Best, Anthony James; (1996) Biological functions of N-chimaerin and Rac1 in mammalian cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The GTPase Rac1 regulates actin cytoskeleton re-organization and membrane ruffling in response to a number of external stimuli. Neuronal-specific chimaerin is a Rac1 GTPase-activating protein (GAP) in vitro, but its cellular functions are not well understood. To investigate whether chimaerin regulates Rac1 function in a cellular environment the morphological effects of over-expressing full length chimaerin and its GAP domain were examined in non-neuronal (COS-7) and neuroblastoma (N1E-115) cell lines. Transiently transfected COS-7 cells, which expressed a cytoskeletal 38kDa protein, were small and rounded with long processes. In contrast cells expressing the cytosolic 20kDa GAP domain were flattened and did not produce membrane ruffles in response to growth factors. Stably transfected N1E-115 cells, expressed a truncated 20kDa protein with GAP activity, did not produce neurites or produce membrane ruffles, peripheral microspikes and flatten in response to DMSO- or butyrate-induced differentiation respectively. Full length and GAP domain proteins were expressed by clones transfected with a modified cDNA construct; DMSO-induced differentiation resulted in increased production of neurites, possibly in response to expression of full length chimaerin. To facilitate comparison of the morphological effects of Rac1 and chimaerin, N1E-115 cells were transfected with wild-type and mutant Rac1 cDNAs. Cells moderately over-expressing wild-type or dominant positive Rac1 flattened, even in the absence of butyrate. In contrast, cells expressing dominant negative Rac1 did not flatten in the presence or absence of butyrate. Inhibition of morphological changes by both dominant negative Rac1 and the GAP domain is consistent with chimaerin acting as a down-regulator of active Rac1-GTP. However, the phenotypes of cells expressing full length chimaerin cells suggests that chimaerin may have activities other than down-regulation of Rac1-GTP. α- and β-tubulin were found to interact with Rac1 when tissue extracts were immobilized onto nitrocellulose and probed with labelled GTPases. Rac1 bound only in its GTP bound form whilst an 'effector site' mutation abolished tubulin binding, characteristics consistent with tubulin being an effector for Rac1. Thus, in addition to actin microfilaments, other cytoskeletal elements may be affected by Rac1 whose GAP, chimaerin, may have a diversity of morphological roles.

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