Evaluation of Protein Markers and Development of Reporter Cell Lines for Detecting Mitochondrial Stress in the Context of Splicing Kinase Loss

Abstract

Mitochondrial homeostasis is essential for cellular function, and its dysregulation is implicated in cancer and neurodegenerative disease. I evaluated methods for assessing mitochondrial stress to study how splicing kinase loss affects homeostasis. Mitochondrial health relies on fission, fusion, and mitophagy. Based on prior work linking PRP4K to autophagy regulation, I hypothesized that PRP4K loss might cause damaged mitochondria accumulation. Bulk protein assays (western blotting for TOM20, ATP synthase γ, MTCO2) showed no significant changes. To overcome this, I used MitoTracker imaging in cells with CCCP, a mitophagy inducer, and found PRP4K depletion consistently elevated MitoTracker intensity, suggesting increased mitochondrial mass. Using CRISPR/Cas9, I generated a mitophagy reporter MCF7 cell line expressing Mito-Rosella, a tandem pHluorin/dsRed mitochondrial reporter. This assay allowed measurement of mitophagy changes following PRP4K depletion, establishing a platform to assess the role of PRP4K and its regulated splicing events in mitophagy and mitochondrial homeostasis.