Connecting Exosomes and Connexins

Abstract

Intercellular communication is accomplished by passage of ions and small molecules through gap junction channels in directly contacting cells or by secretion and response to transmitters, hormones and extracellular vesicles in cells that are distant from each other. Recent studies have suggested that there may be overlap of these processes; specifically, small extracellular vesicles may contain subunit gap junction proteins, connexins. We isolated and analyzed extracellular vesicles secreted by cultured microvascular endothelial cells. These vesicles had a diameter of ~120 nm. They contained four exosomal proteins (flotillin-1, CD63, CD81 and Alix) and the gap junction protein, connexin43. They did not contain an endoplasmic reticulum protein (Grp94) or an adherens junction protein (VE-cadherin). Secretion of vesicles was increased by treatment of the cells with staurosporine. Our data confirm that the gap junction protein, connexin43, can be secreted in vesicles with the properties of exosomes. Although the role of vesicular connexin is not clearly known, we speculate that it might participate in docking/fusion of the exosomes with the recipient cell, transmission of vesicular contents, or cellular signaling.

Keywords: connexin; endothelial cell; exosome; extracellular vesicle; gap junction; intercellular communication.

Figure 1

Microvascular endothelial cells have intercellular junctions containing Cx43 and VE-cadherin. (A,B) Immunolocalization of Cx43 (yellow in A, red in B). (C,D) Immunolocalization of VE-cadherin (green). Panels B and D were also stained with DAPI to visualize nuclei (blue). Bar, 10 µm.

Figure 2

Vesicles secreted from cultured microvascular endothelial cells have properties consistent with identification as exosomes and contain Cx43. (A) Screen shot showing single frame of a video from the nanoparticle tracking analysis of the 100,000× g material. (B) Graph shows a representative profile of the extracellular vesicles in the 100,000× g pellet from the nano-tracking analysis. For this example, the mode was 120.7 nm; mean ± SD was 154 ± 54 nm; 10% of the vesicles were <98 nm; 50% were <141 nm; and 90% were <226 nm. (C) Immunoblots were performed on an hCMEC/D3 cell homogenate and the pellets obtained by sequential centrifugation of conditioned medium at increasing forces (300× g, 2000× g, 10,000× g and 100,000× g for the times noted in Materials and Methods). To facilitate comparisons, the gels were loaded with comparable percentages of the pellets from the sequential spins. The 100,000× g material contained the exosomal markers (flotillin-1, CD63, CD81 and Alix) as well as Cx43, but it did not contain detectable amounts of the endoplasmic reticulum protein Grp94 or VE-cadherin.

Figure 3

Cx43-expressing primary cells and cell lines secrete extracellular vesicles containing Cx43. Cell homogenates and the pellets from 100,000× g centrifugation of conditioned media were prepared as described for hCMEC/D3 cells in Materials and Methods from (1) human dermal microvascular endothelial cells, (2) human dermal fibroblasts, (3) human oligodendroglioma (HOG) cells, and (4) human glioblastoma (A172) cells. Samples were resolved by SDS-PAGE and transferred to membranes that were reacted with anti-Cx43 antibodies as described in Materials and Methods. Immunoblots show the presence of Cx43 protein in all cell homogenates and secreted vesicles.

Figure 4

Treatment with staurosporine increases secretion of vesicles containing exosomal proteins and Cx43. (A) Cells were treated for 18 h with different concentrations of staurosporine (as indicated). The 100,000× g pellets were isolated by centrifugation of the culture medium, they were resuspended, and, for each blot, identical volumes were immunoblotted to detect Cx43, flotillin-1 and CD63. (B) Graph shows the profile from the nanoparticle tracking analysis of the extracellular vesicles in the 100,000× g pellet prepared from cells treated with 10 nM staurosporine. For this profile, the mode was 141.6 nm; mean ± SD was 161 ± 57 nm; 10% were <101 nm; 50% were <148 nm; and 90% were <241 nm.