Rol of Lfc, a guanosine-nucleotide exchange factor for Rho, in axón formation.

CONDE CB; SUNG CH; CÁCERES A

Buzios, Brasil

Congreso; I Congreso IBRO/LARC de Neurociencias de América Latina, Caribe y Península Ibérica; 2008

IBRO / LARC

Rho GTPases play an important role in the regulation of many cellular processes. Lfc was identified as a microtubule-associated GEF with activity towards RhoA. Microtubules also have a crucial role in neuronal polarization by signaling to the actin cytoskeleton. We identified Tctex-1, a dynein light chain, as a linker between microtubules and microfilaments, and Lfc as a protein that interacts with Tctex-1. Objectives:  Evaluate the subcellular distribution, function, and interaction with Tctex-1 of Lfc during neuronal polarization. Methods: All experiments were carried out in cultures of embryonic hippocampal pyramidal neurons. Transfected neurons were processed for immunofluorescence, and visualized by confocal and TIRF microscopy. Assays of Rho-GTPase activity by G-LISA were performed in transfected CHO cells. Results: The results obtained revealed that Lfc associates with microtubules and the actin cytoskeleton in developing neurites. RNAi suppression of Lfc induces the extension of multiple axon-like neurites, while ectopic expression of GFP-Lfc prevents axon formation, but not the initial extension of minor processes. A yeast two-hybrid screen and immunoprecipitation experiments revealed an interaction between Lfc and Tctex-1. Co-expression of both proteins revealed that Tctex-1 could rescue the inhibitory effect of Lfc on axon formation, and that the C-terminal domain of Tctex-1 is required for such interaction. In accordance with this, Rho-GTPase activity assays revealed that Tctex-1 reduces Lfc-induced activation of RhoA. Finally, we decided to evaluate whether inhibition of a downstream effector of Rho, such as Rho Kinase, was capable of rescuing the inhibitory effect of Lfc on axon formation. The results obtained showed that treatment with Y27632 was able to induced axon formation in Lfc-treated neurons. Conclusions: The spatial and temporal control of signaling by Rho GTPases is thought to be determined by regulating their localization and activation at specific sub-cellular sites. Our results suggest that axon formation could have a "tone" of RhoA activity, mediated by Lfc and modulated by an Lfc-Tctex-1 interaction.