Visualization of gap junction mobility in living cells

Abstract

In order to study the dynamics of gap junctions in living cells, a cDNA was expressed in hepatocellular carcinoma-derived PLC cells coding for chimerical polypeptide Cx.EGFP-1, which consists of rat connexin32 and enhanced green fluorescent protein (EGFP). Cx.EGFP-1 was integrated into gap junctions, and the emitted epifluorescence reliably reported the distribution of the chimera. Therefore, stably transfected PLC clone PCx-9 was used to examine the dynamic behavior of gap junctions by time-lapse fluorescence microscopy. The pleomorphic fluorescent junctional plaques were highly motile within the plasma membrane. They often fused with each other or segregated into smaller patches, and fluctuation of fluorescence was detected within individual gap junctions. Furthermore, the uptake of junctional fragments into the cytoplasm of live cells was documented as originating from dynamic invaginations that form long tubulovesicular structures that pinch off. Endocytosis and subsequent lysosomal degradation, however, appeared to contribute only a little to the rapid gap junction turnover (determined half-life of 3.3 h for Cx.EGFP-1), since most cytoplasmic Cx.EGFP-1 fluorescence did not colocalize with the endocytosed fluid phase marker horseradish peroxidase or the receptor-specific endocytotic ligand transferrin and since it was distinct from lysosomes. Disassembly of gap junctions was monitored in the presence of the translation-inhibitor cycloheximide and showed increased endocytosis and continuous reduction of junctional plaques. Highly motile cytoplasmic microvesicles, which were detectable as multiple, weakly fluorescent puncta in all movies, are proposed to contribute significantly to gap junction morphogenesis by the transport of small subunits between biosynthetic, degradative, and recycling compartments.

Cx43 has distinct mobility within plasma-membrane domains, indicative of progressive formation of gap-junction plaques

“…The reduction in Cx43 mobility within GJ-like clusters could further reflect differences in Cx43 interactions with scaffolding or cytoskeletal elements, or progressive increases in the state of channel or connexon interactions. These studies are consistent with the findings in which lateral diffusion and joining of connexon aggregates promotes GJ growth (Jordan et al, 1999;Ryerse et al, 1984;Windoffer et al, 2000).…”

ZO-1 alters the plasma membrane localization and function of Cx43 in osteoblastic cells

“…While our data strongly suggest that the Cx43/ZO-1 interaction plays a role in the formation of Cx43 gap junction plaques, in a number of studies altered or epitope-tagged connexins that should not bind ZO-1 have been shown to make functional gap junctions (Windoffer et al, 2000;Giepmans and Moolenar, 1998;Jordan et al, 1999). However, this does not eliminate the possibility that ZO-1 plays a role in gap junction formation, as recent studies suggested that Cx43-GFP fusion proteins do not interact with ZO-1 and that the aberrantly large gap junction plaques made by the Cx43-GFP constructs are mostly non-functional (Hunter et al, 2003;Bukauskas et al, 2000).…”

The keratin-filament cycle of assembly and disassembly

“…For assessment of drug-induced keratin alterations, cells were seeded in glassbottom Petri dishes (MatTek) and treated with 17 M cycloheximide (Windoffer et al, 2000) (Sigma) or 1-2 g/ml puromycin (Sigma).…”