Date of Degree

9-2019

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Derrick Brazill

Committee Members

Diana Bratu

Pablo Peixoto

Daniel Weinstein

Jason Rauceo

Purnima Bhanot

Subject Categories

Cancer Biology | Cell Biology | Computational Biology | Developmental Biology | Genetics | Molecular Biology | Molecular Genetics | Other Cell and Developmental Biology

Keywords

BLEBS, PAXILLIN, SCAR/WAVE, WASP, TALIN, MYOSIN II

Abstract

A variety of biological functions depend on actin organization. The organization of actin is tightly regulated by a plethora of extracellular and intracellular signaling, scaffolding, and actin-binding proteins. Dysfunctions in this regulation lead to immune diseases, increased susceptibility to pathogens, neurodegenerative diseases, developmental disorders, and cancer metastasis. A variety of actin-dependent processes, including cell motility, are regulated by several proteins of interest: Paxillin, a scaffolding protein; WASP, an actin nucleating protein; SCAR/WAVE, another WASP family actin nucleating protein; Talin, a cortex-to-membrane binding protein; Myosin II, an F-actin contracting motor protein; and Protein Kinase C, a protein kinase. D. discoideum cells have orthologues of all these proteins: PaxB, WasA, SCAR, TalA, MhcA, and PkcA, respectively. Like in mammalian cells, PaxB, WasA, and SCAR all regulate similar actin-based processes. These proteins may regulate each other to execute these actin-based processes. Here, we evaluated WasA and SCAR’s epistatic relationship with PaxB in several actin-based processes using classical, genetic epistasis analysis, and further investigated PaxB, WasA, SCAR, TalA, MhcA, and PkcA’s roles in regulating three dimensional motility where cells endure compression from the environment from other cells and tissue. In three dimensional environments, cells use blebs where the membrane of the cell detaches from the cortex, resulting in a fluid filled blister-like protrusion that dominates motility instead of using filopodia, pseudopodia, and lamellipodia, actin-based structures, that predominate two dimensional motility where cells crawl freely on slides. Most of what we know about these proteins were from studies on slides. Little is known about proteins that regulate blebs and the transition from using actin-based motility structures for blebs.

For epistasis, we found that WasA is downstream of PaxB for plaque formation, endocytosis, cell-substrate adhesion, and for cell autonomy. However, mixing experiments revealed that WasA null cells developmental defect could not be rescued by the addition of wild-type cells. We found that SCAR is downstream of PaxB for plaque formation, endocytosis, development, and cell-sorting throughout development. For development specifically, we found that SCAR is a suppressor of PaxB’s null phenotype. In contrast to WasA, SCAR and PaxB separately regulate adhesion.

For three dimensional motility and blebs, we used an under agarose assay where null lines of all the actin regulators of interest expressing LifeAct-GFP chemotaxed towards cAMP while being compressed by a RITC-dextran laced gel to induce and visualize blebs. We compared motility under two different agarose concentrations. We found that WT cells increase their use of blebs at the expense of actin-based motility structures from low pressure to high pressure. We found that PaxB negatively regulates blebs and the total number of structures a cell can generate. WasA null cells do not respond to cAMP and would not go under the gel. SCAR positively regulates blebs and negatively regulates pseudopodia under lower pressures. TalA is needed to allow the cells to bleb efficiently under any pressure. MhcA is needed for blebs to form and negatively regulates pseudopodia and lamellipodia. PkcA negatively regulates pseudopodia and lamellipodia under either pressure. Lastly, through our software, we found that negative curvature was a predictor of bleb site location only fifty percent of the time. This investigation has allowed us to advance the fields of actin regulation, signal transduction, cytoskeletal reorganization, cell motility, and their contributions to disease.

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