In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors

Alfredo G. Fort, John W. Murray, Nadine Dandachi, Michael W. Davidson, Rolf Dermietzel, Allan W. Wolkoff, David C. Spray

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. Wehave observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 μm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 μM of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 μM ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 μm/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 μM vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.

Original languageEnglish (US)
Pages (from-to)22875-22885
Number of pages11
JournalJournal of Biological Chemistry
Volume286
Issue number26
DOIs
StatePublished - Jul 1 2011

Fingerprint

Kinesin
Connexins
Microtubules
Liver
Adenylyl Imidodiphosphate
Dyneins
Hepatocytes
Anti-Idiotypic Antibodies
Adenosine Triphosphate
Microtubule Proteins
Nocodazole
Proteins
Rhodamines
Vanadates
Antibodies
Gap Junctions
Tubulin
Rats
Actins
Assays

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors. / Fort, Alfredo G.; Murray, John W.; Dandachi, Nadine; Davidson, Michael W.; Dermietzel, Rolf; Wolkoff, Allan W.; Spray, David C.

In: Journal of Biological Chemistry, Vol. 286, No. 26, 01.07.2011, p. 22875-22885.

Research output: Contribution to journalArticle

Fort, Alfredo G. ; Murray, John W. ; Dandachi, Nadine ; Davidson, Michael W. ; Dermietzel, Rolf ; Wolkoff, Allan W. ; Spray, David C. / In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 26. pp. 22875-22885.
@article{1df8e7198ade4480ba7d3f95257d9ea7,
title = "In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors",
abstract = "Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. Wehave observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 μm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 μM of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 μM ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 μm/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 μM vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.",
author = "Fort, {Alfredo G.} and Murray, {John W.} and Nadine Dandachi and Davidson, {Michael W.} and Rolf Dermietzel and Wolkoff, {Allan W.} and Spray, {David C.}",
year = "2011",
month = "7",
day = "1",
doi = "10.1074/jbc.M111.219709",
language = "English (US)",
volume = "286",
pages = "22875--22885",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "26",

}

TY - JOUR

T1 - In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors

AU - Fort, Alfredo G.

AU - Murray, John W.

AU - Dandachi, Nadine

AU - Davidson, Michael W.

AU - Dermietzel, Rolf

AU - Wolkoff, Allan W.

AU - Spray, David C.

PY - 2011/7/1

Y1 - 2011/7/1

N2 - Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. Wehave observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 μm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 μM of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 μM ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 μm/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 μM vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.

AB - Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. Wehave observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 μm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 μM of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 μM ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 μm/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 μM vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.

UR - http://www.scopus.com/inward/record.url?scp=79959545839&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79959545839&partnerID=8YFLogxK

U2 - 10.1074/jbc.M111.219709

DO - 10.1074/jbc.M111.219709

M3 - Article

C2 - 21536677

AN - SCOPUS:79959545839

VL - 286

SP - 22875

EP - 22885

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 26

ER -