Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells

Abstract

Certain membrane channels including acetylcholine receptors, gap junction (GJ) channels, and aquaporins arrange into large clusters in the plasma membrane (PM). However, how these channels are recruited to the clusters is unknown. To address this question, we have investigated delivery of GJ channel subunits (connexons) assembled from green fluorescent protein (GFP)-tagged connexin 43 (Cx43) to the PM and GJs in living cells. Fluorescence-photobleaching of distinct areas of Cx43-GFP GJs demonstrated that newly synthesized channels were accrued to the outer margins of channel clusters. Time-lapse microscopy further revealed that connexons were delivered in vesicular carriers traveling along microtubules from the Golgi to the PM. Routing and insertion of connexons occurred predominantly into the nonjunctional PM. These PM connexons can move laterally as shown by photobleaching and thus, can reach the margins of channel clusters. There, the apposing PMs are close enough to allow connexons to dock into complete GJ channels. When connexon delivery to the PM was inhibited by brefeldin A, or nocodazole pretreatment, the PM pool initially enabled connexon accrual to the clusters but further accrual was inhibited upon depletion. Taken together, our results indicate that GJ channel clusters grow by accretion at their outer margins from connexon subunits that were delivered to the nonjunctional PM, and explain how connexons in the PM can function in intra-͞extracellular signaling before GJ channel formation and direct cell-cell communication.Cx43 ͉ GFP ͉ photo-bleaching ͉ secretion ͉ time-lapse microscopy

Internalization of Large Double-Membrane Intercellular Vesicles by a Clathrin-dependent Endocytic Process

“…About 12 h posttransfection, GJ plaques were detectable in the PMs between contacting cells (marked with arrows in Figure 1A, left). In the Golgi region (marked with asterisks in Figure 1A, left), we also observed vesicular Cx43-GFP signal, which we have characterized previously as secretory Cx cargo destined for delivery to the PM (Lauf et al, 2002). At later time points (24 -40 h posttransfection), the secretory Cx cargo signal was diminished.…”

“…However, previous studies analyzed the assembly and turnover of channels within GJ plaques (Gaietta et al, 2002;Lauf et al, 2002), or only small fragments of GJ plaques were observed to internalize within seconds, or a few minutes (Jordan et al, 2001; our unpublished data). Although the process of GJ channel removal from plaques remains unclear, published work indicates that channels within a plaque are turned over continuously.…”

The Second PDZ Domain of Zonula Occludens-1 Is Dispensable for Targeting to Connexin43 Gap Junctions

“…However, the molecular mechanisms that govern GJ remodeling are starting to emerge. Elegant fluorescence imaging has indicated that accretion of connexin channels (connexons) likely occurs predominately at plaque edges (Gaietta et al 2002;Lauf et al 2002). Given the topology of GJs, peripheral accretion makes sense, and points to the GJ edge as 56 A. W. HUNTER AND R. G. GOURDIE a specialized assembly domain.…”

New roles for astrocytes: Gap junction hemichannels have something to communicate

“…Brefeldin A, which disrupts the Golgi apparatus, prevents the trafficking of Cx32 and Cx43; however, Cx26 still reaches the cell surface. Now, refined methods of labeling indicate that new channels are transported to the surface in an undirected manner and reach existing junctions by lateral diffusion, where they form new channels at their periphery [29,30]; channels being retired are removed from the interior of the junction [29]. This picture does not address the question of the initial formation of junctions [7].…”