Cytoplasmic dynein and dynactin coordinate microtubule dynamics at the growing plus end

Microtubules are cytoskeletal polymers that serve as long-distance tracks for intracellular transport. However, microtubules are not static tracks; they undergo dynamic instability, a non-equilibrium behavior which allows them to continually remodel. Microtubules must remodel to adapt to the changing needs of the cell, yet they must also be stable enough to allow long-distance transport by kinesins and by cytoplasmic dynein and its partner complex dynactin. Microtubule dynamics are strongly influenced by microtubule-associated proteins, which bind to microtubules and bias them toward growth or shrinkage. A pool of dynein and dynactin localizes to the microtubule plus end and to the cortex, where we hypothesized that they might not be solely poised to initiate retrograde runs, but might also act to modify microtubule dynamics. To test this, we reconstituted microtubule dynamic instability in vitro and visualized it using TIRF microscopy. We show that dynein, when immobilized in an orientation recapitulating its membrane recruitment, can tether growing microtubules, reducing their lateral diffusion and delaying catastrophe. This effect does not represent mere microtubule binding; dynein tethers microtubules more effectively than kinesin or the plus end-tracking protein EB1, and we show that this ability of dynein is dependent on its ATPase activity. Modeling suggests that dynein may delay catastrophe by actively applying tension to straighten, and thus stabilize the microtubule. We also have examined the effects of the p150Glued subunit of dynactin on microtubule dynamics. We find that to modify dynamics, p150Glued must be dimerized, and it must bind tubulin, an interaction that requires its CAP-Gly and basic domains in tandem. p150Glued is alternatively spliced in vivo, with the isoform including both these domains expressed primarily in neurons. Accordingly, depletion of p150Glued in a non-polarized cell line does not alter microtubule dynamics, while p150Glued RNAi in neurons leads to a dramatic increase in microtubule catastrophe. Strikingly, a Parkinson syndrome-associated mutation blocks this microtubule-stabilizing activity both in vitro and in neurons. Together, these data provide novel mechanistic insight into how cytoplasmic dynein and dynactin, the principle minus enddirected motor complex in the cell, also act at the plus end to coordinate microtubule dynamics. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Erika L. Holzbaur Subject Categories Biochemistry | Biophysics | Cell Biology This dissertation is available at ScholarlyCommons: http://repository.upenn. edu/edissertations/655 CYTOPLASMIC DYNEIN AND DYNACTIN COORDINATE MICROTUBULE DYNAMICS AT THE GROWING PLUS END Jacob Eric Lazarus A DISSERTATION In Cell and Molecular Biology Presented to the Faculties of the University of Pen nsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2012 Supervisor of Dissertation _____________________ Erika LF Holzbaur Professor, Physiology Graduate Group Chairperson ________________________ Erfei Bi, Professor of Cell and Developmental Biolo gy Dissertation Committee Morris Birnbaum, Professor of Medicine Kathryn Ferguson, Associate Professor of Physiology Michael Ostap, Professor of Physiology CYTOPLASMIC DYNEIN AND DYNACTIN COORDINATE MICROTUBULE DYNAMICS AT THE GROWING PLUS END