טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentStarosvetsky Elina
SubjectA Genetic Analysis of the Signaling Pathways that Mediate
Chemokine induced Chemotaxis and Migration in T
Cells
DepartmentDepartment of Medicine
Supervisor Dr. Deborah Yablonski
Full Thesis textFull thesis text - English Version


Abstract

      Chemokines are a superfamily of small peptides, which selectively attract and activate leukocytes. Relatively little is known about the intracellular components that mediate chemokine effects. SDF-1α  is a chemokine that binds to CXCR4, a 7-transmembrane G protein coupled receptor, leading to a change in cell shape and induction of directional movement towards the SDF-1α gradient, defined as polarization and chemotaxis, correspondingly.    
       We established a system that allows generation and isolation of Jurkat cell line-derived mutants that fail to respond to SDF-1α stimulation by migration. Separate populations of Jurkat T-cells were randomly mutagenized and subsequently enriched for migration-defective mutant cells. We successfully isolated several independently generated mutant cell lines, lacking the ability to respond by migration to SDF-1α. This work is a proof of concept that generation and isolation of Jurkat cell-derived migration defective mutants is possible.

We present functional characterization of three mutant cell lines, as compared to wild-type cells. The mutants express normal levels of CXCR4 receptor, and its sequence and internalization process is normal. Mutants respond to SDF-1α stimulation by time and dose-dependent phosphorylation of Erk1/2. Several mutant cell lines respond normally to TCR stimulation by antigen-presenting cells, suggesting SDF-1α pathway-specific defects. Based on the experiments measuring receptor-proximal events, we suggest that CXCR4 receptor-proximal signaling is functional and the defects reside at the level of the cytoskeletal machinery of the mutant cells.

       We found that SDF-1α-induced migration of the mutant cells can be rescued by pharmacologically-induced microtubule disassembly, suggesting that the cytoskeletal defect is microtubule-related. Microscopic analysis revealed that one of the mutant cell lines (mutant 3.12) was unable to contract upon microtubule disassembly. We hypothesize that cell line 3.12 exhibits impairment in a contractile function, due to an impairment in the GEF-HI-RhoA-Rock-MLC signaling pathway that is triggered in wild type cells upon release of GEF-HI from disassembled microtubules. Cell lines 1.45 and 5.5 have a microtubule- related cytoskeletal defect that damages their ability to diminish rigidity upon SDF-1α stimulation. In these cell lines the contractility is intact, but lack of flexibility is the responsible for loss of migration.

We attempted to characterize our mutant cell lines by several means. We established a method for genetic characterization of these mutant cell lines by means of complementation with retroviral cDNA libraries In the future, attempts will be made to overcome spontaneous reversion of the mutants by elevation of screening efficiency and verification of cDNA library quality.