Rab11-like GTPase associates with and regulates the structure and function of the contractile vacuole system in Dictyostelium

E. Harris, K. Yoshida, J. Cardelli, J. Bush

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

Screening of a cDNA library revealed the existence of a Dictyostelium cDNA encoding a protein 80% identical at the amino acid level to mammalian Rab11. Subcellular fractionation and immunofluorescence studies revealed that DdRab11 was exclusively associated with the ATPase proton pump-rich contractile vacuole membrane system, consisting of a reticular network and bladder-like vacuoles. Video microscopy of cells expressing GFP-DdRab11 revealed that this Rab was associated with contractile vacuolar bladders undergoing formation, fusion and expulsion of water. The association of DdRab11 with contractile vacuole membranes was disrupted when cells were exposed to either hypo-osmotic conditions or an inhibitor of the ATPase proton pump. Cells that overexpressed a dominant negative form of DdRab11 were analyzed biochemically and microscopically to measure changes in the structure and function of the contractile vacuole system. Compared with wild-type cells, the dominant negative DdRab11-expressing cells contained a more extensive contractile vacuole network and abnormally enlarged contractile vacuole bladders, most likely the result of defects in membrane trafficking. In addition, the mutant cells enlarged, detached from surfaces and contained large vacuoles when exposed to water, suggesting a functional defect in osmotic regulation. No changes were observed in mutant cells in the rate of fluid phase internalization or release, suggesting the DdRab11-mediated membrane trafficking defects were not general in nature. Surprisingly, the rate of phagocytosis was increased in the dominant negative DdRab11-expressing cells when compared with control cells. Our results are consistent with a role for DdRab11 in regulating membrane traffic to maintain the normal morphology and function of the contractile vacuole.

Original languageEnglish (US)
Pages (from-to)3035-3045
Number of pages11
JournalJournal of cell science
Volume114
Issue number16
StatePublished - Sep 17 2001

Fingerprint

Dictyostelium
GTP Phosphohydrolases
Vacuoles
Membranes
Urinary Bladder
Adenosine Triphosphatases
Proton Pumps
Video Microscopy
Water
Proton Pump Inhibitors
Gene Library
Phagocytosis
Fluorescent Antibody Technique
Complementary DNA
Amino Acids

Keywords

  • Contractile vacuole
  • DdRab11
  • Dictyostelium

ASJC Scopus subject areas

  • Cell Biology

Cite this

Rab11-like GTPase associates with and regulates the structure and function of the contractile vacuole system in Dictyostelium. / Harris, E.; Yoshida, K.; Cardelli, J.; Bush, J.

In: Journal of cell science, Vol. 114, No. 16, 17.09.2001, p. 3035-3045.

Research output: Contribution to journalArticle

@article{6af0942002a84ab7bbd9eb22b8c840b3,
title = "Rab11-like GTPase associates with and regulates the structure and function of the contractile vacuole system in Dictyostelium",
abstract = "Screening of a cDNA library revealed the existence of a Dictyostelium cDNA encoding a protein 80{\%} identical at the amino acid level to mammalian Rab11. Subcellular fractionation and immunofluorescence studies revealed that DdRab11 was exclusively associated with the ATPase proton pump-rich contractile vacuole membrane system, consisting of a reticular network and bladder-like vacuoles. Video microscopy of cells expressing GFP-DdRab11 revealed that this Rab was associated with contractile vacuolar bladders undergoing formation, fusion and expulsion of water. The association of DdRab11 with contractile vacuole membranes was disrupted when cells were exposed to either hypo-osmotic conditions or an inhibitor of the ATPase proton pump. Cells that overexpressed a dominant negative form of DdRab11 were analyzed biochemically and microscopically to measure changes in the structure and function of the contractile vacuole system. Compared with wild-type cells, the dominant negative DdRab11-expressing cells contained a more extensive contractile vacuole network and abnormally enlarged contractile vacuole bladders, most likely the result of defects in membrane trafficking. In addition, the mutant cells enlarged, detached from surfaces and contained large vacuoles when exposed to water, suggesting a functional defect in osmotic regulation. No changes were observed in mutant cells in the rate of fluid phase internalization or release, suggesting the DdRab11-mediated membrane trafficking defects were not general in nature. Surprisingly, the rate of phagocytosis was increased in the dominant negative DdRab11-expressing cells when compared with control cells. Our results are consistent with a role for DdRab11 in regulating membrane traffic to maintain the normal morphology and function of the contractile vacuole.",
keywords = "Contractile vacuole, DdRab11, Dictyostelium",
author = "E. Harris and K. Yoshida and J. Cardelli and J. Bush",
year = "2001",
month = "9",
day = "17",
language = "English (US)",
volume = "114",
pages = "3035--3045",
journal = "Journal of Cell Science",
issn = "0021-9533",
publisher = "Company of Biologists Ltd",
number = "16",

}

TY - JOUR

T1 - Rab11-like GTPase associates with and regulates the structure and function of the contractile vacuole system in Dictyostelium

AU - Harris, E.

AU - Yoshida, K.

AU - Cardelli, J.

AU - Bush, J.

PY - 2001/9/17

Y1 - 2001/9/17

N2 - Screening of a cDNA library revealed the existence of a Dictyostelium cDNA encoding a protein 80% identical at the amino acid level to mammalian Rab11. Subcellular fractionation and immunofluorescence studies revealed that DdRab11 was exclusively associated with the ATPase proton pump-rich contractile vacuole membrane system, consisting of a reticular network and bladder-like vacuoles. Video microscopy of cells expressing GFP-DdRab11 revealed that this Rab was associated with contractile vacuolar bladders undergoing formation, fusion and expulsion of water. The association of DdRab11 with contractile vacuole membranes was disrupted when cells were exposed to either hypo-osmotic conditions or an inhibitor of the ATPase proton pump. Cells that overexpressed a dominant negative form of DdRab11 were analyzed biochemically and microscopically to measure changes in the structure and function of the contractile vacuole system. Compared with wild-type cells, the dominant negative DdRab11-expressing cells contained a more extensive contractile vacuole network and abnormally enlarged contractile vacuole bladders, most likely the result of defects in membrane trafficking. In addition, the mutant cells enlarged, detached from surfaces and contained large vacuoles when exposed to water, suggesting a functional defect in osmotic regulation. No changes were observed in mutant cells in the rate of fluid phase internalization or release, suggesting the DdRab11-mediated membrane trafficking defects were not general in nature. Surprisingly, the rate of phagocytosis was increased in the dominant negative DdRab11-expressing cells when compared with control cells. Our results are consistent with a role for DdRab11 in regulating membrane traffic to maintain the normal morphology and function of the contractile vacuole.

AB - Screening of a cDNA library revealed the existence of a Dictyostelium cDNA encoding a protein 80% identical at the amino acid level to mammalian Rab11. Subcellular fractionation and immunofluorescence studies revealed that DdRab11 was exclusively associated with the ATPase proton pump-rich contractile vacuole membrane system, consisting of a reticular network and bladder-like vacuoles. Video microscopy of cells expressing GFP-DdRab11 revealed that this Rab was associated with contractile vacuolar bladders undergoing formation, fusion and expulsion of water. The association of DdRab11 with contractile vacuole membranes was disrupted when cells were exposed to either hypo-osmotic conditions or an inhibitor of the ATPase proton pump. Cells that overexpressed a dominant negative form of DdRab11 were analyzed biochemically and microscopically to measure changes in the structure and function of the contractile vacuole system. Compared with wild-type cells, the dominant negative DdRab11-expressing cells contained a more extensive contractile vacuole network and abnormally enlarged contractile vacuole bladders, most likely the result of defects in membrane trafficking. In addition, the mutant cells enlarged, detached from surfaces and contained large vacuoles when exposed to water, suggesting a functional defect in osmotic regulation. No changes were observed in mutant cells in the rate of fluid phase internalization or release, suggesting the DdRab11-mediated membrane trafficking defects were not general in nature. Surprisingly, the rate of phagocytosis was increased in the dominant negative DdRab11-expressing cells when compared with control cells. Our results are consistent with a role for DdRab11 in regulating membrane traffic to maintain the normal morphology and function of the contractile vacuole.

KW - Contractile vacuole

KW - DdRab11

KW - Dictyostelium

UR - http://www.scopus.com/inward/record.url?scp=0034851193&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034851193&partnerID=8YFLogxK

M3 - Article

C2 - 11686306

AN - SCOPUS:0034851193

VL - 114

SP - 3035

EP - 3045

JO - Journal of Cell Science

JF - Journal of Cell Science

SN - 0021-9533

IS - 16

ER -