Role of Cytoskeletal Elements in the Recruitment of Cx43-GFP and Cx26-YFP into Gap Junctions

Thomas, Tamsin; Jordan, Karen; Laird, Dale W.

doi:10.3109/15419060109080729

Cited by 50 publications

(36 citation statements)

References 13 publications

(5 reference statements)

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“…In these studies, these pathways were distinguished by their differential pharmacologic sensitivity to brefeldin A which disassembles the Golgi (but did not block Cx26 transport) and nocodazole, a microtubule disrupting reagent (which blocked Cx26) Martin et al, 2001). However, these conclusions are controversial and not consistent with other published data (Thomas et al, 2001;Johnson et al, 2002). We tested these agents in our coexpressing cells.…”

Section: Oligomerization Of Cx26 and Cx43 In Co-expressing Cellscontrasting

confidence: 47%

Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells

Gemel

Valiūnas

Brink

et al. 2004

Journal of Cell Science

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solubilized connexons from co-expressing cells by centrifugation through sucrose gradients or by affinity purification using a Ni-NTA column showed no evidence of mixing of Cx26 and Cx43. These results contrast with our observations in cells co-expressing other connexins with Cx43 and suggest that Cx26 and Cx43 do not form heteromeric hemichannels. Moreover, the incorporation of Cx26 and Cx43 into oligomers and into the membrane were similarly affected by treatment of co-expressing cells with brefeldin A or nocodazole, suggesting that the lack of mixing is due to incompatibility of these connexins, not to differences in biosynthetic trafficking.

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Section: Oligomerization Of Cx26 and Cx43 In Co-expressing Cellscontrasting

confidence: 47%

Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells

Gemel

Valiūnas

Brink

et al. 2004

Journal of Cell Science

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“…After disrupting microtubules with nocodazole, Cx43 assembly was hardly affected in transfected HeLa cells, whereas Cx26 assembly was inhibited [Martin et al, 2001]. Nocodazole treatment also affected Cx43 recruitment in GJs, whereas Cx26 transport and clustering remained nearly unchanged [Thomas et al, 2001]. Inhibition of Cx transport in the Golgi by brefeldin A and inhibition of microtubules by nocodazole in Cx43-transfected HeLa cells blocked GJ assembly after the pool of non-junctional hemichannels in the plasma membrane was depleted.…”

Section: Shuttling To the Plasma Membrane And Intramembrane Movementmentioning

confidence: 99%

Connexins, cell motility, and the cytoskeleton

Olk

Zoidl

Dermietzel

2009

Cell Motil. Cytoskeleton

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Connexins (Cx) comprise a family of transmembrane proteins, which form intercellular channels between plasma membranes of two adjoining cells, commonly known as gap junctions. Recent reports revealed that Cx proteins interact with diverse cellular components to form a multiprotein complex, which has been termed “Nexus”. Potential interaction partners include proteins such as cytoskeletal proteins, scaffolding proteins, protein kinases and phosphatases. These interactions allow correct subcellular localization of Cxs and functional regulation of gap junction‐mediated intercellular communication. Evidence is accruing that Cxs might have channel‐independent functions, which potentially include regulation of cell migration, cell polarization and growth control. In the current review, we summarize recent knowledge on Cx interactions with cytoskeletal proteins and highlight some aspects of their role in cellular motility. Cell Motil. Cytoskeleton 66: 1000–1016, 2009. © 2009 Wiley‐Liss, Inc.

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“…We previously showed that this treatment condition completely depolymerized microtubules in NRK cells (Thomas et al, 2001). We quantified the movement of vesicles containing fluorescent-protein-tagged Cx43 or Cx26 and found that in both cases there was an ~80% decrease in vesicle movement when microtubules were disrupted (Table 1 and supplementary material Movie 7).…”

Section: Differential Mobility Of Cx43 and Cx26 Within Gap Junction Pmentioning

confidence: 99%

“…To analyze the role of microtubules in vesicular connexin transport, NRK cells expressing fluorescentprotein-tagged Cx43 or Cx26 were first time-lapse imaged (32 second image acquisition intervals) for up to 18 minutes. After treating the cells for 45 minutes with nocodazole (10 M) to disrupt the microtubules (Thomas et al, 2001), the same field of cells was again time-lapse imaged over an additional period of up to 18 minutes. LSM 510 software was used to calculate the individual vesicle displacement over 1 minute from time-lapse images of cells before and after microtubule disruption as modified from the procedure described previously (Johnson et al, 2002).…”

Section: Drug Treatments: Bfa and Nocodazolementioning

confidence: 99%

Mechanisms of Cx43 and Cx26 transport to the plasma membrane and gap junction regeneration

Thomas

Jordan

Simek

et al. 2005

Journal of Cell Science

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118

106

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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-protein-tagged 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 Golgi-to-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 fluorescent-protein-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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Role of Cytoskeletal Elements in the Recruitment of Cx43-GFP and Cx26-YFP into Gap Junctions

Cited by 50 publications

References 13 publications

Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells

Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells

Connexins, cell motility, and the cytoskeleton

Mechanisms of Cx43 and Cx26 transport to the plasma membrane and gap junction regeneration

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