RabD, a Dictyostelium Rab14-related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion

Edward Harris, James Cardelli

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

RabD, a Dictyostelium Rab14-related GTPase, localizes in the endo-lysosomal pathway and contractile vacuole system of membranes. Cell lines expressing dominant-negative RabD were defective in endocytosis, endosomal membrane flow and homotypic lysosome fusion. In support of a role for RabD in fusion, cells overexpressing constitutively active RabDQ67L accumulated enlarged hydrolase-rich acidic vesicles ringed with GFP-RabD, consistent with RabD directly regulating lysosome fusion. To determine whether RabD also regulated phagocytosis and/or homotypic phagosome fusion (a. process stimulated by many intracellular pathogens), cells overexpressing dominant-active (RabDQ67L) or dominant-negative (RabN121I) RabD were analyzed microscopically and biochemically. The rate of phagocytosis was increased two-fold in RabDQ67L-expressing cells and reduced by 50% in RabDN121I-expressing cells compared with control cells. To examine the role of RabD in the formation of multiparticle phagosomes, we performed a series of pulse-chase experiments using fluorescently labeled bacteria and fluorescent latex beads. The rate of fusion of newly formed phagosomes was five times higher in the RabDQ67L-expressing cells and reduced by over 50% in RabDN121I-expressing cells as compared with control cells. GFP-RabDQ67L was found to ring multiparticle spacious phagosomes, which supports a direct role for this protein in regulating fusion. Inhibition of PI 3-kinase activity, which is known to regulate phagosome fusion in the wild-type cells, reduced the rate of phagosome fusion in RabDQ67L+ cells, indicating that RabD acted upstream of or parallel with PI 3-kinase. We hypothesize that RabD and, possibly, Rab14, a related GTPase that associates with phagosomes in mammalian cells, are important regulators of homotypic phagosome and endo-lysosome fusion.

Original languageEnglish (US)
Pages (from-to)3703-3713
Number of pages11
JournalJournal of cell science
Volume115
Issue number18
DOIs
StatePublished - Sep 15 2002

Fingerprint

Phagosomes
Dictyostelium
GTP Phosphohydrolases
Lysosomes
Phagocytosis
Phosphatidylinositol 3-Kinases
Membranes
Cell Fusion
Hydrolases
Endocytosis
Vacuoles
Microspheres
Bacteria
Cell Line

Keywords

  • Dictyostelium
  • Phagocytes
  • Phagosome fusion
  • Rab14

ASJC Scopus subject areas

  • Cell Biology

Cite this

RabD, a Dictyostelium Rab14-related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion. / Harris, Edward; Cardelli, James.

In: Journal of cell science, Vol. 115, No. 18, 15.09.2002, p. 3703-3713.

Research output: Contribution to journalArticle

@article{1a770e67112e4627b45fb78b444f34e2,
title = "RabD, a Dictyostelium Rab14-related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion",
abstract = "RabD, a Dictyostelium Rab14-related GTPase, localizes in the endo-lysosomal pathway and contractile vacuole system of membranes. Cell lines expressing dominant-negative RabD were defective in endocytosis, endosomal membrane flow and homotypic lysosome fusion. In support of a role for RabD in fusion, cells overexpressing constitutively active RabDQ67L accumulated enlarged hydrolase-rich acidic vesicles ringed with GFP-RabD, consistent with RabD directly regulating lysosome fusion. To determine whether RabD also regulated phagocytosis and/or homotypic phagosome fusion (a. process stimulated by many intracellular pathogens), cells overexpressing dominant-active (RabDQ67L) or dominant-negative (RabN121I) RabD were analyzed microscopically and biochemically. The rate of phagocytosis was increased two-fold in RabDQ67L-expressing cells and reduced by 50{\%} in RabDN121I-expressing cells compared with control cells. To examine the role of RabD in the formation of multiparticle phagosomes, we performed a series of pulse-chase experiments using fluorescently labeled bacteria and fluorescent latex beads. The rate of fusion of newly formed phagosomes was five times higher in the RabDQ67L-expressing cells and reduced by over 50{\%} in RabDN121I-expressing cells as compared with control cells. GFP-RabDQ67L was found to ring multiparticle spacious phagosomes, which supports a direct role for this protein in regulating fusion. Inhibition of PI 3-kinase activity, which is known to regulate phagosome fusion in the wild-type cells, reduced the rate of phagosome fusion in RabDQ67L+ cells, indicating that RabD acted upstream of or parallel with PI 3-kinase. We hypothesize that RabD and, possibly, Rab14, a related GTPase that associates with phagosomes in mammalian cells, are important regulators of homotypic phagosome and endo-lysosome fusion.",
keywords = "Dictyostelium, Phagocytes, Phagosome fusion, Rab14",
author = "Edward Harris and James Cardelli",
year = "2002",
month = "9",
day = "15",
doi = "10.1242/jcs.00050",
language = "English (US)",
volume = "115",
pages = "3703--3713",
journal = "Journal of Cell Science",
issn = "0021-9533",
publisher = "Company of Biologists Ltd",
number = "18",

}

TY - JOUR

T1 - RabD, a Dictyostelium Rab14-related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion

AU - Harris, Edward

AU - Cardelli, James

PY - 2002/9/15

Y1 - 2002/9/15

N2 - RabD, a Dictyostelium Rab14-related GTPase, localizes in the endo-lysosomal pathway and contractile vacuole system of membranes. Cell lines expressing dominant-negative RabD were defective in endocytosis, endosomal membrane flow and homotypic lysosome fusion. In support of a role for RabD in fusion, cells overexpressing constitutively active RabDQ67L accumulated enlarged hydrolase-rich acidic vesicles ringed with GFP-RabD, consistent with RabD directly regulating lysosome fusion. To determine whether RabD also regulated phagocytosis and/or homotypic phagosome fusion (a. process stimulated by many intracellular pathogens), cells overexpressing dominant-active (RabDQ67L) or dominant-negative (RabN121I) RabD were analyzed microscopically and biochemically. The rate of phagocytosis was increased two-fold in RabDQ67L-expressing cells and reduced by 50% in RabDN121I-expressing cells compared with control cells. To examine the role of RabD in the formation of multiparticle phagosomes, we performed a series of pulse-chase experiments using fluorescently labeled bacteria and fluorescent latex beads. The rate of fusion of newly formed phagosomes was five times higher in the RabDQ67L-expressing cells and reduced by over 50% in RabDN121I-expressing cells as compared with control cells. GFP-RabDQ67L was found to ring multiparticle spacious phagosomes, which supports a direct role for this protein in regulating fusion. Inhibition of PI 3-kinase activity, which is known to regulate phagosome fusion in the wild-type cells, reduced the rate of phagosome fusion in RabDQ67L+ cells, indicating that RabD acted upstream of or parallel with PI 3-kinase. We hypothesize that RabD and, possibly, Rab14, a related GTPase that associates with phagosomes in mammalian cells, are important regulators of homotypic phagosome and endo-lysosome fusion.

AB - RabD, a Dictyostelium Rab14-related GTPase, localizes in the endo-lysosomal pathway and contractile vacuole system of membranes. Cell lines expressing dominant-negative RabD were defective in endocytosis, endosomal membrane flow and homotypic lysosome fusion. In support of a role for RabD in fusion, cells overexpressing constitutively active RabDQ67L accumulated enlarged hydrolase-rich acidic vesicles ringed with GFP-RabD, consistent with RabD directly regulating lysosome fusion. To determine whether RabD also regulated phagocytosis and/or homotypic phagosome fusion (a. process stimulated by many intracellular pathogens), cells overexpressing dominant-active (RabDQ67L) or dominant-negative (RabN121I) RabD were analyzed microscopically and biochemically. The rate of phagocytosis was increased two-fold in RabDQ67L-expressing cells and reduced by 50% in RabDN121I-expressing cells compared with control cells. To examine the role of RabD in the formation of multiparticle phagosomes, we performed a series of pulse-chase experiments using fluorescently labeled bacteria and fluorescent latex beads. The rate of fusion of newly formed phagosomes was five times higher in the RabDQ67L-expressing cells and reduced by over 50% in RabDN121I-expressing cells as compared with control cells. GFP-RabDQ67L was found to ring multiparticle spacious phagosomes, which supports a direct role for this protein in regulating fusion. Inhibition of PI 3-kinase activity, which is known to regulate phagosome fusion in the wild-type cells, reduced the rate of phagosome fusion in RabDQ67L+ cells, indicating that RabD acted upstream of or parallel with PI 3-kinase. We hypothesize that RabD and, possibly, Rab14, a related GTPase that associates with phagosomes in mammalian cells, are important regulators of homotypic phagosome and endo-lysosome fusion.

KW - Dictyostelium

KW - Phagocytes

KW - Phagosome fusion

KW - Rab14

UR - http://www.scopus.com/inward/record.url?scp=0037106577&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037106577&partnerID=8YFLogxK

U2 - 10.1242/jcs.00050

DO - 10.1242/jcs.00050

M3 - Article

C2 - 12186956

AN - SCOPUS:0037106577

VL - 115

SP - 3703

EP - 3713

JO - Journal of Cell Science

JF - Journal of Cell Science

SN - 0021-9533

IS - 18

ER -