Vertebrate photoreceptors are highly specialized sensory neurons that utilize a modified cilium known as the outer segment to detect light. Proper trafficking of proteins to the outer segment is essential for photoreceptor function and survival and defects in this process lead to retinal disease. In this dissertation I focus on two aspects of protein trafficking, intracellular vesicular trafficking in photoreceptors and retinal pigmented epithelial (RPE) cells and how it relates to the human disease choroideremia (CHM), and the trafficking of proteins through the photoreceptor cilium. The human retinal degenerative disease choroideremia (CHM) is caused by mutation of the Rab escort protein-1 (REP1) gene, which is required for proper intracellular vesicular trafficking. However, it was unclear whether photoreceptor degeneration in this disease is cell-autonomous, due to defective opsin transport within the photoreceptor, or is noncell-autonomous and a secondary consequence of defective RPE. Utilizing the technique of blastomere transplantation and a zebrafish line with a mutation in the rep1 gene, I show that photoreceptor degeneration in CHM is noncell-autonomous and is caused by defective RPE. The molecular machinery responsible for protein trafficking through the photoreceptor cilium remained unclear for a long time. Recent studies found Intraflagellar Transport (IFT) is the process that mediates cilia formation and transport of proteins through a cilium, and further analyses showed IFT is important for trafficking proteins to the outer segment. However, many details about how IFT works in photoreceptors remained unclear. By analyzing zebrafish harboring a null mutation in the ift57 gene, I show that Ift57 is only required for efficient IFT, and that the Ift57 protein plays a role in the ATP-dependent dissociation of kinesin II from the IFT particle. Lastly, I investigate the role of retrograde IFT in photoreceptors, a process that had yet to be investigated. By utilizing antisense morpholino oligonucleotides to inhibit expression of cytoplasmic dynein-2 (the molecular motor that mediates retrograde IFT) , I show that retrograde IFT is required for outer segment extension and the recycling of IFT proteins.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 118-130.

The cell relies on molecular motor proteins for long range transport of vesicles and organelles to maintain the organization required within the cell as it changes over time. Cargos move bidirectionally along microtubules due to the presence of multiple copies of opposite polarity motors. Individual motor properties have been teased out in vitro, but understanding how multiple motors cooperate in vivo has thus far been limited by many obstacles. The goal of this work is to study how multiple similar and dissimilar motors operate together in vivo. Since the function of motors is to generate force to haul cargos, I designed a novel optical trapping system capable of precisely measuring the forces exerted by molecular motors in their native environment, a living cell. Using this system, I find evidence that motors do not fight against each other, supporting the regulation model over the tug-of war model for bidirectional transport. I then study motor regulation in axons in the context of Alzheimer’s disease. I find that GSK-3, a kinase found in abnormal amounts in Alzheimer’s brains, is a negative regulator of transport. I show that GSK-3 regulates motor activity rather than cargo binding. Finally, I also use the optical trap to probe the viscosity of cytosol in vivo and investigate its implications on the cooperation of multiple motors. / text

T cells play pivotal roles in the immune system and focused secretion of either cytokines or cytotoxic molecules toward its target is crucial for T cell functions. This directional secretion involves two critical steps: the movement of the microtubule organizing center (MTOC) up to the cell-cell contact site and the directed movement of secretory vesicles towards the MTOC. The minus end-directed microtubule motor protein dynein was previously shown in our studies and those of others to accumulate and anchor at the contact site where it then draws the MTOC up to the contact site. A variety of studies led to the suggestion that there were two functionally different pools of dynein in Jurkat cells, one a ring-like structure that pulled the MTOC to the contact site and the other one uniquely corresponding to the distribution of dynactin. This led to the hypothesis that the second pool of dynein drove vesicle transport. To address this possibility, we used siRNA to deplete the cell of dynactin. These studies showed that almost complete knockdown of dynactin (p150[superscript Glued]) had little effect on MTOC translocation but it also had little effect on a panel of Golgi vesicle markers, whose movement the literature suggested was dynein dependent. As an alternative, a Jurkat cell line expressing fluorescent CTLA4, a known marker for the secretory lysosomes was generated. CTLA4 accumulated at the contact site when Jurkat cells made contact with synthetic target cells. When we repeated the p150[superscript Glued] knockdown in these cells, we found that vesicle transport was blocked, whereas MTOC polarization remained normal. These studies suggest that dynein serves critical roles in both aspects of T cell effector function, the movement of the MTOC up to the cell-cell contact site and the movement of a special class of secretory vesicles up to the MTOC. By the combined processes of MTOC translocation and the minus end-directed movement of vesicles, T cells make it so that a concentrated pool of secretory vesicles are aimed to secrete locally only towards target cells. This ensures that the antigen-specificity of T cell activation is followed by a localized response aimed at the intended target cell. / text

Lfc is a guanine nucleotide exchange factor (GEF) that activates the small GTPase RhoA, and its GEF activity is tightly regulated through protein-protein interactions, phosphorylation, and cellular localization. Lfc is anchored to microtubules through its interaction with the dynein light chain Tctex-1, which results in inhibition of Lfc's GEF activity. Here we present a crystallographic structure of Tctex-1 in complex with Lfc with residues 143-155 of Lfc bound at the Tctex-1 dimer interface. Structural alignment of our structure with Tctex-1 in complex with the dynein intermediate chain (DIC) shows the binding site of the DIC peptide and Lfc substantially overlap. Biochemical evidence, NMR perturbations assays and intrinsic fluorescence provide structural validation and support an extension of the Lfc binding site to the α-helices that may accommodate additional contact points with Tctex-1. We postulate a potential mechanism for Lfc’s recruitment to the microtubules through a tripartite complex with Tctex-1 and DIC.

Lfc is a guanine nucleotide exchange factor (GEF) that activates the small GTPase RhoA, and its GEF activity is tightly regulated through protein-protein interactions, phosphorylation, and cellular localization. Lfc is anchored to microtubules through its interaction with the dynein light chain Tctex-1, which results in inhibition of Lfc's GEF activity. Here we present a crystallographic structure of Tctex-1 in complex with Lfc with residues 143-155 of Lfc bound at the Tctex-1 dimer interface. Structural alignment of our structure with Tctex-1 in complex with the dynein intermediate chain (DIC) shows the binding site of the DIC peptide and Lfc substantially overlap. Biochemical evidence, NMR perturbations assays and intrinsic fluorescence provide structural validation and support an extension of the Lfc binding site to the α-helices that may accommodate additional contact points with Tctex-1. We postulate a potential mechanism for Lfc’s recruitment to the microtubules through a tripartite complex with Tctex-1 and DIC.

No description available.

The microtubule cytoskeleton plays a vital role in the spatial-temporal organization of subcellular cargo required to maintain homeostasis and direct cell division. Cytoplasmic dynein and kinesin are opposite-polarity, microtubule-based motors that transport a wide variety of cargo throughout eukaryotic cells. While much is known about the stepping mechanism of kinesin from decades of study, cytoplasmic dynein's size and complexity has limited our understanding of its underlying motor mechanism. Here, a minimal, artificially-dimerized dynein motor was observed with two-color, near-simultaneous, high-precision, single-molecule imaging, which reveals the stepping pattern of each motor domain as dynein moves along the microtubule. Although the stepping behavior appeared highly irregular and erratic, with large variability in step sizes, side stepping behavior, and back stepping behavior, dynein did show evidence of tension-based, coordinated stepping. Furthermore, advances in DNA nanotechnology enabled us to engineer a synthetic motor-cargo system, referred to as a chassis, to investigate how multiple cytoskeletal motors work in teams to produce the myriad of motile behaviors observed in vivo. Specifically, the mechanisms that coordinate motor ensemble behavior was examined using three-dimensional DNA origami to which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing control of motor type, number, spacing, and orientation in vitro. Ensembles of 1-7 identical-polarity motors displayed minimal interference with respect to directional velocity, while ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species. This experimental system allowed us to test directly the tug-of-war proposed to occur during dynein's delivery to the microtubule plus-end by the kinesin Kip2. This work led to the mechanistic understanding that Lis1/Pac1, CLIP170/Bik1, and EB1/Bim1 proteins function to enhance kinesin's processivity, allowing it to win a tug-of-war and transport dynein toward the microtubule plus-end. Overall, this work elucidated mechanisms of ensemble motor function and dynein's stepping mechanism in addition to building significant tools to further pave the way for future studies to elucidate how cytoskeletal motors function to organize cellular cargos.

Microtubule motor proteins play fundamental roles in transporting a broad range of cellular cargoes in most eukaryotic cells. While there are over 40 kinesins helping to accommodate these diverse cellular demands, there is only one major form of cytoplasmic dynein (dynein, hereafter) carrying out almost all aspects of microtubule (MT) minus end-directed cargo transport. Dynein achieves this versatility by utilizing a wide range of adaptor proteins. Bicaudal D2 (BicD2) is a dynein adaptor protein responsible for linking cytoplasmic dynein to multiple forms of subcellular cargo. These include Rab6A, which contributes to Golgi function (Grigoriev et al., 2007; Matanis et al., 2002); the nucleoporin RanBP2 (Splinter et al., 2010); and the nucleus-cytoplasmic linker LINC complex protein Nesprin-2 (Goncalves et al., 2020). The latter two proteins were found to play important specific roles in the developing brain, respectively in the oscillatory interkinetic nuclear migration (INM) behavior characteristic of Radial Glial Progenitor (RGP) cells and in the directed nuclear migration in postmitotic neurons traveling to the expanding cortical plate. The BICD2 gene was implicated in the autosomal dominant forms of neuromuscular diseases such as Spinal Muscular Atrophy with Lower Extremity Dominance 2 (SMA-LED2) (Neveling et al., 2013; Oates et al., 2013; Peeters et al., 2013; Synofzik et al., 2014) and, more recently in at least three developmental brain pathologies: polymicrogyria (Ravenscroft et al., 2016), cerebellar hypoplasia (Fiorillo et al., 2016), and Lissencephaly (Tsai et al., 2020) expanding the clinical spectrum from neuromuscular only to potentially the entire central nervous system. It is largely unknown how these diverse clinical presentations have any relations to the BicD2 mutational sites. To investigate the genotype-phenotype relationship, it is inevitable to study each point mutation molecularly and characterize the mutational effects in in vivo setting. To investigate the mutational effects of BicD2, I used five different BicD2 fragments and nine missense mutants and characterized their behavior using biochemical and cellular approaches. Four of the missense mutations were further tested in the rat embryonic brain system, in which our lab has previously elucidated BicD2's roles during neurogenesis and post-mitotic neuronal migration. The chapters are organized by mutational effects on the BicD2-cargo interactions (Chapter 2) and the BicD2-dynein interactions (Chapter 3). In Chapter 4, I include supplemental materials to Chapters 2 and 3. In Chapter 5, I summarize, discuss, and provide my perspectives on all the mutational phenotypes found in the previous chapters. Lastly, all experimental procedures and reagents are described in Chapter 6. Here, I describe the first identification of novel gain-of-function (GoF) defects in BicD2- nuclear cargo interaction associated with two pathogenic mutations, R690C and E770G (Chapter 2). Furthermore, I characterize the GoF defects in the embryonic rat brain by in utero electroporation. The cell-type specific expression of R690C or E770G constructs revealed mutation-specific impairment of nuclear migrations in the developing cerebral cortex. In addition, I found GoF defects in BicD2-dynein interaction associated with three pathogenic mutations, L679R, R690C, and T699, surprisingly, in the C-terminal cargo binding domain of BicD2 (Chapter 3). I demonstrate that these missense mutations cause a defect in BicD2 autoinhibition control, which in turn results in abnormally enhanced dynein association. I provide evidence for hyper-activation of BicD2 for dynein binding contributes to Golgi fragmentation, which has been associated with many neuromuscular diseases (Martinez-Menarguez et al., 2019), including SMA-LED2 (Martinez-Carrera and Wirth, 2015). The entirety of this work describes molecular defects in 9 representative BicD2 mutations in vitro and demonstrates the mutational effects in vivo. I propose that the differential mutational effects associated with the BicD2 mutations might contribute to the broad spectrum of clinical phenotypes seen in patients with BicD2 diseases.

Dynein is a motor protein complex that transports various types of intracellular cargos from the cell periphery towards the cell center. Dynein mutations are linked to several neurodegenerative diseases, including Charcot-Marie-Tooth disease (CMT). A mouse model of CMT was generated with a knock-in H304R dynein allele. This mutation at position 304 corresponds to the H306R mutation found in humans that can cause CMT. Here, a behavioral test was developed to study the onset and progression of CMT symptoms in these mice. In the tail suspension test, mice were suspended briefly by their tails and the posture of their hind limbs was scored. Wildtype mice spread their hind limbs outwards in a characteristic splayed posture, whereas heterozygous and homozygous mutants display abnormal phenotypes. In further investigation, the neuromuscular junctions of these mice were analyzed in order to understand the histological effects of the mutation and how the potential differences could result in the behavioral effects observed. The extent of neuromuscular junction innervation was examined along with the size and complexity of the neuromuscular junctions themselves through multiple criteria. This, when combined with the effects observed during the tail suspension behavioral test, seeks to establish the H304R mutant mouse as a successful model for CMT.