Myosins are cytoskeleton-based molecular motors that display unique spatial and temporal distribution across the eukaryotic cytoplasm and nucleus. Moreover, they are regulated by post-translational modifications, specific interactions with actin isoforms, and light chain activation mechanisms. To better define and understand the regulation of nonsarcomeric myosins in mammalian cells, we implement an approach that examines myosin enzymology, post-translational modifications, cellular interactions with specific actin isoforms and how these interactions affect chemo-mechanical properties. Myosin cellular functionality is strongly determined by enzymatic adaptation to its biological function. Although detailed knowledge of the generic myosin ATPase cycle and motor domain structure has been accumulated, much less is known about the interactions between specific nonsarcomeric myosin and actin isoforms, their respective localization and the effect of post transcriptional modifications on their enzymology. We hypothesize that allosteric effects and their impact on the function of supramolecular complexes in vitro and in the cellular environment is crucial for their cellular role. The actomyosin system is particularly suitable for this type of work, since it allows monitoring the spectroscopic, chemical and mechanical consequences of any modification. Specifically, we study how non-muscle myosin-2C and nuclear myosin-1C are enzymatically behaving in vitro and in their mammalian cellular environment (Zattelman et al., J. Biol. Chem. 2017). We apply the concept of Accurate Allosteric Awareness (AAA) as a precondition for elucidating the effects of the differential expression of splice-isoforms, post-translational modifications and disease-causing mutations (Behrens et al., Sci. Rep. 2017). Our collaboration with Arnon Henn’s group in Israel is funded by a grant from the VolkswagenStiftung.
(PhD projects of Sven Giese and Patrick Reinke)
Collaboration with Prof. Dietmar Manstein (BPC, MHH), Dr. Sharissa Latham (Network Biology Group, Garvan Institute for Medical Research, Sydney, Australia), and Prof. Arnon Henn (Technion, Haifa, Israel)
Funding: VolkswagenStiftung - Niedersächsisches Vorab - Niedersächsisch-israelische Gemeinschaftsvorhaben (funded project partners: Manuel H. Taft, Dietmar J. Manstein, Arnon Henn)
Mutation-induced dysfunction or misfolding of heart muscle sarcomere components have been linked to dilated and hypertrophic cardiomyopathies. In our group, we have previously shown that the synthetic small molecule EMD57033 increases β-cardiac myosin activity and acts as a pharmacological chaperone that stabilizes and refolds the motor domain (Radke, Taft et al., eLife 2014). Following up on this finding, we investigate now whether metabolites such as fatty acids can have a similar effect on β-cardiac myosin. The polyunsaturated omega-6 fatty acid Arachidonic acid (AA) was previously reported as an activator of smooth muscle myosin. We found that among all fatty acids tested, AA induced the largest effect on actin-activated ATPase activity of β-cardiac myosin. We now aim to elucidate the consequences of AA-induced myosin activation in the context of reconstituted human cardiac actin-troponin-tropomyosin complexes for wild-type and mutated sarcomeric proteins. In addition, the effect of selected synthetic and physiological small molecules on the cellular level is studied in neonatal rat cardiomyocytes and human iPSC-derived differentiated cardiomyocytes.
Collaboration with Prof. Dietmar Manstein (BPC, MHH), Dr. Sharissa Latham (Network Biology Group, Garvan Institute for Medical Research, Sydney, Australia), Prof. Vincenzo Lombardi (Department of Biology, Florence, Italy), Prof. Denise Hilfiker-Kleiner (Molecular Cardiology, MHH) and Dr. Robert Zweigerdt (HTTG/LEBAO/REBIRTH, MHH)
Class-18 myosins challenge our established view about myosins acting as molecular motors. No member of this class appears to have a significant ATPase activity, which is a prerequisite for motor activity. Humans express two myosin-18 isoforms, myosin-18A and myosin-18B. Recent studies of our and other groups shed some light on the biochemical and cellular mode of action of myosin-18A (Taft et al., J. Biol. Chem. 2013, Billington et al., Curr. Biol. 2015). The molecular function of myosin-18B remains poorly understood. Class-18 myosins contain protein interaction domains outside their generic motor domain. In the case of myosin-18A this includes a large, N-terminal extension comprising a PDZ module and a KE-rich region, whereas for myosin-18B, the N-terminal extension shows no similarity to any known protein domain. We are interested to unravel the molecular and cellular mechanisms by which class-18 myosins interact with the actin cytoskeleton and how they make use of their unique functional subdomains in the context of cytoskeleton organization. Therefore, we express and purify numerous constructs of both myosin-18 isoforms for in vitro functional and kinetic assays as well as structural studies. Furthermore, we follow the expression and localization pattern of both myosin-18 isoforms in different cell lines to understand their specific roles during cellular processes such as cytokinesis and cardiomyogenesis.
Collaboration with Prof. Dietmar Manstein (BPC, MHH), Dr. Sharissa Latham (Network Biology Group, Garvan Institute for Medical Research, Sydney, Australia), and Dr. Robert Zweigerdt (HTTG/LEBAO/REBIRTH, MHH)