Cytoplasmic dynein is a unique motor that transports a variety of intracellular cargo towards the microtubule minus-end in eukaryotic cells. Mutations lead to neurodegenerative diseases in humans. However, a structural dissection of its mechanism has not been undertaken. By using budding yeast cells to genetically manipulate and express the dynein motor, we perform single molecule FRET experiments to measure interactions between the internal domains in an active protein and investigate how these interactions are correlated with the movement of the motor. The locations of the rings and their rotations will be detected by single molecule microscopy as dynein walks along microtubules. We aim to understand the role of each structural domain in dynein stepping and force generation.

Cytoplasmic dynein’s predicted structure. Each motor head (blue) of a dynein dimer is composed of roughly 3000 amino acids. It contains six AAA+ ATPase domains forming a hexagonal ring, 15 nm in diameter. Dynein motor head binds to microtubules through ~10 nm long stalk domain (purple) with a small MT binding domain at the tip (violet).  The motor heads are held together by a linker region which is presumably composed of multiple short stretches of a coiled-coil. The linker has been proposed as a mechanical element that coordinates the two rings to power dynein’s motion. At the tail, large number of light chains interacts with dynein for regulatory purposes and cargo recognition.

To elucidate how cargoes are transported in vivo, we are using Chlamydomonas cells as a model system. By using this system, we aim to understand how opposite polarity motors function together to move cargoes back and forth along the microtubules and how cells control their activity by associated enzymes. We use mutated strains of Chlamydomonas to externally control motor activity and manipulate cargo transport by applying forces via optical tweezers. Our studies will reveal how cells can rapidly grow and maintain cilia and flagella.

(Left) Chlamydomonas is a single cell green alga which extends two flagella for cell motility and sensory functions. (Right) In order to extend and maintain flagella, particles are moved from cell body to the tip of the flagellum and vice versa continuously by kinesin and dynein motors. Picture shows the bright field imaging of individual IFT particles along Chlamydomonas flagellum.

Model for the control of IFT. Cargoes reverse the direction in turnaround zones located at the tip and base of the flagellum. As IFT particles move anterogradely, kinesin II is driving the movement and dynein is detached from microtubules and a passive cargo. During retrograde transport, dynein is active and kinesin is deactivated. Motor activity can be regulated by phosphorylation by enzymes attached to transition fibers located at turnaround zones.

Telomeric DNA repeats protect the ends of linear chromosomes in eukaryotic cells against degradation. Because of the “3’-end replication problem”, telomeres constantly shorten upon each cell division and critically short telomeres lead to cell cycle arrest. Both aging and human cancers have been related to this important system. Although telomeres and telomere binding proteins have been extensively studied by genetic manipulations and biochemical methods, understanding the formation of functional telomere and its interaction with telomerase and other binding partners needs more sensitive measurements. We aim to develop a single molecule assay by reconstituting human telomere complex in vitro to observe how telomeres form loops for end capping of chromosomes and how these loops open and close as a replication fork reaches to the telomere.

(Left) Telomeres (white) are repeated sequences located at linear ends of chromosomes (blue). (Right) Electron Micrograph shows that telomeres form a large t-loop which may help protection of DNA ends against DNA repair machinery and maintain genetic stability.