Date of Award

Spring 2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology, Molecular Biology and Biochemistry

First Advisor

Robert L Gaehlen

Committee Chair

Robert L. Gaehlen

Committee Member 1

Stephen Konieczny

Committee Member 2

Laurie Parker

Committee Member 3

Kavita Shah

Abstract

The Syk protein-tyrosine kinase, a well-characterized modulator of immune recognition receptor signaling, also plays important, but poorly characterized, roles in tumor progression, acting as an inhibitor of cellular motility and metastasis in highly invasive cancer cells. Multiharmonic atomic force microscopy (AFM) was used to map nanomechanical properties of live MDA-MB-231 breast cancer cells either lacking or expressing Syk. The expression of Syk dramatically altered the cellular topography, reduced cell height, increased elasticity, increased viscosity, and allowed visualization of a more substantial microtubule network. The microtubules of Syk-expressing cells were more stable to nocodazole-induced depolymerization and were more highly acetylated than those of Syk-deficient cells. Silencing of MAP1B, a major substrate for Syk in MDA-MB-231 cells, attenuated Syk-dependent microtubule stability and reversed much of the effect of Syk on cellular topography, stiffness, and viscosity. This study illustrates the use of multiharmonic AFM both to quantitatively map the local nanomechanical properties of living cells and to identify the underlying mechanisms by which these properties are modulated by signal transduction machinery. Proteomic analyses of Syk-binding proteins identified several interacting partners also found to be recruited to stress granules. Treatment of cells with inducers of stress granule formation leads to the recruitment of Syk to these protein-RNA complexes. This recruitment requires the phosphorylation of Syk on tyrosine and results in the phosphorylation of proteins at or near the stress granule. Grb7 is identified as a Syk-binding protein involved in the recruitment of Syk tothe stress granule. This recruitment promotes the formation of autophagosomes and the clearance of stress granules from the cell once the stress is relieved, enhancing the ability of cells to survive the stress stimulus.

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