Microtubules are essential for many dynamic processes occurring within eukaryotic cells including organelle and vesicular trafficking, motility of cilia and flagella, and mitosis. Microtubules operate in conjunction with the kinesin superfamily of microtubule-dependent motor proteins, which use the energy from ATP hydrolysis to "walk" along microtubule tracks, and in doing so generate force for the transport of cellular cargo and mitosis. The goal of this project was to define the microtubule interactions and regulation of two kinesin-like proteins (KLPs), the Homo sapiens mitotic kinesin-like protein-1 (HsMKLP-1) and the Arabidopsis thaliana kinesin-like calmodulin-binding protein (KCBP). Functional domains of HsMKLP-1 and KCBP were heterogeneously expressed in insect cells (HsMKLP-1) and/or E. coli (HsMKLP-1, KCBP) and used to examine the microtubule binding and ATPase activity of HsMKLP-1 and KCBP catalytic domains. Overall, the HsMKLP-1 catalytic domain was found to operate in a similar fashion to other KLPs with respect to microtubule binding and ATP hydrolysis, but HsMKLP-1 exhibited enhanced microtubule binding of the dimer and weaker affinity for ATP that functionally distinguishes it from other KLPs. HsMKLP-1 proteins were also used to generate HsMKLP-1 specific antibodies to be used as a tool for characterizing native HsMKLP-1. To define the role of nuclear localization in regulating the activity of HsMKLP-1 during interphase, sequences directing nuclear localization of HsMKLP-1 were identified. Mutation of the nuclear localization sequence 799PNGSRKRR806 to 799PNGSRTSR806 or removal of AA's 830-856 of HsMKLP-1, which contains the nuclear localization sequence 851PKRKKP856, were sufficient to abolish nuclear localization. In the absence of a functional nuclear localization sequence HsMKLP-1 localized to microtubule plus ends, suggesting that nuclear localization serves to limit the interaction of HsMKLP-1 with the interphase microtubule array. The KCBP catalytic domain, which contains a calmodulin-binding site, was used to determine the effect of Ca2+/calmodulin on the microtubule binding and ATPase activity of KCBP. Ca2+/calmodulin was found to inhibit the binding of KCBP to microtubules and reduced the motor's microtubule-stimulated ATPase activity, which suggests that Ca2+/calmodulin may modulate the activity of KCBP in vivo by regulating the motor's association with microtubules. / Ph. D.

UA62784 is a novel fluorenone identified in a biologic screen of compounds that are selectively cytotoxic in DPC4 (deleted in pancreatic cancer)-deleted pancreatic cancer cells. We sought to determine the mechanism of action of UA62784, and discovered it to be a potent mitotic inhibitor. UA62784 affects the ATPase activity of the mitotic kinesin centromere protein E (CENP-E), but does not affect other known mitotic kinesins. This inhibition of ATPase activity is not caused by an inhibition of microtubule binding nor is it caused by a failure of the kinesin to translocate to the nucleus during mitosis. Despite the anti-cancer properties of this drug, UA62784 is relatively insoluble and is not suitable as a lead compound for further development.Once we determined the mechanism of action of UA62784, we sought to determine if analogs would demonstrate the same potent mitotic inhibition while also offering properties such as increased solubility. A small library of chemical analogs was generated wherein each compound was a slight variation of UA62784 (termed the DPC series). Several potential leads were identified which exhibited increased solubility and/or increased cytotoxic activity. When tested for CENP-E ATPase inhibition, some compounds were noted to inhibit other kinesins as well. We therefore created a screen where each of the DPC compounds was tested for activity in Eg5, CENP-E, MKLP-1, MCAK, and KIF3C kinesins. Within these data, there is a correlation between cellular IC50 and kinesin ATPase inhibition for CENP-E and MKLP-1. A few compounds emerged from these studies, including DPC046, which has a low cellular IC50 and inhibits all five kinesins to some degree. DPC046 was used in a mouse xenograft study to determine in vivo efficacy, but no significant tumor shrinkage was seen, likely due to solubility limitations affecting the amount of bioavailable compound.From these studies we conclude that the cytotoxic effects seen in UA62784 and its analogs are due, at least in part, to their inhibition of kinesin proteins. We demonstrate that compounds that inhibit CENP-E and other kinesin proteins hold promise in cytotoxically targeting pancreatic cancer cells. Further development is needed to optimize DPC046 compound solubility in order to increase in vivo efficacy.

Neurons are among the most highly polarized cells in the human body. This polarization allows the neuron to participate in the transfer of chemical and electrical signals which are crucial to the survival of the organism. As part of polarization, each neuron develops a dendritic arbor and an axon. To ensure the survival of the cell, materials synthesized in the cell body must be trafficked through the axon for delivery throughout ultimately ending at the synaptic termini. The bulk of this cargo transport is microtubule-based fast axonal transport which is molecular motor mediated and tightly regulated though many pathways. Motor based transport is established early in development and maintained for the life of the cell. The kinesin motor protein family plays an integral role in fast axonal transport and the regulation of these motors is essential to proper cargo delivery. Regulation occurs through auto-inhibition, motor interactions with microtubule associated proteins (MAPs) and complex signaling pathways which control the post-translational modification of MAPs, the microtubule track and the motors. The disruption of cargo transport is linked to neurodegeneration and disease state development. Of particular interest in this process is the MAP Tau which has been implicated in a number of neurodegenerative diseases including Alzheimer’s Disease. Tau is expressed at all stages of neural development and has been shown to participate in signaling cascades, modulate microtubule dynamics and preferentially inhibit kinesin-1 motility. Though Tau is involved in these processes, the non-disease state regulation of this MAP and it’s inhibition of kinesin-1 is not well understood. Tau has been shown to bind the microtubule surface in a static-diffusive state equilibrium which differs with isoform and lattice. Previous work demonstrates that the static state is more inhibitory to kinesin-1 than the diffusive state. These different binding behaviors with their different effects on kinesin-1 motility, suggest that cellular regulation of Tau’s static-diffusive binding equilibrium may control inhibition of kinesin-1 and that structural changes may underlie Tau binding to the microtubule surface. Cellular regulation of Tau’s structure and therefore its behavior on the microtubule surface points to a means by which Tau is regulated in the non-disease state. Additionally, this would highlight how early changes lead to disease state development. Using a combination of molecular biology, biochemical techniques and imaging strategies including Total Internal Reflection Fluorescence, single molecule Fluorescence Resonance Energy Transfer (smFRET) and Alternating Laser Excitation, we show that Tau’s static-diffusive state equilibrium is regulated by non-disease state phosphorylation at tyrosine 18. Phospho-mimetics are shifted towards diffusive binding and have decreased affinity for the microtubule surface which in turn reduces inhibition of kinesin-1 motility. These results further demonstrate that Tau undergoes long range structural change while bound to the microtubule surface. We performed smFRET assays and found that Tau binds the microtubule surface in distinct conformations which underlie static and diffusive binding. This work ties the regulation of Tau’s structure and binding behavior to its function and paves the way for our understanding of how cellular regulation acts on multiple levels to fine tune axonal transport.

Alzheimer's Disease is one of the most fearsome diseases worldwide. The study of Alzheimer's Disease (AD) is broad and many have focused on investigating the various proteins involved in neurons. A popular hypothesis of the cellular mechanism of AD is the accumulation of beta-Amyloid. Kinesin is a large group of motor proteins, which plays an extensive role in mitosis and intracellular cargo transport, including that of the Amyloid Protein Precursor. In the present study we have performed fear conditioning behaviour tests on Kif5b conditional knockout (CKO) mouse. Kif5b CKO mouse shows an impair contextual memory compared to the wild type, but does not display an impaired auditory memory. Heterozygous Kif5b knock out mouse shows no significant difference to the wild type. The study has also generated Kif5b fragments and used them to pull-down proteins in mouse brain lysate. The study has identified Clathrin and alpha-Adaptin as binding partners of Kif5b in mouse neuronal cells. The binding domain of Kif5b for these proteins is between amino acid residue 891-935. Finally this study has made a number of recommendations for further study. / published_or_final_version / Biochemistry / Master / Master of Medical Sciences

The cytoskeletal molecular motors kinesin-1 and cytoplasmic dynein drive many diverse functions within eukaryotic cells. They are responsible for numerous spatially and temporally dependent intracellular processes crucial for cellular activity, including cytokinesis, maintenance of sub-cellular organization and the transport of myriad cargos along microtubule tracks. Cytoplasmic dynein and kinesin-1 are processive, but opposite polarity, homodimeric motors; they each can take hundreds of thousands of consecutive steps, but do so in opposite directions along their microtubule tracks. These steps are fueled by the binding and hydrolysis of ATP within the homodimer's two identical protomers. Individual motors achieve their processivity by maintaining asynchrony between the stepping cycles of each protomer, insuring that at least one protomer always maintains contact with the track. How dynein coordinates the asynchronous stepping activity of its protomers is unknown. We developed a versatile method for assembling Saccharomyces cerevisiae dynein heterodimers, using complementary DNA oligonucleotides covalently linked to dynein monomers labeled with different organic fluorophores. Using two-color, single-molecule microscopy and high-precision, two-dimensional tracking, we found that dynein has a highly variable stepping pattern that is distinct from all other processive cytoskeletal motors, which use "hand-over-hand" mechanisms. Uniquely, dynein stepping is stochastic when its two motor domains are close together. However, coordination emerges as the distance between motor domains increases, implying that a tension-based mechanism governs these steps. Many cellular cargos demonstrate bidirectional movement due to the presence of ensembles of both cytoplasmic dynein and kinesin-1. To investigate the mechanisms that coordinate the interactions between motors within an ensemble, we constructed programmable synthetic cargos using three-dimensional DNA origami. This system enables varying numbers of DNA oligonucleotide-linked motors to be attached to the synthetic cargo, allowing for control of motor type, number, spacing, and orientation in vitro. In ensembles of one to seven identical- polarity motors, we found that motor number had minimal effect on directional velocity, whereas ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Signaling systems regulate the flow of cellular information by organizing proteins in space and time to coordinate a variety of cellular activities that are critical for the proper development, function, and maintenance of cells. Signaling molecules can exhibit several levels of complexity through the utilization of modular protein interactions, which can generate simple linear behaviors or complex behaviors such as ultrasensitivity. Protein modularity also serves as the basis for the vast protein networks that form the regulatory networks that govern several biological activities. My work focuses on the importance of protein modularity in complex biological systems, in particular the regulatory pathways of spindle positioning. The first part of my work involves the construction of a synthetic regulatory network using modular protein interactions in an effort to understand the complex behavior of the natural spindle orientation regulator Pins. Utilizing well-characterized protein domains and their binding partners, I built an autoinhibited protein switch that can be activated by a small protein domain. We found that the input-output relationship of the synthetic protein switch could be tuned by the simple addition of "decoy" domains, domains that bind and sequester input signal, thereby impeding the onset of the output response to generate an input threshold. By varying the number and affinities of the decoy domains, we found that we could transform a simple linear response into a complex, ultrasensitive one. Thus, modular protein interactions can serve as a source of complex behaviors. The second part of my work focuses on elucidating the molecular mechanisms underlying spindle positioning in the Drosophila neuroblast. I found that Pins orients the mitotic spindle by coordinating two opposite-polarity microtubule motors Dynein and Kinesin-73 through its multiple domains. Kinesin-73 also relies on its modular domain architecture to perform its duties in Pins-mediated spindle positioning, where its N-terminal half functions in coordinating cortical-microtubule capture while its C-terminal half functions as a region necessary for the activation of Dynein. Thus, modular protein design allows for the organization of spindle orientation regulators in space to achieve the complex biological activity that is spindle positioning. This dissertation includes previously published and unpublished coauthored material. / 10000-01-01

Microtubules are the most rigid element of the cytoskeleton. They are responsible for the structure of cells and make up the tracks for intracellular cargo transport. Interactions between microtubules, motor proteins, and microtubule-associated proteins drive important mechanisms in the cell, such as cell division, cell motility, cell homeostasis, and cell signaling. I seek to understand how such complex, energy-consuming non-equilibrium biological networks self-organize by studying in vitro microtubules bundled by microtubule-associated protein 65 (MAP65), in kinesin-1 gliding assays. I found that large networks can break into smaller, cell-like networks that can mimic types of cell motility. Dynamics of these networks change with varying concentrations of MAP65 and microtubules.

Chinese hamster ovary cells (CHO) were arrested with hydoxyurea at the beginning of DNA synthesis. Subsequent treatment with caffeine induced cells to bypass S-phase and undergo mitosis with unreplicated genomes (MUG). Treated cells built a normal spindle and distributed unattached kinetochores to daughter cells. To determine if MUG cells obey the metaphase checkpoint, we used immunoflourescence to detect and localize known metaphase checkpoint and motor proteins. In addition, the drug taxol was used to stabilize microtubules in MUG cells. The localization of CENP- E, the presence of anaphase A, taxol arrest, and taxol release acted in a similar manner as in controls. The localization of kinesin differed from the controls and that of MAD2 was inconclusive. These results imply that MUG kinetochores behave similarly to controls and probably have an operational metaphase checkpoint.

Drosophila nonclaret disjunctional (Ncd) is a kinesin-like C-terminal motor protein that is involved in spindle assembly in oocytes during meiosis and in spindle maintenance in early embryos during mitosis. Ncd interacts with both "highway" and "cargo" microtubules (MTs) in meiotic and mitotic spindles through the action of ATP-dependent and ATP-independent MT binding sites in the head and tail domains, respectively. Through the action of these binding sites, Ncd bundles and, perhaps, slides MTs relative to each other. These functions are important for the in vivo role of Ncd in the formation of the bipolar spindle and maintenance of the spindle assembly. Despite the high homology of the Ncd head domain to the kinesin head domain, the Ncd tail domain is unique among kinesin-like motor proteins. Characterization of ATP-independent interactions of Ncd with cargo MTs and identification of MT binding sites (located in amino acid residues 83-100 and 115-187) in the tail region by MT co-sedimentation assays revealed that the Ncd tail has functional similarities to microtubule-associated proteins, especially to tau and MAP2, that regulate MT assembly. Like tau MT binding motifs, MT binding sites of the tail domain are rich in basic amino acids that are flanked by proline residues. Cross-linking and MT co-sedimentation assays with subtilisin-digested MTs demonstrated that Ncd tail binding sites (located at the extreme C-terminus and in the H11-H12 loop / H12 helix of each tubulin monomer) on tubulin correspond to tau binding sites. Further, the Ncd tail domain, like tau, can promote and stabilize MT assembly under conditions that induce MT disassembly. Taken together, these results suggest that the Ncd tail functions both in the transport of cargo MTs to spindle poles for the formation of the spindle assembly during meiosis, and in maintenance of spindle assembly during mitosis. How these different functions of Ncd are regulated still remains unknown, however further understanding of the regulation of Ncd function should contribute to our knowledge of cell cycle regulation in both meiotic and mitotic cells. / Ph. D.