In vitro reconstitution of ARF-regulated cytoskeletal dynamics on Golgi membranes

Ji-Long Chen; Weidong Xu; Mark Stamnes

doi:10.1016/S0076-6879(05)04030-9

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In vitro reconstitution of ARF-regulated cytoskeletal dynamics on Golgi membranes

Journal article

Peer reviewed

In vitro reconstitution of ARF-regulated cytoskeletal dynamics on Golgi membranes

Ji-Long Chen, Weidong Xu and Mark Stamnes

Methods in enzymology, Vol.404, pp.345-358

2005

DOI: 10.1016/S0076-6879(05)04030-9

PMID: 16413281

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Abstract

Function of the secretory pathway is intimately connected to the cytoskeleton. Cytoskeletal dynamics and molecular motors are involved in organelle morphology and positioning, as well as the formation and translocation of trafficking intermediates such as vesicles. At least three classes of small GTPases, the ADP-ribosylation factor (ARF), Rho, and Rab families, have been implicated in regulating cytoskeletal dynamics and molecular motor function within the secretory pathway. We have used the reconstitution of transport vesicle formation on isolated Golgi membranes to characterize mechanisms of ARF1 regulated actin polymerization. ARF1 affects cytoskeletal function in part by recruiting a complex between the vesicle-coat protein, coatomer, and the Rho-related GTPase, Cdc42, to the Golgi apparatus. Cdc42 can activate actin polymerization on Golgi membranes through an Arp2/3-dependent mechanism. Coatomer-bound Cdc42 plays a further role in regulating vesicle motility via the motor protein, dynein. Future studies elucidating the molecular mechanisms connecting vesicular transport with actin dynamics will provide important clues to the overall contribution of the cytoskeleton and molecular motors to protein transport. This article describes methods and reagents for characterizing cytoskeletal regulation at the Golgi apparatus through the cell-free reconstitution of vesicle formation.

Actins - metabolism

ADP-Ribosylation Factors - physiology

Animals

Brain - cytology

Cattle

Centrifugation, Isopycnic

Coatomer Protein - metabolism

COP-Coated Vesicles - physiology

Cytoskeleton - drug effects

Cytoskeleton - physiology

Cytosol - metabolism

Golgi Apparatus - physiology

Liposomes - metabolism

Polymers - metabolism

Rats

rho GTP-Binding Proteins - antagonists & inhibitors

Details

Title: Subtitle: In vitro reconstitution of ARF-regulated cytoskeletal dynamics on Golgi membranes
Creators: Ji-Long Chen - University of Iowa
Weidong Xu
Mark Stamnes
Resource Type: Journal article
Publication Details: Methods in enzymology, Vol.404, pp.345-358
DOI: 10.1016/S0076-6879(05)04030-9
PMID: 16413281
ISSN: 0076-6879
eISSN: 1557-7988
Grant note: R01 GM068674 / NIGMS NIH HHS
Language: English
Date published: 2005
Academic Unit: Molecular Physiology and Biophysics; Internal Medicine
Record Identifier: 9984297493002771

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