Skip to main content
eScholarship
Open Access Publications from the University of California

Studies of Inter-and Intra-Molecular Coordination in Cytoplasmic Dynein

  • Author(s): DeWitt, Mark Alan
  • Advisor(s): Yildiz, Ahmet
  • et al.
Abstract

Cytoplasmic dynein is one of the principle motors in eukaryotic cells, responsible for most microtubule (MT) minus end-directed motility and force generation processes, including vesicular and organelle transport, and mitotic spindle MT organization. Despite being essential to the maintanence of cellular structure in higher eukaryotes, many aspects of dynein's stepping mechanism have remained enigmatic. In this Dissertation, I directly observe several fundamental aspects of dynein's mechanism, and build a strong framework for future studies. I outline in detail the methods for the fluorescent tracking of motor proteins, including advanced two-color and super-resolution techniques. To study cytoplasmic dynein, I developed a two-color fluorescent tracking assay to simultaneously measure the positions of both heads of the motor while it walks along the MT. The results of this experiment clearly show that dynein steps through a unique mechanism: the heads remain widely separated, and do not appear to coordinate with each other to a significant extent. However, the leading head is less likely to take a step at large inter-head separations, indicating some degree of residual coordination remains. I hypothesized that tension along the linker connecting the two heads is the source of this coordination. Using a high-speed two-color assay, I examined the motility of a homodimeric dynein with a flexible linker between the two heads that decreases inter-molecular tension. Despite this alteration, this homodimer has identical stepping and overall motility properties to wild-type dynein, but has reduced coordination, indicating that coordination is dispensable to motility. Coordination could be achieved by gating one of the heads. I investigated gating using a heterodimeric dynein with a wild-type head and an inactive, tightly-bound head. In this heterodimer, we find that the WT head is gated when widely separated from its inactive partner, but becomes un-gated when immediately behind it, indicating that the gating is caused by extension-dependent changes in linker tension as predicted. I next I turned from inter- to intra-molecular communication. Dynein has six distinct AAA subdomains on its motor head, of which only two AAA1 and AAA3, are essential to robust motility. By reducing MT affinity with added salt, I found that AAA3 is required for robust MT release, and that MT release in turn promotes hydrolysis at AAA1. To investigate coordination between AAA1 and AAA3, I analyzed motility in the presence of a slowly-hydrolyzable ATP analog, ATPγS. I found that ATPγS selectively inhibits the AAA3 site. In the presence of saturating analog, the motor can still take long runs of fast motility, indicating that the hydrolysis cycle of AAA3 is much shorter than AAA1. These results show that AAA1 and AAA3 do not coordinate. Instead, AAA3 acts as a "switch" that controls AAA1-directed release from the MT.

Main Content
Current View