Role of Cdc42 and related GTPases in T cell migration
Lead Research Organisation:
King's College London
Department Name: Randall Div of Cell and Molecular Biophy
Abstract
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Technical Summary
The migration and accumulation of T cells in tissues contribute to the development of inflammatory and autoimmune diseases, as well as being responsible for the recognition and rejection of allograft transplants.
During inflammation, T cells polarise and migrate in response to adhesion molecules and inflammatory chemokines presented on vascular endothelium and in the tissues. Changes to the T cell cytoskeleton are critical for this process resulting in the formation of a distinct leading edge and retracting tail or uropod. These morphological changes are coupled with the asymmetrical polarisation of polymerised actin at the leading edge and intercellular adhesion molecules and the microtubule organizing centre at the rear.
Rho proteins are key regulators of the actin and microtubule cytoskeletons and thus of cell migration. In the GTP-bound active conformation they bind to and activate downstream effectors responsible for mediating their effects. We aim to determine which Rho GTPases regulate T cell adhesion to and migration across endothelial cells (extravasation), and T cell migration in 3-D through extracellular matrix, using primary human peripheral blood lymphocytes (PBLs), human T cell lines and primary human endothelial cells. Most studies have only investigated the roles of 3 Rho GTPases, RhoA, Rac1 and Cdc42, in migration on 2-D substrates. We will initially use RNAi screening to identify which of the 22 human Rho GTPases affects transendothelial migration and migration through Matrigel. Subsequently, we will follow up on selected Rho GTPases to investigate in detail how they affect T cell adhesion, polarisation, transendothelial migration and 3-D invasion, using a combination of time-lapse microscopy and confocal microscopy. In parallel, we will study their localisation and activation in T cells as they polarise and migrate, and examine their effects on the T cell actin and microtubule cytoskeletons. These experiments will provide the first complete analysis of the contribution of Rho family members to migration, and give new insight into their involvement in T cell migration by studying physiologically relevant human cell models. This should assist the rational design of therapies aimed at inhibiting T cell recruitment in human diseases.
During inflammation, T cells polarise and migrate in response to adhesion molecules and inflammatory chemokines presented on vascular endothelium and in the tissues. Changes to the T cell cytoskeleton are critical for this process resulting in the formation of a distinct leading edge and retracting tail or uropod. These morphological changes are coupled with the asymmetrical polarisation of polymerised actin at the leading edge and intercellular adhesion molecules and the microtubule organizing centre at the rear.
Rho proteins are key regulators of the actin and microtubule cytoskeletons and thus of cell migration. In the GTP-bound active conformation they bind to and activate downstream effectors responsible for mediating their effects. We aim to determine which Rho GTPases regulate T cell adhesion to and migration across endothelial cells (extravasation), and T cell migration in 3-D through extracellular matrix, using primary human peripheral blood lymphocytes (PBLs), human T cell lines and primary human endothelial cells. Most studies have only investigated the roles of 3 Rho GTPases, RhoA, Rac1 and Cdc42, in migration on 2-D substrates. We will initially use RNAi screening to identify which of the 22 human Rho GTPases affects transendothelial migration and migration through Matrigel. Subsequently, we will follow up on selected Rho GTPases to investigate in detail how they affect T cell adhesion, polarisation, transendothelial migration and 3-D invasion, using a combination of time-lapse microscopy and confocal microscopy. In parallel, we will study their localisation and activation in T cells as they polarise and migrate, and examine their effects on the T cell actin and microtubule cytoskeletons. These experiments will provide the first complete analysis of the contribution of Rho family members to migration, and give new insight into their involvement in T cell migration by studying physiologically relevant human cell models. This should assist the rational design of therapies aimed at inhibiting T cell recruitment in human diseases.
