Hochauflösende Fluoreszenz-Messstation mit konfokaler Mikroskopie und TIRF-Mikroskopie
Final Report Abstract
Many types of cellular motility are based on motor proteins, ranging from muscle contraction to exo- and endocytosis, cytokinesis, cell locomotion or signal transduction in hearing. In order to understand the basic mechanisms of cellular motility from the molecular to the cellular level it is necessary to study the basic mechanisms of chemo-mechanical energy transduction of the motor proteins at the single molecule level in vitro, as well as the mechanisms of motor activation and mobilization in vivo inside living cells. The fluorescence workstation for both TIRF- and confocal microscopy enables us to study the dynamics of the acto-myosin cytoskeleton, both on isolated protein complexes in vitro and in vivo in mammalian cell lines and in single cell parasites. The TIRF-mode allows us to localise and track the formation and movement of single fluorescently labeled myosin motor complexes in vitro and to study the molecular mechanisms of their mechanical function and regulation. We applied TIRF-microscopy to perform the first molecular studies on myosin XXI, the only and vitally important myosin expressed in Leishmania major, a protozoan parasite responsible for a variety of severe human diseases including Kalaazar, African sleeping sickness and South American Chagas disease. Using TIRF microscopy, Förster resonance energy transfer and electron microscopy we could uncover a novel mechanism of myosin regulation in human parasites, where calmodulin binding regulates the dimerisation, motility and lipid binding of the potential therapeutic target myosin XXI in Leishmania. The confocal mode of the workstation enabled us to reveal the localization and dynamics of the different oligomerization states of Leishmania myosin XXI in living cells. The TIRF microscope was crucial in investigating for the first time the mechanisms of activation and mobilization of myosin VI, the only myosin directed towards the minus end of actin filaments in the human genome. Myosin VI plays a critical role in the formation of stereocilia in cells of the auditory system, in intracellular membrane transport and is an early marker of cancer development and aggressiveness. The mechanical assays in TIRF-mode revealed a new mechanism of motor activation where calcium-regulation of a myosin motor uncouples the process of target/cargo binding from the mechanical activation of the motor. Such a two-step mechanism might represent a paradigm for the long-known problem of recruitment and timing of activation and inactivation of cytoskeletal motors in general.
Publications
- Cloning, expression and characterization of a novel molecular motor, Leishmania myosin XXI. Journal of Biological Chemistry 287, 27556-2766 (2012)
Batters, Woodall, Toseland, Hundschell and Veigel
- Calmodulin regulates dimerization, motility and lipid binding of Leishmania myosin XXI. PNAS 111, E227-E236 (2014)
Batters, Ellrich, Helbig, Woodall, Hundschell, Brack and Veigel
(See online at https://doi.org/10.1073/pnas.1319285110) - Calcium can mobilize and activate myosin-VI. PNAS March 1, 2016 113 (9) E1162-E1169
Batters, Brack, Ellrich, Averbeck and Veigel
(See online at https://doi.org/10.1073/pnas.1519435113)
