Richard B. Vallee - US grants

High molecular weight MAPs from brain tissue will be characterized with regard to ultrastructure, phosphorylation, and function. The ultimate goal is to understand how microtubules function in the cell. Polyclonal and monoclonal antibodies to MAP 1A, MAP 1B, and MAP 2 were produced in the previous project period. One of the antibodies recognizes a phosphorylated epitope. Further hybridoma production will be carried out to obtain monoclonal antibodies to MAP 1C and to other phosphorylated or dephosphorylated epitopes of each of the MAPs. MAPs will be characterized in vitro by rapid-freeze, deep-etch electron microscopy. Individual molecules and molecules associated with microtubules will be examined. The position of the epitopes recognized by the anti-MAP antibodies will be determined directly by electron microscopy. Immunoelectron microscopy, using thin section, whole mount, and rapid-freeze, deep-etch electron microscopy, will be used to localize the MAPs in cells. This information will be compared with the results of the in vitro electron microscopic analysis to define the interactions mediated by the MAPs between microtubules and other cellular structures. Evaluation of changes in MAP composition, distribution, and phosphorylation state in brain and in cultured cells will be examined. PC 12 cell will be investigated to determine the effect of NGF and other effectors on MAP phosphorylation state and distribution. Antibodies recognizing phosphorylated epitopes will be used to identify and, ultimately, isolate MAP kinases. Several approaches will be taken to investigate MAP function directly. Antibodies to the MAPs will be injected into cultured cells to determine effects on neurite formation, mitosis, organelle transport, and other microtubule related functions. In addition, attempts will be made to develop a system for reconstituting organelle transport in crude and purified microtubule preparations. These are basic studies that should be of considerable importance for understanding the structure and functional properties of the neuronal cytoskeleton. Such information should be of profound importance for understanding Alzheimer's disease and other neuropathological conditions. In addition, because the MAP 1 polypeptides are components of the mitotic spindle in a wide variety of cells, the project should provide important new information of relevance to understanding both normal and abnormal cell division.

Microtubule associated proteins (MAPs) from sea urchin eggs will be characterized. Preliminary studies have yielded a new procedure for isolating microtubules from this source which involves the use of taxol to promote assembly. Several MAPs were identified in the purified microtubules with molecular weight values of 80,000, 105,000, 140,000 and approximately 300,00 to 350,000 (HMW MAPs). Fine arms were observed on the surface of the microtubules, quite similar in appearance to arms observed on microtubules in the mitotic spindle. The arms isolated in vitro appeared to cross-link microtubules in to bundles similar to arrays of microtubules observed in the spindle. Monoclonal antibodies were raised against the MAPs. So far two cloned hybridoma cell lines producing antibody respectively to a component of the HMW MAPs and to a minor 40,000 molecular weight MAP have been isolated. In addition, two hybridoma lines producing antibody to proteins of about 55,000 molecular weight that appear to be distinct from tubulin have also been isolated. All of the antibodies stain the mitotic spindle of dividing sea urchin eggs. The antibodies to the 40,000 molecular weight and HMW MAPs also stain the spindle of mouse 3T3 cells. We plan to continue our isolation of hybridomas to obtain antibodies against other MAPs. We will determine by immunofluorescence microscopy and immunoelectron microscopy which classes of microtubules the individual MAPs are associated with during development and within the mitotic spindle. We will microinject the antibodies into living cells to determine their effect on mitosis and other cellular functions, such as nuclear migration and ciliary movement. We will analyse the MAPs by biochemical means for microtubule assembly promoting activity, microtubule cross-linking activity, and ATPase activity, and will determine which MAP species represent arms. Since the major function of sea urchin microtubules seems to be in mitosis, this study should yield information primarily regarding MAPs in the mitotic spindle. Preliminary results have so far borne out this supposition. It is hoped that by the approach proposed here we will be able to obtain information on how cells divide, something of potentially great value for controlling normal or abnormal cell division in humans.

The goal of this project is to understand the role of the brain microtubule associated proteins (MAPs) in axonal transport, neuronal differentiation, cell division and other aspects of cell behavior. During the preceding project period MAP 1C was found to be a retrograde force producing ATPase and to represent a cytoplasmic form of the ciliary and flagellar ATPase dynein. An additional ATPase, dynamin, was also identified, with properties indicating a role in sliding between microtubules. The proposed work deals with the cellular function of dynein and dynamin and with the identification of functional domains in the fibrous MAPs MAP 1A, MAP 1B, and MAP 2. The cytoplasmic dynein and its many component subunits will be examined for co-localization with membranous organelles and kinetochores by immunocytochemistry. A dynein receptor in organelle membranes will be sought, and modification of the mechanochemical and membrane-binding activities of dynein by phosphorylation and fatty acylation will be investigated. Exogenous labelled dynein will be introduced into cultured cells and squid axoplasm to evaluate the behavior of the enzyme in vivo. The structure of dynamin will be investigated by high resolution electron microscopy. cDNA's will be cloned encoding the 100 kD dynamin polypeptide, and will be sequenced to obtain the predicted amino acid sequence of the polypeptide. The dynamin activator will be purified. The location of the dynamin ATPase site will be identified from the predicted amino acid sequence for the 100 kD polypeptide and by photoaffinity labelling of the dynamin holoenzyme. The direction of force production by dynamin will be determined by microtubule gliding using proteolytic fragments or bacterially expressed segments of the 100 kD polypeptide. The orientation of microtubules within dynamin-induced bundles will be determined from the polarity of the constituent microtubules. Together with immunocytochemical evidence on the distribution of dynamin, a model for the function of the protein in the cell will be constructed. The RII-binding domain of MAP 2 will be further defined by RII binding to truncated portions of MAP 2 expressed in E. coli, and the ability of RII dimers to cross-link microtubules will be evaluated. Efforts to clone and sequence cDNAs encoding the MAP 1 light chains will continue in order to determine whether these polypeptides contain regions of homology with the microtubule binding domains of other MAPs. This work is of direct relevance to the control of abnormal cell division and a variety of neurodegenerative diseases.

The goal of this project is to understand the role of the brain microtubule associated proteins (MAPs) in axonal transport, neuronal differentiation, cell division and other aspects of cell behavior. During the preceding project period MAP 1C was found to be a retrograde force producing ATPase and to represent a cytoplasmic form of the ciliary and flagellar ATPase dynein. An additional ATPase, dynamin, was also identified, with properties indicating a role in sliding between microtubules. The proposed work deals with the cellular function of dynein and dynamin and with the identification of functional domains in the fibrous MAPs MAP 1A, MAP 1B, and MAP 2. The cytoplasmic dynein and its many component subunits will be examined for co-localization with membranous organelles and kinetochores by immunocytochemistry. A dynein receptor in organelle membranes will be sought, and modification of the mechanochemical and membrane-binding activities of dynein by phosphorylation and fatty acylation will be investigated. Exogenous labelled dynein will be introduced into cultured cells and squid axoplasm to evaluate the behavior of the enzyme in vivo. The structure of dynamin will be investigated by high resolution electron microscopy. cDNA's will be cloned encoding the 100 kD dynamin polypeptide, and will be sequenced to obtain the predicted amino acid sequence of the polypeptide. The dynamin activator will be purified. The location of the dynamin ATPase site will be identified from the predicted amino acid sequence for the 100 kD polypeptide and by photoaffinity labelling of the dynamin holoenzyme. The direction of force production by dynamin will be determined by microtubule gliding using proteolytic fragments or bacterially expressed segments of the 100 kD polypeptide. The orientation of microtubules within dynamin-induced bundles will be determined from the polarity of the constituent microtubules. Together with immunocytochemical evidence on the distribution of dynamin, a model for the function of the protein in the cell will be constructed. The RII-binding domain of MAP 2 will be further defined by RII binding to truncated portions of MAP 2 expressed in E. coli, and the ability of RII dimers to cross-link microtubules will be evaluated. Efforts to clone and sequence cDNAs encoding the MAP 1 light chains will continue in order to determine whether these polypeptides contain regions of homology with the microtubule binding domains of other MAPs. This work is of direct relevance to the control of abnormal cell division and a variety of neurodegenerative diseases.

dynein ATPase; protein structure function; regulatory gene; neuronal transport; microtubule associated protein; cell cycle; Golgi apparatus; cytoplasm; organelles; chemical binding; adenylate kinase; gene expression; phosphorylation; electron microscopy; laboratory rabbit; laboratory mouse; genetic mapping; molecular cloning; point mutation; image processing; oligonucleotides; fusion gene; nucleic acid sequence; complementary DNA; antisense nucleic acid;

Dynamin is a 100 kD GTPase identified in this laboratory which is involved in the initial stages of endocytosis. Mutations in dynamin and shibire,its Drosophila homologue, block the formation of coated and non-coated vesicles at the plasma membrane and the internalization of cell surface ligands and receptors. Mutations in shibire also block the reformation of synaptic vesicles at neuromuscular junctions and interneuronal synapses once the vesicles have discharged their contents. Dynamin has been found to interact with a number of macromolecular factors, most recently the SH3 domains of proteins involved in signal transduction, but which of these factors are involved in dynamin function in the cell remains uncertain. The long-term objective of this project is to understand the mechanism of action of dynamin, and, in particular, the steps in its GTPase cycle. To address this problem, we will take advantage of our extensive mutational analysis of the protein, and we will use a variety of molecular genetic, biochemical, ultrastructural and genetic approaches. We will seek to identify novel dynamin-interacting proteins and to characterize known dynamin interactions further. We will determine the dependence of dynamin interactions on the state of the guanine nucleotide in the dynamin active site, to help define the functional cycle of the protein. We will determine whether dynamin self-associates in the cell, and whether self- association activates the dynamin GTPase. We will characterize the association of dynamin with coated pits and other endocytic precursor structures by ultrastructural and biochemical means. Finally, we will attempt to identify a true dynamin homologue in yeast. An understanding of the dynamin functional cycle has a number of medical implications. This knowledge may ultimately lead to methods to control the entry of viruses, LDL, and other potentially deleterious agents into the cell. it may also provide a means to control the lifetime of activated growth factor receptors on the cell surface, and, therefore, a means to control normal and abnormal cell growth. Finally, because temperature-sensitive shibire mutations mimic certain conditional paralytic conditions of humans, an understanding of how dynamin functions could be of value in understanding and controlling these conditions.

DESCRIPTION (Adapted from applicant's abstract): The lissencephalopathies are a class of human brain development diseases thought to result from defects in neuronal cell body migration. Mutations in the LIS-1 gene are responsible for Miller-Dieker Lissencephaly and Isolated Lissencephaly Sequence. LIS-1 encodes a polypeptide which is homologous throughout its length to fungal proteins implicated in cytoplasmic dynein function but which also copurifies with PAF acetylhydrolase, an enzyme responsible for inactivating the lipid mediator PAF (platelet activating factor). The overall goal of this study is to determine the role of LIS-1 in cytoplasmic dynein function and the mechanism through which LIS-1 controls neuronal migration. In preliminary studies in cultured mammalian cells, overexpression of LIS-1, exposure to LIS-1 antisense oligonucleotides, and microinjection of anti-LIS-1 antibody were observed to produce pronounced phenotypic effects. Noteworthy among these were a dramatic increase in mitotic index, defects in chromosome attachment to mitotic spindle, alterations in the structure and organization of mitotic microtubules, and changes in the distribution of dynein and the dynein-associated complex dynactin at the cell cortex and at microtubule ends. Specific Aim 1 seeks to define the physiological function of the LIS-1 polypeptide (i) by monitoring these effects in real time; (ii) by determining the subcellular distribution of LIS-1; and (iii) by comparing LIS-1 and cytoplasmic dynein phenotypes, including effects on nuclear migration, in primary neurons, brain slices and nonneuronal cells. Specific Aim 2 seeks to define the role of PAF in cytoplasmic dynein function (i) by examining the effects of PAF on organelle distribution and microtubule dynamics; (ii) by determining how these effects are altered by changes in LIS-1 expression; and (iii) by determining how PAF affects the post-translational modification of dynein. Specific Aim 3 seeks (i) to define further the physical interaction of LIS-1 with cytoplasmic dynein identified in preliminary studies, and (ii) to determine the relationship of this interaction with those involving PAF acetylhydrolase and other proteins. These studies should shed important new light on the mechanism of a serious brain developmental disease, and provide critical new insight into the role of microtubules and motor proteins in neuronal migration, a fundamental feature of brain development.

dynein ATPase; protein structure; biomedical resource; macromolecule; cytoplasm; clinical research; mass spectrometry;

This proposal was received in response to Nanoscale Science and Engineering initiative, NSF 04-043, category NIRT.

This Nanoscale Interdisciplinary Research Team (NIRT) project will create nanofabricated, biofunctionalized arrays to study the fundamental relationship between spatial order and function in biochemical systems. These 'nanoscale bioarrays' will be fabricated using leading edge nanofabrication technology to allow investigation of structure-function relationships on the molecular scale, i.e. a few to tens of nanometers. The arrays will be organized hierarchically, with unit cells (comprising one or more biofunctionalized 2-10 nm metal dots) organized into micron-sized domains, and patterned in mm-size areas. Each domain will comprise identical unit cells, and the unit cell geometry will be systematically varied from domain to domain, in order to allow for straightforward assay techniques on the micron scale. The nanoscale bioarray technique will be applied to three biological investigations: 1, the study of the dependence of binding of large cytoskeletal proteins on the spatial arrangement of ligands; 2, the study of the effect of ordering cytoplasmic dynein molecules on microtubule-dependent motility; 3, the seeding of protein crystals.

Intellectual Merit:
This project attempts to push the limits of nanofabrication technology in order to interface with biological systems. It will employ a "top-down" approach to study and control "bottom-up" assembly, function and synthesis of these systems. Each sub-project will use nanofabrication technology to extend a biological question or problem beyond where currently available techniques will allow. The project is organized around a central theme and fabrication technique, which will allow for rapid technology development. The project addresses the research and education theme "Biosystems at the Nanoscale."

Broader Impacts:
This project will have many broader impacts. The advances in the use of solid-state nanofabrication to address biological and chemical problems will enhance research infrastructure in ways that will impact areas of biological science beyond just cytoskeleton-based motility or protein crystallization. The participation of young scientists-in-training in this interdisciplinary environment will foster development of a cadre of future scientists with the necessary knowledge and cultural and technical skills to successfully pursue novel multidisciplinary science and technology. Undergraduate students will also participate in the research, including students drawn from institutions in the New York City area with significant minority enrollment. The Principal Investigator will draw on his experience as a New York City public high school teacher to extend outreach to the K-12 level.

[unreadable] DESCRIPTION (provided by applicant): The dyneins are a class of motor protein involved in many of aspects of microtubule-dependent movement. Cytoplasmic dynein, in particular, is responsible for diverse forms of vesicular, macromolecular, and mitotic movement, as well as organization of the microtubule cytoskeleton and directed cell movement. How the dyneins function to produce force, and how cytoplasmic dynein is selectively targeted to a diversity of subcellular structures represent two of the major outstanding issues in the field. This proposal pursues Aims directed at each or these issues based on progress during the preceding project period. Aim I deals with the stalk, a 15 nm long projection extending from the motor domain, which is responsible for microtubule binding. This Aim will determine the structure of the stalk at atomic resolution; it will further identify conformational changes within its putative antiparallel coiled-coil D-helix responsible for long-range allosteric coupling of microtubule binding and ATP hydrolysis. Aim II addresses mechanisms involved in cytoplasmic dynein cargo binding. Our recent studies have revealed a general role for the ZW10 protein complex in linking dynactin and dynein to diverse subcellular structures. We will define the mechanism responsible for ZW10 membrane binding; focusing on the Golgi apparatus, we will determine the role of ZW10 relative to Rab6, spectrin, and other factors implicated in dynein cargo binding, with the goal of a complete elucidation of the Golgi cargo binding pathway. Aim III will involve dynein-mediated virus motility. We will focus on adenovirus as a structurally simple pathogenic form of dynein cargo. We will extend and complete our efforts to identify the interacting partners between dynein and the adenovirus capsid. Together these studies have important implications for understanding many aspects of normal and abnormal cell and developmental biology. The final aim has particular significance for the control of acute viral infection and the design of gene delivery vectors and strategies. [unreadable] [unreadable] [unreadable]

Type I lissencephaly is a severe brain developmental disease which involves defects in neural progenitor cell migration during the formation of the cerebral cortex. It is caused by LIS1 haploinsufficiency, resulting from sporadic mutations in the LIS1 gene. LIS1 has been implicated in the platelet activating factor and cytoplasmic dynein pathways, but current evidence suggests that defects in the latter are responsible for the brain developmental disease. Work from this lab has indicated that LIS1 functions together with cytoplasmic dynein at kinetochores and the cortex of dividing cells, at the leading edge of migrating fibroblasts, and during growth cone remodeling. Recent results from this lab have identified severe effects of LIS1 RNAi on the behavior of neural progenitor cells from neurogenesis through the entire course of radial migration by live imaging of brain slices. Aim I of this project will be to characterize further functional interactions between LIS1 and cytoplasmic dynein and their mutual binding partners NudE, and NudEL. The role of protein phosphorylation in regulating these interactions, and the effects of LIS1, NudE, and NudEL on dynein mechanochemical activity will be'determined. Aim II will be to define the role of LIS1, NudE and NudEL in the morphogenesis and locomotion of bipolar neural progenitors. Aim III will be to define the role of LIS1, NudE and NudEL in the interkinetic nuclear oscillations and cell division cycle of neural progenitor cells at the radial glial stage. Live imaging of embryonic rodent brain slices and isolated neural progenitors will be performed on control, mutant, or RNAi-treated animals to evaluate effects on nucleokinesis, cell division, control of division by nuclear position, glial guided migration, and process dynamics. The behavior of the microtubule cytoskeleton, centrosomes, nuclei, LIS1, NudE, and NudEL will be monitored to understand the underlying cellular causes for observed brain phenotypic effects. These studies are of considerable relevance to understanding normal and abnormal brain development, cell migration, cell division, the causes of mental retardation, and stem cell behavior.

DESCRIPTION (provided by applicant): Defects in neocortical neurogenesis and migration cause severe brain developmental disease. LIS1, mutations in which cause lissencephaly (smooth brain), was the first neuronal migration gene to be identified. LIS1 functions in the cytoplasmic dynein pathway, indicating that microtubule motor proteins play a role in brain development. In earlier work supported by this grant we identified multiple discrete LIS1- and dynein- requiring stages in neurogenesis and migration, leading to a comprehensive model for the cellular basis of classical (type I) lissencephaly. We also found LIS1 to be required for the long-mysterious cell-cycle- dependent interkinetic nuclear migration (INM), a general feature of neuroepithelial and radial glial progenitor cell (RGPC) behavior. We have determined further that INM requires the activity of opposite-directed microtubule motor proteins, the plus end-directed unconventional kinesin Kif1a and cytoplasmic dynein. This model appears to explain the underlying mechanism for INM, and should allow us to address further basic and long-standing questions regarding its function and purpose. The Specific Aims are to determine the mechanism of nuclear transport by Kif1a; to determine how specific inhibition of basal and apical INM affect cell cycle progression and cell fate; and to determine the mechanisms for cell cycle control of INM using small molecule protein kinase inhibitors and other reagents. These issues have important implications for understanding how brain size, composition, and organization are controlled, and how stem cell proliferation is regulated under normal or neoplastic conditions. The analysis of genes responsible for INM and the use of small molecule cell cycle inhibitors will also identify potential targets for modulating neurogenesis and migration during early brain development. PUBLIC HEALTH RELEVANCE: This proposal addresses the mechanisms responsible for neural progenitor cell behavior in the developing brain. We will test the consequences of inhibiting specific genes and protein kinases on the developmental fate of progenitor cells. These studies will identify important new therapeutic targets for developmental conditions, and elucidate the causes of microcephaly, lissencephaly, and heterotopic disorders.

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Cytoplasmic dynein is a microtubule motor protein involved in a very wide array of cellular functions, including vesicular transport, chromosome segregation, and cell migration. A single major form of cytoplasmic dynein is responsible for almost all aspects of these activities, but how it is adapted to such a diversity of functions at a broad range of subcellular sites remains a major question. Among the known dynein interactors, two complexes have emerged with prominent roles in dynein cargo binding and motor regulation, NudE-LIS1 and dynactin. We have recently reported NudE-LIS1 to have a novel and unique ability to interact with the dynein motor domain during its powerstroke and adapt the motor protein for high load functions. We also found LIS1-NudE to compete with dynactin for dynein binding, suggesting that the two complexes may serve in alternative regulatory roles. This proposal is to test this hypothesis and to bring to bear on dynactin the approaches we have successfully used to determine the functions and mechanism of action of LIS1 and NudE. Dynactin is known to enhance dynein processivity in vitro. However, its interaction with dynein has been difficult to control, hampering progress toward a complete understanding of its mechanochemical functions. Because dynactin is also important in dynein cargo recruitment, its specific in vivo role in motor regulation has also been difficult to define. Preliminary results have identified conditions controlling the dynein-dynactin interaction, and have revealed potent, long-range allosteric effects for dynactin fragments on dynein force production and processivity. The Aims of this proposal are (1) to determine how the dynein-dynactin interaction is regulated and to produce and define cocomplexes for further analysis; (2) to determine the complete scope of regulatory functions for the dynactin complex, its major regulatory subunit p150Glued, and its subfragments to understand the underlying mechanisms for dynein regulation; and (3) to determine the specific roles of dynactin in dynein motor regulation in vivo by high resolution particle tracking and force analysis. These studies are of broad relevance for understanding basic mechanisms of cell behavior. In addition, they should shed important new light into the mechanisms underlying brain developmental disease, motor neuron degeneration, cell division, and other physiological and pathophysiological functions.

A number of viruses exhibit bidirectional transport along microtubules following cell entry. Cytoplasmic dynein has been implicated in virus transport toward the nucleus, but the role of kinesins is poorly understood. They might either aid in virus infection, or serve in host defense. Adenovirus provides a simple model for addressing these questions and is the focus of this proposal. Prior studies from this lab worked out the mechanism for dynein recruitment to the adenovirus capsid, and identified roles for protein kinase A (PKA) in virus transport as well as in host cell defense. The Aims of the proposed studies are 1) to identify kinesins involved in plus end-directed adenovirus transport during the early stages of infection; 2) to determine the role of kinesins in host-adenovirus interactions; and 3) to test the effect of microtubule stabilization and posttranslational modifications on live adenovirus transport. The latter Aim will make use of high-resolution live adenovirus particle tracking analysis, and is designed to take advantage of and complement the Aims of other projects described in this Program Project application. The proposed investigation of adenovirus alone has important implications for understanding the cellular mechanisms involved in virus infection, identifying novel mechanisms for host cell defense, and improving the design of gene targeting vectors.

Microtubules (MTs) form complex intracellular networks that not only provide mechanical stability for cell structure and motility, but also from the tracks along which molecular motors traffic cargos to specific sub- cellular sites. While most MT networks have a relatively short half-life, recent work has shown that a subset (-10%) of post-translationally modified MTs are highly stable and function in a various biological processes. However, their role and regulation during viral infection remains poorly understood. Our screens for host factors that determine cellular susceptibility to infection by various viruses, in collaboration with Gundersen (Project 1), Walsh (Project 3) and Goff (Project 4) identified a number of novel regulators of stable MT formation, highlighting the potential role of this stable subset of MTs as specialized tracks for viral trafficking. Our collaborative effort with Gundersen to explore this more directly resulted in identification of the kinesin, Kif4 as an EBl-interacting regulator of MT stability and HIV-1 infection. In addition, Kif4 is also bound by Gag, a retroviral structural polyprotein that has been suggested to promote MT stabilization. Combined with our preliminary data showing that HIV-1 infection promotes stable MT formation and incoming viral cores colocalize with this stable MT subset, this suggests that Kif4:EB1 complexes represent a specific MT tip- binding complex specifically targeted by HIV-1. In this proposal we will map the domains in Kif4 and EBl that mediate their effects on stability and trafficking of HlV-1 during both early stage (inward-directed) movement of viral cores and late stage (outward-directed) movement and budding of viral particles. We will also determine the effects of infection or Gag expression on Kif4:EB1 complex formation and function in mediating MT stabilization during distinct phases of infection, and define the domains in Gag that mediate interactions with Kif4:EB1 complexes to determine how this viral protein affects complex formation and/or activity. Finally, we will determine whether infection or Gag expression manipulates localized signaling nodes to regulate MT formation and/or Kif4:EB1 function as virions enter the cell. This work takes advantage of working closely with members of this PPG with expertise in cytoskeletal regulation, motor-based virion movement, live imaging and cell signaling, as well as using the Imaging Core to develop a real-time image of the dynamic events involved in viral interactions with host MT networks and the role of highly specialized MT regulators in these events. This will provide detailed mechanistic insight into the role of MT regulators and subsets during HIV-1 infection, with important implications for our general understanding of MT regulation and cargo transport.

Microtubules and their nnotors mediate the intracellular movement of cargos and control of cell structure that underlie processes such as cell polarization and motility. Consisting of a/|3-tubulin heteropolymers, MT minus-ends are anchored in the perinuclear MT Organizing Center (MTOC) while their plus-ends radiate toward the cell periphery. Most MTs dynamically grow and shrink rapidly. However, a subset (<10%) become stabilized in response to various physiological signals, acquiring post-translational modifications such as de- tyrosination or acetylation, and are thought to act as specialized tracks for vesicle transport. The end-binding protein, EBl tracks MT plus-ends and regulates both MT dynamics and stabilization by recruiting a variety of plus-end binding regulatory proteins (+TIPs). +TIP binding responds to signal pathways such as Rho-mDia and Akt-GSK-3p. MTs also play a critical yet poorly understood role in the trafficking of pathogens. Herpes Simplex Virus type 1 (HSV-1) infects ~60-90% of the world population, establishing life-long infections that result in periodic disease recurrence, ranging from cold sores to fatal encephalitis. While it is established that HSV-1 induces a-tubulin rearrangements and exploits MT motors for intracellular movement, the precise nature of MT modifications and their role in infection, as well as potential roles for specialized MT regulatory factors remain unknown. Our preliminary data shows that HSV-1 causes centrosomal MTOC dispersal and MT disorganization early in infection of primary normal human dermal fibroblasts. This occurred concomitant with the loss of EB1 comet staining, indicative of MT plus-end tracking, and was dependent on viral gene expression. Co-immunoprecipitation identified a candidate EBI-binding protein synthesized in infected cells while RNAi-mediated depletion of EBl prevented MT reorganization and potently inhibited infection. As infection progressed, EBl comet staining returned as extensive MT re-growth and stabilization occurred. Depletion of EBl or CLASP2, a GSK-3P-regulated +TIP that controls MT growth from the Trans Golgi Network (TGN), a known alternate MTOC, significantly impaired HSV-1 infection. In cells infected with HSV 1 lacking the viral kinase, Us3, which stimulates GSK-3p phosphorylation, MTs remained bundled around the TGN, the site of secondary virus envelopment. We aim to determine the role of specialized host MT end- binding proteins and viral factors in regulating MT dynamics and stabilization at discrete phases of HSV-1 infection in different natural target cell types, defining their role in mediating virion trafficking and spread. This will provide important insight into not only the lifecycle of this ubiquitous human pathogen but also more general mechanisms of MT regulation and macromolecular transport. RELEVANCE (See instructions): Microtubules regulate intracellular cargo movement and changes in cell structure that play critical roles in various biological processes, yet their regulation remains poorly understood in many contexts including viral infection. In this proposal we aim to determine the role of specialized MT regulatory proteins and MT subsets in HSV-1 infection of a variety of natural target cell types. This will not only provide important insight into the replication of this ubiquitous human pathogen, with the potential to uncover novel antiviral targets, but will also add to our broader understanding of fundamental mechanisms of MT regulation and cargo transport.

The intracellular movement of viral particles requires directed motor-based transport on long-range microtubule (MT) networks. However, the molecular details by which these dynamic host networks function during infection remain poorly understood. Members of this Program Project Grant (PPG) have observed diverse changes in MT organization and stability during infection with distinct RNA or DNA viruses. In addition, we have identified roles for specialized MT regulatory factors in mediating MT rearrangements and viral infection as well as key motors involved in Adenovirus bi- directional motility. As part of our Overall Aims to develop a truly detailed understanding of how these motile invaders interact with and exploit the equally dynamic MT networks of their host over the course of infection, we will establish the Imaging Core. Run by an expert in live imaging of organelle and virus motility (Dr. Richard Vallee), the Imaging Core will provide state of the art high temporal and high spatial resolution dual-fluorescence live imaging to support the individual Aims of each of the five members of this PPG. The capacity of this equipment will allow real-time tracking of viral particle movements and their interactions with host MTs at various stages of infection in living cells. This imaging system will also be used to develop new ways of visualizing stable MTs in living cells that should allow for the first time direct tests of trafficking of cargos on specific MT subtypes, as well as developing new ways of generating post-translationally modified MTs in vivo to define viral and motor protein interactions with specific MT subsets. We will also test the effects of plus- end tracking MT regulators on MT dynamics and stabilization and their influence on virion movement in living cells, as well as imaging the effects of infection on MT plus-end tracking protein activity. This will provide an unprecedented level of analysis of these highly dynamic processes and the role of specialized MT regulatory factors and motors, using novel approaches to explore the potential for selective use of dynamic MT capture versus trafficking on stable MT subsets by distinct viruses. To this end, the Imaging Core will provide a central state of the art resource to each PPG member and which plays a fundamental role in addressing the Overall Aims of this PPG to understand MT networks and virus trafficking in diverse viral systems.

Moloney murine leukemia virus (MLV) is a prototypical gammaretrovirus that replicates to high titer in neariy all mitotic rodent cells, causes a persistent viremia in infected mice, and induces a T-cell leukemia at a high incidence through insertional activation of host protooncogenes. Much of what we know about retrovirus replication was first learned through the study of the simple viruses such as the MLVs. In the eariy phases of infection, these viruses enter the cell through specific receptors, synthesize a DNA copy of the viral RNA genome by reverse transcription in the cytoplasm, direct the movement ofthe resulting preintegration complex (PIC) into the nucleus, and integrate the viral DNA into the host genome to form the provirus. In the late phases this DNA is expressed to form viral RNAs and proteins, and progeny virions are assembled at the plasma membrane and released to begin a new infectious cycle. In this project we propose to examine the early post-entry events of infection, the most pooriy characterized portion ofthe viral life cycle, focusing on a key host protein, IQGAP, microtubules, and dynein motors. We have previously identified IQGAPI as a major host protein interacting with the Moloney MuLV Gag matrix protein (MA), and have documented the critical importance of that interaction for virus replication. The IQGAPs are large cytoskeletal scaffolding proteins involved in the regulation of cell motility and morphology, and are noteworthy in binding and regulating both actin and microtubule networks. They integrate multiple inputs (especially from small GTPases) and produce multiple outputs, including stabilization of microtubules and capture of microtubule ends. We will determine the role ofthe IQGAPs in MLV infection, and the precise time and step in the life cycle at which they act. Using live-cell imaging of fluorescence-tagged virions, we will examine the trafficking of MLV mutants that do not bind IQGAP, and of wild-type virus blocked by dominant-negative fragments of IQGAP, to determine whether virions fail to be properly delivered to microtubules. We will test for the importance of phosphorylation of IQGAP, thought to be mediated by PKCe, in normal virus trafficking. Finally, we will test the key subunits of the dynein motor and dynactin for their roles in movement of the MLV PICs along both stable and dynamic microtubules. With the help of our cell biologist colleagues in this program, we hope to fill in a majorgap in our current understanding of the MLV life cycle. RELEVANCE (See instructions): Retroviruses are agents of serious human diseases, including leukemias and AIDS, and conversely hold out great promise as tools for gene therapy. Recent work has shown that these viruses are critically dependent on the cytoskeleton and microtubules (MTs) for their intracellular trafficking. In this proposal we aim to define the role of particular MT regulators and motors in eariy steps of MLV infection. Deeper understanding of these processes will provide new insights into retrovirus replication, potentially define new antiviral targets and increase our knowledge of trafficking of cargos on MTs.

The overall goal of this project Is to understand the contribution of microtubules (MTs) to viral infectivity. MTs exist in dynamic and stabilized states. Upon stabilization, tubulin subunits comprising MTs become post- translationally modified (PTM). While both dynamic and stable MTs may play roles in viral infection, stable, PTM MTs may be particularly important. Viral proteins bind MT stabilizing factors, host cell factors critical for viral infectivity affect levels of stable MTs in cells and some viruses even enhance the formation of stable, PTM MTs in cell they infect. While we understand a number of the factors involved in MT stabilization pathways in cells, we have relatively little understanding of the relationship between viral infectivity and the functions that stable, PTM MTs provide during viral infection. In this project, we pursue three aims to enhance our understanding of the relationship between MT stability, tubulin PTMs and viral infectivity. We will conduct a screen for new factors involved in MT stability and viral infectivity by examining a family of MT interacting proteins implicated in MT stability. We will critically test the relationship between viral infectivity, tubulin PTMs, and MT stability, by developing strategies to generate cells lacking or overexpressing the two PTMs of tubulin that commonly occur in stable MTs of cells infected by viruses. And, we will examine the spatial relationships between viruses and different classes of MTs by developing new methods to image stable and PTM MTs in living ceils infected with fluorescent viruses and by testing the motility of viral motors on PTM MTs. These aims will be conducted in collaboration with the Goff, Naghavi, Walsh and Vallee groups. The results of these studies will provide new mechanistic information into how different classes of MTs contribute to viral infection and will provide new insights into how viruses manipulate and take advantage of host cell factors to augment their infection. These studies will also identify new host cell factors that influence viral infection and hence have important implications forthe development of anti-viral and viral- based gene targeting strategies.

DESCRIPTION (provided by applicant): Upon entry into cells, many diverse viruses exploit their hosts' cytoskeletal transport networks to reach their sub-cellular site of replication, and nw viral progeny use these same networks to return to the cell surface and spread. Viruses frequently move along actin at the cell periphery before transitioning onto host microtubule (MT) networks that mediate long-range intracellular transport. MTs are highly dynamic heteropolymers of ?/? tubulin (half-lives <5 min), which radiate from the perinuclear MT Organization Center (MTOC) towards the cell surface. Subsets of MTs become stabilized (half-life >1h) in response to various environmental and developmental signals, and are thought to act as specialized networks for vesicle transport during events such as cell polarization and motility. MT dynamics and stabilization are controlled by a number of highly specialized regulators, including actin-MT crosslinking factors and MT plus-end binding proteins (+TIPs), whose accumulation at MT ends is facilitated by the MT plus-end tracking protein, EB1. Movement of cargos on these MT networks involves motor proteins; generally, dynein directs minus-end and kinesins direct plus-end transport. However, our understanding of the role of MTs, their regulators and motors in the movement of viral particles during infection is severely limited. This Program Project Grant (PPG) nucleates expertise in cytoskeletal regulation, motors and MT-based motility, cell signaling and infection by diverse viruses to address these fundamental questions in mechanistic detail in a variety of systems. As a group, our interactions to date have established that both RNA and DNA viruses cause distinct MT modifications and require a range of specialized MT regulatory factors for efficient infection, including actin-MT crosslinkers, +TIPs and EB1, as well as identifying specific host motors used for virion trafficking to the nucleus. In this PPG we aim to determine the mechanistic details underlying these highly dynamic interactions between MT subsets, motors and invading virions, including the use of state-of-the-art dual-color imaging to analyze these events in real time. This integrated and interactive approach not only greatly enhances each individual project's potential by leveraging the strengths of other members, but also serves to focus our cumulative expertise on addressing key aspects of the Overall Aims of this PPG, Microtubule Networks and Virus Trafficking. This efficient group approach has the potential to uncover fundamental new insights in MT function and regulation during viral infection that will likely be important in broader biological contexts and may lead to the development of novel therapeutic approaches.

Microtubule motor proteins are responsible for numerous transport functions in cells. This proposal focuses on a novel mechanism for the motor protein cytoplasmic dynein in mammalian autophagy. Autophagy is a critical cellular function responsible for recycling old or damaged proteins and organelles, and for clearing toxic protein aggregates. Autophagy is also implicated in neurodevelopmental and neurodegenerative diseases. Cytoplasmic dynein is a major motor protein responsible for a broad range of basic cellular roles, including retrograde axonal transport, cell division, and nuclear and cell migration. We have now found that the cytoplasmic dynein regulator, RILP (Rab-interacting Lysosomal Protein) acts as a novel master regulator of neuronal and nonneuronal autophagy. We find that RILP recruits dynein to autophagosomes at a succesion of stages throughout this process via a sequence of distinct recruitment mechanisms involving interactions with the autophagosomal proteins LC3 and ATG5, as well as the late endosomal/lysosomal protein Rab7. We find RILP mediates not only autophagosome transport, but has a surprising role in autophagosome biogenesis as well. Of further interest we find RILP expression to be controlled by the mTOR kinase, which plays a central role in the cellular response to nutrient deprivation, injury, and toxic protein aggregation. We find further that RILP is necessary for processing of p62(/SQSTM1), direct evidence for a physiological role in clearance of protein aggregates. RILP appears, therefore, to represent a missing link in understanding how mTOR regulates the cellular machinery in response to diverse forms of insult or stress. This proposal is to work out the detailed mechanisms for RILP regulation and function, especially in neurons. Aim 1 will test the role of mTOR in controlling RILP expression, and of PKA in controlling RILP/dynein-mediated autophagosome transport. Aim 2 Will define the roles of RILP in autophagosome biogenesis and maturation. Aim 3 will define the role of a novel RILP-dynein-dynactin-LIS1 supercomplex we have isolated in regulating autophagosome transport. The proposed studies should provide important insight into a basic new autophagy pathway, with fundamental implications for understanding the etiology and control of neurodegenerative and neurodevelopmental diseases.

Mutations in the human kinesin gene KIF1A cause a variety of neurological defects. This syndrome has remained poorly defined because of the rarity of the condition. This proposal brings together the very different, but highly complementary expertise of Dr. Wendy Chung at Columbia University Medical School, a specialist in human genetic disease; Dr. Richard Vallee, also at Columbia, an expert in the role of Kif1a in neuronal development and physiology; and Dr. Arne Gennerich, at Albert Einstein College of Medicine, an expert in motor protein biophysics. Dr. Chung's lab has developed clinical and computational methods to compile information from patients locally and worldwide on the range, severity, and variety of symptoms associated with this condition, which her lab has termed KAND KIF1A Associated Neurological Disorders. This is a heterogeneous group of severe neurodegenerative conditions, including spastic paraplegia, peripheral neuropathy, optic nerve atrophy, cerebral and cerebellar atrophy, cognitive impairment, and seizures. The condition available. The over-all goals of this project are to obtain sufficient clinical information to understand the full-range of KAND symptoms; to determine how mutations at diverse sites within the Kif1a motor domain impact clinical outcome; to understand the cellular and developmental causes of the syndrome; and to identify small molecule reagents to treat it. Aim 1 will be to define the natural history of KAND based on a rapidly increasing patient database and correlate clinical severity and rate of progression with KIF1A genotype. Aim 2 will be to use advanced single molecule biophysical and in vivo axonal transport approaches to determine the molecular and cellular consequences of the Kif1a mutations. Aim3 will be to use Kif1a mutant mice to determine the longitudinal and cross-sectional effects of the condition in a model organism, and to test more completely the role of BDNF in KAND and the value of small molecule BDNF mimetics as KAND therapeutic agents. These studies are of great importance for a number of reasons. They will dramatically extend our capability to identify and characterize rare diseases. They will provide detailed insight into the molecular basis of a motor protein-associated disease. They will provide extensive new information on the progression of the disease and the relationship of mutation site to prognosis. And, they will take advantage of our new molecular and physiological insights into gene function to develop targeted therapies. can be fatal, and there is at present no treatment