Access Type

Open Access Dissertation

Date of Award

January 2021

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Karen A. Beningo

Abstract

Cell migration is a fundamental physical process for wound repair, innate immune response, embryonic development, inflammatory responses as well as cancer metastasis. Biochemical and mechanical cues present inside and outside the cell can modulate the mechanical aspects of cell migration. During cell migration, traction forces are generated by the contraction of the actomyosin cytoskeleton and transmitted to the extracellular matrix through integrin-mediated focal adhesions, facilitating the forward movement of the cell. Although regulating traction force is central to achieving successful cell migration, the detailed mechanism of the force production is still largely unclear. The calpain family of proteases has been implicated in various aspects of cell migration, including the regulation of cell spreading and focal adhesion dynamics. calpains 1 and 2 (Capn1 and 2) are the most widely characterized holoenzymes within the calpain family that are proteolytically active only when the catalytic subunits are heterodimerized with the small subunit calpain 4 (Capn4). To address whether calpains participate in the mechanism of traction force production or mechanosensing, traction force microscopy and mechanosensing experiments were utilized. Previous studies from our lab have demonstrated that Capn1, 2, and 4 are all responsible for mechanosensing of mouse embryonic fibroblasts (MEFs) against homeostatic and locally applied tension whereas Capn4 promotes the production of traction forces, independently of the large subunit’s catalytic activity. The following study has further identified that Capn4 indirectly modulates the tyrosine phosphorylation status and secretion of galectin-3 (Gal3), a β-galactoside-binding protein known to cluster and activate the transmembrane receptors such as integrins.As extracellular functions of Gal3 in cell migration have extensively been reported, we tested if extracellular Gal3 is involved in the Capn4-mediated traction forces. Not only did we find the involvement of Gal3 in the traction force pathway but its function in Capn4-regulated focal adhesion maturation and strengthening, migration rate and persistence were also determined. However, extracellular Gal3 failed to regulate the mechanosensing mechanism, suggesting that the identified Gal3 functions are associated solely with Capn4 but not with other catalytic subunits. In the course of elucidating the traction force pathway involving Capn4 and extracellular Gal3, we have demonstrated that Abelson (c-Abl) kinase is responsible for the tyrosine phosphorylation of Gal3, mainly on tyrosine-107 residue, that facilitates Gal3 secretion and traction force production. Furthermore, we have proposed a possible traction force mechanism through a myosin-II-dependent/FAK-independent pathway based on our data and previously reported data: 1) Capn4 assists to maintain the activation status of c-Abl kinase, 2) active c-Abl kinase tyrosine-phosphorylates Gal3 with Y107 as the main target, 3) tyrosine-phosphorylated Gal3 is secreted through exosomes, 4) extracellular Gal3 forms pentamers and clusters and activates β1 integrins, and 5) active β1 integrins triggers intracellular signaling pathway in a microtubule-associated, myosin-II-dependent, and FAK-independent manner. Further studies that unravel the gaps in each step in the signaling pathway will help to understand how cell migration is mechanically regulated.

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