Date of Award

Fall 2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Immunology

First Advisor

Dr. Janis K. Burkhardt

Abstract

Numerous aspects of T cell function, including TCR signaling, migration, and execution of effector functions, depend on the actin cytoskeleton. Cytoskeletal rearrangements are driven by the action of actin-regulatory proteins, which promote or antagonize the assembly of actin filaments in response to external cues. In this work, we have examined the regulation and function of HS1, a poorly-understood actin regulatory protein, in T cells. This protein, which becomes tyrosine phosphorylated upon T cell activation, is thought to function primarily by stabilizing existing branched actin filaments. Loss of HS1 results in unstable actin responses upon TCR engagement and defective Ca2+ responses, leading to poor activation of the IL2 promoter. TCR engagement leads to phosphorylation of HS1 at Tyr 378 and Tyr 397, creating binding sites for SH2 domain-containing proteins, including Vav1 and Itk. Phosphorylation at these residues is required for Itk-dependent recruitment of HS1 to the IS, Vav1 IS localization, and HS1-dependent actin reorganization and IL2 production. Therefore, in addition to directly interacting with branched actin filaments, HS1 regulation of Vav1 localization provides another mechanism by which HS1 signals to the actin cytoskeleton. Analysis of Ca2+ responses in HS1-/- T cells reveals that the defect in these cells lies at the level of release from intracellular stores. Phosphorylated HS1 interacts with PLCγ1, and mediates its association with the cytoskeleton and regulates microcluster dynamics. Although HS1 is clearly important for signaling downstream of the TCR, HS1-/- mice exhibit normal T cell development and normal peripheral populations. Surprisingly, HS1 is not required for T cell homing to lymphoid organs, for TCR endocytosis or for CD8+ T cell effector function. However, T cells from HS1-/- mice produce reduced amounts of Ifnγ, and are subsequently less likely to become Ifnγ-producing TH1 effector cells. These data demonstrate that HS1 functions as a cytoskeletal adaptor protein and plays specific roles downstream of TCR engagement.

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Additional Files

Supplemental movie 2.1.mov (531 kB)
GFP-actin Jurkat cells transfected with control vector, spreading on anti-CD3-coated coverslips

Supplemental movie 2.2.mov (870 kB)
GFP-actin Jurkat cells transfected with control vector, spreading on anti-CD3-coated coverslips

Supplemental movie 2.3.mov (1565 kB)
GFP-actin Jurkat cells transfected with control vector, spreading on anti-CD3-coated coverslips

Supplemental movie 2.4.mov (1264 kB)
GFP-actin Jurkat cells transfected with HS1 suppression vector, spreading on anti-CD3-coated coverslips

Supplemental movie 2.5.mov (802 kB)
GFP-actin Jurkat cells transfected with HS1 suppression vector, spreading on anti-CD3-coated coverslips

Supplemental movie 2.6.mov (652 kB)
GFP-actin Jurkat cells transfected with HS1 suppression vector, spreading on anti-CD3-coated coverslips

Supplemental movie 2.7.mov (1374 kB)
GFP-actin Jurkat cells transfected with HS1 suppression vector, spreading on anti-CD3-coated coverslips

Supplemental Movie 2.8.mov (115 kB)
Venus-HS1 cells, spreading on anti-CD3-coated coverslips

Supplemental Movie 2.9.mov (475 kB)
Venus-HS1 cells, spreading on anti-CD3-coated coverslips

Supplemental Movie 3.1.mov (681 kB)
GFP-actin Jurkat cells transfected with control vector, spreading on anti-CD3-coated coverslips

Supplemental Movie 3.2.mov (2001 kB)
GFP-actin Jurkat cells transfected with control vector, spreading on anti-CD3-coated coverslips

Supplemental Movie 3.3.mov (1195 kB)
GFP-actin Jurkat cells transfected with Itk suppression vector, spreading on anti-CD3-coated coverslips

Supplemental Movie 3.4.mov (1003 kB)
GFP-actin Jurkat cells transfected with Itk suppression vector, spreading on anti-CD3-coated coverslips

Supplemental Movie 3.5.mov (1230 kB)
GFP-actin Jurkat cells transfected with HS1 suppression vector, spreading on anti-CD3-coated coverslips

Supplemental Movie 3.6.mov (691 kB)
GFP-actin Jurkat cells transfected with HS1 suppression vector, spreading on anti-CD3-coated coverslips

Supplemental Movie 3.7.mov (3326 kB)
PLCgamma1-YFP cells transfected with control vector, spreading on anti-CD3-coated coverslips

Supplemental Movie 3.8.mov (453 kB)
PLCgamma1-YFP cells transfected with control vector, spreading on anti-CD3-coated coverslips

Supplemental Movie 3.9.mov (511 kB)
PLCgamma1-YFP cells transfected with HS1 suppression vector, spreading on anti-CD3-coated coverslips

Supplemental Movie 3.10.mov (1954 kB)
PLCgamma1-YFP cells transfected with HS1 suppression vector, spreading on anti-CD3-coated coverslips

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