Visualization of Annular Gap Junction Vesicle Processing: The Interplay Between Annular Gap Junctions and Mitochondria

Abstract

It is becoming clear that in addition to gap junctions playing a role in cell⁻cell communication, gap junction proteins (connexins) located in cytoplasmic compartments may have other important functions. Mitochondrial connexin 43 (Cx43) is increased after ischemic preconditioning and has been suggested to play a protective role in the heart. How Cx43 traffics to the mitochondria and the interactions of mitochondria with other Cx43-containing structures are unclear. In this study, immunocytochemical, super-resolution, and transmission electron microscopy were used to detect cytoplasmic Cx43-containing structures and to demonstrate their interactions with other cytoplasmic organelles. The most prominent cytoplasmic Cx43-containing structures-annular gap junctions-were demonstrated to form intimate associations with lysosomes as well as with mitochondria. Surprisingly, the frequency of associations between mitochondria and annular gap junctions was greater than that between lysosomes and annular gap junctions. The benefits of annular gap junction/mitochondrial associations are not known. However, it is tempting to suggest, among other possibilities, that the contact between annular gap junction vesicles and mitochondria facilitates Cx43 delivery to the mitochondria. Furthermore, it points to the need for investigating annular gap junctions as more than only vesicles destined for degradation.

Keywords: annular gap junction vesicle; connexin; gap junction; lysosome; mitochondria.

Figure 1

Localization of gap junction plaques and annular gap junction vesicles. (A) Immunocytochemistry of endogenous Cx43 (red) and cortical actin (green). Cortical actin (green) staining was used to define the boundaries of the cell and to aid in distinguishing intracellular annular gap junction puncta (red, arrow) from surface gap junction plaques (red, arrowhead). (B) Pseudocolored confocal microscopic image of Cx43-GFP from a Time-lapse frame with gap junction plaques (arrowhead) and annular gap junction vesicles (arrow) demonstrate the differences in Cx43-GFP intensities. (C) Pseudocolored super-resolution microscopic image demonstrating Cx43-GFP intensities of annular gap junction (arrow) and secretory vesicles (broken arrow). The Look Up Table (LUT) scale in B and C indicates the intensities of GFP in the gap junction structures. (D,E) TEM of a gap junction plaque (D) and annular gap junction vesicles (E) identified, depending on the plane of focus, by typical pentalaminar membrane (white arrow head or arrow) or the appearance of the densely stained double membrane (black arrowhead and arrow). (F) Bizarre annular gap junction vesicle with annular structure and other unknown material within the lumen. The pentalaminar membrane (white arrows) and immuno-electron microscopic Cx-43 Q-dots labeling (black arrows) was used to identify the annular gap junction vesicle. Scale Bars = (A,B):10 µm, (C): 5 µm, (D–F): 100 nm.

Figure 2

Time-lapse imaging of transfected cells expressing Cx43-GFP. (A,B) Differential interference contrast (DIC)/fluorescein isothiocyanate (FITC) overlay images of cell populations expressing Cx43-GFP. (A) An annular gap junction vesicle (arrow) and gap junction plaque (green) are evident. (B) Annular gap junctions (green) that were in contact with unidentified dense cytoplasmic structures (arrows) can be seen. (C–H) Montage of selected Time-lapse images collected at 1 min intervals. The gap junction plaque between two cells is evident as well as the cytoplasmic annular gap junction (arrows, (G,H)). The invagination of the gap junction plaque (arrows, (C–F)) and the subsequent release of an annular gap junction vesicle into the cytoplasm of one of the two contacting cells could be followed over time and the cell morphology appreciated by the DIC/FITC over lay image (A). (I) Flow chart summary of Time-lapse observations depicting annular gap junction formation and behavior. Scale Bars (A–H): 10 µm.

Figure 3

Localization of annular gap junction vesicles and lysosomes. (A) Immunocytochemical localization of endogenous Cx43 (green) and lysosomes detected with LAMP1 (red) demonstrates the colocalization (yellow) of an annular gap junction vesicle and lysosome (arrow). (B–G) The intimate contact between annular gap junction (AGJ) and lysosomes, which are in various stages of degradation is shown. In B (and enlarged in D) the outer membrane of the annular gap junction can be seen to be continuous with that of the lysosomal membrane (white arrows). Clathrin, identified by the bristle coat (arrows, (C)) or quantum dot label (black arrows, (E)), can be seen associated with annular gap junction vesicles that are fused with lysosomes. Typical clathrin-coated vesicles also can be seen decorated with clathrin Q dots (dashed white arrows in (E)). (F) (enlarged from (G)) and (D) (enlarged from (B)) have been magnified to allow the annular gap junction pentalaminar membranes to be clearly seen (black arrows) and the change in this membrane at the annular/lysosomal junction to be appreciated (white arrows in (F,D)) N = Nucleus; Scale Bars = (A): 10 µm, (B,C,E–G): 100 nm, (D): 20 nm.

Figure 4

Mitochondria associate with annular gap junctions as seen with standard light (A), super-resolution STED (B), and TEM microscopy (C). (A,B) Immunocytochemical analysis of mitochondria, with Mitotracker (red in (A)) or with antibody to TOM (red in (B)) and endogenous Cx43 gap junction antigen detected with antibody staining (green in (A,B)). The colocalization (yellow, white arrows) of mitochondria (red) and annular gap junctions (green) can be seen. (B) Note the gap junction plaque as well as small puncta at the cell surface (arrowheads) that are indicative of gap junction plaque formation. The image in (B) has been magnified three times (boxes) to show the lumen of an annular gap junction vesicle and the contact between mitochondria and annular gap junctions yellow, white arrows). (C) The intimate association between the mitochondria and annular gap junction (white box) seen with TEM. Scale bars = (A): 10 µm, (B): 1000 nm, (C) 100 nm.

Figure 5

Transmission electron microscopy of mitochondria closely associated with annular gap junctions. (A–E) Areas of contact between annular gap junctions (AGJ) and mitochondria (M) are indicated within the black boxes (A,B,D,E). (C) The annular gap junction vesicle seen in (B), but imaged at a different section plane, is enlarged to better show the contact areas. Scale Bars: 500 nm.