Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1 - PubMed (original) (raw)

Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1

J Matthew Rhett et al. Mol Biol Cell. 2011 May.

Abstract

Connexin 43 (Cx43) is a gap junction (GJ) protein widely expressed in mammalian tissues that mediates cell-to-cell coupling. Intercellular channels comprising GJ aggregates form from docking of paired connexons, with one each contributed by apposing cells. Zonula occludens-1 (ZO-1) binds the carboxy terminus of Cx43, and we have previously shown that inhibition of the Cx43/ZO-1 interaction increases GJ size by 48 h. Here we demonstrated that increases in GJ aggregation occur within 2 h (∼Cx43 half-life) following disruption of Cx43/ZO-1. Immunoprecipitation and Duolink protein-protein interaction assays indicated that inhibition targets ZO-1 binding with Cx43 in GJs as well as connexons in an adjacent domain that we term the "perinexus." Consistent with GJ size increases being matched by decreases in connexons, inhibition of Cx43/ZO-1 reduced the extent of perinexal interaction, increased the proportion of connexons docked in GJs relative to undocked connexons in the plasma membrane, and increased GJ intercellular communication while concomitantly decreasing hemichannel-mediated membrane permeance in contacting, but not noncontacting, cells. ZO-1 small interfering RNA and overexpression experiments verified that loss and gain of ZO-1 function govern the transition of connexons into GJs. It is concluded that ZO-1 regulates the rate of undocked connexon aggregation into GJs, enabling dynamic partitioning of Cx43 channel function between junctional and proximal nonjunctional domains of plasma membrane.

PubMed Disclaimer

Figures

FIGURE 1:

FIGURE 1:

ZO-1 regulates GJ accretion. (A) Representative confocal images of Cx43-HeLa cells treated with either vehicle (Veh), αCT-1, or reverse control peptide (Rev) for 2 h followed by fixation and labeling for the nucleus (blue), cytoskeleton (red), and Cx43 (green). These representative images are maximum projections of Z stacks from three separate experiments in which 15 optical fields were imaged. Cell borders are indicated by dashed lines. (B) Confocal immunofluorescent images of ZO-1–overexpressing (ZO-1OE) HeLa cells stained for the nucleus (blue), ZO-1 (red), and Cx43 (green). ZO-1OE cells are clearly distinguishable from sister cells by bright red staining. Arrows indicate examples of Cx43 GJ staining, and arrowheads highlight visible GJs in ZO-1OE cells. This representative image is from 10 optical fields taken from a single experiment. The rightmost panels in (A) and (B) are expanded views of the boxed regions in the adjacent panels to the left. (C) Quantification of GJ area from confocal images of vehicle (Veh)–, αCT-1–, and reverse control peptide (Rev)–treated cells shows that disruption of the Cx43/ZO-1 interaction acutely and significantly increases GJ size. *P < 0.05 vs. all other groups; N = 3. (D) Quantification of GJ area from confocal images of ZO-1OE cells shows that ZO-1OE decreases GJ size in comparison to “control” sister cells. GJ size was determined in control and ZO-1OE cells within the same image. (E) Quantification of Cx43 colocalized with ZO-1 showed increased superposition of Cx43 and ZO-1 signal in ZO-1OE cells. (F) Representative blots of ZO-1 (top), Cx43 (middle), and actin (bottom) immunoprecipitated with ZO-1 or control IgG. Scale bars = 10 μm.

FIGURE 2:

FIGURE 2:

ZO-1 interacts with Cx43 in the perinexus. (A) Representative confocal images of the same fields in Figure 1A, omitting nuclear fluorescence and adding Duolink fluorescence (Cx43/ZO-1 interaction; red). Actin is labeled in blue, and Cx43 is labeled in green. Vehicle (Veh), αCT-1, and reverse control peptide (Rev) are shown on the left, center, and right, respectively. For each treatment an expanded view of the boxed region in the top left panel is given in the other three panels, which show Cx43 plus actin staining (top right), Duolink alone (bottom left), and merged images (bottom right). Arrows indicate Duolink fluorescence that is adjacent to GJs but does not appear to overlap with actin. Arrowheads indicate Duolink spots that overlap with Cx43 and, frequently, actin fluorescence. (B) Representative confocal image of RECs expressing Cx43-EGFP (green) and labeled for plasma membrane (WGA; blue) and Duolink fluorescence (Cx43/ZO-1 interaction; red). The boxed region of the leftmost image is expanded in the other three panels, showing (from left to right) Cx43 plus plasma membrane fluorescence, Duolink fluorescence, and a merged image. In the rightmost panel, the dashed white line indicates the extent of Duolink stain, and the dotted black line indicates the perimeter of the GJ. We have termed the intervening perijunctional space the perinexus. (C) An analysis of the staining pattern of Duolink fluorescence colocalized with Cx43 fluorescence in the experiment represented in (A). The ratio of Duolink fluorescence overlapping with Cx43 fluorescence to total Duolink fluorescence was significantly reduced in αCT-1 as compared with vehicle. *P < 0.05 vs. Veh; N = 3. The scale bars = 10 μm except for the enlarged images in (B), which equal 5 μM. Error bars represent the standard error of the mean (SEM) for all panels.

FIGURE 3:

FIGURE 3:

The proportion of Cx43 hemichannels contributing to GJ accretion is increased by Cx43/ZO-1 disengagement. (A) Representative blot of Triton X-100–insoluble (junctional, top) and –soluble (nonjunctional, bottom), cell-surface, biotin-tagged Cx43 collected immediately after tagging (0 h) or following 2 h of treatment with Veh, αCT-1, or Rev posttagging. (B) The ratio of Triton X-100–insoluble to Triton X-100–soluble Cx43 is significantly increased by αCT-1 treatment. *P = 0.002 vs. 0 h; #P = 0.011 vs. Rev; N = 3 experiments, which represent densitometry from one blot per experiment. Error bars represent SEM.

FIGURE 4:

FIGURE 4:

αCT-1 increases GJIC as assayed by scrape loading. (A) Representative images of neurobiotin (NB; green) in scrape-loaded wild-type HeLa cells (i.e., not expressing Cx43) and Veh-, αCT-1–, and Rev-treated Cx43-Hela cells (correspondingly labeled WT, Veh, αCT-1, and Rev in the images). The nuclear labeling has been changed to red for clarity; scale bars = 25 μm. (B) Cx43-HeLa cells from all treatment groups transfer dye further than WT HeLa cells: *P < 0.03. N = 8 for WT, Veh, and αCT-1; N = 3 for Rev. Additionally, αCT-1–treated cells display significantly greater communication than control Cx43-HeLa cells: #P = 0.016 vs. Veh; †P = 0.032 vs. Rev. N numbers indicate the number of experiments performed in which the distance of dye spread was calculated from measurements taken from two replicates per treatment. Four images were examined from each replicate, and two measurements were taken from each image (one from each side of the scratch). Error bars represent SEM.

FIGURE 5:

FIGURE 5:

Inhibition of Cx43/ZO-1 increases the rate of recovery in a gap-FRAP model of GJIC. (A) Fluorescent images of calcein green fluorescence in wild-type (WT) and Cx43-HeLa cell monolayers before bleaching (Prebleach), just after bleaching (t = 0 s), and halfway through and at the end of the recovery period (t = 150 and 300 s, respectively). The Cx43-HeLa cells are treated with either vehicle (Veh), αCT-1, reverse control peptide (Rev), heptanol (Hep), or heptanol with αCT-1 (Hep + αCT). Arrows indicate the location of the bleached cell. Scale bar = 15 μm. (B) Gap-FRAP plot of bleached cell fluorescence during 300 s postbleaching. (C and D) Nonlinear regression of fluorescence data to an exponential decay function yielded values for (C) F∞ (maximal predicted fluorescence recovery) and (D) k (rate of recovery). *P < 0.02 vs. all other groups; N = 4 experiments, which are determined from three bleach fields for each treatment in each experiment. Error bars represent SEM for all panels.

FIGURE 6:

FIGURE 6:

ZO-1 siRNA knockdown mimics the effects of αCT-1. (A) Representative blots of ZO-1, Cx43, and glyceraldehyde_-_3_-_phosphate dehydrogenase from Cx43-HeLa cells under control conditions or transfected with either Sc siRNA or ZO-1 siRNA. (B and C) Densitometric analysis of (B) Cx43 protein levels shows no statistical difference between Sc and ZO-1 siRNA–transfected cells, whereas (C) ZO-1 levels are significantly reduced by ZO-1 siRNA, *P = 0.002; N = 4 separate cultures transfected with ZO-1 siRNA. (D) Gap-FRAP plot of bleached cell fluorescence during 175 s postbleaching from Sc siRNA– or ZO-1 siRNA–transfected Cx43-HeLa monolayers. (E and F) Nonlinear regression to fluorescence data yielded values for (E) maximal predicted recovery, F∞, and (F) the rate constant, k. *P = 0.05; N = 3 experiments performed on three cultures transfected in parallel with those analyzed in A–C. Measurements were determined from three separate bleach fields in each experiment from each treatment condition. Error bars represent SEM for all panels.

FIGURE 7:

FIGURE 7:

Disruption of the Cx43/ZO-1 interaction reduces the rate of EtdBr uptake in Cx43-HeLa cell monolayers but not individual, noncontacting cells. (A) Representative images from a live-cell EtdBr uptake experiment performed on vehicle (Veh)–treated Cx43-HeLa cell monolayers or single cells during 20 min. Scale bar = 15 μm. (B and C) Plots of measured EtdBr fluorescence change over a 20 min period from (B) monolayers or (C) individual wild-type HeLa (WT) cells and Cx43-HeLa cells treated with Veh, αCT-1, or reverse control peptide (Rev). (D and E) Statistical analysis of rate data from B and C, respectively. (D) *P < 0.002 vs. WT; #P = 0.001 vs. Veh; †P = 0.009 vs. Rev; N = 3. (E) *P < 0.02 vs. WT; N = 3. Error bars represent SEM for all panels. N numbers for both monolayer and noncontacting cultures indicate the number of separate experiments performed in which six different fields were monitored and EtdBr uptake was quantified.

FIGURE 8:

FIGURE 8:

ZO-1 knockdown and αCT-1 both inhibit EtdBr uptake in a hemichannel-specific manner. (A) EtdBr taken up after 15 min in monolayers of Cx43- and wild-type HeLa (WT) cells treated with vehicle (Veh), αCT-1, or reverse control peptide (Rev). *P < 0.004 vs. any treatment of WT HeLa cells; #P = 0.0002 vs. Veh; †P = 0.011 vs. Rev; N = 4. (B) EtdBr taken up after 15 min in monolayers of Cx43- and WT HeLa cells treated with Veh, BGA, or MFQ. *P = 0.037 vs. Veh; #P < 0.0004 vs. Veh; N = 3. (C) EtdBr taken up after 15 min in monolayers of Cx43- and WT HeLa cells treated with Veh or EGTA. *P < 0.0001 vs. any treatment of WT HeLa cells; #P = 0.02 vs. Veh; N = 15, 11, and 4 for Cx43-HeLa Veh, Cx43-HeLa EGTA, and both WT HeLa treatments, respectively. (D) EtdBr taken up after 15 min in monolayers of Cx43-HeLa cells treated with either Sc siRNA or ZO-1 siRNA. *P = 0.02; N = 3. N numbers indicate the number of experiments performed in which measurements were taken on five images per experiment for each treatment type. Error bars represent SEM for all panels.

FIGURE 9:

FIGURE 9:

The connexon switch. When ZO-1 is bound to Cx43 at the periphery of GJ plaques and hemichannels in the perinexus—“connexon switch inhibited”—hemichannel addition to the plaque perimeter is restricted. When Cx43/ZO-1 interaction is inhibited (as with αCT-1)—“connexon switch uninhibited”—GJ accretion from hemichannels is unchecked, resulting in larger GJs and fewer hemichannels. The predicted functional consequences of disengaging Cx43 and ZO-1 are greater GJIC (red arrows) and reduced hemichannel activity (blue arrows).

References

    1. Abbaci M, Barberi-Heyob M, Blondel W, Guillemin F, Didelon J. Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication. Biotechniques. 2008;45:33–52–56–62. - PubMed
    1. Abbaci M, Barberi-Heyob M, Stines JR, Blondel W, Dumas D, Guillemin F, Didelon J. Gap junctional intercellular communication capacity by gap-FRAP technique: a comparative study. Biotechnol J. 2007;2:50–61. - PubMed
    1. Akoyev V, Takemoto DJ. ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43. Cell Signal. 2007;19:958–967. - PMC - PubMed
    1. Baker SM, Kim N, Gumpert AM, Segretain D, Falk MM. Acute internalization of gap junctions in vascular endothelial cells in response to inflammatory mediator-induced G-protein coupled receptor activation. FEBS Lett. 2008;582:4039–4046. - PMC - PubMed
    1. Bastide B, Herve JC, Cronier L, Deleze J. Rapid onset and calcium independence of the gap junction uncoupling induced by heptanol in cultured heart cells. Pflugers Arch. 1995;429:386–393. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources