Visualizing the effect of dynamin inhibition on annular gap vesicle formation and fission

Abstract

SummaryAlthough gap junction plaque assembly has been extensively studied, mechanisms involved in plaque disassembly are not well understood. Disassembly involves an internalization process in which annular gap junction vesicles are formed. These vesicles undergo fission, but the molecular machinery needed for these fissions has not been described. The mechanoenzyme dynamin has been previously demonstrated to play a role in gap junction plaque internalization. To investigate the role of dynamin in annular gap junction vesicle fission, immunocytochemical, time-lapse and transmission electron microscopy were used to analyze SW-13 adrenocortical cells in culture. Dynamin was demonstrated to colocalize with gap junction plaques and vesicles. Dynamin inhibition, by siRNA knockdown or treatment with the dynamin GTPase inhibitor dynasore, increased the number and size of gap junction 'buds' suspended from the gap junction plaques. Buds, in control populations, were frequently released to form annular gap junction vesicles. In dynamin-inhibited populations, the buds were larger and infrequently released and thus fewer annular gap junction vesicles were formed. In addition, the number of annular gap junction vesicle fissions per hour was reduced in the dynamin-inhibited populations. We believe this to be the first report addressing the details of annular gap junction vesicle fissions and demonstrating a role of dynamin in this process. This information is crucial for elucidating the relationship between gap junctions, membrane regulation and cell behavior.

Redistribution of connexin 43 during cell division

“…Here, we have demonstrated that the cytoplasmic Cx43 punctate staining observed with immunofluorescence correlated in size, with the double membrane annular gap junction vesicles detected with Q‐dot Cx43 immunoelectron microscopic techniques, and not with single membrane vesicles such as endosomes or secretory vesicles. We and others have demonstrated with time lapse microscopy that annular gap junction vesicles result from the internalization of gap junction plaques into one of two contacting cells (Murray et al, ; Jordan et al, , ; Lauf et al, ; Nickel et al, ). In this study, the annular gap junction vesicle membranes were labeled with Cx43 Q‐dots.…”

“…The loss of gap junction plaques could reflect either the disaggregation of gap junction plaques by lateral movement of channels in the membrane or the recovery of channels from an intracellular site, both events independent of Cx43 synthesis. It has been well established, however, that portions or entire gap junction plaques can be removed from the cell surface (termed “endoexocytosis”) to form spherical, cytoplasmic, double‐membraned annular gap junction vesicles in one of the two contacting cells (Larsen and Hai, ; Larsen et al, ; Jordan et al, ; Murray et al, ; Piehl et al, ; Nickel et al, , ). We suggest that the removal of gap junctions from the cell surface during mitosis also occurs by an endoexocytotic process that results in the formation of annular gap junctions.…”

Connexin 43 is an emerging therapeutic target in ischemia/reperfusion injury, cardioprotection and neuroprotection

“…A gap junction can be internalized in its entirety via formation of a double membrane structure termed an annular junction (Archard & Denys, 1979; Fong et al, 2012; Johnson et al, 2013; Jordan et al, 2001; Laird, 2006; Leithe et al, 2006; Nickel et al, 2013; Piehl et al, 2007; Severs et al, 1989) or via loss of extracellular Cx interactions followed by endocytosis of the remaining connexon from a single membrane. During internalization, Cx43 can be phosphorylated by c-Src and inhibition of c-Src activity via protein phosphatase 2 blocks growth factor-induced gap junction turnover (Gilleron et al, 2008; Spinella et al, 2003).…”

“…The formation of double membrane endocytic vesicles (i.e., annular junctions) appears to be fairly specific to gap junctions though there are a few reports of “trans-endocytosis” occurring in dendritic cells (Spacek & Harris, 2004) and in response to receptor ligand complex formation during neural (Marston et al, 2003) and Drosophila development (Klueg et al, 1998). Cx43-containing annular junctions have been well-documented (Archard & Denys, 1979; Fong et al, 2012; Johnson et al, 2013; Jordan et al, 2001; Laird, 2006; Leithe et al, 2006; Nickel et al, 2013; Piehl et al, 2007; Severs et al, 1989) but the role Cx43 phosphorylation plays is still unclear.…”

Fast structural responses of gap junction membrane domains to AB5 toxins

“…Formation of GJs requires vesicular transport and in some cases Golgi-dependent posttranslational modifications of connexins (10). GJ turnover involves endocytosis and proteasomal/lysosomal degradation (11)(12)(13)(14). During infection and under stress conditions the level of signaling molecules is elevated dramatically, and how signaling via GJs is modulated between affected cells remains unclear.…”