Evidence for the participation of actin microfilaments and bristle coats in the internalization of gap junction membrane.

Abstract

Thin sections of rabbit granulosa, human SW-13 adrenal cortical adenocarcinoma, and mouse B-16 melanoma cells revealed an apparent single-layered basket of 4-to 7-nm filaments surrounding cytoplasmic gap junction vesicles . This interpretation was based upon apparent longitudinal, cross, and en face sections of structures surrounding the vesicle profiles in tissue treated with tannic acidglutaraldehyde . In granulosa cells incubated with the S-1 fragment of heavy meromyosin, arrowhead-decorated filaments were observed at the periphery of the gap junction vesicles, suggesting that these filaments contained actin . In addition, we found that small gap junctional blebs and vesicles at the cell surface were coated with short electron-dense bristles similar in appearance to the clathrincontaining coat of coated vesicles of nonjunctional membrane . The role of these and other cytoskeletal elements in the possible endocytosis of gap junction membrane is discussed .

Connexins, cell motility, and the cytoskeleton

“…3). Thin-section electron microscopy of rabbit granulosa, human SW-13 adrenal cortical adenocarcinoma, and mouse B-16 melanoma cells showed that cytoplasmic GJ vesicles are surrounded by actin-containing filaments [Larsen et al, 1979]. Consistent with these data were further electron microscopical studies characterizing the plasma membrane and its cytoskeletal undercoat at sites of GJ invagination.…”

Internalization of Large Double-Membrane Intercellular Vesicles by a Clathrin-dependent Endocytic Process

“…Early ultrastructural analyses revealed large vesicular double-membrane GJ structures, AGJs, in the cytoplasm of cells derived from many different tissues (Ginzberg and Gilula, 1979;Larsen et al, 1979;Pfeifer, 1980;Leach and Oliphant, 1984;Mazet et al, 1985;Severs et al, 1989). Here, we demonstrate by expressing Cx43-GFP (a major GJ subunit protein) in HeLa cells, that entire GJ plaques can be internalized into one of the coupled cells to form these cytoplasmic, double-membrane GJ vesicles (Figure 1).…”

“…In an early ultrastructural study, clathrin had been speculated to be involved in GJ internalization (Larsen et al, 1979); however, a robust colocalization of clathrin with GJs, M. Piehl et al…”

“…In addition, this internalization process occurs on lateral membranes that contact neighboring cells. Recent consistent reports have shown clathrin-related internalization of large particles, such as viruses and pathogenic bacteria (Ehrlich et al, 2004;Rust et al, 2004;Veiga and Cossart, 2005) and of large latex beads (Aggeler and Werb, 1982).In an early ultrastructural study, clathrin had been speculated to be involved in GJ internalization (Larsen et al, 1979); however, a robust colocalization of clathrin with GJs, M. Piehl et al …”

“…Additional studies have shown that docked connexons cannot be separated under physiological conditions (Goodenough and Gilula, 1974;Ghoshroy et al, 1995), suggesting that GJ degradation could occur via the internalization of complete double-membrane spanning GJ plaques. Structural and ultrastructural analyses of differentiating tissues and cells in culture have shown cytoplasmically located, double-membrane GJ vesicles that were termed annular gap junctions (AGJs; Ginzberg and Gilula, 1979;Larsen et al, 1979;Leach and Oliphant, 1984;Mazet et al, 1985;Jordan et al, 2001), leading to the hypothesis that AGJ vesicles represent internalized GJs.To test this hypothesis, we investigated the spatiotemporal process of GJ degradation in living and fixed HeLa cells that transiently or stably expressed fluorescent proteintagged Cx43 (Cx43-green fluorescent protein [GFP], -cyan fluorescent protein [CFP], and -yellow fluorescent protein [YFP]). We then examined these cells by time-lapse fluorescence microscopy combined with ultrastructural analyses.…”