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| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
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MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 588 |
Mechanisms
of Cx43 and Cx26 transport to the
plasma membrane and gap junction regeneration |
Tamsin
Thomas, Karen Jordan, Jamie Simek, Qing Shao, Chris Jedeszko, Paul Walton and Dale W. Laird |
Connexin,
Gap junctions, Post-Golgi carriers, Timelapse
imaging, Fluorescence recovery after photobleaching |
BICR-M1Rk cells transiently expressing
Cx43-GFP or stably expressing Cx26-YFP were plated in 35 mm glass-bottom tissue culture dishes
for live imaging (MatTek Corporation, MA).
|
inverted
confocal microscope |
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BICR-M1Rk |
| Abstract |
| |
Previous reports have suggested that Cx26 exhibits
unique intracellular transport pathways en route to the cell surface compared with other members
of the connexin family. To directly examine and compare nascent and steady-state delivery of
Cx43 and Cx26 to the plasma membrane and gap junction biogenesis we expressed fluorescent-proteintagged
Cx43 and Cx26 in BICR-M1Rk and NRK cells. Static and time-lapse imaging revealed that both connexins
were routed through the Golgi apparatus prior to being transported to the cell surface, a process
inhibited in the presence of brefeldin A (BFA) or the expression of a dominant-negative form
of Sar1 GTPase. During recovery from BFA, time-lapse imaging of nascent connexin Golgito- plasma
membrane delivery revealed many dynamic post-Golgi carriers (PGCs) originating from the distal
side of the Golgi apparatus consisting of heterogeneous vesicles and long, tubular-like extensions.
Vesicles and tubular extensions were also observed in HBL-100 cells expressing a human, disease-linked,
Golgi-localized Cx26 mutant, D66H-GFP. A diffuse cell surface rim of fluorescentprotein- tagged
wild-type connexins was observed prior to the appearance of punctate gap junctions, which suggests
that random fusion of PGCs occurred with the plasma membrane followed by lateral diffusion of
connexins into clusters. Fluorescence recovery after photobleaching studies revealed that Cx26-YFP
was more mobile within gap junction plaques compared with Cx43-GFP. Intriguingly, Cx43-GFP delivery
and gap junction regeneration was inhibited by BFA and nocodazole, whereas Cx26-GFP delivery
was prevented by BFA but not nocodazole. Collectively, these studies suggest that during gap
junction biogenesis two phylogenetically distinct members of the connexin family, Cx43 and Cx26,
share common secretory pathways, types of transport intermediates and turnover dynamics but
differ in their microtubule-dependence and mobility within the plasma membrane, which might
reflect differences in binding to protein scaffolds. |
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