Cytoskeletal Structure and Evolution
| Project Leader: | Felipe Merino | |
| Department: | Protein Evolution - Lupas | |
| Assistant: | María José Prieto | |
| Phone: | +49 7071 601-340 | |
| Fax: | +49 7071 601-349 |
Introduction
Cytomotive filaments couple their spontaneous polymerisation to force generation, which can be harnessed by diverse processes like cell shape remodeling or low-copy plasmid segregation. How has this elementary motor function emerged in evolution? We aim at understanding this from a structural perspective, using a combination of electron cryo-microscopy, structural modeling and simulations and bioinformatic tools.
Research interest
Although once thought as an eukaryotic character, we now know that cytoskeletal proteins are also ubiquitous in bacteria and archaea. Like their eukaryotic counterparts, many of these can couple their polymerisation to force production; they are bona fide cytomotive filaments. Just like actin’s polymerisation and remodelling propels cell motility, prokaryotic actin-like proteins (ALPs) can form cytomotive filaments used to actively segregate different cargo within cells. We seek to understand, from a structural perspective, how has this function emerged in evolution.
Actin-like plasmid segregation filaments
Figure 1. Schematic representation of the ParMRC plasmid segregation machinery. (A) Genes for the filament forming protein (ParM) and the centromere-binding protein (ParR) are located directly after the plasmids centromere (ParC). (B) ParR couples ParM’s polymerisation to plasmid segregation. Figure modified from Merino and Raunser (2018) PNAS 115: 3205-3206.
We are particularly interested in a group of ALPs – the ParM family – in charge of the proper segregation of low-copy-number plasmids (Fig. 1). Unlike actin, these protein are extremely diverse in sequence and, to a lesser extent, in structure. In spite of this, their biological function is the same. How was this function preserved? We combine biochemical characterisation with Cryo-EM to study the function of these filaments and their interaction with key regulators, and use molecular dynamics simulations to understand their function in atomic detail.
Tools to study the actin cytoskeleton
Figure 2. Schematic representation of the process of actin-probe design. A Cryo-EM structure of actin decorated with a known actin-binding protein (lifeAct in this case) can be used to search, in silico, for mutations to improve its affinity. The best candidates are later tested experimentally.
Actin visualization is key to characterise eukaryotic cells. While many actin probes exist, they are prone to artifacts as actin’s function depends on its interaction with regulatory proteins. Recent Cryo-EM work from us and others uncovered the binding mode of the most common of these probes, allowing for the selection of specific tools for particular cytoskeletal structures. Could new ones be designed or discovered? We combine protein design and Cryo-EM to tune the properties of current actin probes and to search for proteins new actin-binding modes (Figure 2).
Selected Publications
1. Funk, J.*, Merino, F.*, Matthias Schaks, M., Rottner, K., Raunser, S., and Bieling, P. (2021) A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks. Nat. Comm. 12: 5329.
2. Belyy, A., Merino, F., Sitsel, O., and Raunser, S. (2020) Structure of the Lifeact–F-actin complex. PLOS Biol. 18: e3000925.
3. Funk, J., Merino, F., Venkova, L., Vargas, P., Raunser, S., Piel, M., and Bieling, P. (2019) Profilin and formin constitute a pacemaker system for robust actin filament growth. eLife 8: e50963.
4. Gatsogiannis, C.*, Merino, F.*, Roderer, D.*, Balchin, D., Schubert, E., Kuhlee, A., Hayer-Hartl, M., and Raunser, S. (2018) Tc toxin activation requires unfolding and refolding of a β-propeller. Nature 563: 209–213.
5. Merino, F.*, Pospich, S.*, Funk, J., Wagner, T., Küllmer, F., Arndt, H-D., Bieling, P.,and Raunser, S. (2018) Structural transitions of F-actin upon ATP hydrolysis revealed at near-atomic resolution by electron cryo-microscopy. Nat. Struct. Mol. Biol. 25: 528–537.
6. Gatsogiannis, C., Merino, F., Prumbaum, D., Roderer, D., Leidreiter, F., Meusch, D., and Raunser, S. (2016) Membrane insertion of a Tc toxin in near-atomic detail. Nat. Struct. Mol.Biol. 23: 884–890.