Monitoring mitochondrial translation with spatial resolution and high throughput strategies

Abstract

Nearly all mitochondrial proteins are encoded in the nuclear genome, synthesized in the cytosol, and imported into mitochondria. However, mitochondria are semi-autonomous and possess their own genome, the mitochondrial DNA (mtDNA). In humans, mtDNA encodes 13 proteins, all of which are essential components of the oxidative phosphorylation system (OXPHOS). Despite the limited number of genes on the mitochondrial genome, gene expression and maintenance are of great importance, and dysfunction in these processes is associated with diseases. All mitochondrial proteins responsible for mitochondrial genome maintenance and expression, including mitochondrial ribosomes, are nuclear-encoded and synthesized in the cytosol. Therefore, close coordination between the nuclei, cytosol, and mitochondria is critical for proper synthesis of mitochondrial-encoded proteins and biogenesis of OXPHOS complexes. In addition, each mitochondrion contains multiple copies of mtDNA, resulting in heterogeneity among mitochondria in a single cell. How mitochondrial heterogeneity affects protein synthesis and how mitochondrial gene expression is regulated in response to cellular requirements is not fully understood. In this project, we explored two questions. First, given the strong coordination between mitochondrial gene expression and numerous nuclear-encoded proteins, we asked whether mitochondria express their genome differently in regions spatially distant from the nucleus, such as in the synaptic regions of neurons. To answer this question, we extended the use of a fluorescence-based technique to label nascent protein synthesis in mitochondria. In this method, an alkyne-containing amino acid was incorporated into the sequence of proteins encoded by mitochondria and then reacted with an azide-containing fluorophore. This provided visualization of nascent mitochondrial-encoded proteins using a fluorescence microscope (mitochondrial-FUNCAT). The spatial resolution of this technique allowed us to detect heterogeneous protein synthesis in mitochondria of different cell types and disease models. Moreover, we applied this technique in primary hippocampal cultures and detected new protein synthesis along axons and dendrites as well as in the pre- and postsynapse of hippocampal neurons. Thus, we provided further evidence for local biogenesis of mitochondria at the synapse. These results are presented in Chapter 2 as a published article. Given the high specificity and ability of mitochondrial-FUNCAT to detect small changes in mitochondrial protein synthesis, in the second part of the project we investigated whether it could be used to screen novel factors and pathways in the regulation of mitochondrial gene expression. To this end, we optimized mitochondrial FUNCAT in a high-throughput setup with a low error. Labeling mitochondrial protein synthesis in a 96-well format opened a new possibility to screen different factors and to find potential regulators of mitochondrial gene expression. Next, we combined high throughput mitochondrial-FUNCAT and transfection with a siRNA library of human kinases to explore the potential cellular players of mitochondrial gene expression. We introduced 65 kinases whose downregulation altered the level of mitochondrial protein synthesis. Among them, several kinases were associated with mitochondria in different databases with less-described functions. Preliminary investigation of these candidates opened new possibilities for understanding the gene expression regulation in mitochondria and the associated pathologies. These results are presented in Chapter 3 as a manuscript prepared for submission.