Characterizing the Spatial Variance of Autophagic Cargo Receptor, OPTN

Abstract

The mitochondria are required for various cellular processes spanning from cellular energy generation to signal transduction, growth and survival. Not surprisingly, cumulative mitochondrial damage is tightly linked to the development of multiple human diseases including various neurodegenerative diseases such as Parkinson's and Huntington's disease. In order to maintain functional mitochondria, cells have coped with multiple mitochondrial quality control pathways such as mitochondrial Unfolded Protein Response (mt‐UPR), the ubiquitin proteasome system, and mitochondrial autophagy (mitophagy). The most well‐characterized mitophagy is the PINK1‐PARKIN pathway, where damaged mitochondria are selectively marked with ubiquitin chains by the PINK1 kinase and PARKIN ubiquitin E3 ligase, engulfed by autophagosome, and cleared by the lysosome. While we now have a better understanding on this pathway, it is still unclear how autophagic cargo adaptor proteins, such as optineurin (OPTN), get recruited to damaged mitochondria, synthesized or recycled throughout the process. We hypothesized that the availability of OPTN would be the rate‐limiting factor to achieve complete removal of damaged mitochondria and this would require synthesis of new OPTN molecule. To test this hypothesis, we generated a cell line containing a genomic Halo tag at OPTN N‐terminus by utilizing CRISPR/cas9, where differentially fluorescent labeled Halo tag can distinguish older versus newer OPTN populations over time. After successfully generating a CRISPR/Cas9 knock‐in cell line for OPTN, single cell imaging was performed to determine the spatial variance and emergence of OPTN recruited to mitochondria. We were able to conclude that OPTN localizes to a fraction of damaged mitochondria despite the fact that the entire mitochondria are coated with ubiquitin chains, suggesting specificity of this recruitment. Additionally, our preliminary results indicated that newly synthesized OPTN is recruited to mitochondria for the continuation of mitophagy. Together, these observations suggest that OPTN is the rate‐limiting factor and that in order to completely remove all dysfunctional mitochondria, cells need to synthesize new OPTN. In the future, we would like to further explore the underlying mechanism of OPTN's binding specificity and determine what drives this selective recruitment of OPTN to mitochondria.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.