Ensemble Microscopy Reveals Nanoscale Patterns of Molecular Motors on Microtubules

The precise localization of proteins is of great importance to understand their biological function and to quantify molecular interactions. Here, we present an imaging strategy to obtain nanoscale localization in biochemical reconstitution experiments. This is achieved by imaging a large ensemble of molecules using a conventional total internal reflection fluorescence (TIRF) microscope with automated image acquisition in conjunction with post-acquisition alignment of the individual observations. The strategy bypasses typical limitations of time-lapse microscopy such as small sample size and bleaching. We apply our approach to study the localization of molecular motors on microtubules in a concentration range that spans from single molecule to macroscopic traffic jams. We find that motor proteins, dynein as well as kinesin, show stripe patterns on stabilized microtubules as well as strongly correlated intensity profiles on the nanoscale. The strategy of ensemble microscopy hence allows for spatial high-resolution imaging and is widely applicable in biochemical reconstitution experiments that probe interactions of proteins with the cytoskeleton or DNA for example.