Myosin VI facilitates connexin 43 gap junction accretion

Figures

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  Fig. 1.. Myosin VI localizes to Cx43 GJs. (A) Heart sections and (B) isolated NRVMs were processed for confocal immunofluorescence microscopy to assess endogenous Cx43 (green) and myosin VI (MVI, red) colocalization. Nuclei were labeled with Hoechst 33342 (blue) and F-actin was labeled with Alexa-Fluor-647–phalloidin (white). (C) Live NRK cells stably expressing GFP–myosin-VI (green) and Cx43–TagRFP-t (red) were imaged on a Nikon spinning disk microscope. Arrowheads identify myosin VI localization to Cx43 plaques at intercellular junctions. Scale bars: 10 µm.

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  Fig. 2.. Myosin VI targets to Cx43 GJs through the RRL motif in the myosin VI cargo-binding tail domain. (A) Cartoon depicting the domain structure of myosin VI (MVI). Myosin VI contains a catalytic motor domain, a unique insert (green), an IQ calmodulin-binding motif (orange), a tail region containing a three-helix bundle and a single α-helix (SAH) domain (light blue), and a cargo-binding tail domain (dark blue). The cargo-binding tail contains protein interaction motifs (RRL and WWY) as well as binding regions for ubiquitin and phosphatidylinositol 4,5-bisphosphate. Alternative splicing can give rise to insertions of both a large and small insert (LI, SI). (B) NRK cells stably expressing Cx43–TagRFP-t (red) were transfected with full-length GFP–myosin-VI (green) constructs with point mutations, fixed and processed for confocal microscopy. Visually, colocalization was lost in the ΔRRL and ΔWWY/ΔRRL mutants (arrows) compared to the wild type (WT) and ΔWWY mutant (arrowheads). (C) 4-µm line plots for GFP and TagRFP-t fluorescence were then drawn across all Cx43 GJs (illustrated for the boxed region from B) toward the center of the GFP–myosin-VI -expressing cell. (D) Channel intensity for…

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  Fig. 3.. The loss of myosin VI results in reduced GJ size. (A) Whole-heart sections from wild-type (WT) and sv (SV) mice were processed for confocal immunofluorescence microscopy and immunostained for Cx43 (green) and stained for F-actin (white). (B) Cx43 GJ plaques were identified and quantified for two pairs of hearts (26,000–47,000 structures per heart). ****P<0.0001; ns, not significant. (C) Confluent monolayers of HeLa cells stably expressing Cx43–GFP (green) were transfected with non-targeting or myosin VI siRNA, stained for F-actin and imaged. (D) GJ plaque area was quantified using ImageJ (mean±s.d., n=3). ****P<0.0001. (E) Immunoblot for Cx43 and myosin VI in WT and sv MEFs. (F) Quantification of western blot Cx43 intensity normalized by Bio-Rad Stain-Free tryptophan labeling (total protein) (mean±s.d., n=5). *P<0.05. (G) WT and sv MEFs were processed for confocal immunofluorescence microscopy and immunostained for Cx43 (green). Arrowheads indicate GJ plaques in WT and SV cells. (H) Cx43 GJ plaque area was quantified using ImageJ. (mean±s.d., n=3). **P<0.01. Scale bars: 10 µm.

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  Fig. 4.. Gap junction intercellular communication is reduced in myosin VI-null MEFs. (A) Cartoon detailing an assay to measure GJIC. We used the BioPen microfluidic pipette to administer 20 µM calcein AM and 1 µg/ml WGA to monolayers of wild-type (WT) and sv (SV) MEFs for 10 min. The total spread of calcein AM through GJs was measured 60 min later. (B) Calcein AM (green) and WGA (red) loading was imaged using live-cell spinning disk microscopy over 10 min. (C) Tiled images surrounding the site of administration (WGA, grayscale) were obtained 60 min later to visualize the extent of transfer of calcein (Fire LUT). (D) Cartoon depicting the quantification method in which line scans were drawn across cell monolayers. A rotated image from C is re-shown for demonstrative purposes. Signal intensities across each sample were averaged and plotted for WGA–647 (E) and Calcein AM (F) (dashed lines indicate ±s.e.m., n=3). Scale bars: 10 µm.

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  Fig. 5.. Loss of myosin VI compromises GJ intercellular transport. (A) Wild-type (WT) and sv (SV) MEFs were loaded with calcein AM and cells were photobleached for FRAP analysis. Fluorescence recovery as a function of time was plotted (mean, ±s.e.m. is indicated by the region between the dashed lines, n=3). (B) Quantification of fluorescence intensity at t, F∞ as calculated by linear regression analysis (mean±s.d., n=3). *P<0.05. (C) Quantification of recovery rate. (mean±s.d., n=3). ****P<0.0001.

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  Fig. 6.. Cx43 GJ plaque formation is reduced in myosin VI-null MEFs, but Golgi trafficking remains intact. (A) Wild-type (WT) and sv (SV) MEFs were treated for 6.5 h with 5 µg/ml Brefeldin A (BFA). Cells were fixed after washout periods of 0, 1, 2 or 3 h, processed for confocal immunofluorescence microscopy and immunostained for Cx43 (grayscale) to identify GJ plaques (arrowheads). (B) GJ plaque area was quantified using ImageJ (mean±s.d., n=3). *P<0.05, **P<0.01. (C) Cartoon depicting the RUSH trafficking assay. Upon the addition of biotin to the medium, GFP–Cx43 fused to streptavidin-binding peptide (SBP–GFP–Cx43) is displaced from the ER retention hook (core streptavidin anchored to an invariant chain of the major histocompatibility complex, Ii–STV). SBP–GFP–Cx43 then freely traffics from the ER, through the Golgi, to the plasma membrane. (D) Wild-type and Snell's waltzer MEFs were transiently transfected with SBP–GFP–Cx43 – a bicistronic construct that also contains the ER hook. Biotin was added to the medium and cells were imaged on a Nikon spinning disk microscope to follow SBP–GFP–Cx43 trafficking. Scale bars: 10 µm.

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  Fig. 7.. Plasma membrane-localized Cx43 exhibits an accretion defect in myosin VI-null MEFs. (A) The surface of wt and sv (SV) MEFs was biotinylated at 4°C and biotinylated proteins were isolated using NeurtrAvidin beads. Whole-cell lysates and biotinylation fractions were immunoblotted with an antibody against Cx43. MVI, myosin VI. (B) Quantification of western blot Cx43 intensity normalized by Bio-Rad Stain-Free tryptophan labeling (total protein) (mean±s.d., n=3). ****P<0.0001; ns, not significant. (C) Wild-type and sv MEFs were transfected with mEOS3–Cx43 and imaged on a Nikon TIRF microscope for single-particle-tracking photoactivation localization microscopy analysis. The diffusion coefficient of mobile mEOS3–Cx43 (Cx43 D250ms; mean±s.d.) was quantified for at least seven cells for each phenotype. (D) NRK cells stably expressing Cx43–GFP (green) and HT–myosin-VI were incubated with the JF646-HaloTag ligand (red) before imaging using TIRF microscopy to identify peripheral recruitment of myosin VI to Cx43 GJ plaques (arrowheads). (E) WT and sv MEFs were transfected with Cx43–GFP (Fire LUT) and imaged using a 3i Spinning Disk microscope for FRAP experiments. Square regions of Cx43…

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  Fig. 8.. Myosin VI facilitates Cx43 GJ accretion. Our experiments showed that myosin VI did not localize together with Cx43 along the biosynthetic pathway, and that loss of myosin VI did not affect Cx43 delivery to or trafficking within the plasma membrane. We also did not observe clathrin and Dab2 localization colocalization with Cx43 at the plasma membrane. Instead, by using TIRF microscopy, we visualized myosin VI at the periphery of Cx43 GJ plaques, and by using FRAP, we observed an Cx43 accretion defect when myosin VI was lost. We hypothesize that it is at the GJ plaque edge that myosin VI aids in actin-mediated Cx43 accretion.