Abstract
Caveolae are plasma membrane invaginations that are enriched in cholesterol-binding proteins called caveolins. The presence of caveolae and caveolins in mixed cultures of human neurons and glia has not been investigated. Here, we sought to determine the presence of caveolae and caveolins in human NTera-2 (NT2/D1) cells, differentiated with retinoic acid into neuron-like (NT2/N) and astrocyte-like (NT2/A) cells. We found that while caveolin-3 mRNA levels remained relatively constant, caveolin-1 and -2 levels were upregulated in NT2/A and downregulated in NT2/N. No caveolin-1 immunoreactivity was detected in NT2/N. Electron microscopy revealed numerous flask-shaped invaginations (~86–102 nm in diameter) in the plasma membrane of NT2/A and NT2/N cells, while only few were detected in NT2/D1 cells. Immunoelectron microscopy localized caveolin-1 gold particles in the flask-shaped structures on plasmalemma and cytoplasmic vesicles of NT2/A cells. Furthermore, NT2/A endocytosed Alexa 488 conjugated-cholera toxin B subunit (CTX-B) through a caveolae- and clathrin-dependent pathway, whereas NT2/N endocytosed CTX-B through a caveolae-independent pathway. We have established that while NT2/A expressed functional caveolae, the molecular identity of the plasma membrane invaginations in NT2/N is unknown. The expression of caveolin proteins was differentially regulated in these cells. Taken together, our findings support the usefulness of the human NT2 model system to study the role of caveolins in neuron–glia communication, and their involvement in brain health and disease.
Highlights
Caveolae are non-clathrin coated, 50–100 nm in diameter, omega- or flask-shaped invaginations of the plasma membrane which are highly enriched in glycosphingolipids, cholesterol and sphingomyelin
The analysis revealed a deletion in exon-2 which resulted in a 1314 bp mRNA, compared to the 3332 bp mRNA encoded by the full length caveolin-2 gene
Protein extracts were prepared from various NT2 cell types, subjected to SDS-PAGE and probed with anti-caveolin-1 or -3 antibodies, respectively. β-actin was used as a loading control. (H) Western blot analysis of caveolin-1 in subcellular fractions of NT2/A
Summary
Caveolae (little caves) are non-clathrin coated, 50–100 nm in diameter, omega- or flask-shaped invaginations of the plasma membrane which are highly enriched in glycosphingolipids, cholesterol and sphingomyelin. These plasma membrane pits are decorated by a family of 18–24 kDa cholesterol binding and scaffolding proteins known as caveolins. The caveolin proteins are expressed at varying levels in different cell types and tissues and perform highly specialized functions. Caveolin-2 has not been extensively studied, but has a similar distribution to caveolin-1 [2], whereas caveolin-3 is highly expressed in muscle cells (skeletal, cardiac, and smooth). The lowering of caveolin-1 expression is associated with a reduction in the number of caveolae [6]
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