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Molecular motors hydrolyze ATP to produce mechanical work by stepping along the cytoskeleton network and carrying cargos. Dynein has many cellular roles which require its minus end-directed motility and force generation along the microtubules (MTs). All dynein activity needs to be tightly regulated by its many associated factors. Lis1 is the only associated factor that directly binds to dynein’s ATP hydrolyzing AAA ring, and it is involved in most, if not all, cellular processes that require dynein activity. In my thesis work, working with both mammalian and yeast proteins, I showed how dynein motility and force generation is regulated by Lis1 and its yeast homolog Pac1 (both Lis1 from here on). Mammalian dynein is mostly autoinhibited, that is, it cannot take many steps before detaching from the microtubules, a property that is essential for dynein-mediated cargo transportation. For processive motility, dynein needs to be relieved from this autoinhibition and needs to bind to dynactin and a cargo adapter. Using protein engineering, single molecule motility, optical trapping, and biophysical characterization assays, we have shown that Lis1 relieves dynein from autoinhibition, thus allowing the formation of dynein dynactin cargo adapter complex (DDX). Through the same mechanism, Lis1 increases the copy number of dynein in DDX complexes which enables faster motility and higher force generation. However, even after the formation of these complexes Lis1 can remain bound to dynein. In that case, we see an inhibitory effect of Lis1. In my thesis, I have shown the mechanism by which Lis1 binding affects dynein motility I switched my research to S. cerevisiae cytoplasmic dynein which has inherently processive motility without needing any cofactors, unlike mammalian dynein. I showed that Lis1 binding to the motor domain slows down dynein motility thus confirming previous studies done on yeast dynein and Lis1. Through multicolor TIRF colocalization assays, I have demonstrated

This thesis presents work toward understanding how the obligate intracellular pathogen, Chlamydia trachomatis, modulates the host cell to establish a protected replicative niche. In order to evade host-cell innate immune surveillance, internalized Chlamydia develop within a membrane-bound compartment referred to as the inclusion. Given that C. trachomatis relies on host-cell derived nutrients and energy, this bacterial pathogen must avoid globally inhibiting host-cell function while building what is essentially a novel organelle. Through strategic deployment of effectors into the host cytosol and inclusion membrane, C. trachomatis actively remodels host-cell structures from within the inclusion. This enables the bacteria to obtain the required metabolites and regulate specific organelle functions. This work focuses on: understanding which host proteins and cellular structures are repositioned around the growing inclusion; identifying which bacterial effectors are responsible for these modifications; and elucidating the mechanisms by which C. trachomatis calibrates organelle function to divert specific resources to the replicating bacteria while maintaining host viability.Until recently, the host targets of only a few Incs had been identified. We utilized high-throughput affinity purification-mass spectrometry to comprehensively define the Inc-human protein interactome, and discovered putative binding partners for 38/58 of the predicted C. trachomatis Incs. Using confocal immunofluorescence microscopy, we screened ~200 of the 335 identified high-confidence Inc-human protein-protein interactions for localization at the inclusion membrane, and we identified the recruitment of many host proteins involved in host processes consistent with Chlamydia’s intracellular life cycle. Thus, Chlamydia effectors recruit distinct subsets of host proteins to the inclusion, and mediate precise changes to the landscape of the host cell. In the first project, we characterized an interac

This thesis presents work toward understanding how the obligate intracellular pathogen, Chlamydia trachomatis, modulates the host cell to establish a protected replicative niche. In order to evade host-cell innate immune surveillance, internalized Chlamydia develop within a membrane-bound compartment referred to as the inclusion. Given that C. trachomatis relies on host-cell derived nutrients and energy, this bacterial pathogen must avoid globally inhibiting host-cell function while building what is essentially a novel organelle. Through strategic deployment of effectors into the host cytosol and inclusion membrane, C. trachomatis actively remodels host-cell structures from within the inclusion. This enables the bacteria to obtain the required metabolites and regulate specific organelle functions. This work focuses on: understanding which host proteins and cellular structures are repositioned around the growing inclusion; identifying which bacterial effectors are responsible for these modifications; and elucidating the mechanisms by which C. trachomatis calibrates organelle function to divert specific resources to the replicating bacteria while maintaining host viability.Until recently, the host targets of only a few Incs had been identified. We utilized high-throughput affinity purification-mass spectrometry to comprehensively define the Inc-human protein interactome, and discovered putative binding partners for 38/58 of the predicted C. trachomatis Incs. Using confocal immunofluorescence microscopy, we screened ~200 of the 335 identified high-confidence Inc-human protein-protein interactions for localization at the inclusion membrane, and we identified the recruitment of many host proteins involved in host processes consistent with Chlamydia’s intracellular life cycle. Thus, Chlamydia effectors recruit distinct subsets of host proteins to the inclusion, and mediate precise changes to the landscape of the host cell. In the first project, we characterized an interac

This thesis presents work toward understanding how the obligate intracellular pathogen, Chlamydia trachomatis, modulates the host cell to establish a protected replicative niche. In order to evade host-cell innate immune surveillance, internalized Chlamydia develop within a membrane-bound compartment referred to as the inclusion. Given that C. trachomatis relies on host-cell derived nutrients and energy, this bacterial pathogen must avoid globally inhibiting host-cell function while building what is essentially a novel organelle. Through strategic deployment of effectors into the host cytosol and inclusion membrane, C. trachomatis actively remodels host-cell structures from within the inclusion. This enables the bacteria to obtain the required metabolites and regulate specific organelle functions. This work focuses on: understanding which host proteins and cellular structures are repositioned around the growing inclusion; identifying which bacterial effectors are responsible for these modifications; and elucidating the mechanisms by which C. trachomatis calibrates organelle function to divert specific resources to the replicating bacteria while maintaining host viability.Until recently, the host targets of only a few Incs had been identified. We utilized high-throughput affinity purification-mass spectrometry to comprehensively define the Inc-human protein interactome, and discovered putative binding partners for 38/58 of the predicted C. trachomatis Incs. Using confocal immunofluorescence microscopy, we screened ~200 of the 335 identified high-confidence Inc-human protein-protein interactions for localization at the inclusion membrane, and we identified the recruitment of many host proteins involved in host processes consistent with Chlamydia’s intracellular life cycle. Thus, Chlamydia effectors recruit distinct subsets of host proteins to the inclusion, and mediate precise changes to the landscape of the host cell. In the first project, we characterized an interac

Cytoplasmic dynein is an AAA+ motor that drives the transport of many intracellular cargoes towards the minus end of microtubules (MTs). Previous in vitro studies characterized isolated dynein as an exceptionally weak motor that moves slowly and diffuses on an MT. Recent studies altered this view by demonstrating that dynein remains in an autoinhibited conformation on its own, and processive motility is activated when it forms a ternary complex with dynactin and a cargo adaptor. This complex assembles more efficiently in the presence of Lis1, providing an explanation for why Lis1 is a required cofactor for most cytoplasmic dynein-driven processes in cells. This review describes how dynein motility is activated and regulated by cargo adaptors and accessory proteins.

Lissencephaly ('smooth brain') is a severe brain disease associated with numerous symptoms, including cognitive impairment, and shortened lifespan. The main causative gene of this disease - lissencephaly-1 (LIS1) - has been a focus of intense scrutiny since its first identification almost 30 years ago. LIS1 is a critical regulator of the microtubule motor cytoplasmic dynein, which transports numerous cargoes throughout the cell, and is a key effector of nuclear and neuronal transport during brain development. Here, we review the role of LIS1 in cellular dynein function and discuss recent key findings that have revealed a new mechanism by which this molecule influences dynein-mediated transport. In addition to reconciling prior observations with this new model for LIS1 function, we also discuss phylogenetic data that suggest that LIS1 may have coevolved with an autoinhibitory mode of cytoplasmic dynein regulation.

Cytoplasmic dynein-1 is a minus-end-directed motor protein that transports cargo over long distances and organizes the intracellular microtubule (MT) network. How dynein motor activity is harnessed for these diverse functions remains unknown. Here, we have uncovered a mechanism for how processive dynein-dynactin complexes drive MT-MT sliding, reorganization, and focusing, activities required for mitotic spindle assembly. We find that motors cooperatively accumulate, in limited numbers, at MT minus-ends. Minus-end accumulations drive MT-MT sliding, independent of MT orientation, resulting in the clustering of MT minus-ends. At a mesoscale level, activated dynein-dynactin drives the formation and coalescence of MT asters. Macroscopically, dynein-dynactin activity leads to bulk contraction of millimeter-scale MT networks, suggesting that minus-end accumulations of motors produce network-scale contractile stresses. Our data provide a model for how localized dynein activity is harnessed by cells to produce contractile stresses within the cytoskeleton, for example, during mitotic spindle assembly.

Oligodendrocytes in the central nervous system produce myelin, a lipid-rich, multilamellar sheath that surrounds axons and promotes the rapid propagation of action potentials. A critical component of myelin is myelin basic protein (MBP), expression of which requires anterograde mRNA transport followed by local translation at the developing myelin sheath. Although the anterograde motor kinesin KIF1B is involved in mbp mRNA transport in zebrafish, it is not entirely clear how mbp transport is regulated. From a forward genetic screen for myelination defects in zebrafish, we identified a mutation in actr10, which encodes the Arp11 subunit of dynactin, a critical activator of the retrograde motor dynein. Both the actr10 mutation and pharmacological dynein inhibition in zebrafish result in failure to properly distribute mbp mRNA in oligodendrocytes, indicating a paradoxical role for the retrograde dynein/dynactin complex in anterograde mbp mRNA transport. To address the molecular mechanism underlying this observation, we biochemically isolated reporter-tagged Mbp mRNA granules from primary cultured mammalian oligodendrocytes to show that they indeed associate with the retrograde motor complex. Next, we used live-cell imaging to show that acute pharmacological dynein inhibition quickly arrests Mbp mRNA transport in both directions. Chronic pharmacological dynein inhibition also abrogates Mbp mRNA distribution and dramatically decreases MBP protein levels. Thus, these cell culture and whole animal studies demonstrate a role for the retrograde dynein/dynactin motor complex in anterograde mbp mRNA transport and myelination in vivo.

The Golgi apparatus is a dynamic organelle involved in processing and sorting of lipids and proteins. In neurons, the Golgi apparatus is important for the development of axons and dendrites and maintenance of their highly complex polarized morphology. The motor protein complex cytoplasmic dynein has an important role in Golgi apparatus positioning and function. Together, with dynactin and other regulatory factors it drives microtubule minus-end directed motility of Golgi membranes. Inhibition of dynein results in fragmentation and dispersion of the Golgi ribbon in the neuronal cell body, resembling the Golgi abnormalities observed in some neurodegenerative disorders, in particular motor neuron diseases. Mutations in dynein and its regulatory factors, including the dynactin subunit p150Glued, BICD2 and Lis-1, are associated with several human nervous system disorders, including cortical malformation and motor neuropathy. Here we review the role of dynein and its regulatory factors in Golgi function and positioning, and the potential role of dynein malfunction in causing Golgi apparatus abnormalities in nervous system disorders.

Intracellular trafficking pathways, including endocytosis, autophagy, and secretion, rely on directed organelle transport driven by the opposing microtubule motor proteins kinesin and dynein. Precise spatial and temporal targeting of vesicles and organelles requires the integrated regulation of these opposing motors, which are often bound simultaneously to the same cargo. Recent progress demonstrates that organelle-associated scaffolding proteins, including Milton/TRAKs (trafficking kinesin-binding protein), JIP1, JIP3 (JNK-interacting proteins), huntingtin, and Hook1, interact with molecular motors to coordinate activity and sustain unidirectional transport. Scaffolding proteins also bind to upstream regulatory proteins, including kinases and GTPases, to modulate transport in the cell. This integration of regulatory control with motor activity allows for cargo-specific changes in the transport or targeting of organelles in response to cues from the complex cellular environment.

Autophagy is a spatially regulated process in axons; autophagosomes form preferentially in the distal axon tip then move actively and processively toward the cell body. Despite the primarily unidirectional transport observed in live-cell imaging experiments, both anterograde-directed KIF5/kinesin-1 motors and retrograde-directed dynein motors are tightly associated with axonal autophagosomes. Here, we discuss our recent work identifying the scaffolding protein MAPK8IP1/JIP1 (mitogen-activated protein kinase 8 interacting protein 1) as a key regulator of autophagosome transport in neurons. MAPK8IP1 tightly coordinates motor activity to ensure the fidelity of retrograde autophagosome transport in the axon.

The spatial organization of the cell depends upon intracellular trafficking of cargos hauled along microtubules and actin filaments by the molecular motor proteins kinesin, dynein, and myosin. Although much is known about how single motors function, there is significant evidence that cargos in vivo are carried by multiple motors. While some aspects of multiple motor function have received attention, how the cargo itself - and motor organization on the cargo-affects transport has not been considered. To address this, we have developed a three-dimensional Monte Carlo simulation of motors transporting a spherical cargo, subject to thermal fluctuations that produce both rotational and translational diffusion. We found that these fluctuations could exert a load on the motor(s), significantly decreasing the mean travel distance and velocity of large cargos, especially at large viscosities. In addition, the presence of the cargo could dramatically help the motor to bind productively to the microtubule: the relatively slow translational and rotational diffusion of moderately sized cargos gave the motors ample opportunity to bind to a microtubule before the motor/cargo ensemble diffuses out of range of that microtubule. For rapidly diffusing cargos, the probability of their binding to a microtubule was high if there were nearby microtubules that they could easily reach by translational diffusion. Our simulations found that one reason why motors may be approximately 100 nm long is to improve their 'on' rates when attached to comparably sized cargos. Finally, our results suggested that to efficiently regulate the number of active motors, motors should be clustered together rather than spread randomly over the surface of the cargo. While our simulation uses the specific parameters for kinesin, these effects result from generic properties of the motors, cargos, and filaments, so they should apply to other motors as well.

The spatial organization of the cell depends upon intracellular trafficking of cargos hauled along microtubules and actin filaments by the molecular motor proteins kinesin, dynein, and myosin. Although much is known about how single motors function, there is significant evidence that cargos in vivo are carried by multiple motors. While some aspects of multiple motor function have received attention, how the cargo itself - and motor organization on the cargo-affects transport has not been considered. To address this, we have developed a three-dimensional Monte Carlo simulation of motors transporting a spherical cargo, subject to thermal fluctuations that produce both rotational and translational diffusion. We found that these fluctuations could exert a load on the motor(s), significantly decreasing the mean travel distance and velocity of large cargos, especially at large viscosities. In addition, the presence of the cargo could dramatically help the motor to bind productively to the microtubule: the relatively slow translational and rotational diffusion of moderately sized cargos gave the motors ample opportunity to bind to a microtubule before the motor/cargo ensemble diffuses out of range of that microtubule. For rapidly diffusing cargos, the probability of their binding to a microtubule was high if there were nearby microtubules that they could easily reach by translational diffusion. Our simulations found that one reason why motors may be approximately 100 nm long is to improve their 'on' rates when attached to comparably sized cargos. Finally, our results suggested that to efficiently regulate the number of active motors, motors should be clustered together rather than spread randomly over the surface of the cargo. While our simulation uses the specific parameters for kinesin, these effects result from generic properties of the motors, cargos, and filaments, so they should apply to other motors as well.

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