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An initial exposure to high concentrations of free fatty acid increased the transfer of albumin across cultured endothelial monolayers. The rate and amount of albumin transfer was dependent on the oleic acid concentration to which the cultures were initially exposed, with 300 p.M producing the maximum transfer. The albumin transfer also increased with the increasing time of exposure to oleic acid, the maximum effect occurring during the first 24 hours. An exposure to 300 juM linoleic acid produced an even greater increase in albumin transfer than did 300 /uM oleic acid. The increased albumin transfer observed when cells were exposed to high concentrations of free fatty acid was largely reversible after reincubation of the cell monolayers in free fatty acid-poor media. In parallel experiments, radioactive oleic acid incorporation into cell triglycerides increased linearly as the fatty acid concentration was raised, with cell triglyceride content increasing up to sevenfold after incubation in a medium containing 300 /nM oleic acid. A significant amount of oleic acid was incorporated into phospholipids, and the fatty acid composition of the endothelial triglycerides and phospholipids was modified. All these effects of oleic acid occurred without altering the incorporation of leucine into the cell protein. These results indicate that exposure to high concentrations of free fatty acid can alter endothelial cell lipid composition, and that this increases the albumin transfer across endothelium. This process might permit more macromolecules to enter the arterial wall. (Arteriosclerosis 4:489-497, September/October 1984)

To monitor the interaction of cell surface acetylcholine (AcCho) receptors with the cytoskeleton, cultured muscle cells were labeled with radioactive or fluorescent a-bungarotoxin and extracted with Triton X-100, using conditions that preserve internal structure. A significant population of the AcCho receptors is retained on the skeletal framework remaining after detergent extraction . The proportion of nonextracted AcCho receptors increases during myotube development. Both photographic images and quantitative fluorescence measurements indicate that AcCho receptors in patched or aggregated areas are retained on the cytoskeleton while the diffuse receptors are partially extracted by detergent. The skeleton organization responsible for restricting AcCho receptors to a patched region may also result in their retention after detergent extraction .

To examine how nascent myosin heavy chains associate with the cytoskeletons of developing muscle cells, we used pulse labeling, cell fractionation, and immunoprecipitation. More than 80% of nascent myosin heavy chains associate with the cytoskeleton. More than one-third of these nascent chains are not released by puromycin and/or RNase. The fraction of nascent heavy chains that resists release increases during development of muscle cells in culture. Treatment with cytochalasin D but not nocodazole decreases myosin heavy chain cotranslational assembly. These results indicate that (i) cotranslational assembly of myosin heavy chains is developmentally regulated, (ii) structures containing actin and not microtubules may mediate initial association of the heavy chains with the cytoskeleton, and (iii) the site of translation dictates where a significant fraction of the heavy chains will be inserted into the cytoskeleton.Eukaryotic cells contain a complex intracellular network, the cytoskeleton. Most cytoskeletons include the three major filament systems and numerous associated proteins (1-5). Cells have characteristic cytoskeletons with specific protein compositions and a high degree of spatial order (refs. 6 and 7; reviewed in refs. 8 and 9). Complex structures of cells, in which different regions not separated by membranes retain different compositions, present major problems for assembly. Earlier studies (10,11) showed that many cytoskeletal proteins associate with the Triton-resistant cytoskeleton shortly after or during translation; such proteins were found near polyribosomes and redistributed with time. Cytoskeletal assembly studied in an in vitro translational system indicated that over three-quarters of cytoskeletal protein formed puromycin-resistant contacts during translation; these contacts required initiation in vivo and low calcium concentration (12). More recent studies (13) show that some cytoskeletal proteins are coordinately expressed. Coordinate synthesis is one simple mechanism for cotranslational assembly.These studies did not address whether a given protein assembles into the cytoskeleton during translation. In the systems in which association during synthesis was examined, no one specific protein could be identified, since nascent polypeptides differ in isoelectric point and molecular weight from completed proteins. Clearly, one must identify nascent polypeptides of a specific protein to address this question further.Such identification is possible with monoclonal antibodies (mAbs) that recognize proteins near their amino terminus. A mAb that recognizes muscle myosin heavy chain (MHC) 30 kDa from the amino terminus has permitted us to examine the extent of cotranslational assembly of MHC into the cytoskeleton during muscle development. MATERIALS AND METHODSMuscle Culture. Thigh muscle of 12-day-old chicken embryos was cultured as described (ref. 14; further details will be published elsewhere). Twenty-four hours after plating, cultures were subjected to limited divalent cation depletion ...