Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Karen A. Beningo

Abstract

Cell migration plays a vital role in many physiological events including: morphogenesis, wound healing, and immune response. Dysfunctional cell migration results in multiple disease states including chronic inflammation, vascular disease, and tumor metastasis, to name a few. Progress in understanding the mechanism of cell migration had been slow until the turn of the century when rapid technological advances in microscopy and omics burst to the forefront. These advances led to the realization that physical factors (dimensions, fluid shear stress, hydrostatic pressure, compression stress, environmental stiffness, and topography) have profound effects on cell migration. This study of cell mechanics has expanded extensively in the past 20 years as with the application of multidisciplinary approaches in nanotechnology, biophysics, and modern cell biology.

Given the importance of focal adhesion dynamics in migration and mechanics, we focused on the function of calpain proteases on cell migration. Previously we discovered that when compared to wildtype MEFs, Capn4-/- cells displayed reduced traction force and this was not observed when the large catalytic subunits were silenced respectively or when the endogenous inhibitor calpastatin was overexpressed. In comparison, mechanosensing of localized tension was defunct in cells lacking the large subunits, or calpain 4, or when the holoenzyme activity was inhibited by calpastatin. These results together formed our conclusion that the regulatory small subunit calpain 4 must modulate the production of traction forces independent of the catalytic activity of the calpain holoenzymes, but function together to regulate the mechanosensing of localized tension.

In gaining understanding of the mechanics of traction force and mechanosensing of cell migration, we asked how calpain 4 protein regulates traction force. By overexpressing each domain in Capn4-/- cells, we have found that only the overexpression of domain V in Capn4-/- cells rescues the traction force defect, the reduced migration rate, and the abnormal focal adhesion organization. However, only the overexpression of domain VI in Capn4-/- cells restores both the ability to sense mechanical stimuli and the proteolytic activity. These results suggest that domains of calpain 4 function independently in regulating the traction force and sensing the external stimuli. We also asked what other players also function in regulating traction force through calpain 4. We performed a yeast two-hybrid assay and identified basigin to be one of the binding proteins. Further results indicated that inhibition of basigin in MEFs resulted in reduced level of traction force and defective adhesion strength without interfering with the sensing of external stimuli and homeostatic tension of the substrate.

Together these results further elucidate the mechanism of cell migration and interplay of traction force and mechanosensing, and establish calpain 4 to be a critical player in the regulation of traction force. Further investigation into this signaling pathway will greatly expand our scope of the mechanical aspects of cell migration and further benefit cell migration related diseases studies.

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