In Vitro Motility of Liver Connexin Vesicles along Microtubules Utilizes Kinesin Motors

Abstract

Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin-and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 m/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 M of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 M ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4 -0.5 m/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 M vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.Gap junction channels form direct intercellular passageways that allow the exchange of ions and second messenger molecules between cells, thereby spreading signals and unifying cellular responses within a tissue. Gap junctions are formed by the connexin family of proteins that are co-translationally inserted into endoplasmic reticulum membranes (1) and assembled into hexameric connexons or hemichannels within the endoplasmic reticulum or Golgi compartments depending on connexin, cell type, or both (2-6). Connexins are tetra-spanning transmembrane proteins with both termini (N termini and C termini) facing the cytoplasm. This conformation results in two extracellular loops required for connexon docking and one cytoplasmic loop.Knowing the mechanisms of delivery is crucial to understanding connexin regulation because insertion into the plasma membrane is a prerequisite to connexin function. The route of delivery of connexons from the trans-Golgi to the surface membrane has been the subject of many studies, most recently focusing on exogenously expressed connexins with carboxylterminally tagged fluorescent proteins. These studies have indicated that the vesicular structures in which connexins are delivered to the surface occur in a variety of sizes and are motile. Moreover, although delivery appears to be facilitated by microtubules, intact cytoskeleton is not absolutely required for trafficking, as evidenced by the variable effects reported for microtubule-disrupt...

Trafficking Highways to the Intercalated Disc: New Insights Unlocking the Specificity of Connexin 43 Localization

“…Using live-cell imaging, we determined the time course and directionality of Cx43 cargo movement relative to actin. Cx43 cargo slowed down when associated with actin, with the majority traveling at speeds slower than that of microtubule-based transport (<0.25 μm/s) (Fort et al, 2011; Shaw et al, 2007), but consistent with myosin-based transport on actin (Howard, 1997). This finding is consistent with previous studies of actin–myosin based transport of melanosomes, slowing of endocytic vesicles at actin-rich regions in the cell cortex (Aschenbrenner et al, 2004; Ross et al, 2008), and Cx32 pausing at actin structures en route to the hepatocyte cell surface (Fort et al, 2011).…”

Glucose Regulates Mitochondrial Motility via Milton Modification by O-GlcNAc Transferase

“…1) The locus of the inhibitory regulation is not in the motors themselves but in Milton, which is specific to mitochondria. 2) Changing extracellular glucose from 5.5 to 30mM has been calculated to increase ATP three-fold in hippocampal neurons (Huang et al, 2007) and raising ATP levels enhances the activity of kinesin and dynein, which require ATP hydrolysis for force generation (Fort et al, 2011; Visscher et al, 1999). Therefore, in the absence of the overriding influence of Milton GlcNAcylation, cargo transport will increase with increased glucose availability, a phenomenon we also observed for mitochondria when the action of OGT was prevented by expression of hMilton1 Qmut (Figure 6).…”

GJA1-20k Arranges Actin to Guide Cx43 Delivery to Cardiac Intercalated Discs

“…It is understood that Cx43 hemichannels oligomerize in the trans-Golgi network 12 , and are subsequently packaged into vesicles transported by motor proteins along microtubules 13–15 . Our previous work identified a targeted delivery paradigm 16–18 , by which the specificity of Cx43 trafficking is in part achieved by the interaction between microtubule plus-end binding proteins and the membrane scaffolding proteins of the intercalated disc 17, 18 .…”