Force Generation by Cytoplasmic Dynein and Development of PhotoGate Microscopy
- Author(s): Belyy, Vladislav
- Advisor(s): Yildiz, Ahmet
- et al.
Cytoskeletal motors play key roles in the organization and division of eukaryotic cells. Although detailed mechanistic understanding has been achieved for motors in the myosin and kinesin families, the mechanochemical cycle of cytoplasmic dynein remained a subject of debate. Understanding the mechanism of dynein motility has been difficult due to its large size, unusual architecture, irregular stepping pattern, and complex regulation by a number of auxiliary proteins. In my doctoral work, I showed that the two heads of dynein utilize a load-sharing mechanism that allows them to work against hindering forces larger than the maximal force produced by a single head. Next, I demonstrated that the regulatory proteins dynactin and Bicaudal-D homolog 1 (BICD) dramatically increase the force production of human dynein and allow it to defeat a human kinesin-1 motor in a tug-of-war competition.
In addition, I developed the PhotoGate method for imaging single fluorescent molecules in the crowded environment of a living cell. This method eliminates the need for fluorophore photoactivation, enabling longer single-particle tracking times and direct measurement of stoichiometry of macromolecular complexes. This technique was used to measure ligand-induced dimerization of epidermal growth factor (EGF) receptors on the cell membrane at densities of >50 molecules per μm2. It was also applied to monitor the arrivals and departures of single Adaptor Protein Phosphotyrosine Interaction PH domain and Leucine Zipper-containing-1 (APPL1) molecules at early endosomes. PhotoGate will be broadly applicable to the study of macromolecular complex formation in the densely packed conditions of the cytoplasm.