Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules

Ross G. Johnson; Rita A. Meyer; Xin Ren Li; Doris M. Preus; Lana Tan; Haiying Grunenwald; Alicia F. Paulson; Dale W. Laird; Judson D. Sheridan

doi:10.1006/excr.2002.5480

Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules

Ross G. Johnson, Rita A. Meyer, Xin Ren Li, Doris M. Preus, Lana Tan, Haiying Grunenwald, Alicia F. Paulson, Dale W. Laird, Judson D. Sheridan

Genetics, Cell Biology and Development (TMED)

Research output: Contribution to journal › Article › peer-review

72 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	67-80
Number of pages	14
Journal	Experimental Cell Research
Volume	275
Issue number	1
DOIs	https://doi.org/10.1006/excr.2002.5480
State	Published - 2002

Bibliographical note

Funding Information:
The authors are grateful to Pete Lefebvre and Carolyn Silflow for advice regarding relevant work on the cytoskeleton, to Tom Hays for providing the tubulin antibodies, and to Mike Atkinson and Erica TenBroek for helpful comments on the manuscript. This work was supported by Grant GM-46277 from the National Institutes of Health to R.G.J. and Grant MT-12241 from the Medical Research Council of Canada to D.W.L.

Keywords

Actin filament
Cell communication
Connexin43
Dye injection
Freeze-fracture
Gap junction
Microtubule

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title = "Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules",

abstract = "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.",

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note = "Funding Information: The authors are grateful to Pete Lefebvre and Carolyn Silflow for advice regarding relevant work on the cytoskeleton, to Tom Hays for providing the tubulin antibodies, and to Mike Atkinson and Erica TenBroek for helpful comments on the manuscript. This work was supported by Grant GM-46277 from the National Institutes of Health to R.G.J. and Grant MT-12241 from the Medical Research Council of Canada to D.W.L.",

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T1 - Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules

AU - Johnson, Ross G.

AU - Meyer, Rita A.

AU - Li, Xin Ren

AU - Preus, Doris M.

AU - Tan, Lana

AU - Grunenwald, Haiying

AU - Paulson, Alicia F.

AU - Laird, Dale W.

AU - Sheridan, Judson D.

N1 - Funding Information: The authors are grateful to Pete Lefebvre and Carolyn Silflow for advice regarding relevant work on the cytoskeleton, to Tom Hays for providing the tubulin antibodies, and to Mike Atkinson and Erica TenBroek for helpful comments on the manuscript. This work was supported by Grant GM-46277 from the National Institutes of Health to R.G.J. and Grant MT-12241 from the Medical Research Council of Canada to D.W.L.

PY - 2002

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N2 - 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.

AB - 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.

KW - Actin filament

KW - Cell communication

KW - Connexin43

KW - Dye injection

KW - Freeze-fracture

KW - Gap junction

KW - Microtubule

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SN - 0014-4827

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EP - 80

JO - Experimental Cell Research

JF - Experimental Cell Research

IS - 1

ER -

Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules

Abstract

Bibliographical note

Keywords

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