Author

Ashrifa Ali

Date of Award

January 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Dr. Bryon Grove

Abstract

A functional endothelium must maintain an equilibrium among processes such as endothelial cell (EC) migration, barrier function and angiogenesis. The endothelium is constantly exposed to external factors that that result in a complex network of signaling wired to several possible cellular outcomes. A-kinase anchoring proteins (AKAPs) have a critical role in regulating cell signaling by providing spatial and temporal control of signaling proteins to increase downstream target specificity. Several studies have linked gravin (also called SSeCKS or AKAP12), an AKAP, to migration, barrier function, and angiogenesis. However, the functional significance of each gravin variant in ECs has not been investigated before. We hypothesize that each gravin variant has a different role in regulating endothelial cell migration and proliferation.

To test this hypothesis, we knocked out each variant from immortalized human umbilical vein ECs using CRISPR/Cas9. First, gravin variant 1 (v1) was knocked out by targeting the gRNA/Cas9 complex to exon 3, a region unique to gravin v1. Insertions/deletions (Indels) created at the double stranded break region (DSB) via non homologous end joining resulted in the loss of gravin v1 and created a cell line expressing only gravin variant 2 (v2). Cells lacking gravin v1 showed increased invasion across a VEGF gradient and increased migration in a scratch wound assay. However, this enhanced migratory phenotype was not evident in a dispersed population of cells. In addition, ECs lacking gravin v1 did not show differences in cell cycle phases compared to WT cells. Attempts to knockout gravin v2 targeted the gRNA/Cas9 complex to exon 4, the only exon specific to variant 2. Single cell clones positive for CRISPR/Cas9 mediated indel, identified by a restriction fragment length polymorphism assay, showed poor cell proliferation and sparse growth. However, the loss of gravin v2 could not be confirmed with a western blot due to insufficient number of cells. Hence, investigation into the role of gravin v2 will require an alternate approach such as using homology directed repair methods during CRISPR/Cas9 editing or transfecting gravin v2 in double knock out ECs.

These results from the variant 1 knockout study are consistent with the literature where overexpression of gravin v1 in immortalized rodent cells inhibited migration while elevated expression of gravin v2 is linked to increased metastasis of melanoma cells. We propose that both variants may be working together to regulate migration and proliferation.

Available for download on Thursday, June 08, 2023

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