Stephen J. Doxsey - US grants

The long term objective of this work is to develop a molecular understanding of the process by which centrosomes organize microtubules. The spatial orientation of microtubules is crucial for their function and is controlled by the centrosome. The centrosome has been recalcitrant to investigation so the basis for its function and regulation are unknown. We have discovered a highly conserved centrosome protein called pericentrin that is essential for cell division and organization of the microtubule cytoskeleton. Pericentrin provides us with a unique opportunity to understand centrosome function at the molecular level. A recent advance in our laboratory suggests that we will be able to identify and reconstitute the components of nucleation. We are now in an excellent position to: 1. Test the hypothesis that pericentrin is involved in centrosome assembly and define the sequence requirements for function. 2. Test the hypothesis that pericentrin assembles onto centrosomes in a cell cycle-specific phosphorylation-dependent manner. 3. Test the hypothesis that pericentrin binding proteins are involved in microtuble nucleation. Significant progress has been made toward th goals of this proposal. We have raised antibodies that block pericentrin function, produced mutant and wild type pericentrin cDNAs and fusion proteins and developed a novel in vitro assay for reconstituting centrosome function. Since that first submission of this proposal, we identified a pericentrin binding partner, perturbed centrosome structure using mutant pericentrin, and made the remarkable observation that pericentrin antibody-beads promote assembly of microtubules. These recent advances, molecular tools, and assays will allow us to make rapid progress in understanding the properties and function of pericentrin and centrosomes in general. Understanding centrosome function is important for several reasons. As the organizer of the microtubule cytoskeleton, the centrosome is a pivotal player in many fundamental cellular processes. Given its importance in the assembly of the mitotic spindle during cell division, the centrosome is a potential regulator of cell growth and a coordinator of morphogenesis. Centrosome- mediated microtubule polarity is critical for organizing the cytoplasm for intracellular vesicular transport and positioning of organelles. Despite its central position at the focus of microtubules and its central role in the life of a cell, the centrosome is poorly understood. Our preliminary results suggest that pericentrin will allow us to make important contributions in an area of biology that has been difficult to study. We are especially excited about the future prospect of realizing our long term goal of reconstituting centrosome function in vitro from individual components.

Centrosomes play several fundamental roles in the cell including the nucleation and organization of microtubules for spindle assembly and molecular motor-driven processes. They also anchor important regulatory activities that control centrosome and spindle function and may contribute to tumorigenesis through the organization of dysfunctional spindles that fail to segregate chromosomes properly. The overall objective of our research is to understand the molecular basis of centrosome function. Our general strategy is to focus on the function of pericentrin, a centrosome protein I identified in six years ago, using a combination of molecular, biochemical and morphological strategies. Over the past budget period, we have made significant progress in understanding pericentrin function. We determined that pericentrin forms a complex with microtubule nucleating proteins including gamma tubulin and is in close proximity with gamma tubulin at the centrosome. We found that pericentrin interacts directly with cytoplasmic dynein light intermediate chain 1 and that dynein mediates assembly of pericentrin and gamma tubulin onto centrosomes. Moreover, pericentrin overexpression disrupts dynein localization, causes spindle defects and creates aneuploid cells. In collaboration with Dr. J. Scott (Vollum Inst.), we showed that pericentrin anchors kinase A to centrosomes and that this interaction is important for spindle function. Based on the observations outlined above, we have formulated a model in which pericentrin serves to specifically transport, anchor and organize important functional and regulatory activities at the centrosome. Over the next budget period we will continue to study the role of pericentrin and pericentrin- interacting proteins in centrosome and spindle function using both in vivo approaches and in vitro reconstitution. In the first aim of this proposal we will investigate the role of pericentrin in the assembly of microtubule nucleating complexes onto centrosomes. We will use cytoplasmic extracts prepared from Xenopus eggs to test the ability of protein complexes containing pericentrin to mediate the assembly of gamma tubulin complexes onto centrosomes in vitro. The second objective is to determine the significance of the interaction between pericentrin and dynein light intermediate chain 1. Specifically, we will use a dominant negative form of pericentrin to uncouple the pericentrin-dynein light intermediate chain interaction and examine the role of this interaction in centrosome assembly and spindle organization. The third aim is to identify and characterize other pericentrin-interacting proteins and to characterize a novel centriole protein which, like pericentrin, was identified using autoimmune sera.

The long term objective of this work is to understand how centrosomes contribute to tumorigenesis. Centrosomes are poorly understood organelles required for organization of mitotic spindles and accurate segregation of chromosomes during cell division. Thus, centrosomes are critical players in the redistribution and reorganization of the genome as it is assembled into nascent nuclei following mitosis. There is no other single cellular event that has a greater impact on the quantity, composition and organization of chromatin within the nucleus. We recently made the striking observation that malignant tumors had increased levels of the centrosome protein pericentrin, abnormal centrosomes, aberrant mitotic spindles and missegregated chromosomes. Moreover, artificial elevation of pericentrin in normal cells induced nearly identical features including aneuploidy, a condition linked to tumor malignancy, metastasis and fatality. Pericentrin over-expression also appeared to abrogate the mitotic checkpoint that normally induces mitotic arrest in the presence of unattached chromosomes. Over- expressed pericentrin bound and mislocalized cytoplasmic dynein, a molecular motor known to function in spindle organization and possibly checkpoint control. Based on these observations, we propose a model in which centrosome defects contribute to tumorigenesis by causing improper segregation of the genome and creating aneuploid cells. The discovery of centromsomes as potential contributors to malignant tumor progression provides us with a unique opportunity to elucidate a novel mechanism for tumorigenesis. The specific aims of this proposal are: 1. To determine whether proteins of the centrosome and nucleus are altered in tumors. 2. To test whether pericentrin has oncogenic potential. 3. To determine how elevated levels of pericentrin cause aneuploidy.

Centrosomes are best known for their ability to organize microtubules to form interphase arrays and mitotic spindles. Recent evidence suggests that centrosomes are involved hi cytokinesis and cell cycle progression. However, little is known about the centrosome proteins that contribute to these processes. In the previous funding period, we examined the molecular basis for these under-explored centrosome functions. We identified a novel centrosome protein called centriolin, which is essential for cytokinesis in mammalian cells. Centriolin interacts with proteins implicated or involved in cytokinesis including snapin (SNARE), sec 15 (exocyst), MKLP-1 (centralspindlin) and a novel GTPase activating protein for Rho GTPases. Many of these proteins localize to the midbody in dividing cells and induce cytokinesis defects when downregulated. Based on these and other data, we propose a model in which centriolin serves as a scaffold protein at the midbody to coordinate vesicle fusion, microtubule depolymerization, furrow ingression and cell separation late in cytokinesis. Defective cytokinesis induced by centriolin downregulation was followed by Gl arrest. We unexpectedly found that many centrosome proteins induced GI arrest when downregulated. However, the arrest did not correlate with defects in cytokinesis or other centrosome functions, or in centrosome structure or composition. We postulate that cell cycle arrest is triggered by a checkpoint that monitors defects in a common function not yet identified, or 'centrosome damage' induced by reduction of individual proteins at centrosomes. GI arrest requires p53 and p38 MAP kinase and induces recruitment of activated p53 and p38 to centrosomes. Based on these observations, we propose two specific aims. In Aim 1 we will determine the role centriolin and associated proteins hi cytokinesis. More specifically, we will test whether centriolin anchors these proteins at the midbody and if a complex of these proteins controls vesicle fusion, microtubule organization and cell separation at the midbody. Aim 2 focuses on the role of centrosome proteins in cell cycle progression and checkpoint activation. We will determine the mechanism of GI arrest and identify the signal transduction pathway that connects centrosome proteins to cell cycle arrest.

Centrosomes are involved in mitotic spindle organization and an increasing number of other fundamental[unreadable] cellular processes. Defects in centrosomes contribute to spindle defects, chromosome missegregation and[unreadable] aneuploidy. Recent work initially from our labraotory and one other showed that centrosome defects are a[unreadable] characteristic feature of human tumors. The long-term goal of our laboratory is to gain insight into the[unreadable] function of centrosomes and centrosome proteins in fundamental cellular processes and human cancer,[unreadable] toward this goal, we recently discovered that the centrosome protein pericentrin interacts with proteins of[unreadable] the nucleosome remodeling and deacetylase (NuRD) complex. Our preliminary results show that NuRD[unreadable] complex components are at the centrosome and that alteration of their levels affects centrosome integrity,[unreadable] spindle organization and genetic stability. We unexpectedly found that pericentrin localizes to the nucleus[unreadable] and that functional abrogation of pericentrin has dramatic effects on nuclear structure. We have also shown[unreadable] that pericentrin induces the formation of multipolar spindles when elevated in cells and mislocalizes dynein[unreadable] from spindles. Other work confirms that loss of dynein from mitotic spindles induces multipolarity and shows[unreadable] that re-establishing dynein binding to spindles results in clustering of spindle poles and restoration of spindle[unreadable] bipority. Based on these observations we propose three specific aims:[unreadable] Aim 1. To determine the mechanism of centrosome disruption and spindle dysfunction in cells with[unreadable] abrogated NuRD components.[unreadable] Aim 2. To determine the mechanism of nuclear disruption and functional changes in nuclei following[unreadable] abrogation of pericentrin function.[unreadable] Aim 3. To restore spindle bipolarity in tumor cells that have multipolar spindles and elevated levels of[unreadable] pericentrin as was accomplished in tumor cells overexpressing another dynein-interacting protein, NuMA.[unreadable] If successful this work will increase our basic understanding of centrosomes and centrosome proteins. In[unreadable] addition, it will provide information on how cells develop genetic changes that accompany human[unreadable] tumorigenesis. This could prove useful in cancer diagnosis and prognosis, and provide new targets for[unreadable] designing cancer therapeutics.

[unreadable] DESCRIPTION (provided by applicant): The Digital Imaging Core Facility (DICF) of the University of Massachusetts (UMass) Medical School and a group of seven NIH funded principal investigators request funds to purchase a spinning disk confocal microscope system for live cell imaging. The Digital Imaging Core Facility was established by the UMass Medical School Office of Research and Scientific Council to provide multimode digital imaging microscopy facilities and digital deconvolution to the UMass research community. The requested instrument will be integrated into this campus wide research resource and would be available to the entire UMass Medical School research community when it is not being used by the major and minor users. There is a great need by many NIH funded UMass Medical School researchers for a confocal microscope system designed for live cell imaging in order to accomplish the objectives of their NIH funded research. All of the researchers requesting this instrument are involved in qualitative and quantitative studies of dynamic cellular processes, and four are members of the UMass Cellular Dynamics Program. There is currently no spinning disk confocal microscope system for live cell imaging at UMass Medical School available for general use as part of a core facility. This spinning disk confocal microscope system will allow UMass researchers to expand their research in new directions and advance their biomedical research programs. The requested spinning disk confocal microscope system will be used to conduct basic research in cancer cell biology, cell signaling, developmental biology and cancer, cell cycle regulation, intraflagellar transport as related to polycystic kidney disease and retinitis pigmentosa, Bardet-Biedl syndrome and Senior-Loken syndrome, molecular motor proteins, sensory transduction and immunology. The Digital Imaging Light Microscopy Core Facility of the University of Massachusetts (UMass) Medical School and a group of seven UMass faculty members request funds to purchase a spinning disk confocal microscope system to advance their NIH funded biomedical research. The requested instrument will be used to conduct basic research in cancer cell biology, cell signaling, developmental biology and cancer, cell cycle regulation and intraflagellar transport as related to polycystic kidney disease and other diseases. [unreadable] [unreadable] [unreadable]

community; molecular /cellular imaging; neoplasm /cancer; university

community; molecular /cellular imaging; university

community; molecular /cellular imaging; university

SUMMARY (ABSTRACT) Centrosomes are organelles of diverse structure that share a common ability to organize microtubule arrays for various cellular functions. They contribute to mitotic spindle organization and orientation in dividing cells. In nondividing cells of many organisms, centrosomes contribute to cell polarity and epithelial morphogenesis, and serve as templates for assembly of motile cilia and sensory (primary) cilia. Both primary and motile cilia are assembled, maintained, and regulated by specific proteins, which have been enumerated in a number of proteomic analyses. One class of ciliary proteins is involved in the process of intraflagellar transport (IFT), where proteins required for cilia assembly are transported by protein carriers (IFT complexes) to the tip of the cilium/flagellum by plus-end-directed motors (kinesin-2), whereas disassociated material is returned to the cilia base by minus-end-directed motors (dynein 1b). Defects in cilia proteins are associated with a number of human diseases termed ciliopathies, and by definition all exhibit defects in cilia structure and/or function. However, ciliopathies are complex disorders characterized by a diversity of cellular abnormalities that make it difficult to precisely define the disease etiology. This complexity is manifest in recent studies suggesting that defects other than cilia may contribute to these disorders. For example, the Doxsey laboratory and others have recently shown that cilia proteins localize to centrosomes and mitotic spindle poles, though mitotic roles of these proteins is poorly understood. During the previous grant period, the Doxsey laboratory began to investigate IFT protein function in mitotic cells. IFT proteins localize to spindle poles as well as kinetochores, midbodies and spindle microtubules. RNAi-mediated depletion of the cilia protein IFT88 in zebrafish embryos and cultured cells disrupts centrosome/spindle pole integrity, astral microtubule organization and spindle organization;depletion of other IFT proteins (IFT20, 52, 57) induces related and additional mitotic defects. A novel mitosis-specific IFT88 complex was identified, which contains proteins linking microtubules to the cell cortex. This event is required for orientation of mitotic spindles and the plane of cell division, and is implicated in cystogenesis and ciliopathy. Based on this work we propose the following specific aims: Aim 1. Determine the role of IFT88 complexes in mitosis, particularly spindle organization, and test whether IFT88 zebrafish morphants and mouse mutants exhibit mitotic defects prior to cilia expression in early embryos. Aim 2. Determine functional interactions of IFT88-BPs that contribute to spindle orientation and organization. Aim 3. Test whether the global ciliogenesis machinery is redirected in dividing cells to perform multiple mitotic functions through the identification of additional mitotic phenotypes and mitosis-specific protein complexes in several different classes of ciliary proteins. This work has the potential to identify new functions for ciliary proteins (mitotic), characterize their molecular underpinnings and provide insights into the etiology of ciliopathies through the study of mitotic abnormalities.

DESCRIPTION (provided by applicant): Centrosomes contribute to mitotic spindle organization and orientation in mitosis, and to the assembly of primary cilia in nondividing cells. Centrosome anomalies affect the fidelity of spindle and cilia function and are associated with ciliopathies, microcephaly, dwarfism, cancer and other human disorders. Cilia proteins are found at centrosomes in mitotic cells (spindle poles) and some are involved in the orientation of cell division. However, the function of these cilia proteins in mitotic cells is not known. Preliminary results from the Doxsey laboratory suggest that cilia proteins required for transporting material up and down cilia in noncycling cells (intraflagellar transport, IFT) also transport material to and from spindle poles in mitotic cells. Disruption of these cilia proteins induces defects in mitotic spindle orientation, astral microtubule organization, spindle pole function and mitotic progression. Other cilia proteins localize to additional mitotic structures such as kinetochores and midbodies, suggesting additional mitotic functions of this class of proteins. The overall goal of this proposal is to test the hypothesis that IFT complexes involved in cilia formation and function in noncycling cells, are re-directed, at least in part, to perform previously unanticipated mitotic functions. To test this, we will address the molecular mechanism of IFT protein complex function in mitotic spindles. Our preliminary studies indicate that IFT88 forms particles in cells that transport microtubule-nucleating proteins to spindle poles using the dynein motor. The specific aims are designed to test if IFT protein complexes serve as carriers of mitotic cargoes and if dynein provides the force for their movement. Novel aspects of the work include the identification of a novel mechanism for spindle pole assembly and spindle orientation, the use of super- resolution microscopy to image the dynamics of novel IFT protein-containing particles in living mitotic cells, the characterization of new mitotic IFT protein-dynein complexes using new affinity systems and the use of in vitro assays to study dynein-based motility. This work has the potential to identify a new molecular pathway for spindle pole assembly and to define novel functions of cilia proteins in mitotic cells. PUBLIC HEALTH RELEVANCE: We seek to uncover a novel molecular pathway for spindle organization, which is relevant to a number of human disorders including cancer, developmental abnormalities, ciliopathies, microcephalies and primordial dwarfisms. A long-term goal of this work is to develop new therapeutic strategies for prevention or treatment of disorders arising from spindle dysfunction.