Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Physiology

Supervisor

Dr. Lina Dagnino

Abstract

The epidermis consists of a basal layer of undifferentiated keratinocytes and multiple suprabasal layers of differentiated keratinocytes. Undifferentiated keratinocytes are adherent and highly motile, whereas differentiated keratinocytes are comparatively less motile and downregulate cell attachments to the extracellular matrix. Integrin-linked kinase (ILK) is a scaffold protein implicated in the regulation of many cellular functions in keratinocytes, including cell attachment, migration, phagocytosis, and protein trafficking. To determine the mechanisms by which ILK is involved in these processes, I sought to identify other proteins which may interact with ILK in keratinocytes. I identified Engulfment and Cell Motility 2 (ELMO2) to interact with ILK in undifferentiated and differentiated keratinocytes. Similar to ILK, ELMO2 is a scaffold protein involved in migration, phagocytosis, and protein trafficking.

In undifferentiated keratinocytes, I determined that ILK and ELMO2 localize to lamellipodia in migrating keratinocytes stimulated with the extracellular matrix protein laminin 332. I determined that ELMO2 recruits ILK to lamellipodia and that they regulate front-rear polarity as well as directional cell migration. ELMO2 localization and function is determined, in part, by the small GTPase RhoG; active RhoG is can bind and recruit ELMO2 to the plasma membrane induce the lamellipodia formation. I identified RhoG in complexes containing ILK and ELMO2. Moreover, I determined that ELMO2 acts as a bridge between ILK and RhoG and that interaction between ELMO2 and RhoG is critical for ILK-associated front-rear polarity and cell migration. Importantly, I determined that ILK-ELMO2 complexes mediate polarization and directed cell migration upon stimulation of the epidermal growth factor receptor, a receptor that activates RhoG and is important in epithelial wound healing and cancer metastasis.

I determined that ILK and ELMO2 localize to puncta in differentiated keratinocytes. I identified these puncta to be recycling endosomes but not early or late endosomes and are positive for b1-integrin and E-cadherin, cell surface proteins that are regulated during differentiation. My data would suggest ILK and/or ELMO2 may be involved in protein trafficking in differentiated keratinocytes. Taken together, this would suggest ILK and ELMO2 localize to distinct subcellular compartments in undifferentiated and differentiated keratinocytes and are involved in multiple cellular functions.

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