Quantifying ER-mitochondria contact points using 3D-EM imaging

Abstract

We study peripheral neuropathies such as Charcot-Marie-Tooth (CMT), a genetic disorder leading to progressive degeneration of the peripheral nerves, causing muscle weakness and atrophy amongst other symptoms. Despite over 60 different genes having been identified, little progress has been made in understanding the pathomechanism and the development of effective cures. Of these textrangle60 genes, a disproportionate number plays a role in mitochondria or at the endoplasmic reticulum (ER)-mitochondria interface known as Mitochondria-Associated Membrane (MAM). We aim to develop a new method to quantify contact points between the ER and mitochondria, using a 3D electron (EM) microscopy approach. To this end, we are imaging fixed cells using the Zeiss Auriga Focused-Ion-Beam Scanning Electron Microscope (FIB-SEM). This method provides high-resolution image stacks of the cell, allowing precise 3D reconstruction and unbiased analysis of the organelles of interest. Besides optimising the imaging itself, we have experimented with several approaches to analyse these complex datasets, using both manual 3D reconstruction as well as automated 3D rendering with software such as Imaris. In addition to these approaches, we have also applied a stereologic method to estimate mitochondrial volume and surface within our image volume and determine the fraction of mitochondrial surface that is in contact with the ER membrane. We wish to use these methods to examine effects on contact point formation in cells expressing WT and mutant genes in the context of peripheral neuropathy.

Publication
Bioimage Data Analysis, Abstracts