSAT, cell substratum attachment; ECM, extracellular matrix; FITC, fluorescein isothiocyanate; Fn, fibronectin; GP-3, third glycoprotein band; Lm, laminin; Mab, monoclonal antibody; Vn, vinculin.

We have used the CSAT (cell substrate attachment) monoclonal antibody (Mab), which is directed against a putative laminin and fibronectin receptor, to examine its role in the adhesive phenomena of neurons. This antibody was previously found to disturb the adhesion of several classes of fibroblasts and muscle. Here we report its effects upon neuronal-substrate adhesion. Two sources of neurons were investigated--the dorsal root and ciliary ganglia. Both responded similarly. Neurons plated in the presence of the CSAT Mab did not adhere to the substratum and process formation was inhibited completely for at least 24-48 hr. In explant cultures, when neurons were first allowed to extend processes prior to addition of the CSAT Mab, the results depended on the particular substrate. With some substrates, the neurites bundled and detached from the substratum; with others, they retracted and regrew to form large fascicles or bundles of processes. In dissociated cultures that already had extended processes, neurites fasciculated and cell bodies aggregated in response to the presence of the CSAT Mab. The magnitude of this response varied, depending upon the substrate. The antigen was localized, using immunofluorescence, on neuronal cell bodies, axons, and growth cones. This distribution correlated with its biological effects on all parts of the neuron. The antigen was isolated from neuronal cultures by immunoaffinity purification. It migrated in the molecular weight range of 140 kDa on reducing SDS-PAGE. This antigen is very similar to that isolated from fibroblasts, which is an integral membrane glycoprotein complex. The data presented implicate the participation of the CSAT antigen in neurite extension and fasciculation.

We have cultured myogenic cells derived from primary explants and a cell line (L6) in a lipid-depleted medium (LDM) and produced large alterations of the fatty acyl and polar headgroup composition and of the cellular sterol levels. These alterations were produced by altering the composition of the media as follows: removing biotin and providing exogenous fatty acid; removing choline and providing exogenous ethanolamine or choline analogues; and by adding 25-OH cholesterol, an inhibitor of 3-hydroxy-3-methylglutarate (HMG)-CoA reductase. Relatively small, secondary alterations of other lipid classes accompany the large primary alteration. In general, they are not obviously compensatory for the primary alteration by retaining some physical property.We have explored the influence of these lipid alterations on myoblast proliferation and fusion into myotubes. In general, considerable variability appears tolerated, but there also appear to be limits. Long-term cultures grown in media containing a single fatty acid do not proliferate indefinitely, and the fatty acid does not become the sole fatty acyl component of the phospholipids. This phenomenon is also observed for cultures enriched in phosphatidylethanolamine (PE) or phosphatidyldimethylethanolamine (PDME).The influence of the lipid alterations on fusion is particularly interesting. The inclusion of 25-OH cholesterol inhibits fusion. Enrichment of the fatty acyl chains with elaidate or the polar headgroups with PE also inhibits fusion, but in contrast to that by 25-OH cholesterol, a significant fraction of the myoblasts are aligned and interacting with each other. Oleate enrichment enhances the rate of fusion. KEY WORDS membranes lipids fusionMyogenic cells derived from primary explants or available as continuous cell lines can be grown in culture while retaining many of their differentiated membrane properties. When grown under suitable culture conditions, for example, they sort out and fuse into multinucleate myotubes, synthesize acetylcholine receptors, become electrically and chemically excitable, cluster their receptors into "hot spots", and form synapses with appropriate neurons (17-20, 22, 25, 34, 37, 41,43, 50, 56, 57, 59, 61). It is likely that the synthesis and assembly of the relevant molecular 334J. CELL BIOLOOV 9 The Rockefeller University Press 9 0021-9525/78/0501-033451.00 on

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The coordination of the syntheses of the several cellular lipid classes with one another and with cell cycle control were investigated in proliferating L6 myoblasts and fibroblasts . Cells cultured in lipid-depleted medium containing one of two inhibitors of hydroxymethylglutaryl-CoA reductase, 25-hydroxycholesterol or compactin, display a rapid, dose-dependent inhibition of cholesterol synthesis. Inhibition of the syntheses of each of the other lipid classes is first apparent after the rate of sterol synthesis is depressed severalfold . 24 h after the addition of the inhibitor, the syntheses of DNA, RNA, and protein also decline . The inhibition of sterol synthesis leads to a threefold reduction in the sterol : phospholipid ratio that parallels the development of proliferative and G, cell cycle arrests and alterations in cellular morphology . All of these responses are reversed upon reinitiation of cholesterol synthesis or addition of exogenous cholesterol .A comparison of the timing of these responses with respect to the development of the G, arrest indicates that the primary factor limiting cell cycling is the availability of cholesterol provided either from an exogenous source or by de novo synthesis. The G, arrest appears to be responsible for the general inhibition of macromolecular synthesis in proliferating cells treated with 25-hydroxycholesterol . In contrast, the apparent coordinated inhibition of lipid synthesis is not a consequence of the G, arrest but may in fact give rise to it .Sequential inhibition of lipid syntheses is also observed in cycling cells when the synthesis of choline-containing lipids is blocked by choline deprivation and is observed in association with G, arrests caused by confluence or differentiation . In the nonproliferating cells, the syntheses of lipid and protein do not appear coupled .The study of the mechanism and control of membrane biogenesis is still in its infancy. One of the many unexplored areas is the coordination between the synthesis of membranes and the synthesis of DNA and cell cycling. We have begun to study the coupling between membrane lipid and macromolecular syntheses by culturing cells in lipid-depleted medium (LDM)' 'Abbreviations used in this paper: LDM, lipid-depleted medium ; 25-OH, 25-hydroxycholesterol; CMF-PBS, calcium, magnesium-free, 810 containing inhibitors of the synthesis of selective lipid pathways . In this way the cells are deprived of both the exogenous and endogenous source of a particular lipid.By this approach evidence for coordinated control of membrane and DNA syntheses has been found (7). The lipid phosphate-buffered saline ; Versene, 0.02% disodium EDTA in CMF-PBS; DMEM, Dulbecco's minimal essential medium; GLC, gas-liquid chromatography .