Single molecule imaging
To explain the functions of the individual proteins we must be able to visualize single molecules in our experimental system and track them in space and time. We tag the proteins of interest by fluorescent molecules and attach the experimental system to a functionalized cover-slip surface. Limiting the excitation light to a thin layer using total internal reflection illumination and using highly sensitive detectors we typically localize single molecules with a spatial resolution of tens of nanometers at a time resolution of tens of milliseconds. When higher space- and time-resolution is necessary, we use interferometric scattering (iSCAT) microscopy in collaboration with the Nano Optics group at the Institute of Photonics and Electronics CAS.
To probe the forces exerted by the cytoskeletal systems or to manipulate these systems or perturb them physically on single molecule level we use optical tweezers. Attaching the protein of interest to a micron-sized bead, we can trap and move the bead in the measurement solution by a focused laser beam. In our setup, we can simultaneously use up to four traps. Combined with confocal imaging with single molecule resolution we can correlate the exerted forces with the molecular composition of the experimental system.
In vitro reconstitution
To disentangle the roles of the individual players in the highly orchestrated action of cytoskeletal self-assembly we reconstitute functional parts of the cytoskeletal systems in vitro. We combine the individual players – isolated proteins – to build these simplified systems. The main advantage of this approach is that we have full control over the composition of the system. We can thus easily perturb the system, which combined with the fact that the systems comprise a distinct set of proteins, enables us to experimentally observe features, which would be inaccessible during in vivo experiments, such as in cells or tissues.
Neuronal cell culture
To assess if the mechanisms observed in our reconstitution systems apply in vivo, we fluorescently label in living neurons our proteins of interest. For these experiments we employ neurons grown from X. laevis embryonal neural tube explants and, in collaboration with the Laboratory of Molecular Genetics of Development, Faculty of Science, Charles University, motor neurons differentiated from human induced pluripotent stem cells.