Comparative study of Miro1 and Miro2 proteins through tagging of the endogenous genes

Abstract

Mitochondria, known as powerhouse of the cells, are highly dynamic organelles capable of traversing long distances within neurons to supply energy to remote regions such as synaptic terminals. Research has shown that mitochondria show a diversity in their motility behavior. This diversity caused by factors such as directionality of their movement, stop and run time and so on. Kinesin/Dynein microtubule motor proteins and TRAK adaptor proteins are involved in long range mitochondrial movement. Mitochondrial Rho GTPases (Miro) protein as an outer mitochondrial membrane protein link the motor /adaptor protein complex to the mitochondria to facilitate mitochondrial trafficking. Miro1 and Miro2 are the two known Miro isoforms in mammalian cells. Miro protein not only play a key role in mitochondrial movement but also involves in shaping mitochondrial morphology, generating energy, mitophagy and more. Although many studies have discovered a diversity in expression level of Miro1 and Miro2 in different cell types, the functional significance of Miro1 and Miro2 is still not clear. Therefore, to gain deeper understanding of Miro1 and Miro2 functional differences in the context of mitochondrial trafficking, the objectives of this study were set as follows: first, to determine whether Miro1 and Miro2 are equally distributed across the entire mitochondrial population. Second, to study whether the Miro isoforms interact with distinct protein complexes, or do they share common binding partners and present in same protein complex as heterodimers. To achieve these goals, I generated stable cell lines using Crispr Cas9 technology: Flag-GFP-Miro1 knock-in SH-SY5Y cells, MYC-mRFP-Miro2 knock-in HeLa cells, and a double knock-in of Miro1 and Miro2 HeLa cells. These cell lines enable the study of not only the localization and distribution of endogenous Miro1 and Miro2 among different mitochondrial populations but also their biochemical characteristics using mass spectrometry analysis. Preliminary data showed that Miro1 and Miro2 were not only localized on mitochondria using immunocytochemistry but some of them were also found in close proximity to each other, as demonstrated by the Proximity Ligation Assay (PLA). Additionally, immunoprecipitation analysis did not detect any interactions between Miro2, Kinesin, MYO19, and TRAK proteins, indicating that miro2 may not interact with motor-adaptor complex at the endogenous level. Immunoprecipitation studies related to Miro1's interaction with the motor-adaptor complex protein are still ongoing, and further protocol optimization is necessary. There are two main experiments in the list for future. First, to immunoprecipitate Miro1 and Miro2 and send sample for mass spectrometry analysis in order to identify their binding partners. Second, to isolate mitochondria containing each isoform separately and subject them to proteomic and lipidomic analyses. This investigation aims to determine whether labeling a subset of mitochondria with Miro1 or Miro2 leads to alterations in the proteome and lipidome composition of that subset or not. The results of this research are expected to provide insights into about functional differences between Miro1 and Miro2 proteins and how these differences affect mitochondrial movement-related characteristics. Considering that mitochondrial motility impairment is associated with the onset and progression of some neurodegenerative diseases such as Alzheimer's and Parkinson’s and given the important role of Miro proteins in mitochondrial transport, we hope that these findings can be useful in the discovery of a novel therapeutic approach for neurodegenerative diseases.