Superresolution Studies to Reveal the Interactions between Motor Proteins and Individual Cargos in Chlamydomonas Flagella

Abstract

Our research aims to understand the molecular mechanism of cytoplasmic dynein, which is involved in the transport of cargo towards the microtubule minus end of eukaryotic cells. Specifically, we use intraflagellar transport (IFT) in Chlamydomonas cells as a model system to study interaction of IFT dynein with kinesin II, opposite polarity motor that moves cargos toward the plus end. To detect the distribution and cargo interaction of single motor proteins along the flagellum, we employed Stochastic Optical Reconstruction Microscopy (STROM) microscopy. This technique has proven to construct superresolution images of precisely positioned fluorophores from single-molecule images. In order to facilitate STORM imaging, photoswitchable cyanine reporter and an activator molecule were coupled to antibodies against kinesin II and IFT dynein. The quantitative analysis of the STORM images will allow us to determine how many kinesin and dynein motors actively work on a cellular cargo, and how the cargo direction is determined by kinesin-dynein interactions. IFT is a universal process in all eukaryotic cilia and flagella. Defects in this process are the primary causes of polycystic kidney disease and retinal degeneration.