Biophysical and in vivo characterization of the dynein-dynactin interaction; implications for multiple modes of regulation
Dynein is a large 1.2 MDa multimeric complex involved in fundamental cellular processes such as retrograde transport, cell division and positioning of organelles. Nearly all known dynein functions are coupled through its interaction with dynactin, a 1.2 MDa complex that increases dynein's processivity along microtubules. In addition to their role in normal biological function, abnormalities in both dynein and dynactin have been implicated in numerous disease states, such as neuropathologies, cancer and are required for viral infection. Despite their importance in both normal and aberrant cell function, essentially nothing is known about the regulation of this fundamental interaction. The dynein-dynactin interaction proceeds through the dynein intermediate chain (IC) and the p150Glued subunit of dynactin. Biochemical and biophysical methods were utilized to understand the IC-p150Glued interaction. We show that the IC-p150Glued interaction is mediated by residues 1-44 of the IC and 415-530 of p150Glued. This interaction forms a heterotetramer in solution, is governed by electrostatics and undergoes a disorder-toorder transition upon binding. Based on these studies, the p150Glued binding site on the IC is in close proximity to the light chain binding region, therefore we hypothesize that the LCs regulate the dynein-dynactin interaction through an allosteric mechanism. Using S. cerevisiae as a model system, we found that abrogation of the dynein light chain LC8-IC interaction in vivo results in dynein specific defects. While our data indicate there are two LC8 binding sites on the S. cerevisiae IC, this differs from other eukaryotic dynein complexes in that the dynein light chain TcTex-1 is absent. Taken together, these data indicate that light chain occupancy regulates dynein-dynactin function. Finally, we leverage these studies to design an inducible IC-trap targeted specifically to the p150Glued binding region of the IC. Induction of the IC-trap results in Golgi disruption, endosomal dispersal and disruption of the microtubule organizing center. The rapid kinetics of disruption indicates that the dynein-dynactin interaction is extremely dynamic. Taken together, these studies provide significant insight into the mechanism and regulation of the dynein-dynactin interaction and enable the design of new approaches for studying the dynein-dynactin interaction in vivo.
Siglin, Amanda E, "Biophysical and in vivo characterization of the dynein-dynactin interaction; implications for multiple modes of regulation" (2010). ProQuest ETD Collection - Thomas Jefferson University. AAI3398133.