21001SP Subcellular organisation in mammalian cells

In this project we aim to investigate the interplay of the cytoskeleton with lipid substructures of endoplasmic reticulum origin using correlative super resolution fluorescence microscopy and soft X-ray tomography.

A key element in the development of a cell is the constant and balanced production, upkeep and distribution of structures and vesicles tasked with particular functions such as digestion, energy production and waste disposal. The provision and distribution of these structures depend on interactions between components of the cytoskeleton (such as tubulin and actin) and the endoplasmic reticulum and they in turn respond to environmental triggers and intracellular requirements. Although evidence is accumulating on the role of the tubulin network in ER architecture, much less is known of the role of filamentous actin in ER remodelling. To date, there is no comprehensive 3D imaging data of filamentous actin architecture in action, because of the transient nature of its interactions and the difficulty in capturing relevant 3D imaging data in cells under physiological conditions. 

Here we propose to use the newly developed correlative imaging platform at B24 (super resolution cryo-Structured Illumination Microscopy (cryoSIM) and cryo-Soft X-ray Tomography (cryoSXT)) to visualise the actin cytoskeleton and its contacts with the ER in established mammalian cell lines. We will work with cells that express fluorescent filamentous actin and will subject these to chemical and biological cues to study their effects on the role of the cytoskeleton.

The two microscopes we will use provide a unique imaging tool that delivers both chemical localisation through visible light fluorescence and cellular ultrastructure mapping through soft X-ray imaging.