Joanna B. Olmsted - US grants

The long term objective of the proposed research is to understand how the cytoskeleton of neuroblastoma cells is assembled and organized. The role of 215,000 dalton microtubule associated protein (215 K MAP) that is present in neurite-bearing neuroblastoma cells but absent in non-differentiated cells will be examined in detail. In vivo analyses will establish the synthetic pattern of this protein during stages of cell attachment and neurite formation. Immunofluorescence microscopy with antisera against the 215 K MAP and tubulin will indicate the distribution of these proteins at various stages of microtubule organization. These results will be compared to those obtained using cellular fractionation to analyze the distribtuion and amount of the 215 K MAP relative to soluble and assembled tubulin. In vitro assays will establish the mechanisms by which the 215 K MAP interacts with tubulin, and biochemical procedures will demonstrate whether this protein is post-translationally modified. Peptide mapping and monoclonal antibodies will be used to investigate the extenet of homology between the 215 K MAP and other microtubule associated proteins in neuroblastoma cells, as well as to microtubule associated proteins from other cell types. Limited studies will be undertaken to examine the relationship between microtubules and neurofilaments during neurite development, and to establish whether any of the microtubule associated proteins coordinate these two cytoskeletal systems. These investigations should provide basic information on the biological role of microtubule associated proteins in the organization of microtubules, and on the biochemical properties of this class of specialized proteins.

The long range goal of the proposed research is to understand how microtubule-associated proteins that are not motors participate in the function and organization of microtubules. These studies will focus on MAP 4, a microtubule-associated protein that is expressed during early development and in specific adult tissues in mice. Coding sequences for mouse and human MAP 4 have been obtained, a structural model for the protein defined, and novel transcripts observed in striated muscle and testis. A variety of approaches, including transfection and/or microinjection of protein fragments or fluorescently derivatized probes will be applied to deduce the regions of MAP 4 that are essential for function, and to ask whether or not there are domains that interact with cellular structures other than microtubules. Myogenic cell lines, in which induction of muscle-specific MAP 4 transcripts is observed, will be employed to examine the relationship between MAP 4 and tubulin isotype expression and distribution during myotube formation. Antisense technology will be used to ask whether MAP 4 is essential for normal cellular function, or events such as myogenesis or pre-implantation development. MAP 4 expression during mouse myogenesis, spermatogenesis and early development will be studied with correlative immunocytological and in situ hybridization analyses. Further molecular characterization of the coding sequences will be undertaken to determine the number, relationship, and mechanism of generation of the multiple MRNAS which presumably give rise to multiple protein isoforms. A minor project will be the completion of experiments that indicate the RII subunit of CAMP-dependent protein kinase mediates interaction of MAP 2 with the Golgi.

This application requests funds for the purchase of a Bio-Rad confocal system integrated with a Nikon microscope. The seven major users of this facility are junior and senior faculty in the Department of Biology at the University of Rochester, and all are funded from national granting agencies (NSF, NIH, USDA). The current programs of the users involve carrying out studies on fate specification, cell-cell interactions and morphogenesis in developing organisms (Drosophila, sea urchins, Arabidopsis); the role of cytoskeletal elements in cellular and organismal functions; the mechanisms of nuclear transport; and regulation of genome packaging. These projects are unified in using molecular, genetic and/or biochemical techniques to ask how specific gene products influence cellular and developmental processes. In order to carry out quantitation, dynamic analysis and co-localization studies, all of these projects now require a confocal microscope system which is: 1) reliable and easily used by a number of individuals; 2) has multiple types of optics; 3) has flexibility in the excitation and emission wavelengths; and 4) has excellent resolution in the Z and X/Y axes. Image analysis software that allows further quantification and manipulation of the confocal data is also requested. As members of the Department of Biology, all of the users are also involved in education of undergraduate and graduate students in both formal and informal settings. In the context of each of the research projects described, the requested instrumentation will be used by graduate students and postdoctorals in training. The instrumentation will also be employed for demonstrations in undergraduate courses and outreach programs, and for hands-on experiences- in advanced laboratory courses and independent undergraduate research projects.