Regulation of early endocytic vesicle motility and fission in a reconstituted system

Eustratios Bananis, John W. Murray, Richard J. Stockert, Peter Satir, Allan W. Wolkoff

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

We previously established conditions to reconstitute kinesin-dependent early endocytic vesicle motility and fission on microtubules in vitro. The present study examined the question whether motility and fission are regulated in this system. Screening for proteins by immunofluorescence microscopy revealed that the small G protein, Rab4, was associated with 80% of hepatocyte-derived early endocytic vesicles that contain the ligand asialoorosomucoid (ASOR). By contrast, other markers for early endocytic vesicles including clathrin, Rab5 and EEA1 were present in the preparation but did not colocalize with the ASOR vesicles. Guanine nucleotides exchanged into the Rab4 present on the vesicles as shown by solubilization of Rab4 by Rab-GDI; solubilization was inhibited by incubation with GTP-γ-S and promoted by GDP. Pre-incubation of vesicles with GDP increased the number of vesicles moving on microtubules and markedly increased vesicle fission. This increase in motility from GDP was shown to be towards the minus end of microtubules, possibly through activation of the minus-end-directed kinesin, KIFC2. Pre-incubation of vesicles with GTP-γ-S, by contrast, repressed motility. Addition of exogenous GST-Rab4- GTP-γ-S led to a further repression of motility and fission. Repression was not seen with addition of GST-Rab4-GDP. Treatment of vesicles with Rab4 antibody also repressed motility, and repression was not seen when vesicles were pre-incubated with GDP. Based on these results we hypothesize that endogenous Rab4-GTP suppresses motility of ASOR-containing vesicles in hepatocytes and that conversion of Rab4-GTP to Rab4-GDP serves as a molecular switch that activates minus-end kinesin-based motility, facilitating early endosome fission and consequent receptor-ligand segregation.

Original languageEnglish (US)
Pages (from-to)2749-2761
Number of pages13
JournalJournal of Cell Science
Volume116
Issue number13
DOIs
StatePublished - Jul 1 2003

Fingerprint

Transport Vesicles
Guanosine Triphosphate
Kinesin
Microtubules
Hepatocytes
Ligands
Clathrin
Guanine Nucleotides
Monomeric GTP-Binding Proteins
Endosomes
Fluorescence Microscopy
Antibodies
asialoorosomucoid
Proteins

Keywords

  • Endocytosis
  • Kenesin
  • Microtubules
  • Motility
  • Rabs

ASJC Scopus subject areas

  • Cell Biology

Cite this

Regulation of early endocytic vesicle motility and fission in a reconstituted system. / Bananis, Eustratios; Murray, John W.; Stockert, Richard J.; Satir, Peter; Wolkoff, Allan W.

In: Journal of Cell Science, Vol. 116, No. 13, 01.07.2003, p. 2749-2761.

Research output: Contribution to journalArticle

Bananis, Eustratios ; Murray, John W. ; Stockert, Richard J. ; Satir, Peter ; Wolkoff, Allan W. / Regulation of early endocytic vesicle motility and fission in a reconstituted system. In: Journal of Cell Science. 2003 ; Vol. 116, No. 13. pp. 2749-2761.
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