Gary G. Borisy, PhD - US grants

The purpose of this proposal is to identify and study the function of components of the mitotic spindle at the molecular level. Although tubulin is the most abundant component of the spindle, it serves primarily a structural role. The activity of microtubules is likely to be understood through the molecules that govern their assembly and interaction with poles, chromosomes, the midbody and perhaps other spindle structures. This proposal will focus on specific molecules recently identified as components of the spindle. As determined by reaction with monoclonal antibodies specific for a phosphorylated epitope (MPM), a subset of these components has been shown to become phosphorylated at mitosis and dephosphorylated upon the return to interphase. The phosphorylation/dephosphorylation of specific spindle components may serve important regulatory functions for spindle formation, chromosome movement and spindle disassembly. This proposal seeks to determine the MPM-reactive phosphoprotein composition of mitotic MTOCs as well as to identify important non-MPM reactive components; to define biochemically the phosphorylated epitope, to determine the cellular distribution of identified spindle molecules in the dephospho as well as phospho forms in interphase as well as mitosis; to assay the function of phosphorylation/dephosphorylation events in vitro by assaying the effect of phosphorylation on the microtubule nucleating capacity of centrosomes and in vivo by microinjection of phosphorylation/dephosphorylation inhibitors; to test the function of selected spindle components by microinjection of antibodies; to begin the purification of putative protein kinase(s) and phosphorprotein phosphatase(s) responsible for the events; and finally, to attempt to identify and clone genes for selected spindle molecules. These basic studies on cell division may reveal control mechanisms important for understanding the biochemical defects which result in the uncontrolled division of cancer cells.

Our project goal is to elucidate the mechanism of ribosome self-assembly. Two different approaches are being used to reach this objective. First, we are using a newly discovered inhibitor of ribosome assembly. This inhibitor, a dye called Cibacron Blue F3GA, can effectively stop assembly at any point in time without damage to the particle that has assembled up to that stage. This allows us to determine in detail the actual kinetic pathway of ribosome assembly. Furthermore, we should be able to determine the effect of various parameters such as temperature on the kinetics of assembly of each protein. This work is being carried out on both the 30s and the 50s ribosomal subunits. Our second approach involves the production of various fragments of individual proteins. We have used both enzymatic and chemical methods of cleavage. In addition, we have available a number of mutant strains of E. coli which are comprised of ribosomes carrying one of the proteins in a fragmented form. We have used a number of these selected fragments in the assembly reaction in place of the parent protein. The effect of the missing section on the mechanism of assembly and ribosome function is then assessed.

molecular biology; chromosome movement; microtubules; centrosome; neurons; fibroblasts;

DESCRIPTION (provided by applicant): The human mouth is colonized by a microbial community of enormous complexity that plays a key role in human health and disease. Dental plaque is a biofilm constructed and inhabited by microbes, and the oral microbial community is involved in the development of several infectious diseases such as dental caries, periodontal disease, alveolar osteitis, tonsillitis, strep throat and otitis media. The question of which organisms are present in the oral cavity has been extensively studied and over 600 species or phylotypes have been identified, of which only about half can be cultivated at the present time. Recent surveys using 16S rRNA clones indicate that the relative abundance of taxa is uneven, with about 250 taxa accounting for 90% of the clones observed. The DNA microarray or tag sequencing methods currently used to identify which taxa are present require that a sample be homogenized to extract the DNA for further analysis. Although centimeterscale information can be retained (for example, which tooth the plaque was taken from, or whether a sample was from the side or the top of the tongue), spatial information at the micron level--namely the level of microbial community organization--is lost. The objective of this project is to develop a novel, "combinatorial imaging" method for simultaneous imaging and identification of many microbial taxa in samples from the human mouth, so as to obtain micronscale information about the spatial organization of the oral microbiome. The technique of fluorescence in situ hybridization (FISH) targeting ribosomal RNA is widely used to identify microbes and can be specific and sensitive, but as commonly employed it allows the differentiation of only two or three taxa simultaneously. We propose to extend the capabilities of FISH by at least an order of magnitude, employing a combination of fluorescent reporter groups to create "spectral signatures" that allow simultaneous encoding and imaging of tens to potentially hundreds of different microbial phylotypes. Over the course of the project, individual FISH probes, and sets of fifteen or more FISH probes labeled with different combinations of fluorophores, will be designed and tested on cultured cells, in vitro biofilms and on plaque obtained from volunteers. The focus of the project is translational technology development, expanding the set of probes that can be employed to detect oral microbial taxa, obtaining information about where taxa are located relative to each other and relative to host (human) tissues, and pushing the practical limits of this technique as applied to dental microbes in small clusters and in biofilms. The technology has the potential to be used for developing high throughput translational science assays for the rapid and cost-effective monitoring of oral communities. It will lay the foundation for future studies examining the role of the normal microbial flora in both healthy and diseased mouths. PUBLIC HEALTH RELEVANCE: Hundreds of species of bacteria live in the human mouth;dental plaque is a biofilm constructed and inhabited by microbes;and the oral microbial community is involved in the development of many oral diseases including dental caries and periodontitis. This proposal is to develop innovative, "combinatorial imaging" technology to study the precise spatial organization of different kinds of bacteria comprising microbial communities found in the mouth. The technology introduces "spectral signatures" using binary combinations of fluorescently labeled probes targeting intact bacteria and using spectral imaging to differentiate large numbers of labeled probes simultaneously. This method will allow us to determine where taxa are located relative to each other and relative to the host (human) tissues, which will lay the foundation for new, rapid diagnostic assays and for studies of how oral microbial communities work and how pathogenic species invade host tissues and cause disease.

DESCRIPTION (provided by applicant): The Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts, proposes to launch a high-impact, multidisciplinary and unique initiative in regenerative and stem cell biology drawing upon the special advantages of marine invertebrates. Regenerative and stem cell biology is being recognized in medical centers across the country as a critical area of science for establishing the potential of regenerative medicine. In this emerging field, scientists seek to define and understand the natural processes by which damaged or aging tissues and organs can regenerate or be repaired, and to apply that knowledge to developing medical therapies. Although mammalian systems are unquestionably of direct relevance and great importance for human health, non-mammalian models including bacterial, fungal, invertebrate and vertebrate systems have contributed enormously to our understanding of the fundamental cell and tissue biology that underpin medical advances. Among possible model systems, marine invertebrates and lower vertebrate organisms hold great promise specifically as research models for regeneration, particularly as the field progresses in decoding their genomes. The MBL has invested in the cognate areas of cellular dynamics, imaging, genomics, marine husbandry and biodiversity informatics. The MBL is renowned for its advanced research training programs in physiology, neurobiology, embryology and gene regulatory networks. We believe the physical and intellectual infrastructure investments already made at the MBL pre- adapt it to launch a new initiative in Regenerative Biology. The missing element is to establish a critical mass of individuals focused on this area. The P30 program support is vital for recruiting two individuals who, with existing MBL scientists, would provide the critical mass for establishing the Center. We believe that the time is ripe to exploit the special experimental potential of marine organisms and that the MBL is uniquely pre- adapted to serve as the home for a new Center for Regenerative Biology.