Investigation of a potential interaction between PKD3 and MP-GAP utilizing fluorescent microscopy and FRET

Rouhani Ardeshiri, Roumina (2025) Investigation of a potential interaction between PKD3 and MP-GAP utilizing fluorescent microscopy and FRET. Masters thesis, Memorial University of Newfoundland.

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Abstract

Failures in cytokinesis, the final stage of mitosis, can lead to binucleation, which may act as an initiation point for cancer development. Protein kinase D3 (PKD3) is an enzyme that belongs to a family of protein kinases that have key roles in promoting many cellular processes, including proliferation, survival, and adhesion. It has been demonstrated that PKD3 depletion can cause a significant increase in binucleation in mouse embryonic fibroblasts (MEFs). In addition, the M�Phase GTPase-Activating Protein (MP-GAP) is shown to play an important role during the abscission of two daughter cells by inactivating the Ras homolog gene family member A (RhoA). The Leitges group previously showed that MP-GAP is translocated to the cleavage furrow at the late cytokinesis, where it colocalizes with RhoA and PKD3. Considering the effect of PKD3 deficiency on cells and the role of MP-GAP in cytokinesis, we aimed to verify the hypothesis that these two proteins might interact to regulate the final abscission. In this regard, this project was based on fluorescent microscopy imaging to track the dynamics of fluorescent protein-fused PKD3 and MP-GAP to characterize a potential interaction. In conclusion, while some data were collected on the endogenous PKD localization in cells, more experiments are required to establish a definitive strategy to identify a potential interaction.

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