Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

Bejarano, Eloy; Girão, Henrique; Yuste, Andrea; Patel, Bindi; Marques, Célio Gonçalo; Spray, David C.; Pereira, Paulo; Cuervo, Ana María

doi:10.1091/mbc.e11-10-0844

Cited by 120 publications

(180 citation statements)

References 47 publications

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“…To verify that K6W-Ub stabilizes Cx43, we elicited the ubiquitination of Cx43 by phorbol-12-myristate-13-acetate (PMA) treatment or short serum depletion (35) in human lens epithelial cells (HLECs) in vitro (Fig. 5C).…”

Section: Expressing K6w-ub Diminishes Gap Junction Protein Cx46 But mentioning

confidence: 99%

Altered ubiquitin causes perturbed calcium homeostasis, hyperactivation of calpain, dysregulated differentiation, and cataract

Liu

Lyu

Chin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Although the ocular lens shares many features with other tissues, it is unique in that it retains its cells throughout life, making it ideal for studies of differentiation/development. Precipitation of proteins results in lens opacification, or cataract, the major blinding disease. Lysines on ubiquitin (Ub) determine fates of Ub-protein substrates. Information regarding ubiquitin proteasome systems (UPSs), specifically of K6 in ubiquitin, is undeveloped. We expressed in the lens a mutant Ub containing a K6W substitution (K6W-Ub). Protein profiles of lenses that express wild-type ubiquitin (WT-Ub) or K6W-Ub differ by only ∼2%. Despite these quantitatively minor differences, in K6W-Ub lenses and multiple model systems we observed a fourfold Ca2+ elevation and hyperactivation of calpain in the core of the lens, as well as calpain-associated fragmentation of critical lens proteins including Filensin, Fodrin, Vimentin, β-Crystallin, Caprin family member 2, and tudor domain containing 7. Truncations can be cataractogenic. Additionally, we observed accumulation of gap junction Connexin43, and diminished Connexin46 levels in vivo and in vitro. These findings suggest that mutation of Ub K6 alters UPS function, perturbs gap junction function, resulting in Ca2+ elevation, hyperactivation of calpain, and associated cleavage of substrates, culminating in developmental defects and a cataractous lens. The data show previously unidentified connections between UPS and calpain-based degradative systems and advance our understanding of roles for Ub K6 in eye development. They also inform about new approaches to delay cataract and other protein precipitation diseases.

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Section: Expressing K6w-ub Diminishes Gap Junction Protein Cx46 But mentioning

confidence: 99%

Altered ubiquitin causes perturbed calcium homeostasis, hyperactivation of calpain, dysregulated differentiation, and cataract

Liu

Lyu

Chin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…7a). Since internalization of Cx43 is the first step in its degradation 33,34 , we performed IF analyses of control-transduced mNCMs that showed clear staining of Cx43 at the ICD, whereas Tmem65-silenced cells showed less membrane staining and internalization of Cx43 (Fig. 7b).…”

Section: Resultsmentioning

confidence: 99%

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

et al. 2015

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Membrane proteins are crucial to heart function and development. Here we combine cationic silica-bead coating with shotgun proteomics to enrich for and identify plasma membrane-associated proteins from primary mouse neonatal and human fetal ventricular cardiomyocytes. We identify Tmem65 as a cardiac-enriched, intercalated disc protein that increases during development in both mouse and human hearts. Functional analysis of Tmem65 both in vitro using lentiviral shRNA-mediated knockdown in mouse cardiomyocytes and in vivo using morpholino-based knockdown in zebrafish show marked alterations in gap junction function and cardiac morphology. Molecular analyses suggest that Tmem65 interaction with connexin 43 (Cx43) is required for correct localization of Cx43 to the intercalated disc, since Tmem65 deletion results in marked internalization of Cx43, a shorter half-life through increased degradation, and loss of Cx43 function. Our data demonstrate that the membrane protein Tmem65 is an intercalated disc protein that interacts with and functionally regulates ventricular Cx43.

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“…NT-tagged connexins have proven useful in this regard in previous studies (48,(52)(53)(54)(55). If channel activity is required, the msfGFP CT tags are probably the current best choice.…”

Section: Discussionmentioning

confidence: 99%

Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques

Stout¹,

Snapp

Spray³

2015

Journal of Biological Chemistry

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Background: Connexin proteins form gap junction channel clusters termed plaques. Results: Microscopy with fluorescent protein-tagged connexins showed that connexin26 and connexin30 plaque arrangement is dynamic, but gap junctions of connexin43 are stabilized by its C-terminal region. Conclusion:The arrangement of channels in gap junctions depends on connexin type. Significance: These findings help to clarify how gap junction plaque dynamics and composition could modulate intercellular communication.

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Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

Cited by 120 publications

References 47 publications

Altered ubiquitin causes perturbed calcium homeostasis, hyperactivation of calpain, dysregulated differentiation, and cataract

Altered ubiquitin causes perturbed calcium homeostasis, hyperactivation of calpain, dysregulated differentiation, and cataract

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques

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