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

Abstract

Beyond its well-documented role in vesicle endocytosis, clathrin has also been implicated in the internalization of large particles such as viruses, pathogenic bacteria, and even latex beads. We have discovered an additional clathrin-dependent endocytic process that results in the internalization of large, double-membrane vesicles at lateral membranes of cells that are coupled by gap junctions (GJs). GJ channels bridge apposing cell membranes to mediate the direct transfer of electrical currents and signaling molecules from cell to cell. Here, we report that entire GJ plaques, clusters of GJ channels, can be internalized to form large, double-membrane vesicles previously termed annular gap junctions (AGJs). These internalized AGJ vesicles subdivide into smaller vesicles that are degraded by endo/lysosomal pathways. Mechanistic analyses revealed that clathrin-dependent endocytosis machinery-components, including clathrin itself, the alternative clathrin-adaptor Dab2, dynamin, myosin-VI, and actin are involved in the internalization, inward movement, and degradation of these large, intercellular double-membrane vesicles. These findings contribute to the understanding of clathrin's numerous emerging functions. INTRODUCTIONThe role of clathrin in endocytosis is well documented. This protein forms a typical curved lattice around endocytic vesicles that are internalized at the plasma membrane (PM). In addition, the involvement of clathrin in several uncharacteristic endocytic processes has been reported, including the internalization of viruses, pathogenic bacteria, and large latex beads (Aggeler and Werb, 1982;Ehrlich et al., 2004;Rust et al., 2004;Veiga and Cossart, 2005). Here, we describe another function of the clathrin-dependent endocytic machinery that results in the internalization of large, doublemembrane vesicles at lateral PMs of cells that are coupled by gap junctions (GJs).GJs are ubiquitously distributed channels that connect the cytoplasms of two apposing cells each participating in this connection via a half channel termed a connexon to provide direct cell-to-cell communication. Connexons are hexamers of four-pass membrane proteins called connexins (Cxs;Bruzzone et al., 1996;Kumar and Gilula, 1996). Once transported to the PM, GJ channels cluster into two-dimensional arrays termed plaques that can be composed of a few to many thousands of individual channels and vary from a few square nanometers to many square micrometers (Bruzzone et al., 1996; Falk, 2000a;Severs et al., 2001). GJ channels can open and close (gate) and physiological parameters, including intracellular pH, Ca 2ϩ concentration, and Cx phosphorylation, are known to modulate GJ channel gating and the extent of GJ-mediated intercellular coupling (Delmar et al., 2004;Lampe and Lau, 2004;Moreno, 2005). However, the extent of intercellular coupling could also be regulated through altering the number of GJ channels in the PM.Cxs have a surprisingly short half-life of only 1-5 h, leading to a rapid GJ and Cx protein turnover (Fallon and Goodeno...

Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

“…The latter is consistent with findings by Leithe et al (50) that proteasomal degradation occurs after stimulation of PKC with TPA. Cx43 has been shown to co-localize with clathrin, AP-2, Dab2, and dynamin2 at gap junction plaques (51)(52)(53). Additionally, the loss of dynamin GTPase activity or the reduction of dynamin2, clathrin, AP-2, or Dab2 proteins inhibited the internalization of Cx43, as observed by a significant decrease in the number of annular junctions.…”

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

“…The exogenous expression of fluorescently tagged gap junction proteins, in particular, Cx43, has clearly demonstrated that vesicles containing connexons are closely associated with microtubules and actin filaments and move linearly within cells (28). Moreover, cytoskeletal disrupting agents have been shown to interfere with normal connexin delivery to the plasma membrane (5,6,29).…”

Connexins, cell motility, and the cytoskeleton

“…Mechanistic analyses revealed that components of the clathrin-dependent endocytosis machinery are directly involved in the internalization, inward movement, and initial degradation of these intercellular double-membrane vesicles. The components described in this study include clathrin, the alternative clathrin-adaptor Dab2, the GTPase dynamin, the unconventional myosin-VI, and actin filaments [Piehl et al, 2007]. The mechanism by which Cx43 GJ plaques are internalized might involve Src-mediated dissociation of ZO-1 from one side of the GJ plaque [Gilleron et al, 2008].…”