Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

Abstract

We have demonstrated that the plasmalemmal vesicles (caveolae) of the continuous microvascular endothelium function as transcytotic vesicular carriers for protein molecules Ͼ20 Å and that transcytosis is an N-ethylmaleimide-sensitive factor (NSF)-dependent, N-ethylmaleimide-sensitive process. We have further investigated NSF interactions with endothelial proteins to find out 1) whether a complete set of fusion and targeting proteins is present in the endothelium; 2) whether they are organized in multimolecular complexes as in neurons; and 3) whether the endothelial multimolecular complexes differ from their neuronal counterparts, because of their specialized role in transcytosis. To generate the complexes, we have used myc-NSF, cultured pulmonary endothelial cells, and rat lung cytosol and membrane preparations; to detect them we have applied coimmunoprecipitation with myc antibodies; and to characterize them we have used velocity sedimentation and cross-linking procedures. We have found that both cytosolic and membrane fractions contain complexes that comprise beside soluble NSF attachment proteins and SNAREs (soluble NSF attachment protein receptor), rab 5, dynamin, caveolin, and lipids. By immunogold labeling and negative staining we have detected in these complexes, myc-NSF, syntaxin, dynamin, caveolin, and endogenous NSF. Similar complexes are formed by endogenous NSF. The results indicate that complexes with a distinct protein-lipid composition exist and suggest that they participate in targeting, fusion, and fission of caveolae with the endothelial plasmalemma.

Molecular determinants of endothelial transcytosis and their role in endothelial permeability

“…Morphological and biochemical studies demonstrated that in ECs, 25% of the entire amount of caveolin-1 is present in the cytosol. Cytosolic caveolin was found in a protein-lipid complex comprising general components of the membrane fusion-fission machinery (158). The finding is also consistent with a role of caveolin in cholesterol trafficking (46).…”

“…Caveolin-1 is also present, depending on the cell type, in the plasmalemma proper, mitochondria, and elements of the secretory pathway (96). Caveolin-1 can either be an integral membrane or a cytosolic soluble protein (96,156,158). As a soluble protein, caveolin-1 forms protein-lipid complexes containing cholesterol (158,217,238).…”

“…Caveolin-1 can either be an integral membrane or a cytosolic soluble protein (96,156,158). As a soluble protein, caveolin-1 forms protein-lipid complexes containing cholesterol (158,217,238). Morphological and biochemical studies demonstrated that in ECs, 25% of the entire amount of caveolin-1 is present in the cytosol.…”

Nitric oxide promotes endothelial cell survival signaling through S-nitrosylation and activation of dynamin-2

“…Thus, the relationship between dynamin and ensuing downstream signal transduction, which regulates the counterbalances of EC survival and apoptosis, may be significant. This is especially true in EC in which the dynamin-2 isoform is closely associated with key EC survival proteins, including the NO generating enzyme, endothelial NO synthase (eNOS), and the VEGF receptor, KDR (Bhattacharya et al, 2005;Chatterjee et al, 2003;Predescu et al, 2001).…”

Mechanisms Regulating Endothelial Permeability

“…2,3,7,11,12 The transport of macromolecules larger than 3 nm, such as albumin, IgG, and other macromolecules, occurs through the transcellular pathway, which is also called transcytosis or vesicular transport. 3,[12][13][14] Molecules smaller than 3 nm, such as glucose, water, and ions, can pass through interendothelial junctions (IEJs) through the paracellular pathway. 2,3,14 Both transcellular and paracellular pathways work in concert to maintain tissue-oncotic pressure and thereby maintain the endothelial barrier.…”