Molecular mechanisms regulating formation, trafficking and processing of annular gap junctions

Falk, Matthias M.; Bell, Cheryl L.; Andrews, Rachael M. Kells; Murray, Sandra A.

doi:10.1186/s12860-016-0087-7

Cited by 67 publications

(79 citation statements)

References 208 publications

(274 reference statements)

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“…Although the two membrane bilayers making up a ring-shaped gap junction could not be resolved, we were able to distinguish them by their darker thicker membrane, the lucid area just inside the membrane, their round shape and their diameter of 200-800 nm ( Fig. 1C) (Espey and Stutts, 1972;Merk et al, 1973;Falk, et al, 2016). While smaller connexosomes are in the size range of secretory vesicles, they are still distinguishable by their characteristic electron-density pattern.…”

Section: Characterization Of Connexosomesmentioning

confidence: 85%

“…The development of phosphorylation-specific antibodies made it possible to monitor the phosphorylation status of many of these sites by western blotting (Lampe et al, 2006). Patterns and sequences of phosphorylation changes have been observed in wounded skin, ischemic hearts and cultured cells stimulated with growth factors (Smyth et al, 2014;Falk et al, 2016;Solan and Lampe, 2016). In western blots, entire cells are solubilized and probed with an antibody.…”

Section: Introductionmentioning

confidence: 99%

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Localization of phosphorylated connexin 43 using serial section immunogold electron microscopy

Norris

Baena

Terasaki

2017

Journal of Cell Science

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Gap junction turnover occurs through the internalization of both of the plasma membranes of a gap junction plaque, forming a double membrane-enclosed vesicle, or connexosome. Phosphorylation has a key role in regulation, but further progress requires the ability to clearly distinguish gap junctions and connexosomes, and to precisely identify proteins associated with them. We examined, by using electron microscopy, serial sections of mouse preovulatory ovarian follicles that had been collected with an automated tape collecting ultramicrotome (ATUM). We found that connexosomes can form from adjacent cell bodies, from thin cell processes or from the same cell. By immunolabeling serial sections, we found that residue S368 of connexin 43 (also known as GJA1) is phosphorylated on gap junctions and connexosomes, whereas connexin 43 residue S262 is phosphorylated only on some connexosomes. These data suggest that phosphorylation at S262 contributes to connexosome formation or processing, and they provide more precise evidence that phosphorylation has a key role in gap junction internalization. Serial section electron microscopy of immunogold-labeled tissues offers a new way to investigate the three-dimensional organization of cells in their native environment.

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Section: Characterization Of Connexosomesmentioning

confidence: 85%

Section: Introductionmentioning

confidence: 99%

Localization of phosphorylated connexin 43 using serial section immunogold electron microscopy

Norris

Baena

Terasaki

2017

Journal of Cell Science

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“…Together with other known examples of trans-endocytosis, including Notch-Delta, Eph-ephrin, the connexin subunits of gap junctions, and MHC-T-cell receptor complexes [18–24], our findings highlight a pervasive intercellular exchange of transmembrane proteins between cells, and suggest that trans-endocytosis may be rather widespread. Celsr1 internalization bears similarity to gap junction turnover, where large, double-membrane vesicles known as “annular junctions” internalize entire gap junction plaques that go on to be degraded [22, 25]. …”

Section: Discussionmentioning

confidence: 99%

Trans-endocytosis of Planar Cell Polarity Complexes during Cell Division

Heck

Devenport

2017

Current Biology

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SUMMARY To coordinate epithelial architecture with proliferation, cell polarity proteins undergo extensive remodeling during cell division [1–3]. A dramatic example of polarity remodeling occurs in proliferative basal cells of mammalian epidermis whereupon cell division, transmembrane planar cell polarity (PCP) proteins are removed from the cell surface via bulk endocytosis [4]. PCP proteins form intercellular complexes, linked by Celsr1-mediated homophilic adhesion, that coordinate polarity non-autonomously between cells [5, 6]. Thus, the mitotic reorganization of PCP proteins must alter not only proteins intrinsic to the dividing cell, but also their interacting partners on neighboring cells. Here we show that intercellular Celsr1 complexes that connect dividing cells with their neighbors remain intact during mitotic internalization, resulting in uptake of Celsr1 protein from interphase neighbors. Trans-internalized Celsr1 carries with it additional core PCP proteins, including the posteriorly-enriched Fz6 and anteriorly-enriched Vangl2. Cadherin-mediated homophilic adhesion is necessary for trans-endocytosis, and adhesive junctional PCP complexes appear to be destined for degradation upon internalization. Surprisingly, whereas Fz6 and Vangl2 both internalize in trans, Vangl2 proteins intrinsic to the dividing cell remain associated with the plasma membrane. Persistent Vangl2 stabilizes Celsr1 and impedes its internalization, suggesting dissociation of Vangl2 from Celsr1 is a prerequisite for Celsr1 endocytosis. These results demonstrate an unexpected transfer of PCP complexes between neighbors, and suggest that the Vangl2 population that persists at the membrane during cell division could serve as an internal cue for establishing PCP in new daughter cells.

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“…Phosphorylation of Cx43 is well established and has been shown to be a regulatory mechanism for Cx43 trafficking, GJ assembly, gating, plaque internalization, and degradation (reviewed in Falk et al, 2016;Solan and Lampe, 2014;Thevenin et al, 2013). Cx43 phosphorylation by Akt (protein kinase B), PKA (protein kinase A), and CK1 (casein kinase 1)…”

Section: Discussionmentioning

confidence: 99%

Connexin 43 K63-polyubiquitination on lysines 264 and 303 regulates gap junction internalization

Kells-Andrews

Margraf

Fisher

et al. 2017

Preprint

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Statement: Here we show that gap junction (GJ) turnover is mediated by K63polyubiquitination that is triggered by Cx43 phosphorylation. These novel findings significantly contribute to our understanding of GJ patho-/physiology. ABSTRACTGap junctions (GJs) assembled from connexin (Cx) proteins play a pivotal role in cellto-cell communication by forming channels that connect the cytosols of adjacent cells.Connexin 43, the best-studied Cx, is ubiquitously expressed in vertebrates. While phosphorylation is known to regulate multiple aspects of GJ function, much less is known about the role ubiquitination plays in these processes. Here we show by using ubiquitination-type specific antibodies and Cx43 lysine (K) to arginine (R) mutants that a portion of Cx43 in GJs can become K63-polyubiquitinated on K264 and K303. Relevant Cx43 K/R mutants assembled significantly larger GJ plaques, exhibited much longer protein half-lives and were internalization impaired. Intriguingly, ubiquitin-deficient Cx43 mutants accumulated as hyperphosphorylated polypeptides in the plasma membrane, suggesting that phosphorylation triggers K63-polyubiquitination. Phospho-specific Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282, and 255, well-known regulatory PKC and MAPK phosphorylation sites. Together, these novel findings suggest that upon internalization, some Cx43 in GJs becomes K63-polyubiquitinated, ubiquitination is critical for GJ internalization, and that K63-polyubiquitination is induced by Cx phosphorylation as is typical for phosphodegron-regulated protein degradation.

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Molecular mechanisms regulating formation, trafficking and processing of annular gap junctions

Cited by 67 publications

References 208 publications

Localization of phosphorylated connexin 43 using serial section immunogold electron microscopy

Localization of phosphorylated connexin 43 using serial section immunogold electron microscopy

Trans-endocytosis of Planar Cell Polarity Complexes during Cell Division

Connexin 43 K63-polyubiquitination on lysines 264 and 303 regulates gap junction internalization

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