Insights into Mitochondrial Dynamics using CSTET

This study, supported by the Erwin Schrödinger Fellowship and performed in the lab of Prof. Deborah Fass together with the Electron Microscopy Unit at the Weizmann Institute, Israel, aims to elucidate the mechanisms driving mitochondrial dynamics. Mitochondria are widely known as the powerhouses of the cell, but they also carry out diverse signaling and sensing functions. Mitochondria are tubular organelles around 500 nm in diameter. They form a highly interconnected and dynamic network, which is constantly remodeled via the processes of fusion (merging of two mitochondria into one) and fission (splitting of one mitochondrion into two). Fission begins with constriction of the mitochondrion, possibly aided by other organelles and the cytoskeleton. Subsequently, several proteins assemble at the constriction site and finalize the fission step. Most of the proteins involved in mitochondrial fission have been discovered by light microscopy imaging of perturbed mitochondrial dynamics in mutant cells lacking these proteins. However, many questions remain regarding the biophysical mechanism of fission and the participation of the surrounding cellular environment. Light microscopy offers limited insight into the mechanism of fission due to its low resolution. Higher resolution, sufficient for observing details of mitochondrial ultrastructure on the scale of a few nanometers, can be achieved by electron microscopy (EM). In this project I will use a unique EM technique to image intermediates in mitochondrial fission. Cells with particular defects in fission factors will be used to stall the multi- step fission process and enable analysis of states that are usually transient. I will focus on answering two vital biological questions about mitochondrial dynamics. The first is what are the ultrastructures of fission intermediates observed in cells lacking key fission factors, and what do these ultrastructures tell us about the biophysical mechanism of fission? Second, how do perturbations of the fission process influence the interactions of mitochondria with other cellular components? The novel EM technique I will use is known as Cryo-Scanning and Transmission Electron Tomography (CSTET). CSTET is able to illuminate 3D volumes sufficiently thick to include entire mitochondria within intact cells, providing unprecedented insight into both their structure and their intracellular surroundings. CSTET was developed at the EM Unit at the Weizmann Institute and has been recently applied in a collaborative study together with the Fass lab.

This study, supported by the Erwin Schrödinger Fellowship and performed in the lab of Prof. Deborah Fass together with the Electron Microscopy Unit at the Weizmann Institute, Israel, aims to elucidate the mechanisms driving mitochondrial dynamics. Mitochondria are widely known as the "powerhouses" of the cell, but they also carry out diverse signaling and sensing functions. Mitochondria are tubular organelles around 500 nm in diameter. They form a highly interconnected and dynamic network, which is constantly remodeled via the processes of fusion (merging of two mitochondria into one) and fission (splitting of one mitochondrion into two). Fission begins with constriction of the mitochondrion, possibly aided by other organelles and the cytoskeleton. Subsequently, several proteins assemble at the constriction site and finalize the fission step. Most of the proteins involved in mitochondrial fission have been discovered by light microscopy imaging of perturbed mitochondrial dynamics in mutant cells lacking these proteins. However, many questions remain regarding the biophysical mechanism of fission and the participation of the surrounding cellular environment. Light microscopy offers limited insight into the mechanism of fission due to its low resolution. Higher resolution, sufficient for observing details of mitochondrial ultrastructure on the scale of a few nanometers, can be achieved by electron microscopy (EM). In this project I will use a unique EM technique to image intermediates in mitochondrial fission. Cells with particular defects in fission factors will be used to stall the multi-step fission process and enable analysis of states that are usually transient. I will focus on answering two vital biological questions about mitochondrial dynamics. The first is what are the ultrastructures of fission intermediates observed in cells lacking key fission factors, and what do these ultrastructures tell us about the biophysical mechanism of fission? Second, how do perturbations of the fission process influence the interactions of mitochondria with other cellular components? The novel EM technique I will use is known as Cryo-Scanning and Transmission Electron Tomography (CSTET). CSTET is able to illuminate 3D volumes sufficiently thick to include entire mitochondria within intact cells, providing unprecedented insight into both their structure and their intracellular surroundings. CSTET was developed at the EM Unit at the Weizmann Institute and has been recently applied in a collaborative study together with the Fass lab.