Leslie Wilson - US grants

Microtubules are important components of the cytoskeleton of eukaryotic cells, and participate in the development and maintenance of cell shape and in many kinds of intracellular movement. They are prominent in the central nervous system where they are especially important in the organization and function of axonal and dendritic processes of neurons. Microtubules exhibit a wide variety of polymerization behaviors in vitro and in cells, and there is strong reason to believe that the various polymerization behaviors are important in microtubule function. Microtubule functions such as those related to the organization and development of axons and dendrites may be determined and regulated by modulation of the assembly and disassemble reactions at microtubule ends. The role of polymerization dynamics in the function of microtubules at a mechanistic level is not understood. With a focus on brain microtubule systems, the main strategy of this proposal is to investigate the dynamics of tubulin addition and loss at microtubule ends in vitro, with the goals of understanding the mechanisms that govern tubulin addition and loss and identifying and elucidating mechanistically the actions of microtubule- associated proteins that control assembly and disassembly dynamics in neuronal cells. A combination of procedures will be employed that can distinguish tubulin addition and loss events at the individual microtubule ends. The dynamics of tubulin addition and loss at the ends of microtubules composed of unmodified and posttranslationally-modified brain tubulins (detyrosinated, glutamylated, ADP-ribosylated) and specific unmodified and modified (phosphorylated) neuronal MAPs will be examined. The work will focus on the actions of MAP-2 and Tau, two prominent neuronal microtubule-associated proteins that are important in the development, organization, and functions of dendrites and axons, respectively. The work will also focus on the role of GTP hydrolysis in microtubule polymerization. The objective is to characterize the flux and dynamic instability parameters of the microtubules, and to determine to what extent and how specific microtubule-associated proteins, specific tubulin isotypes, posttranslational modifications of tubulin and microtubule- associated proteins, and the drug colchicine, modulate and regulate microtubule polymerization.

This proposal focuses on an in-depth investigation into the mechanism of action of the vinca alkaloids. The vinca alkaloids are a group of chemically related indole-dihydroindole drugs that are currently used in the treatment of a number of forms of cancer. They appear to inhibit cell growth at least in part by the disruption of microtubules. Although a considerable amount has been learned about how the vinca alkaloids inhibit microtubule assembly in vitro, the precise mechanism of action in quantitative kinetic terms is almost completely unknown, as is an understanding of the relationship between the ability of the vinca alkaloids to inhibit the polymerization of microtubules, and to destroy preformed microtubules directly. Further, the antitumor specificities of the individual vinca alkaloids are different, and it is conceivable that the different vinca congeners exert their chemotherapeutic activities on cells by a combination of distinct mechanisms. Our research strategies fall into three specific areas. One major aim is to continue studies begun previously in our laboratory on the quantitative characterization of the mechanism of microtubule assembly inhibition by the vinca alkaloids. These studies will involve the kinetic characterization of tubulin addition and loss at the opposite ends of steady-state microtubules in vitro, using microtubules from several sources and of different molecular compositions. The second goal is to characterize the interactions of the vinca alkaloids that result in the direct depolymerization of microtubules (the peeling of protofilament strands at microtubule ends). The relationship between this interaction of vinblastine with microtubules and the interaction that results in assembly inhibition will be determined. The third goal is to investigate the structure-activity-relationship of the vinca alkaloid derivatives in terms of the abilities of individual congeners to inhibit microtubule polymerization in cells, and the abilities of the congeners to inhibit cell growth, using a human cell line (Hela) whose microtubule properties in vitro have been characterized. The purpose is to determine whether the action of a particular vinca alkaloid derivative in inhibiting cell growth is due exclusively to the disruption of microtubules in cells, or occurs through a concerted ability to inhibit microtubule function and other cellular processes.

Microtubules are important components of the cytoskeleton of eucaryotic cells, and participate in diverse processes such as the development and maintenance of cell shape and in various kinds of intracellular movements (e.g., intraaxonal transport and mitotic chromosome movement during meiosis and mitosis). Microtubule populations differ in cells, from being completely stable such as those found in cilia and flagella, to being extremely dynamic, such as those found in mitotic and meiotic spindles. Microtubules have been found to exhibit a variety of polymerization behaviors in vitro that may reflect the heterogeneous behaviors that they exhibit in cells. Cells may use the various polymerization capabilities of microtubules to accomplish different functions. It seems reasonable to believe that microtubule functions such as those related to the organization and growth of microtubules in cells, and those associated with certain kinds of microtubule-linked motility such as mitotic chromosome movement, are mechanistically determined and regulated through the assembly and disassembly reactions at microtubule ends. Further, it is reasonable to think that diversity in microtubule behavior and function may be related to participation of distinct tubulins and microtubule-associated proteins in different microtubule populations. Thus, the main strategy of this proposal is to investigate the dynamics of tubulin addition and loss at microtubule ends in vitro. A combination of procedures will be employed that can distinguish tubulin addition and loss dynamics at individual microtubule ends, together with analysis by electron microscopy of microtubule length dynamics. Microtubule preparations composed of distinct tubulins and microtubule-associated proteins from brain and sea urchin eggs and sperm will be examined. The goal is to understand the mechanisms responsible for tubulin addition and loss at microtubule ends, and to identify, characterize, and understand the functions of molecules that interact with the surfaces and ends of microtubules and regulate assembly and disassembly dynamics in cells.

This award will help fund a conference in singularity theory. This conference will be held from August 6 to 10, 1990 on the campus of the University of Hawaii, Manoa. This conference will be concerned with all branches of singularity theory, but with emphasis on the following: (1) singularities of mappings (real or complex), (2) singularities of real varieties, (3) foundational properties: differential analysis, subanalytic sets, etc., and (4) applications, e.g., to differential geometry.

[unreadable] DESCRIPTION (provided by applicant): Microtubules are dynamic polymers that are critical in the determination and maintenance of cell shape, polarity, and in many types of cellular movement. They exhibit two unique dynamic behaviors, dynamic instability and treadmilling, which are important determinants of their functions in cells. Both dynamic behaviors are intrinsic properties of the tubulin backbone, while microtubule-associated proteins acting at the surface or ends of microtubules control the dynamics. The expression of microtubule- associated proteins that regulate microtubule behavior is significantly altered during neuronal development, and their mis-regulation contributes to human diseases including cancer and neurodegeneration. This proposal is based upon a considerable body of knowledge indicating that 1) microtubules have a large number of binding sites along their surfaces and at their ends that serve as the targets for cellular regulatory molecules and 2) that the binding of small numbers of such regulatory molecules to their specific sites exerts powerful effects on the dynamic behaviors and, thus, the functions of the microtubules. By using a combination of in vitro mechanistic approaches together with analysis in cells, the focus will be on the mechanisms that determine and control the dynamic instability and treadmilling behaviors of microtubules. It will involve determination how the tau proteins, which stabilize microtubules and may mis-regulate dynamics in neurodegenerative tauopathies such as Frontotemporal Dementia with Parkinsonism linked to chromosome 17 (FTDP-17) and the stathmin/SCG10 family of microtubule destabilizing proteins, act mechanistically to modulate dynamics. The goals are 1) to elucidate at a mechanistic/biochemical level in vitro, and behaviorally in living cells, how the tau proteins regulate dynamic instability and treadmilling, how FTDP-17 mutated forms of tau mis-regulate the dynamics, and how microtubule-targeted drugs can correct the mis-regulation, 2) to determine at a mechanistic/biochemical level in vitro, and behaviorally in living cells, how the stathmin and SCG10 family of microtubule destabilizing proteins regulates dynamic instability, treadmilling, and minus end dynamics, and 3) to determine the size and chemical nature of the stabilizing cap at microtubule ends, and to use microtubule-associated proteins that modulate dynamics as tools to determine how the cap mechanism is regulated. [unreadable] [unreadable]

DESCRIPTION Candida albicans, an opportunistic pathogen, can cause vaginal, oral and lung infections in immunocompromised individuals and systemic tissue damages in acquired immunodeficiency patients. The chemotherapy of C. albicans infections is limited because of the strong similarities between C. albicans cells and human cells. However, the mitotic spindles in mammalian and Candida cells are constructed differently. In addition, significant differences exist in the sequences of fungal and mammalian tubulins, which are the building block units of mitotic spindles. Little information is available at biochemical and functional levels about Candida tubulin, and virtually nothing is known regarding the polymerization and dynamics properties of Candida microtubules. The thinking is that understanding the differences between fungal cell tubulin and mammalian tubulin could lead to development of new and selective drugs for the treatment of fungal diseases. Therefore, it is proposed to develop a large-scale purification strategy for C. Albicans tubulin based upon previous success in this laboratory with tubulin from Saccharomyces cerevisiae. The tubulin will be characterized biochemically, and the polymerization and dynamic properties of Candida microtubules determined. Finally, the mechanism of interaction of two known microtubule-targeted antifungal drugs (benomyl and griseofulvin) with the Candida tubulin will be determined and the mechanisms by which the drugs modulate the polymerization and dynamics properties of the tubulin will be elucidated.

DESCRIPTION (provided by applicant): A broadly focused international research meeting on the cytoskeleton and its associated proteins in human diseases is planned from April 17 through April 20, 2001. The meeting, entitled Colloquium on the Cytoskeleton and Human Disease, will be held on the medical/pharmacy school campus of the University of the Mediterranean, specifically at the Faculte de Pharmacie, which is located in the center of Marseille. We expect approximately 150 participants including graduate students and post doctoral fellows. It has become clear that the major cytoskeletal components, microtubules, intermediate filaments, and actin filaments, are involved in a large number of diverse human diseases. Thus our purpose is to assemble scientists to participate in the first broadly-based research meeting on the cytoskeleton and its associated proteins in human disease. New drugs and new targets related to the cytoskeleton and its associated proteins will be highlighted. The meeting will consist of invited lectures, short oral communications, and poster sessions. Topics to be covered include: 1)microtubules and cancer (microtubule-targeted anticancer drugs, drug resistance, new approaches and new compounds), 2)microtubule-associated proteins and neurodegenerative disease (Alzheimer's disease and other tauopathies, new concepts and new targets), 3)intermediate filaments and disease (keratin and skin diseases, desmin in skeletal and cardiac muscle disease, muscular dystrophy), and 4)microfilaments and disease ()cell adhesion, migration metastasis, infectious diseases, membrane muscle defects, potential new targets). The members of the organizing committee from the United States are Drs. Leslie Wilson, Mary Ann Jordan, Ernest Braguer, Vincent Peyrot, and Bernard Rossignol. Drs. Wilson, Briand, Jordan, Hamel, Marvaldi, and Binder have the primary responsibility for the scientific program, and Drs. Briand, Wilson, Briand, Wilson, Braguer, Peyrot, and Rossignol are in charge of meeting arrangements.

DESCRIPTION (provided by applicant): Microtubules (MTs), tube-shaped polymers composed of ab tubulin heterodimers and a diverse array of MT-associated proteins (MAPs), are critical for the development, structural organization, stability, and functions, of the axonal and dendntic processes of neurons. MTs are not simple equilibrium polymers. Guanosine-5'-triphosphate is irreversibly hydrolyzed to guanosine-5'-diphosphate and orthophosphate during tubulin addition to the MTs, which creates two unique dynamic behaviors, treadmilling and dynamic instability. These behaviors are critical for MT function in cells, and are finely regulated. Both dynamic instability and treadmilling are intrinsic properties of the tubulin backbone of MTs, while MAPs acting at the MT surfaces and ends control the dynamics. One major goal is to elucidate the mechanisms responsible for the MT's unique dynamic behaviors. A second major goal is to determine how important neuronal MAPs regulate dynamics. These studies will involve high-resolution video microscopy and radiolabeled guanine-nucleotide exchange strategies. Studies will focus on the dynamics of reconstituted brain MTs and specific neuronal MAPs in vitro, and on MT dynamics in living neuronal and non-neuronal cells. A main focus will be on the tau proteins and mutated forms of tau, which are involved in Alzheimer's disease and are causally linked to frontotemporal dementias (FTDP-17) in humans.

This collaborative project brings together a strong multi-institutional interdisciplinary team of investigators to study and advance the current understanding of cellular and sub-cellular events. Continuing technological advances in fluorescence and atomic-force microscopy allow scientists to observe molecular function, distribution, and interrelationships in living cells. However, a full understanding of tens of thousands of proteins and the complex molecular processes they engage in requires a voluminous amount of image data, which currently must be analyzed by visual inspection. To facilitate such an analysis, researchers from the four participating institutions are focusing on three main research thrusts. First, next-generation intelligent imaging involves information processing at the sensor level to enable high-speed and super-resolution imaging. The goal is to enable biologists to study cellular processes at resolutions in time and space that are not possible with current technologies. The second research thrust is pattern recognition and data mining as applied to bio-molecular image collections. Salient features that characterize the underlying patterns in cells and tissues need to be computed for the vast volumes of images acquired through automated microscopy. Third, a distributed database of bio-molecular images is being created. The merging of pattern-recognition and data-mining tools with new, powerful methods for indexing, data modeling, and collaboration, is aimed at creating a unique infrastructure that greatly facilitates image bioinformatics, thus complementing recent revolutionary advances in genomics.

The outcome of this research will lead to new and novel information-processing methods for bio-molecular image data. Efficient and effective representation of such data will enable researchers to search and browse through large collections of image and video data and look for similar patterns in such datasets, thus facilitating information discovery. During its five-year duration, this project will develop, test, and deploy a distributed database of bio-molecular image data accessible to researchers around the world. The impact of the distributed database will be through large-scale biology in which the results of a single experiment can be globally correlated with the results from other groups of scientists, thus accelerating discovery of dynamic relationships between structure and function in complex biological systems.

The project will develop new courses, and will facilitate student exchanges, semi-annual meetings, and workshops, benefiting students at all levels. This project will train a new generation of biologists, computer scientists and engineers well versed in the imaging and information-processing sciences at the forefront of next-generation biotechnology. Partnership will be established with institutions with large populations of students from groups underrepresented in science and engineering, such as the California State Universities at Fresno and San Bernardino and the Universidad Metropolitan in Puerto Rico, for undergraduate recruitment and outreach. An effective mode of outreach for students is to educate their teachers, and the project will offer summer fellowships for elementary, high-school, college, and university teachers.