Eukaryotic cells face a considerable challenge organizing a complicated interior with spatial and temporal precision. They do so, in part, through the deployment of the microtubule- based molecular motors kinesin and dynein, which translate chemo-mechanical force production into the movement of diverse cargo. Many aspects of kinesin’s motility mechanism are now known in detail, whereas fundamental aspects of dynein’s motility mechanism remain unclear. An important unresolved question is how dynein couples rounds of ATP binding and hydrolysis to changes in affinity for its track, a requisite for a protein that takes steps. Here we report a sub- nanometer cryo-EM reconstruction of the high affinity state of dynein’s microtubule binding domain in complex with the microtubule. Using molecular dynamics flexible fitting, we determined a pseudoatomic model of the high affinity state. When compared to previously reported crystal structure of the free microtubule binding domain, our model revealed the conformational changes underlying changes in affinity. Surprisingly, our simulations suggested that specific residues within the microtubule binding domain may tune dynein’s affinity for the microtubule. We confirmed this observation by directly measuring dynein’s motile properties using in vitro single molecule motility assays, which demonstrated that single point mutations of these residues dramatically enhance dynein’s processivity. We then sought to understand why dynein has been selected to be a restrained motor, and found that dynein-driven nuclear oscillations in budding yeast are defective in the context of highly processive mutants. Together, these results provide a mechanism for the coupling of ATPase activity to microtubule binding and release by dynein, and the degree to which evolution has fine-tuned this mechanism. I conclude with a roadmap of future approaches to gain further insight into dynein’s motility mechanism, and describe our work developing materials and methods towards this goal.

Cytoplasmic dynein is a molecular motor that moves cargos along microtubules. Dynein, together with its large co-factor dynactin, is responsible for the vast majority of traffic towards the centre of the cell. The largest subunit of the dynein complex is called the dynein heavy chain (DHC). The DHC includes a C-terminal motor domain, which converts ATP hydrolysis into mechanical force, an N-terminal tail domain, and a flexible linker domain to join the two together. An intermediate chain (DIC) and light intermediate chain (DLIC) bind directly to the DHC tail, while light chains (DLCs) bind to the DIC. This tail complex is important for both cargo binding as well as homodimerisation of the DHC, which is necessary for processive movement. Previous studies suggest that the DLCs play an important role in homodimerisation, but it remains unclear how else the DHCs are held together. Using S. cerevisiae as a model system, I co-expressed all four dynein subunits and purified functional dynein motors. In this background, I found that truncating the DHC to include only the first 1004 residues (out of the total 4092) eliminates the motor domain as well as the flexible linker domain, while preserving binding to the DIC, DLIC and DLC. However, truncating just another 50 residues off of the C-terminus led to a loss of all accessory subunits. I developed a protocol for expressing and purifying large quantities of the 1004 residue construct, thus I provide the first description of a recombinant dynein tail domain. Using negative stain electron microscopy (EM), I also present the first 3D structural information for the tail region of the cytoplasmic dynein motor. I then describe a construct including only the first 557 residues of the DHC, which dimerises despite not being able to bind any of the other subunits. I present a crystal structure of this smaller DHC fragment, which shows that the N-terminal 180 residues of the DHC constitute an intricate dimerisation domain made up of a β-sheet sandwiched between α-helices. Not only is this the first crystal structure of any part of the DHC N-terminus, but it reveals a previously undocumented dimerisation domain within the DHC itself. Furthermore, information garnered from this crystal structure allowed for interpretation of a recent cryo-EM structure of a triple complex containing the dynein tail, dynactin and the cargo adaptor BICD2 (TDB) that was solved by my colleagues in the Carter group. Only by docking the DHC N-terminus crystal structure within the TDB EM density did it become clear that the N-terminus of the DHC is responsible for the majority of the contacts the dynein tail makes with both dynactin and BICD2. Therefore the work that I present here sheds new light on the unexpected importance of the DHC N-terminus and allows two important conclusions to be made. First, the N-terminal 180 residues of the DHC constitute a dimerisation domain of its own. Second, the next ~400 residues of the DHC form a domain that plays a key role in the complex interface between dynein, dynactin and BICD2.

No description available.

Cytoplasmic dynein is a microtubule-dependent motor protein which participates in numerous cellular processes. The motor complex consists of two heavy chains, intermediate, light intermediate and 3 families of light chains. Dynein is able to bind to these accessory chains as well as to regulatory proteins which enables the motor protein to fulfil such a variety of cellular processes. The associated light chains participate in long-distance organelle and vesicle transport in interphase and in chromosome segregation during mitosis. However, how these light chains control the activity of the motor protein is still unknown. In this study, I combine molecular genetics and live cell imaging to elucidate the role of the associated dynein light intermediate and light chains in dynein behaviour and early endosome (EE) motility in hyphal interphase cells as well as the anchorage of dynein to the microtubule (MT) plus-end in interphase and mitotic cells. I show that the dynein light intermediate chain (DLIC) as well as the light chain 2 (DLC2, Roadblock) are involved in dynein processivity and EE movement in interphase. The downregulation of either protein results in short hyphal growth which could be caused by a decreased runlength of EE and dynein. In addition, both proteins participate in dynein anchorage to the microtubule plus-end in interphase and mitosis as well as in spindle elongation during mitosis. Each protein causes a decrease of the motor protein dynein at MT plus-ends. Surprisingly, I found only minor or no defects in LC8 or Tctex mutants in the observed functions of dynein. LC8 seems to affect the dynein but not the EE runlength. In this case, dynein is still able to move into the bipolar MT array from where kinesin3 is able to take over EEs and move them towards the cell center. In contrast, Tctex has no effect on dynein or EE runlength or any other observed dynein function in hyphal cells. However, it causes a reduction in spindle elongation. Taken together, DLIC and DLC2 are important for dynein behaviour in long distance transport as well as in spindle positioning and elongation during mitosis. Furthermore, I studied the involvement of the dynein regulators Lis1 and NudE as well as the plus-end binding protein Clip1 (Clip-170 homologue) in the anchorage of dynein to the astral microtubule plus-ends during mitosis. The disruption of the anchorage complex at the astral MT plus-end causes a decrease in dynein number at this site and therefore slower spindle elongation in Anaphase B. Taken together, all three proteins are involved in anchorage of dynein to the astral microtubule tip and the subsequent spindle elongation. Furthermore, these findings also show that Ustilago maydis evolved two different mechanisms to anchor the motor protein to MT plus-ends in hyphal and mitotic cells. The plus-end binding protein Peb1 (EB1 homologue) and the dynein regulator dynactin mediate the dynein anchorage in hyphal cells whereas in mitotic cells the plus-ends binding protein Clip1 and the dynein regulators Lis1 and NudE anchor dynein to astral MT plus-ends.