Heide Schatten, PhD, University of Heidelberg 1977 - US grants

microscopy; growth factor; biomedical resource; Invertebrata;

DESCRIPTION (adapted from the applicant's abstract): This research proposal is designed to enable continued participation by the Mt. Sinai Medical Center (MSMC) on the NSABP trials. This staff is dedicated to achieving the aims of the NSABP which are specifically to improve disease-free survival and survival in patients with primary operable breast and colorectal cancers. Dr. Richard Bornstein has chaired the Breast Committee, and is now a member of the Combined Breast Committee along with their nurse, Sheryl Reynolds. Both Bornstein and Reynolds are on the group's executive committee. Reynolds chairs the Clinical Research Associate Committee. It is their intention to continue to co-author scientific papers and to accrue all eligible patients to these trials. They have been successful in accruing 471 patients thus far, with 350 currently in follow-up. Periodic auditing of their referring doctors' performance sites occur on a regular basis and will continue. They expect accrual to increase rapidly once new protocols are available. Their community outreach program remains productive and is growing.

The purpose of this proposal is to request funds for the purchase of a JEOL JSM-6340F Field Emission Scanning Electron Microscope with key optional capabilities including a field emission (FE) electron gun for increased resolution of sample analysis, and a backscattered electron detector for low voltage imaging and analysis of gold-labeled (3, 5, and 10 nm) surface antigens. This new JEOL JSM 6340F FESEM will serve 7 major and 13 minor users from seven different departments in interdisciplinary research and education efforts at the University of Missouri-Columbia (MU). The requested microscope will be housed in the Electron Microscopy Core Facility (EMCF) which is a recently reorganized centralized multi-user core facility that has as its mission to support research and education within the University community and in the region. It was established in 1995 as a result of combining the resources from the College of Agriculture, Food and Natural Resources, College of Arts and Science, College of Veterinary Medicine, and School of Medicine. The need for more modern capabilities has made it essential to ask for funds to purchase a new versatile, reliable, and user-friendly instrument, as the 20-year old JEOL-JSM35 scanning electron microscope cannot satisfy the demands of the present users. Additionally, because the EMCF serves a diversity of research projects and has as it's mission the training and education of future scientists, modern instrumentation is critically important. The new capabilities requested for this multi-user Core Facility will allow (1) high resolution analysis of minimally coated samples of biological plant and animal materials, and of molecular isolates and spreads; (2) high resolution detection of small gold labels down to 3 and 5nm gold-labeling for immuno-electron microscopy; (3) image acquisition and archiving for post-examination analyses; (4) use of the PC-based, fully digital microscope for training computer-age microscopists and resea rchers; (5) the ability to add a cryo-system; and (6) the ability to add enhanced x-ray microanalysis of minimally coated specimens from an EDS system that is planned for future purchase. These features have become essential for major parts of funded projects in agronomy, entomology, plant pathology, cell and molecular biology, veterinary parasitology, plant and animal reproduction, microbiology, and other basic sciences. The requested microscope will also serve to train users and students in a graduate-level Cell and Molecular Electron Microscopy course offered annually. The new microscope will be supervised by the EMCF director, who is assisted by two permanent staff EM technicians. The EMCF is one of six Core Facilities administered by MU's Molecular Biology Program, a state-funded program, that is committed to provide 50% of the capital costs, long-term salaries for the director and staff of the EMCF, and substantial subsidies for existing equipment as well as laboratory renovations and service contracts for all Core instruments.

Several mammalian cell culture lines (PtK2, PC 12, HeLa) have been utilized to study the structure-function relationship of centrosomal material with microtubule organization during interphase and mitosis employing standard chemical fixation as well as high pressure freezing (BFF). To preserve ultrastructural and immunological details of centrosomes, chromosomes, microtubules, membranes and intermediate filaments several antibodies were used and analyzed with transmission and scanning electron as well as with immunofluorescence microscopy utilizing a human autoinunune antibody against centrosomes (SPJ), a mouse monoconal. antibody against tubulin (M), an intermediate filament antibody against vimentin (Ah-6), and DAR to stain DNA. During interphase in control cells, centrosomal material is closely associated with the nuclear envelope by a fibrous network and becomes gradually dissociated during mitosis where it functions as the microtubule organizing center for the microtubule-based mitotic apparatus. Unlike in sea urchin eggs and embryos where microtubules are needed for cell-cycle specific expansion and compaction of centrosomes (Schatten et al., 1988, Cell Motil. Cytoskel. 11, 248-259), microtubules are not required for cell-cycle specific progression of centrosomes in mammalian cells. These studies extend on previous findings (Joswig and Petzelt, 1990, Cell Motil. Cytoskel. 15, 181-192) and support the notion that different mechanisms for centrosome expansion and compaction behavior are used in different eukaryotic species. By using high pressure freezing, this project is likely to contribute to our understanding on the mechanisms of folding and unfolding of centrosomal material and will also contribute to our understanding on centrosome-cytoskeletal interactions during fertilization, cell division, cell differentiation, and embryo development. SCXENTXFXC SUBPROJECT GRANT NUMBER: P41RR00570-27

The purpose of this project is to understand motility and cytoskeleton-membrane interactions in the intracellular protozoan parasite Toxoplasma gondii. Genetic and cell biological studies have determined that parasite motility and cell invasion is powered by an actin-myosin based motor in the parasite, but it has not been possible so far to localize actin filaments with Fonventional. electron microscopy. This led to the conclusion that actin might exist primarily in globular form. T gondii represents an interesting parasite system in which to study cytoskeletal motility. Unlike bacterial cell uptake, parasite invasion does not involve significant alteration in the host cell cytoskeleton. Instead, invasion is an active process of penetration into the host cell by the parasite. During invasion, actin and myosin that is localized underneath the plasma membrane in the parasite presumably combine to produce the force necessary for motility during invasion. By using the techniques described by Dr. Ris to look at actin and myosin structures with LVSEM we expect to be able to determine the mechanisms underlying the gliding motility in the parasite T gondii and its interactions with host cells during parasite invasion.

This project will test whether genistein?s antiproliferative effects occur through inhibition of abnormal phosphorylation of S-phase centromere proteins and nuclear mitotic apparatus protein NuMA or through reductions in free radical damage to mitochondria.

DESCRIPTION (provided by the applicant): The purpose of this research is to understand cytoskeletal organization and functions during cell division of the intracellular protozoan parasite Toxoplasma gondii, a model for harmful apicomplexan parasites that include Plasmodium falciparum and Cryptosporidium parvum. Despite the importance of the cytoskeleton for cell division in these unicellular organisms the mechanisms that power the separation of the genome remain uncertain, largely because only limited data exist about the organization and composition of the cytoskeleton. Recent data suggest an unconventional division machinery that divides the nucleus and separates the apicoplast, a single non-photosynthetic plastid that is essential for parasite survival. We hypothesize (1) that the parasite's unconventional division apparatus is based on a complex organization of microtubules, their organizing center (the centriole-centrosome complex) and perhaps other cytoskeletal components yet to be determined. Fibers of the division apparatus may associate with the pellicle (the parasite's outer membrane system) to separate the genome and the apicoplast during cell division in T. gondii; we hypothesize (2) that the centriole-centrosome complex plays an important role in the organization of the cytoskeleton and may contain proteins that differ from mammalian cells. Our specific aims are to (1) analyze the parasite's cytoskeleton during cell division with focus on the interactions with the pellicle, the apicoplast and the nucleus, and (2) to characterize the centriole-centrosome complex with focus on proteins that may differ from mammalian cells and may serve as targets to inhibit cell division in apicomplexan parasites. The novel approach of the proposed experiments is to use high-resolution field emission scanning electron microscopy (HRFESEM) on thick-sectioned resin de-embedded material. This method used in preliminary experiments allowed for the first time viewing of actin-like filaments and intermediate-like filaments which had not been possible with any other method available so far. We will further isolate the division apparatus of T. gondii and generate antibodies to determine parasite-specific centrosome proteins. This research will address unresolved questions regarding the cytoskeleton and the centriole-centrosome complex during cell division in T. gondii and, perhaps, the existence of other cytoskeletal fibers. This research will advance our understanding of cell division in apicomplexan parasites and may lead to the identification of targets to control replication of harmful apicomplexan parasites. Pilot data from this RO3 research will be used to apply for funding through the RO1 mechanism.

[unreadable] DESCRIPTION (provided by applicant): The purpose of this research is to explore mitochondrial distribution as markers for developmental potential of cloned pig embryos. Cloning of mammalian embryos has become attractive in recent years because of the high potential for biomedical and agricultural applications. Cloning of pigs as tissue and organ donors is of high interest because of the exceptional physiological compatibility with humans. However, practical applications are not yet feasible because of the low cloning efficiency (ca. 0.2% in pigs) for which causes are only little understood. One reason may be an asymmetrical mitochondria distribution that can result in reduced ATP generating capacity and an inability to support normal cell functions. We propose that specific patterns of mitochondria aggregation and microtubule organization will allow us to predict developmental potential of cloned embryos and increase cloning efficiency. Our specific aims are to analyze: 1a) whether differences in mitochondrial distribution occur among individual blastomeres in cohorts of morphologically normal cleavage stage embryos; 1b) whether changes in intracellular pH are associated with disruption of mitochondrial organization and reduced development in vitro; 2a) whether microtubule organization plays a role in mitochondrial distribution after nuclear transfer in cloned embryos; and 2b) whether unequal centrosome separation after nuclear transfer plays a role in mitochondrial distribution. The distribution of mitochondria will be examined by scanning laser confocal microscopy in fixed and live oocytes, in cleavage stage embryos, and development to the blastocyst stages. MitoTracker Green FM and Mitotracker-X-Rosamine will be used to stain mitochondria. Double and triple immunofluorescence staining with anti-tubulin and anti-centrosome antibodies will determine the relationship between mitochondria and microtubule organization. We will correlate survival to the blastocyst stages with characteristic mitochondria fluorescence patterns. These experiments will provide new information on mitochondria distribution and microtubule organization after nuclear cloning and allow future research aimed at selecting embryos that are most likely to survive and increase live birth of cloned animals. Pilot data from this RO3 small grants program research will be used to apply for funding through the RO1 mechanism. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The purpose of this research is to explore the patterns of apoptosis in cloned pig embryos and possible causes for increased apoptosis in nuclear transfer (NT) embryos as compared to in vitro fertilized embryos. Cloning of pig embryos has become an important new topic in biomedical research because of the high potential for biomedical applications. Genetically modified pigs are being produced to serve as tissue and organ donors for humans and as a model for human disease because of the exceptional physiological similarity of pigs with humans. However, practical applications are difficult because of the low cloning efficiency (ca. 1% in pigs) for which causes are only little understood. Incomplete or abnormal remodeling of the donor cell nucleus following transfer, resulting in imprinting failures and abnormal gene expression throughout development, are thought to be among the primary reasons for developmental failures and embryo defects. It has been proposed that improper reprogramming by epigenetic factors may affect later stages of development that can result in implantation failures, which account for a large percentage of the causes for cloning failures. Misguided apoptosis during preimplantation development results in an imbalance of inner cell mass (ICM) and trophectoderm (TE) cells that negatively affects implantation. Insufficient placentation is the primary cause for cloned fetal loss. To understand the increased frequency of apoptosis in NT embryos we propose the following specific aims: to analyze 1a) whether donor cell mitochondria are distributed unequally and contribute to apoptosis in specific cells resulting in increased apoptosis during development; 1b) whether donor cell nuclei transferred without mitochondria exhibit lower incidences of apoptosis; 2a) whether mistargeting of lamins produces increased cells undergoing apoptosis; and 2b) whether misregulation of the nuclear mitotic apparatus (NuMA) plays a rolls in apoptosis during development. Our studies will employ donor cells with a fluorescent mitochondria recognition signal (Aim 1) to trace donor cell mitochondria throughout development. Cells undergoing apoptosis will be identified with the TUNEL assay. Molecular and imaging methods including Western immunoblotting and immunofluorescence microscopy of fixed oocytes, cleavage stage embryos, and development to the blastocyst stages will be employed. We will correlate mitochondria staining patterns and TUNEL staining with apoptosis and with embryo development to the blastocyst stages. These experiments will provide new information on imbalanced apoptosis patterns in cloned pig embryos during preimplantation development in which the distribution of mitochondria, lamins B, A/C, and NuMA might play a role. Future research is aimed at manipulating embryos to prevent abnormal apoptosis to increase cloning efficiency and normal development of cloned embryos. Pilot data from this R03 small grants program research will be used to apply for funding through the R01 mechanism. [unreadable] [unreadable] [unreadable]