Quantitative dSTORM super-resolution microscopy localizes Aurora kinase A/AURKA in the mitochondrial matrix

Abstract

Background information

Mitochondria are dynamic organelles playing essential metabolic and signaling functions in cells. Their ultrastructure has largely been investigated with electron microscopy (EM) techniques. However, quantifying protein-protein proximities using EM is extremely challenging. Super-resolution microscopy techniques as direct stochastic optical reconstruction microscopy (dSTORM) now provide a fluorescent-based, quantitative alternative to EM. Recently, super-resolution microscopy approaches including dSTORM led to valuable advances in our knowledge of mitochondrial ultrastructure, and in linking it with new insights in organelle functions. Nevertheless, dSTORM is mostly used to image integral mitochondrial proteins, and there is little or no information on proteins transiently present at this compartment. The cancer-related Aurora kinase A/AURKA is a protein localized at various subcellular locations, including mitochondria.

Results

We first demonstrate that dSTORM coupled to GcoPS can resolve protein proximities within individual submitochondrial compartments. Then, we show that dSTORM provides sufficient spatial resolution to visualize and quantify the most abundant pool of endogenous AURKA in the mitochondrial matrix, as previously shown for overexpressed AURKA. In addition, we uncover a smaller pool of AURKA localized at the OMM, which could have a potential functional readout. We conclude by demonstrating that aldehyde-based fixatives are more specific for the OMM pool of the kinase instead.

Conclusions

Our results indicate that dSTORM coupled to GcoPS colocalization analysis is a suitable approach to explore the compartmentalization of non-integral mitochondrial proteins as AURKA, in a qualitative and quantitative manner. This method also opens up the possibility of analyzing the proximity between AURKA and its multiple mitochondrial partners with exquisite spatial resolution, thereby allowing novel insights into the mitochondrial functions controlled by AURKA.

Significance

Probing and quantifying the presence of endogenous AURKA – a cell cycle-related protein localized at mitochondria – in the different organelle subcompartments, using quantitative dSTORM super-resolution microscopy.