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 and tropomyosin 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 and tropomyosin isoforms and how these affect chemo-mechanical properties. The cellular functionality of myosin isoforms is strongly determined by enzymatic adaptation to their biological function. Although detailed knowledge of the generic myosin ATPase cycle and motor domain structure has been accumulated, much less is known about interactions between specific nonsarcomeric myosin and actin isoforms, their regulation by tropomyosin isoforms, the respective subcellular localization of these complexes in specific tissues and cell lines and the effect of post-translational 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 class-II non-muscle myosins and myosin-1C are enzymatically behaving in vitro and in their mammalian cellular environment (Zattelman et al., J. Biol. Chem. 2017; Pathan-Chhatbar and Taft et al., J. Biol. Chem. 2018;). 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; Latham et al., Nat. Commun. 2018;). Our collaboration with Arnon Henn’s group in Israel is funded by a grant by the VolkswagenStiftung.
(PhD projects of Sven Giese and Nadine Weiß)
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 (reviewed in: Taft and Latham, Adv Exp Med Biol, book chapter in: Coluccio L. (eds) Myosins 2020);. 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. Previous 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). 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. 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, 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. Applying this approach, we recently uncovered the molecular and cellular properties of human myosin-18B required for cardiac sarcomere organization (Latham et al., Cell Rep. 2020).
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)