Jeffrey L. Salisbury, Ph.D. - US grants

Striated flagellar roots occur associated with the basal apparatus of flagellated or ciliated eukaryotic cells and have been observed in association with centrioles and primary cilia. Striated flagellar roots are contractile organelles. We have recently demonstrated that striated flagellar roots are composed in large part of a calcium-binding 20,000 mol. wt. contractile phosphoprotein. This protein shares many features of the parvalbumin/troponin c/calmodulin superfamily of calcium-binding proteins. We present preliminary findings which suggest that antigenic homologs of the striated flagellar root protein occur associated with the centrioles and mitotic spindle poles of diverse organisms, including mammalian cells. Our ultrastructural studies indicate that the fibrous material and their condensations (pericentriolar satellites) of mammalian centrosomes is composed at least in part of antigenic homologs of striated flagellar roots. These findings have led to the proposal that striated flagellar root homologs respresent 'primitive' motile systems that are simple in composition and undergoe contraction directly in response to calcium binding. If these ideas are correct they have important implications for our understanding of eukaryotic cell structure and contractile function. We propose to characterize the antigenic homologs of the striated flagellar root protein in mammalian cells. We propose to generate a library of monoclonal antibodies and to use these in epitope mapping, immunofluorescent and immunoelectron microscopic studies of striated flagellar root homologs in mammalian cells. In addition we will characterize calcium-binding to the protein and its proteolytic fragments and study the characteristics of flagellar root protein phosphorylation.

The sperm centrosome is intimately associated with organization and morphogenesis of the spermatocyte, particularly with regard to establishment of extreme cell polarity in mature sperm. In addition, the sperm centrosome is delivered to the oocyte at the time of fertilization where it plays a fundamental role in the organization of the cytoskeleton of the zygote and early embryo through the formation of the microtubule-based sperm aster and the first mitotic spindle. We have recently established that centrin, a 21 kDa calcium-binding protein found in centrosome-associated fibers, is a prominent component of the sperm centrosome. Previous studies in other systems demonstrate a role for centrin-based fibers in centrosome/mitotic spindle pole positioning, segregation, and reorientation. Based on these observations, we present the specific hypothesis that sperm centrin is an essential component of the sperm connecting piece which functions in centrosome dynamics during sperm morphogenesis, and in zygotes and early embryos during spindle assembly and function. The work outlined in this proposal is directed toward achieving a greater understanding of centrosome dynamics in the male reproductive cell during spermatocyte differentiation and following fertilization of the oocyte. For a thorough understanding of the contribution by sperm to the zygotic centrosome it is important to determine the components of the sperm connecting piece that are essential to the process of spermatocyte differentiation, and sperm aster and centrosome formation and function. What proteins are carried by the sperm connecting piece to the egg? Are they essential for centrosome function? What is their relationship to preexisting pools of centrosomal components present in the oocyte? Can their function be perturbed by specific antibodies and/or drug treatments? Our laboratory has developed immunological, biochemical, and molecular tools which will allow us to readily probe the role of a recently discovered and novel calcium-binding cytoskeletal protein, centrin, in these processes. The specific aims of the proposed studies include; 1) characterization of molecular and biochemical properties of sperm centrin through the purification, peptide and epitope mapping, partial peptide sequencing of centrin from sperm, and the molecular cloning of sperm (testicular) centrin. 2) To define the precise structural organization of centrin in developing spermatids, mature sperm, and in fertilized eggs and early embryos by immunofluorescence and immunoelectron microscopy using polyclonal and monoclonal antibodies raised against centrin. And 3), to determine if i centrin plays an essential role in sperm aster formation and centrosome behavior in early fertilized eggs. Finally, the proposed studies are likely to shed light on the nature of several human infertility disease states that involve centrosomal and spermatid malformations including 'decapitated' sperm.

women's health; female; reproductive system; microscopy; neoplasm /cancer; human subject; biomedical resource;

DESCRIPTION: (adapted from the investigator's abstract) The centrosome functions in maintenance of cell polarity and in progression through the cell cycle by determining the number, polarity, and organization of interphase and mitotic spindle microtubules. Defects in centrosome organization and function have profound consequences for the cell, including the characteristic loss of cell polarlity and chromosomal segregation abnormalities seen in many cancer cells. Cell cycle checkpoints regulating centrosome duplication are believed to operate under the influence of p53. In preliminary studies, they have performed a careful examination of centrosomes in human breast tumors to determine if centrosome abnormalities occur in these cells. The preliminary studies have revealed striking and characteristic changes in several centrosome properties in breast tumor cells including: excess accumulation of key centrosomal proteins, supernumerary centrioles, and inappropriate phosphorylation status of centrosome proteins. In addition, they have developed a novel microtubule nucleation assay to assess breast tumor cell centrosome function. Their preliminary studies further demonstrate that breast tumor cells show specific functional centrosome abnormalities characterized by inappropriate numbers of MTOCs that nucleate large microtubule asters. They, therefore, propose to: 1) determine the cell cycle control mechanism for centrosome duplication in normal breast epithelial and breast tumor derived cell lines, 2) to determine the functional relationship between alterations in centrosome structure and the loss of cell polarity and increase in chromosomal segregation abnormalities seen in breast carcinomas, and 3) to systematically and quantitatively characterize molecular and structural markers for centrosome abnormalities in human breast tissues from proliferating and nonproliferating fibrocystic disease, LCIS, DCIS, and invasive ductal and lobular carcinomas. The proposed studies represent a novel approach to understanding the mechanism of loss of both cell polarity and the increased propensity toward chromosomal segregation abnormalities seen in many carcinoma cells, and these studies may provide new targets useful in the development of novel clinical interventions.

[unreadable] DESCRIPTION (provided by applicant): Cells of malignant breast tumors exhibit centrosome defects including an excess number of centrioles, increased microtubule nucleation capacity, and inappropriate phosphorylation of centrosomal proteins, a condition termed 'centrosome amplification Centrosome amplification leads to multipolar mitosis and consequent chromosomal instability, and therefore, is one mechanism by which aneuploidy and phenotypic variability arise in the development of cancer. We propose that centrosome amplification is an early event in the development of breast cancer, and that amplified centrosomes may arise through one of several alternative mechanisms. We propose to characterize the origin of centrosome amplification during the development of breast tumors. We will test the hypothesis that ER and growth factor signaling are mechanistically coupled to centriole separation and centrosome duplication. And finally, we will experimentally disrupt cell cycle progression in normal breast and tumor-derived cell lines to test the hypothesis that the G 1/S and G2/M cell cycle checkpoints are mechanistically coupled to centrosome duplication and that this linkage becomes uncoupled during mammary tumorigenesis. The study of centrosome behavior is of fundamental importance to our understanding of the origin of malignant tumors and may reveal new targets for intervention or prevention of the development of breast cancer.

DESCRIPTION (Applicant's Description): A. Purpose and Program Characteristics - The purpose of this program is to provide postdoctoral training to individuals wishing to pursue basic research in the pathobiology of cancer. The program is designed to achieve maximum faculty participation and specifically strives to foster a working knowledge of the interface between cancer cell biology, cancer diagnostics, and cancer therapy. The applicants' goal is to produce highly focused independent investigators capable of productive collaboration with other basic scientists and with clinical colleagues involved in the study and treatment of cancer. T h e p rimary means of achieving this goal are an interactive and multidisciplinary research faculty, a formal Tumor Biology Curriculum which integrates current concepts in cell growth control with those of human carcinogenesis, and a weekly workshop in which trainees discuss their research with fellow trainees, with members of the training grant faculty, and with the Mayo scientific community at large. B. Trainees - Nine postdoctoral trainees are requested to participate for each of five years. Individuals holding the Ph.D. and/or M.D. degrees are eligible and are selected on the basis of academic record, research experience, career goals, letters of recommendation, and motivation for academic research. C. Training Facilities - The research laboratories of individual investigators constitute the primary training facilities. These are located within the Departments/Divisions of Biochemistry and Molecular Biology, Immunology, Pharmacology, Developmental Oncology Research, Urology Research, Experimental Pathology, and Laboratory Medicine, and Thoracic Diseases Research. These Departments/Divisions are all located within the Guggenheim Building for Basic Biomedical Research and are supported by institutional Shared Research Resource Facilities in Analytical NMR, Electron Microscopy, Mass Spectroscopy, Molecular Biology, Radioimmunoassay, Pharmacology, Cancer Biostatistics, Biomedical Imaging, Flow Cytometry/Optical Morphometry, Mathematical Methods, Protein Sequencing/Peptide Synthesis, Research Computing, Cytogenetics, and Pathology. Separate facilities are available for animal housing, engineering, and classroom and lecture space.

three dimensional imaging /topography; neoplastic cell; breast neoplasms; centrosome; biomedical resource; molecular oncology; bioimaging /biomedical imaging;

The Electron Microscopy Core Facility (EMCF) is a research resource laboratory providing specimen preparation, microscopy, photography and digital imaging services to investigators from both clinical and basic science laboratories within the Mayo Clinic Foundation. The facility is fully equipped to offer standard transmission (TEM) and scanning (SEM) electron microscopy. The facility also performs X-ray probe microanalysis and immuno-gold labeling procedures. Costs associated with this core are recovered primarily through recharges with modest institutional support. The EMCF is requesting support specifically for the development of new techniques and procedures in electron microscopy for Cancer Center Members. In addition, methods in electron tomography, cryomicroscopy, and novel methods for identification and localization at the ultrastructural level are being incorporated into the available services of the EMCF. Incorporation of these methodologies will enable Cancer Center members to study three-dimensional structural features of cells and molecules at a resolution and preservation that were previously unattainable.

The Electron Microscopy Shared Resource (EMSR) is an institutional research resource laboratory providing specimen preparation, microscopy and digital imaging services to investigators within the Mayo Clinic. Centrally located in the Guggenheim Research Building on the 14th floor on the Rochester Campus, the shared resource is fully equipped to offer standard transmission (TEM) and scanning (SEM) electron microscopy. The shared resource also performs X-ray probe microanalysis, electron tomography, cryoultramicrotomy, and immuno-gold labeling procedures. Costs are recovered primarily through recharges to investigator budgets and through institutional support. The facility has been an important shared resource of the Mayo Clinic Cancer Center (MCCC) since 2004. The requested budget will support, in part, current EMSR services utilized by cancer center members, and partially offset the resource's projected yearly operational expenditures of $1,202,039. Usage of the EMSR by MCCC members account for an average utilization of -20% of the EMSR's services and operations during the period from 2003 to 2007. The budget requested for the next five year period will support 8% of the resource's expenses and operations provided to MCCC members. The EMSR provides additional value to Mayo Clinic Cancer Center members in three ways: First the EMSR is continuously developing and implementing new techniques and procedures in electron microscopy directed specifically to the individual needs of MCCC Members. New methods under development include electron tomography, cryo ultramicroscopy, 3-D image analysis, electron diffraction and microwave processing. Second, MCCC member projects are given first priority in the laboratory's specimen handling queue. And finally, MCCC member projects are directly subsidized through discounts.

The Microscopy and Cell Analysis Core is fully equipped and expertly staffed to perform specimen preparation, light and electron microscopy, photography, digital imaging, and multi-parameter Flow and Cell Sorting services to Mayo Clinic Cancer Center investigators from both clinical and basic science laboratories. The facility also performs X-ray probe microanalysis, immuno-gold labeling, 3-D EM reconstruction from serial-sectioned specimens, and high-resolution reconstruction using image averaging of negative stained and cryo-tomography imaged specimens. Instrumentation and expertise are also supported in the core for high-end optical, two-photon, super-resolution, laser confocal, TIRF, ratio imaging, and other optical methods, as well as Flow Cytometry and Live Cell Sorting technologies and analysis. Additionally, the core staff (23 FTEs) and the core director are available for the development and/or implementation of new microscopy techniques and for testing and evaluation of instrumentation that would be of value to individual Mayo Cancer Center investigators and the greater research community. During the last budget year, the Microscopy and Cell Analysis Shared Resource has been widely utilized by each of the Mayo Cancer Center Programs. During the past 12 months, the facility served over 100 Mayo Cancer Center investigators processing 1000-plus specimens for electron microscopy, and logging 5,000 hours of Optical Microscopy and over 5000 hours of Flow Analysis and Cell Sorting. Because of the large expense for purchase and maintenance of high-end optical and electron microscopes, and the expertise required for their productive use, most investigators and their research laboratories find the Microscopy and Cell Analysis Core services to be the most economical, practical, and up-to-date means to achieve their sophisticated microscopy and cell sorting and analysis research needs.

MICROSCOPY AND CELL ANALYSIS SHARED RESOURCE PROJECT SUMMARY Decoding mechanisms in cancer biology requires a fundamental understanding of the relationship between structure and function. This is especially important for assessing tissue/tumor cell context, and at the macomolecular level, for organelle function and integrity, and chromosome structure and behavior. The Microscopy and Cell Analysis Shared Resource (MIC) provides exceptionally maintained high-end equipment for optical and electron microscopy, cytometry and cell sorting, and provides the technical expertise for their use to Mayo Clinic Cancer Center (MCCC) members conducting clinical or basic science cancer research. This shared resource is heavily used with 161 MCCC members using the facility in 2017, from each of the MCCC sites. The facility operates 10 hours/day on weekdays and 6 hours/day on weekends for assisted use, and for most areas 24/7 for unassisted use. The MIC provides access, training, and expertise for a wide array of state- of-the-art light and electron microscopy instrumentation, and for cell sorting and analysis. Our portfolio of instrumentation allows MCCC members to image single molecules, in live cells and tissues, or fixed specimens at optical or electron microscopic resolution, to analyze image data files by a wide variety of image analysis and 3D reconstruction packages, and to perform multi-color flow analysis and sorting of fixed or live cells. We support advanced optical imaging techniques such as FRET, TIRF, PALM, multi-photon, and 3D serial electron microscopy, as well as state-of-the-art flow cytometry and cell sorting instrumentation and analysis methods. The facility has aggressively pursued instrumentation funding which has allowed us to recently obtain a Zeiss Elyra super-resolution microscope, and this past year, a FEI Apreo Serial Block Face Electron Microscope. The MIC also provides cyberinfrastructure for data analysis, transfer, and storage of extremely large image data sets.