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Modulation of intracellular transport by small GTP ases in cultured cells and during mouse development

Objective

The aim of this proposal is to study the in vivo functional properties of five different small GTPases of the RAB family in established cell lines, in primary cultures of foetal hippocampal neurons and in the developing mouse.
The discovery that the large superfamily of small guanosine triphosphatases (GTPases) of the Rab subfamily function as specific regulators of intracellular transport has opened new vistas into the basic mechanisms which generate and maintain cellular organization.
Research has been carried out in order to investigate the regulation of membrane traffic by various rab proteins in the secretory and endocytic pathways in tissue culture cells and to elucidate the basic mechanisms of function of rab GTPases.

Rab5 has been localized to both the apical and basolateral plasma membrane in epithelial cells and to the axonal and dendritic surface in neurons, and appears to control endocytosis from both plasma membrane domains. The function of Rab6 has been investigated by transiently overexpressing in mouse L-cells and human HeLa cells wildtype rab6, and 2 dominant interfering mutants. The results obtained suggest that rab6 controls intraGolgi transport, either acting as an inhibitor in anterograde transport or controlling retrograde transport. The construction of Rab4 mutants analagous to those already constructed for Rab5 and Rab6 has been initiated. Either expressed in vivo or purified and tested in vitro, the wild type and mutant proteins will used to study in detail the function of Rab4 in the endocytic and recycling pathway. The double hybrid system in yeast has been employed to search for factors that interact with, and regulate the activity of Rab5 and Rab6. Wild type and mutated Rab5 and Rab6 complementary deoxyribonucleic acid (cDNAs) have been fused with the deoxyribonucleic acid (DNA) binding domain of the lexA protein and used to screen a mouse brain and HeLa cDNA libraries. Several specific clones have been isolated and are being characterized. The role of Rab proteins in mouse development has also been explored by the use of transgenic mice expressing functional wild type and dominant nonfunctional mutant rab proteins, under constitutive as well as inducible promoters.
The proposed project is aimed at studying several members of the rab sub- family of small GTPases. These proteins are implicated in the regulation of specific steps of intracellular membrane transport. Our four groups will focus on rab1, rab4, rab5, rab6 and rab17. We will focus on three main points: 1. The elucidation of the basic mechanism of function of rab GTPases. This involves the identification and functional characterization of interacting components using biochemical methods. 2. The modulation of membrane traffic by various rab proteins. Using rab proteins as specific transport regulators we plan to either positively or negatively influence the cellular parameters of secretion and endocytosis in established cell lines and in primary cultures of foetal rat hippocampal neurons. 3. The impact of alterations in intracellular transport on mouse development. Functional studies will be performed in transgenic mice expressing functional wild type and dominant non-functional mutant rab proteins. The phenotypic effects during embryonic development and in the adult animals will be investigated. The understanding of the mechanisms controlling intracellular transport is not only relevant for the basic knowledge on how cellular organization is controlled but has also functional implications for biotechnological tasks. The availability of molecules which are playing an important role in the regulation of intracellular transport will hopefully allow the development of animal models which are amenable to the experimental studies of various human diseases and to the improvement of the biology of farm animals.

Funding Scheme

CON - Coordination of research actions

Coordinator

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Address
Meyerhofstrasse 1
69012 Heidelberg
Germany

Participants (2)

Institut Pasteur
France
Address
25 Rue Du Docteur Roux
75724 Paris
UNIVERSITY COLLEGE CORK, NATIONAL UNIVERSITY OF IRELAND, CORK
Ireland
Address
Lee Maltings, Prospect Row
30 Cork