ORCID
0000-0002-1246-3884
Date of Award
2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cellular, Molecular and Biomedical Sciences
First Advisor
Brian Cunniff
Abstract
Subcellular mitochondrial positioning is necessary to support various cellular processes such as cell migration, invasion and signaling involved in cell growth. Mitochondrial trafficking occurs in part via Miro1, an outer mitochondrial membrane protein, which binds to microtubule motor/adaptor complexes. Deletion of Miro1 (Miro1-/-) from mouse embryonic fibroblasts (MEFs) restricts the mitochondria to the perinuclear space, which is rescued upon the stable re-expression of a Myc-tagged Miro1 plasmid (Myc-Miro1). Deletion of Miro1 does not compromise mitochondrial bioenergetics, indicating the mitochondria are not defective. Thus, differences in cellular processes could be linked to mitochondrial positioning rather than mitochondrial defects. Mitochondria are a significant source of reactive oxygen species, specifically hydrogen peroxide (H2O2) a redox signaling molecule. To quantify subcellular H2O2 levels dependent on Miro1 mitochondrial positioning, we used the H2O2-responsive biosensor HyPer7. Miro1-/- had higher levels of H2O2 in their perinuclear area compared to Miro1+/+ and Myc-Miro1 MEFs, and lower levels in the cell periphery. Mitochondrial density correlated with highest levels of subcellular H2O2. The increased peripheral H2O2 was associated with bigger and more abundant focal adhesions in the Miro1+/+ and Myc-Miro1 MEFs. Additionally, the phosphorylation of vinculin and p-130cas was increased in Miro1+/+ compared to Miro1-/- MEFs. Together, we identified loss of Miro1 disrupts subcellular H2O2 gradients and cell migration phenotypes. We next investigated a role for Miro1 in supporting cell proliferation and gene expression. Mitochondria are highly dynamic during the cell cycle and support signaling events governing transitions through the cell cycle. We determined Miro1-/- proliferated slower compared to Miro1+/+ and Myc-Miro1 MEFs. We show that asynchronous Miro1-/- MEFs have double the number of cells in the S/G2 cell cycle phase. We conducted the first ever RNA-sequencing of Miro1-/- MEFs to evaluate possible differential gene expression changes. We identified differentially expressed genes including those in the MAPK pathway. Literature shows that cell cycle progression is supported by dynamic regulation of ERK1/2 phosphorylation. Therefore, we evaluated phosphorylation of the MAPK proteins MEK1/2 and ERK1/2. We found no difference in MEK1/2 phosphorylation. ERK1/2 was hyperphosphorylated in Miro1-/- MEFs, in both the nuclear and cytoplasmic compartments. ERK1/2 phosphorylation was independent of the oxidation and expression of the dual specificity phosphatases (DUSPs). ERK1/2 phosphorylation was constitutive in Miro1-/- MEFs following serum starvation and stimulation independent of DUSP oxidation or expression. Lastly, we uncovered ERK1/2 oxidation to be increased in Miro1-/- MEFs, a post translational modification that supports persistent ERK1/2 phosphorylation. We next evaluated differential gene expression through RNA sequencing of cells following artificial relocalization of mitochondria via optogenetics. Many pathways including the MAPK pathway, focal adhesions, and angiogenesis were revealed to be altered due to mitochondrial positioning. This system allows us to evaluate the role of subcellular mitochondrial positioning independent of Miro1. These data contribute to the growing body of literature that has identified Miro1 and mitochondrial positioning in governing cell processes in normal and disease states.
Language
en
Number of Pages
222 p.
Recommended Citation
Shannon, Nathaniel, "The Role Of Miro1 Mediated Mitochondrial Positioning On Redox Gradients, Subcellular Signaling, And Gene Expression" (2025). Graduate College Dissertations and Theses. 2015.
https://scholarworks.uvm.edu/graddis/2015
