Protocol to study cytoskeleton at molecular level with cryo-electron microscopy (cryo-EM)

Project lead: Antonio Martínez Sánchez, University of Murcia

Participating universities: University of Cologne, University of Konstanz, University of Murcia, Nantes Université

General Overview

Cytoskeletal disorders underlie many of the leading causes of non-infectious disease and death, including cardiovascular disease, neurodegeneration, invasive cancers, cirrhosis, pulmonary fibrosis, and blistering skin diseases, among many others.  This project will bring together a network of researchers who will produce preliminary data and protocols to combat issues of analysing cytoskeletal components, initially in vitro.

Purpose and Significance

The cytoskeleton is an essential and extensive component of cells (over 80% in some cases). Due to the size, dynamics and filamentous nature of cytoskeletal components, traditional methods used to obtain atomic resolution models of protein systems such as X-ray crystallography and NMR do not suffice. Furthermore, although recent advances in cryo-EM allow for the atomic resolution of larger complex assemblies, it remains technically challenging to apply this method to the structural elucidation of complexes decorating cytoskeleton filament networks. Therefore, there is a pressing need for methodological developments within cryo-electron microscopy (cryo-EM), which can tackle issues involved in imaging cytoskeletal complexes ex vivo. Solving these issues is critical for the development of new therapies.

Implementation Method and Timeline

Partners will contribute as follows: analysis by researchers from Nantes of the dynamic complex components will be combined with state-of-the-art cryo-EM methods and infrastructure in Cologne, together with the development of tailored algorithms to solve current methodological bottlenecks by researchers in Murcia. To establish the above protocols and generate data relevant to human health, samples of a novel actin binding protein complex of medical significance will be produced for this network by the University of Konstanz.

Expected Outcomes

The establishment of these techniques and network will allow the analysis of cryo-EM data, with a particular view to analyse specialised cytoskeletal formations such as sarcomere- and axon-based cell signalling assemblies. Regulatory signalosomes in these systems comprise important biomedical targets, but their analyses are complicated by the extensive molecular crowding occurring in these para-crystalline architectures. Thus, developments in this network will help to facilitate and accelerate the application of the latest powerful EM methodologies to a system of high physiological and biomedical significance that currently remains poorly accessible to study.