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Vol. 10, Issue 6, 2033-2050, June 1999
§
and
*Department of Anatomy and Cell Biology, University of Western
Ontario, London, Ontario, Canada N6A 5C1; To examine the trafficking, assembly, and turnover of connexin43
(Cx43) in living cells, we used an enhanced red-shifted mutant of green
fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA
encoding Cx43-GFP was transfected into communication-competent normal
rat kidney cells, Cx43-negative Madin-Darby canine kidney (MDCK)
cells, or communication-deficient Neuro2A or HeLa cells, the fusion
protein of predicted length was expressed, transported, and assembled
into gap junctions that exhibited the classical pentalaminar profile.
Dye transfer studies showed that Cx43-GFP formed functional gap
junction channels when transfected into otherwise
communication-deficient HeLa or Neuro2A cells. Live imaging of
Cx43-GFP in MDCK cells revealed that many gap junction plaques remained
relatively immobile, whereas others coalesced laterally within the
plasma membrane. Time-lapse imaging of live MDCK cells also revealed
that Cx43-GFP was transported via highly mobile transport intermediates
that could be divided into two size classes of <0.5 µm and 0.5-1.5
µm. In some cases, the larger intracellular Cx43-GFP transport
intermediates were observed to form from the internalization of gap
junctions, whereas the smaller transport intermediates may represent
other routes of trafficking to or from the plasma membrane. The
localization of Cx43-GFP in two transport compartments suggests that
the dynamic formation and turnover of connexins may involve at least
two distinct pathways.
Fred Hutchinson
Cancer Research Center, Seattle, Washington, 98109;
Department of Anatomy and Cell Biology, McGill
University, Montreal, Quebec, Canada H3A 2B2; and
Department of Pediatric Dentistry, School of Dental
Medicine, University of Connecticut Health Center, Farmington,
Connecticut 06030
Online version of this article contains video material
for Figures 9-11. Online version available at www.molbiolcell.org.
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