Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules.
The role of cytoskeletal elements in gap junction (GJ) assembly has been studied using Novikoff hepatoma cells treated with cytochalasin B (CB) to disrupt actin filaments or with colchicine or nocodazole to disrupt microtubules. After 60 min of cell reaggregation, freeze-fracture was used to evaluate quantitatively the "initiation," "maturation," and "growth" phases of GJ assembly. The development of junctional permeability to fluorescent dyes was also analyzed. The only effects of CB on the structure or permeability of the developing junctions involved an elongation of GJ aggregates and a small decrease in formation plaque areas. Colchicine (but not the inactive form, lumicolchicine) prevented the enhancement of GJ growth by cholesterol, but its effect on basal growth was equivocal. Nocodazole inhibited the growth of GJ, even under basal conditions, without an effect on initiation. Nocodazole also blocked the forskolin-enhanced increase in the growth of GJs and, in living MDCK cells, reduced the movement of transport intermediates containing green fluorescent protein-tagged connexin43. Thus, neither actin filaments nor microtubules appear to restrict GJ assembly by anchoring intramembrane GJ proteins, nor are they absolutely required for functional GJs to form. However, microtubules are necessary for enhanced GJ growth and likely for facilitating connexin trafficking under basal conditions.
Johnson
Ross G
RG
Department of Genetics, Cell Biology, and Development, University of Minnesota, St. Paul, Minnesota 55108, USA. gaplab@tc.umn.edu
Meyer
Rita A
RA
Li
Xin-Ren
XR
Preus
Doris M
DM
Tan
Lana
L
Grunenwald
Haiying
H
Paulson
Alicia F
AF
Laird
Dale W
DW
Sheridan
Judson D
JD
D000199:Actins / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D000818:Animals; D002449:Cell Aggregation / Q000187:drug effects; D002460:Cell Line; D003078:Colchicine / Q000494:pharmacology; D018031:Connexin 43 / Q000502:physiology; D003571:Cytochalasin B / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D004285:Dogs; D005614:Freeze Fracturing; D017629:Gap Junctions / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D008114:Liver Neoplasms, Experimental; D008164:Luminescent Proteins; D008870:Microtubules / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D015739:Nocodazole / Q000494:pharmacology; D051381:Rats; D014407:Tumor Cells, Cultured
D000199:Actins; D018031:Connexin 43; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D003571:Cytochalasin B; D015739:Nocodazole; D003078:Colchicine