|M.Sc Student||Kurtz Marina|
|Subject||The Role and Regulation of RhoA in SDF-1 Alpha Induced T|
|Department||Department of Medicine||Supervisor||Dr. Deborah Yablonski|
|Full Thesis text - in Hebrew|
Cell migration is a central process in the development and maintenance of multicellular organisms.
Cell migration is a tightly regulated process that occurs in tissue development, chemotaxis and wound healing. Rho family GTPases, including Cdc42, Rac1 and RhoA, play a central role in the signal transduction pathways driving morphogenesis and migration.
The aim of this work was to gain a better understanding of the role and regulation of RhoA in SDF-1α induced T cell chemotaxis.
Amoeboid leukocytes squeeze through narrow gaps by cell deformation. This morphological deformation is dependent on actomyosin contractility and is regulated mainly by RhoA.
To investigate RhoA signaling processes associated with migration, we employed as a model the Jurkat leukemic T cell line. Migration through restrictive gaps was examined in a transwell system towards the chemokine, SDF-1α.
Dr. Elina Starosvetsky, in her PhD work, isolated number Jurkat mutants which were damaged in migration through small pores.
we hypothesized that the mutant cells were defective in migration through restricted pores because of reduced RhoA signaling, which is required for cell contraction.
This work focuses on two mutant clones: clone 1.45 and clone 3.12.
I first calibrated an experimental system to examine RhoA activation in Jurkat cells. Using this system we found that whereas clone 1.45 exhibited normal RhoA activation in response to chemokine, Nocodazole or TCR stimuli, clone 3.12 didn’t show RhoA activation in response to the same stimuli, although it expressed RhoA at a normal level. Following this result, we decided to focus on investigation of clone 3.12.
Isolation and sequencing RhoA cDNA from clone 3.12, revealed two point mutations, A61V and E142K. Each mutation is located on different allele, meaning this clone doesn’t have an intact RhoA allele. Expression of RhoAWT protein in clone 3.12 partially rescued migration of these cells, suggesting that the migration defect can be attributed, at least partially, to the lack of WT RhoA.
Interestingly, the E142K mutation was also observed in WT Jurkat cells, and therefore was carried to clone 3.12. The two mutations we found were not previously characterized and their effect on protein activation and activity is not known. Based on the location of these mutations, we surmise that the A61V mutation, specific to clone 3.12, impacts GTP loading, and the E142K mutation, which is common to Jurkat WT and clone 3.12, may impact activation of downstream effectors and may also impact RhoA activation.