Date of Award

12-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cell Biology and Biochemistry

Research Advisor

Kaushik Parthasarathi, Ph.D.

Committee

Suleiman Bahouth, Ph.D. Randal K. Buddington, Ph.D. Anjaparavanda P. Naren, Ph.D. Fu-Ming Zhou, Ph.D

Abstract

Roles of cyclic nucleotides and cyclic nucleotide-dependent signaling molecules in regulating several signaling pathways including cell migration have long been known. However, the new and revolutionary concept is that it is not just the absence or presence of cyclic nucleotides, but a highly coordinated balance between these molecules regulates cell migration. Multi-drug resistance protein 4 (MRP4), is a member of the large family of ATP binding cassette (ABC) transporter proteins, that localizes to the plasma membrane and functions as a nucleotide efflux transporter and thus plays a pivotal role in the regulation of intracellular cyclic nucleotide dynamics. In our study we used MRP4-expressing fibroblast cells and MRP4 knockout mice as model systems and wound healing assays as the experimental system to explore this unique and emerging concept. Here we demonstrated for the first time that mouse embryonic fibroblasts (MEFs) isolated from Mrp4-/- mice have higher intracellular cyclic nucleotide levels and migrate faster compared to MEFs isolated from Mrp4+/+ mice. Using FRET based sensors specific for cAMP and cGMP we showed that inhibition of MRP4 with MK571 moderately increases both cAMP and cGMP levels and enhances the cell polarization leading to an increased cell migration. Whereas a robust increase in cAMP levels was observed following treatment with forskolin and IBMX, which causes decreased fibroblast migration. Cell migration appeared to be biphasic in response to externally added cell-permeant cyclic nucleotides (cpt-cAMP and cptcGMP). Furthermore we identified actin as an integral part of MRP4-interactome and these two proteins associate with each other predominantly at the plasma membrane. We also observed that actin cytoskeleton restricts MRP4 to the specific micro-domains of plasma membrane. Using FRET based sensor we showed that Mrp4-/- fibroblasts are more polarized compared to the Mrp4+/+ cells with a higher compartmentalized cAMP levels and PKA activities near the leading edge, which in turn lead to an enhanced cortical actin polymerization at the cell front. Taken together our studies suggested a novel cAMP-dependent mechanism for MRP4-mediated regulation of fibroblast migration where PKA and actin play critical roles as downstream targets.

DOI

10.21007/etd.cghs.2014.0293

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