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.

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Advances in the antiretroviral therapy (ART) have dramatically reduced the death rate from human immunodeficiency virus type 1 (HIV-1) induced acquired immune deficiency syndrome (AIDS). However, it is still necessary to develop anti-HIV-1 new drugs. In this study, two projects were conducted and may contribute to the new drug development. The first project is focused on characterizing the anti-HIV activity of a kinase inhibitor Kenpaullone (Ken). We found a cyclin dependent kinase (CDK) and glycogen synthase kinase-3β (GSK-3β) inhibitor named Ken can significantly inhibit HIV-1 replication. Mechanistic analysis by RT-PCR revealed that Ken inhibited HIV-1 replication by disrupting transcription possibly through CDK-dependent pathways. The second project is focused on understanding the association between HIV-1 integrase (IN) and dynein components. Our investigation indicated that HIV-1 IN is associated with DIC1 and DYNLT1. Further investigation this IN/dynein component association may help to reveal new anti-HIV targets. / May 2016

The unicellular parasite Giardia intestinalis is one of the organisms carrying mitochondrion-related organelle known as mitosome, which is adapted to the microaerobic lifestyle. The only known fuction of the mitosome is the synthesis of the iron-sulphur clusters. The research of the mitosomal proteome provides new information on the biogenesis and function of this unusual organelle. One of the means of the mitosome research is the analysis of the interactome of the known mitosomal proteins. The state-of- the-art method of the interactome approach is the use of the chemical crosslinking and the subsequent immunoaffinity isolation of the complexes, containing the protein of interest. In this thesis, the interactomes of GiTom40 and GiMOMP35 were characterized with the bioinformatic tools. The cellular localization of four of the chosen proteins was determined by the fluorescent microscopy. One of the proteins, the predicted dynein intermediate chain DIC6939, was phylogenetically classified as an axonemal dynein. The superresolution microsopy was utilized to observe the possible colocalization of DIC6939 with the mitosomes and blue native PAGE led to the visualization of its native complexes. In this work, the optimal conditions for DIC6939 interactome isolation were succesfully determined. The outcome...

Upon entry into G2 and mitosis (G2/M), dynein dissociates from its interphase cargos and forms mitotic-specific interactions that direct dynein to the nuclear envelope, cell-cortex, kinetochores, and spindle poles to ensure equal segregation of genetic material to the two daughter cells. Although the need for precise regulation of dynein’s activity during mitosis is clear, questions remain about the mechanisms that govern the cell-cycle dependent dynein interactions. Frequently dynein cofactors provide platforms for regulating dynein activity either by directing dynein to specific sites of action or by tuning the motor activity of the dynein motor. In particular the dynein cofactor Nde1 may play a key role in defining dynein’s mitotic activity. During interphase, Nde1 is involved in the dynein-dependent processes of Golgi positioning and minus-end directed lysosome transport (Lam et al., 2009; Yi et al., 2011), but as the cell progresses into G2/M, Nde1 adopts mitotic specific interactions at the nuclear envelope and kinetochores. It is unknown how Nde1’s cell-cycle specific localization is regulated and how, if at all, Nde1 is ultimately able to influence dynein’s recruitment and activity at each of these sites. One candidate is cell-cycle specific phosphorylation of Nde1 by a G2/mitotic specific kinase, cyclinB/Cdk1 (Alkurayaet al. 2011). To study the potential function of the phosphorylation by Cdk1, we assayed the localization of GFP Cdk1Nde1 phospho-mimetic and phospho-mutant constructs at the NE and kinetochores. We demonstrate Cdk1 phosphorylation of Nde1 is required for Nde1 localization to both the NE and to the kinetochore, and also the phosphorylation of Nde1 directly activates physical interactions between Nde1 and its nuclear envelope and the kinetochore-binding partner, CENP-F. Furthermore, physiological studies of Nde1 phosphorylation constructs show that over-expression of GFP Nde1 phospho-mutant causes a significant delay in time from NEBD to anaphase onset, specifically demonstrating a late prometaphase/metaphase arrest. Therefore, we conclude Cdk1 phosphorylation of Nde1 not only regulates its localization to the nuclear envelope and kinetochore but also plays an important functional role in Nde1’s mitotic activity in vivo. In addition to understanding how the cell cycle specific activity of Nde1 is regulated, to fully comprehend how dynein functions during mitosis it is necessary to understand how Nde1 is able to modulate dynein’s activity. Nde1 is typically believed to act as a bridge between dynein and specific cellular cargo by physically interacting both with the cargo and dynein/Lis1 to specify the sites of dynein’s activity. Therefore, to understand how Nde1 functions with Lis1 and dynein during mitosis, we created point mutations in the N-terminal coiled-coil domain that specifically disrupted either the Nde1-Lis1 interaction or the Nde1-dynein interaction. We find that disrupting the Nde1-dynein interaction has more severe phenotypic effects compared to disrupting the Nde1-Lis1 interaction: expression of GFP Nde1 del dynein mutant caused a significant delay in anaphase onset while GFP Nde1 del Lis1 only caused a slight increase in cell cycle duration before anaphase onset. Phenotypic analysis suggests that the effects of abolishing the Nde1-dynein interaction on mitotic progression may be due to defects in maintaining kinetochore-microtubule stability during metaphase. Nde1 plays a role in this dynein-dependent mitotic activity through recruitment of a subfraction of dynein to the kinetochore by Nde1’s coiled-coil domain. While the phenotypic effect of removing the Lis1-Nde1 interaction is less severe than removing the dynein-Nde1 interaction, the interaction between Lis1 and Nde1 plays an important role in Nde1’s mitotic behavior as it is affects Nde1’s localization at the kinetochore, specifically by influencing Nde’1 interaction with its kinetochore recruitment partner, CENP-F. The entirety of this work demonstrates that Nde1 acts as a link between cellular cargo and dynein behavior as phospho-regulation of Nde1 throughout the cell cycle allows Nde1’s to interact with unique mitotic cargoes and influence the recruitment and activity of dynein at the kinetochore.

A number of viruses exhibit microtubule-based bidirectional transport following cell entry. This behavior raises three questions: First, what mediates their transport along microtubules? Second, how do viruses recruit the motor proteins? Finally, how do they go to the right place by bidirectional transport in a variety of cell types with different microtubule organizations? We studied these questions with Adenovirus 5 (Ad5), a virus with well characterized, dynein-mediated minus transport mechanism. One form of plus end directed motor, Kif5C, has been reported to disrupt Ad5 capsids at the Nuclear Pore Complexes(NPC), but the mechanisms and roles of microtubule plus end-directed Ad5 transport prior to this stage are largely unknown. Here we performed a RNAi screen of 38 microtuble plus end-directed kinesins, which implicated Kif5B (kinesin-1 family) in plus-end directed Ad5 transport, along with several other forms of kinesin. Kif5B knockdown caused an accumulation of Ad5 particles near the centrosomes in human pulmonary epithelial A549 cells. This effect was strongly enhanced by blocking Ad5 nuclear pore targeting with Leptomycin B and supports a role for Kif5B in Ad5 transport prior to NPC docking. Kif5B RNAi was rescued by expression of any of the three Kif5 orthologues. We also found that Ad5 directly interacts with kinesin-1 via the capsid subunit Penton Base in a PH-independent manner. Together with our earlier studies, these findings reveal that Ad5 has evolved distinct recruitment mechanisms for cytoplasmic dynein and at least one form of kinesin-1 during early infection. Despite clear evidence for short-range linear microtubule-associated Ad5 transport, we found the overall behavior of most Ad5 particles to be stochastic at a larger time scale, by mean-square-displacement (MSD) analysis. We named this behavior "assisted diffusion''. In consistent with this mechanism, Ad5 was able to maintain a normal nuclear targeting after we displaced centrosomes away from the nucleus by inhibiting CDK1 in late G2 cells. We also directly observed Ad5 switching from microtubule based transport to nuclear targeting from a microtubule near the nucleus. Kif5B RNAi dramatically inhibited this novel microtubule-based random-walk/“assisted-diffusion” mechanism. By super resolution microscopy, we found a more local distribution of NPC attached Ad5 over the entire nuclear surface under conditions of Kif5B knock down. We propose that adenovirus uses independently-recruited kinesin and dynein to fully explore the cytoplasm to search for and dock at the nucleus, a mechanism of potential importance for physiological cargoes as well.

Cilia are polarized extensions of the cells microtubule-based cytoskeleton dedicated to sensory, signaling and motility-related functions. In mammals, there are two main types of cilia, immotile and motile, where motile cilia generate/modulate fluid flow at the embryonic node, in respiratory airways, cerebral ventricles and the oviduct in addition to sperm propulsion via the flagellum. Defects in cilia motility cause a rare genetic disorder called Primary Ciliary Dyskinesia (PCD). In this thesis, I present functional and molecular characterisation of two PCD causing genes HEATR2 and ZMYND10. Core cilia genes are transcriptionally activated by members of the winged-helix transcription factors of the RFX family. The forkhead transcription factor FOXJ1, additionally activates motility genes such as the ones encoding components of axonemal dynein motors which transfer the chemical energy released from ATP hydrolysis to kinetic motion necessary for ciliary motility. I present data in this thesis which show that Heatr2 and Zmynd10 are both targets of the RFX3-FOXJ1 transcriptional module which co-operatively switches on genes required to make motile cilia Mutations in both HEATR2 and ZMYND10 cause the same subtype of PCD (loss of inner and outer arm dyneins in cilia). I characterise a human PCD causing mutation in HEATR2 in this thesis. Additionally, using genetic null mouse models generated using the CRISPR technology, I describe the phenotypic effects of complete loss of Zmynd10 in mice. Zmynd10 mutant mice display characteristic PCD-like features. Adding to my functional studies, I present proteomic data to propose mechanisms by which HEATR2 and ZMYND10 proteins control cilia motility. Mass spectrometry and protein interaction studies support distinct roles for HEATR2 and ZMYND10 in intracellular transport and pre-assembly of axonemal dynein motors. The multisystem approaches described in this thesis to characterise the roles of HEATR2 and ZMYND10 highlight the molecular complexity underlying the assembly and delivery of axonemal dyneins to motile cilia and provide novel functional and molecular insights into the pathophysiology of PCD.

The inside of a eukaryotic cell is a highly organized microscale factory that shuttles components that are created or obtained in one place for use or further modification in another. Diffusion cannot accomplish the feat of translocating an object in the cytoplasm to a particular location that is a micron or more away in a timely fashion, so cells rely instead on processive motor proteins. Microtubule motor proteins are enzymes that harness the chemical energy from ATP hydrolysis to produce force and carry vesicles, membrane-bound organelles, and other cargos along paths in the cell's microtubule filament network to their destinations in the cytoplasm. These proteins recognize the polarity of the microtubule, and different classes of motors walk in different directions with respect to this polarity, giving the cell control over the direction in which a cargo is carried. It has been observed experimentally that many cargos are carried by more than one motor simultaneously, and that these multiple-motor systems can consist both of motors of the same type and of varying numbers of motors of different types. Multiple-motor systems present the possibilities of both enhanced transport performance and of tunable behavior, where the number, type, and arrangement of motors on a group of cargos can be modulated by the cell like an analog-style control to induce those cargos to arrive at a particular distribution of locations in the cytoplasm. In order to resolve the mechanisms by which these things might occur, the combination of experimental and theoretical studies in this thesis focus on the relationship between the basic biophysical properties of the constituent motors in small multiple-motor systems and the degree and nature of the cooperation observed, from the standpoint of several relevant metrics. The results highlight the importance of both the mechanochemistry of the motors and the geometry of the system itself, and offer substantial new insights into why different classes of motors cooperate to different extents, with broad implications.

T cell effector functions depend on focused secretion. This is accomplished by secretory vesicle (SV) clustering around the microtubule organizing center (MTOC) and MTOC translocation to the specialized site of cell-cell contact - the immunological synapse (IS). The dynein molecular motor has been implicated in both processes. To investigate the roles of dynein and dynein-associated proteins we used Jurkat cells expressing fluorescent CTLA-4 for SV tracking and molecular traps targeting dynein subunits to show that dynein intermediate chain (DIC) and the light chain LC8 are needed for both SV clustering and MTOC translocation. We also found that immunostaining with different anti-DIC antibodies labeled different pools of dynein at the IS in activated Jurkat cells. To discern how dynein separately accomplishes both MTOC and SV activities we cloned DIC cDNAs from Jurkat cell mRNA and obtained two isoforms, DIC2B and DIC2C. However, both isoforms were concentrated around the MTOC and formed a ring-like structure at the IS. We also saw little difference in dynein-binding proteins that co-immunoprecipitated with each isoform. We then investigated the roles of the dynactin component p150Glued and Lis1 protein in MTOC translocation and SV clustering. Surprisingly, p150Glued was concentrated around the MTOC but was not present at the IS. SVs marked by CTLA-4 showed clustering defects while MTOC translocation was not significantly affected in p150Glued siRNA knockdown cells. On the other hand, Lis1 immunostaining labeled a ring at the IS where it mimicked the distribution of the dynein ring thought to be involved in MTOC translocation. MTOC translocation was potently blocked in Lis1 siRNA knockdown cells but dynein recruitment was only slightly disrupted and there was no visible effect on actin localization at the IS. Overexpression of Lis1 or expression of Lis1 deletion mutants interfered with MTOC translocation and interfered with dynein recruitment, while actin was still localized at the IS. However, studies of calcium flux in response to T cell receptor (TcR) stimulation showed that these mutant-expressing cells had deficiencies in cell signaling from the TcR. These results suggest that MTOC translocation and SV clustering are mediated by dynein but likely involve different dynein-binding proteins. / text

Molecular motors are responsible for all long range transport and organization of organelles within cells. However, little is known about the interaction of multiple similar and dissimilar motors. In this thesis I describe experiments to probe the coordination of the motors kinesin and dynein which move towards the opposite ends of microtubules. Cargos they haul show bidirectional movement at short scales yet there is net transport in one direction or the other. Two distinct models for the bidirectional transport exist: regulation and a tug-of-war. In order to differentiate between them, kinesin-specific antibodies are injected into Drosophila embryos and the effect on transport of lipid droplets is quantified and compared to unperturbed motion. The function-blocking antibodies resulted in an increased run length of dynein-mediated transport and a decrease in that of kinesin. Furthermore, reduced velocities in both directions and a trend towards shorter pauses were observed. Comparison of these results to predictions the models provide for this scenario supports a tug-of-war model rather than regulation. / text