title: Gap junction assembly: roles for the formation plaque and regulation by the C-terminus of connexin43.
abstract: Using an established gap junction (GJ) assembly system with experimentally reaggregated cells, we analyzed "formation plaques" (FPs), apparent sites of GJ assembly. Employing freeze-fracture electron microscopy methods combined with filipin labeling of sterols and immunolabeling for connexin43 (Cx43), we demonstrated that FPs constitute distinct membrane "domains" and that their characteristic 10-nm particles contain connexin43, thus representing precursors (i.e., GJ hemichannels) engaged in assembly. Analysis of FPs in new systems-HeLa and N2A cells-resolved questions surrounding several key but poorly understood steps in assembly, including matching of FP membranes in apposed cells, reduction in the separation between FP membranes during assembly, and the process of particle aggregation. Findings also indicated that "docking" of GJ hemichannels occurs within FP domains and contributes to reduction of intermembrane separation between FPs. Other experiments demonstrated that FPs develop following a major C-terminal truncation of Cx43 (M257), although assembly was delayed. Particle aggregation also occurred at lower densities, and densities of particles within developing GJ aggregates failed to achieve full-length levels. With regard to regulation, inhibition of assembly following protein kinase C activation failed to occur in the M257 truncation mutants, as measured by intercellular dye transfer. However, several C-terminal serine mutations failed to disrupt inhibition.
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA. gaplab@umn.edu
authors: Ross G
authors: RG
authors: Johnson
authors: James K
authors: JK
authors: Reynhout
authors: Erica M
authors: EM
authors: TenBroek
authors: Bradley J
authors: BJ
authors: Quade
authors: Thomas
authors: T
authors: Yasumura
authors: Kimberly G V
authors: KG
authors: Davidson
authors: Judson D
authors: JD
authors: Sheridan
authors: John E
authors: JE
authors: Rash
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D018031:Connexin 43 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005372:Filipin / Q000737:chemistry; D022001:Focal Adhesions / Q000378:metabolism* / Q000648:ultrastructure; D005614:Freeze Fracturing; D017629:Gap Junctions / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D018031:Connexin 43; D008563:Membrane Lipids; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005372:Filipin; D002784:Cholesterol; D011493:Protein Kinase C