Investigating how kinesin and dynein walk on complex microtubule surfaces

Eukaryotic cells are intricately organized on many length and time scales, from molecules to organelles. Much of this organization is achieved by motor proteins, which directionally transport intracellular components along cytoskeletal tracks (myosins on actin filaments, kinesins and dyneins on microtubules). Intracellular transport takes place in a highly crowded and dynamic cytoplasm, and microtubules are decorated with decorated with obstacles such as microtubule-associated proteins (MAPs), stationary organelles, protein aggregates, microtubule defects, opposing motor traffic and other cytoskeletal filaments. My graduate work focused on how kinesin and dynein respond to the presence of obstacles and MAPs on the microtubule. I chose to approach this question with an in vitro reconstitution approach coupled with single-molecule fluorescence microscopy./nTo understand how obstacles and MAPs on the microtubule (MT) affect cargo transport driven by these motors, I first studied their motility on pure microtubules and then added in various types of obstacles, proteins, and microtubule geometries. Kinesin-1, yeast dynein and the active human dynein complex were challenged to walk on the microtubule in the presence of quantum dots or anti-tubulin antibodies attached to the microtubule surface. I used single-molecule tracking to dissect the motions of these motors to nanometer accuracy on the microtubule. I also used microfabrication to create “microtubule bridges” where the microtubule was raised off the coverslip surface, allowing for the motors to access all sides of the microtubule. I found that dynein motors are capable of bypassing a diversity of obstacles. In comparison, kinesin motors were incapable of walking on obstacle-coated microtubules. This observation, however, was not in line with in vivo observations that both kinesin and dynein can navigate cargos in the complex cellular environment. I discovered that kinesin motors are able to avoid obstacles when working as part of a multi-kinesin team. These results show that multiplicity of motors not only increases the collective force generation and the length of processive runs on an MT but also enables motors to maneuver around obstacles in their path./nNext, I investigated how motors and MAPs interact on the microtubule surface. MAPs have a microtubule-binding domain (MTBD) and a disordered projection domain which extends from the microtubule surface. I monitored kinesin and dynein motility on the microtubule in the absence and presence of the MAPs tau, MAP7 and DCX. With the exception of MAP7 and kinesin, each combination of motor and MAP resulted in inhibition of the motor. In collaboration with Lisa Eshun-Wilson and Mert Gölcük, we dissected the cause of this inhibition using fluorescence imaging, cryo-electron microscopy, and all-atom molecular dynamics. The ability of kinesin to walk on MAP7-coated microtubules was due to the favorable interactions between kinesin and MAP7’s projection domain. Due to the differential effects of MAP7 on kinesin and dynein, we were able to control the directionality of kinesin-dynein assemblies by the addition of MAP7 to the microtubule. Protein engineering revealed that the inhibition of motors was due to the MAP’s MTBD. Surprisingly, we found that motors could be inhibited by a MAP that did not overlap with the motor’s binding site or activated by a MAP that did overlap with the motor’s binding site. Using molecular dynamics, we showed that a large portion of the inhibition of dynein by tau could be linked to positively-charged residues on tau. The MTBDs of MAPs are enriched in lysine and arginine, allowing them to bind to the electronegative microtubule surface. We propose that the inhibition of motors by MAPs is driven by the positively-charged nature of MAP MTBDs, which alters the electrostatic environment of the microtubule and disrupts motor binding. Together, I present a general model for understanding how motors and MAPs interact on the microtubule.

Advisor: Yildiz, Ahmet