Ahna Renee Skop - US grants

The goal of this project is to identify and characterize proteins that comprise the mammalian midbody by matrix assisted laser desorption/ionization (MALDI-MS) to identify novel players during mitotic spindle assembly and cytokinesis. I plan to screen Xenopus spindle extracts for proteins that may affect spindle assembly. I will utilize the C. elegans system to look for proteins that affect cytokinesis utilizing live imaging and RNA interference techniques. In addition, C. elegans and human cell lines will provide excellent systems to study the localization of these proteins during mitosis. Ultimately, I hope to address questions of cellular organization and cytokinesis as an independent investigator at an academic or research institute. Since many cell division processes are perturbed in cancerous cells and birth defects, the understanding of the mechanisms that underlie cell division processes ought to lead to an understanding of the basis for cancer pathology. In the post-genomic era, the discovery of proteins that comprise cellular structures, proteomics, will aid in drug development since all drugs are directed against proteins.

At the University of Wisconsin-Madison and in other universities throughout the state, there are over a thousand faculty performing research within the life sciences. Fundamental to many of these studies is a mass spectrometric-assisted proteomic analysis of the cells, tissues and organs that make up the organisms. The UW -Madison Biotechnology Center Mass Spectrometry/Proteomic Facility was established in 1998 as a centralized facility for the purpose of acquiring mass spectrometers and making them available to the research community on a fee-for-service basis. During the six years since inception, it has proven successful in meeting the proteomic needs of nearly one hundred different academic laboratories in a cost effective, top quality manner. However, a major deficiency for investigators using this facility has been the inability to obtain protein sequence from very small samples of tissue, a common handicap of biological research. A second limitation is the ability to routinely perform quantitative differential proteomic experiments using isotope-assisted tandem mass spectrometry. The MALDI-based tandem mass spectrometer known commonly as a MALDI-TOFTOF is an instrument that is uniquely capable to address both of these problems. Surprisingly, there is not a single MALDI-TOFTOF available in the state. Wisconsin researchers currently must drive/fly to Chicago or Boston to obtain precious instrument time. Lack of ready access to this instrument locally has hindered our researchers in obtaining the critical information needed for converting genomic sequence 'data' into a real understanding of life processes. The purpose of this award is to acquire this instrument and make it available to academic researchers statewide. The instrument will be placed within the existing UWBC Core Mass Spectrometry/Proteomics Facility, ensuring that (i) it will be accessible to a large diverse group of researchers, (ii) that it will heavily used and (iii) that it will be well maintained for optimal sensitivity and overall reliability.

The broader impacts of this project are several-fold. First, the research users represent a large number of different academic disciplines, from Animal/Food Science to Engineering to Prebiotic Chemistry to the 'traditional' Molecular, Cellular and Organismal Biology areas. Six years of prior experience in operating expensive and sophisticated mass spectrometers ensures that the operation of this MALDI-TOFTOF will be well organized, accessible and affordable for the entire community. In addition, the university has strived to ensure that the UWBC core facilities in general, and the Mass Spectrometry/Proteomics in particular, are utilized for education at all levels, including undergraduates and high schools, both locally and in Wisconsin communities with underrepresented groups, such as economically impoverished schools in Milwaukee and Native American Indians in small communities of northern Wisconsin. This instrument will advance discovery and promote teaching and generally enhance the academic infrastructure, not only at UW-Madison, but also, all over the state.

This is a five-year CAREER award.

Intellectual merit: Cytokinesis is achieved by the formation of a cleavage furrow in anaphase that bisects the mitotic spindle between the separated chromosomes, cytoplasm and organelles. The goal of this research activity is to gain insight into the mechanisms required to establish cleavage furrows, identify factors required to regulate furrow formation and to determine how DIP-1, a novel dynamin/DYN-1 interacting protein, coordinates this process. The underlying hypothesis is that the cleavage furrow is established by clustering of lipid raft proteins by an unknown signal(s) from the spindle. This signal triggers DIP-1 to regulate the targeting and assembly of dynamin/DYN-1, a known raft component, at the equatorial membrane of the cell during the metaphase-anaphase transition. Dynamin/DYN-1 subsequently directs actin filament formation, which assemble into the acto-myosin ring and in turn, promotes cleavage furrow invagination and completion. This hypothesis is based on the observations that: 1) The target of the signal for cytokinesis is the equatorial cortex; 2) DYN-1 localizes to equatorial membranes and newly formed cleavage furrows; 3) Dynamin is a known lipid raft component; 4) Dynamin can influence actin assembly; and 5) DIP-1, like DYN-1, regulates the establishment of cleavage furrow formation as well as completion. Based on these observations, the experimental focus of this project is on the role of DIP-1 in establishing the cleavage furrow and regulating DYN-1 function during cytokinesis in animal cells, using the nematode, C. elegans, as a model. Preliminary studies in dip-1 (RNAi) embryos show that (i) the spindle midzone microtubules assemble properly, but buckle and disappear during late anaphase (60%); (ii) the formation of cytoplasmic, spherical tubulin::GFP aggregates are observed (40%); (iii) the actin cortex is disorganized and membrane ruffling during cytokinesis is almost absent; and (iv) DIP-1 contains a BRCT motif commonly used to mediate interactions with other BRCT motif-containing proteins, such as BRCA2 and ECT2, known cytokinesis proteins. The strategy of this study is to: 1) Determine the cellular function of DIP-1; 2) Characterize the membrane-cytoskeletal consequences of DIP-1 depletion during cytokinesis; 3) Determine if DIP-1 directly binds to DYN-1; and 3) Determine the cell cycle localization of DIP-1::GFP and identify protein motifs and additional factors that regulate DIP-1::GFP localization during mitosis. These studies should provide new insight into the regulation and function of the membrane-cytoskeletal events that occur during cytokinesis with an understanding of the specific role of DIP-1 in this process.

Broader Impact: This CAREER project also involves a significant, integrated educational component, designed to benefit both the students involved directly with the work and for the public, especially for local Native American communities. The high school, undergraduate and graduate students involved in or introduced to this research will gain hands-on experience using in vivo microscopy techniques in conjunction with genetics, biochemistry and molecular biology. Since live movies of cell division in C. elegans embryos are quite exciting and easy to comprehend, Dr. Skop's work is especially welcoming to all educational backgrounds. Since "systems biology" will be taught and performed in the laboratory, the introduction to these techniques will be skills that are highly sought after in the fields of genomics, proteomics and biology. Information about Dr. Skop's work and her "systems biology" course (Genetics 875) will be disseminated to the academic and high school communities through online resources related to her laboratory and courses.

DESCRIPTION (provided by applicant): The plasma membrane plays a central role in cytokinesis and polarity, yet we know very little about how it is regulated and maintained during embryonic development. In addition, how the association between the plasma membrane and underlying cytoskeleton is dynamically transformed during the cell cycle is unclear. The plasma membrane-associated protein, dynamin has an essential yet undefined role in cytokinesis and polarity. In recent years, considerable progress has been made demonstrating that dynamin regulates a variety of membrane-cytoskeletal events. While it is evident that dynamin may regulate and even link membrane to the cytoskeleton, it is still entirely unclear how dynamin coordinates and maintains these essential events during development. The long-term goal is to better understand how the plasma membrane is regulated and maintained during embryonic development. The objective of this research program is to define and identify factors that regulate membrane-cytoskeletal events that occur during cytokinesis and polarity and to determine how dynamin coordinate these processes. The central hypothesis of the proposal is that dynamin functions to temporally and spatially control cytokinesis machinery and influence embryonic polarity. Guided by published and strong preliminary data, we plan to test our central hypothesis and accomplish the objective of this application by pursuing the following three aims: 1) Characterize the role of dynamin during cytokinesis in animal cells;and 2) Determine the contribution of dynamin during polarity maintenance;and 3) Identify and characterize cell-cycle factors critical for membrane remodeling and maintenance during embryonic development. The proposed work is innovative, because it capitalizes on a new means of identifying and characterizing the function of distinct plasma membrane domains that are highly regulated during the cell cycle. The proposed research is significant, because it is expected that the results will fundamentally advance the fields of cytokinesis, polarity as well as membrane biology. Relevance to Public Health: Understanding how the plasma membrane is regulated and maintained during development is of utmost interest because the identified factors are anticipated to provide new targets for preventive and therapeutic interventions that will aid in the growing numbers of persons who have cancer, developmental disorders and age-related abnormalities. (End of Abstract)

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our goal is to understand the mechanisms of cytokinesis. We are using proteomic and comparative genomic techniques to identify proteins that are required during specific cell cycle stages. Analyzing protein composition of different cell stages may lead to better understanding of the mechanism of cell division by pinpointing proteins specific to particular cell cycle stage.

INTELLECTUAL MERIT
During embryonic development, the identity of cells in tissues and organs is determined by a series of asymmetric divisions. Cell asymmetry is controlled by two mechanisms: cell polarity and cytokinesis. Errors in these events often lead to changes in cell proliferation and growth. The plasma membrane and underlying cell cortex play an important role in cell polarity and cytokinesis, yet very little is known about how it is regulated and maintained during development. To address this issue, the project is focused on the protein dynamin, which has an essential role in both of these events. It is hypothesized that dynamin influences cell polarity and facilitates the temporal and spatial control of the cytokinesis machinery through its role in endocytosis and actin dynamics. This project will employ an interdisciplinary strategy to reveal the role of the dynamin using a combination of genetics, cell and molecular biology and high-resolution, in vivo microscopy techniques. The project will focus on the early divisions of the nematode, Caenorhabditis elegans.

BROADER IMPACTS
The work outlined in this proposal will be used in numerous minority outreach opportunities and scientific art shows. The P.I., Dr. Ahna Skop has extensive experience with outreach to minority groups at the local, state and national levels. Through her consistent involvement in the SACNAS, AISES and MARC programs at New Mexico State University, she has contributed to a very positive trend in minority graduate student enrollment on the UW-Madison campus. The P.I.'s dedication to increasing and retaining minority, undergraduate and graduate students in the STEM fields is a priority, and part of her mission is to make science accessible to every race and group and welcome these students any chance she gets. In addition, the P.I. has continued to pursue the curation of various scientific art shows nationally and internationally, which inherently promote the beauty of science and provide public access to scientific research.

Cell division involves two fundamental process. In the first step, genetic information is partitioned to opposite sides of the mother cell, while in the second step, the mother cell physically partitions into two separate daughter cells at a region known as the midbody. This project will investigate the observation that some genetic information accumulates at the midbody. The work employs interdisciplinary approaches that can be widely employed for making rapid progress in understanding the functions of poorly understood cell division genes and mRNAs. The Broader Impact activities include a public outreach component in which microscopy images generated from this research will be used to create a moveable 20-foot scientific art installation called "Genetic Reflections". This piece will be installed in a public, high-traffic area in the UW-Madison Biotech Center and then the Wisconsin Science Museum. Portions of this installation will be constructed to travel locally around the state of Wisconsin and then nationally. The goal is to have scientific artwork that is beautiful, informative and promotes future public engagement projects between scientists and the public.

In certain cells types, midbody factor mis-regulation and midbody accumulation leads to altered growth and development, suggesting that the factors (proteins and mRNAs) associated with midbodies when mis-handled by the cell, can lead to dramatic and detrimental proliferative and cell fate changes. Necessary reagents, techniques, and datasets will be generated and shared, providing the foundation to determine how midbody-associated mRNAs are regulated during cell division. With such knowledge, it will be possible to probe the dynamics of midbody-specific mRNAs during mitosis. The PI has made an unexpected preliminary discovery in the midbody transcriptome of an enrichment of the mRNA of an important kinesin widely known to function on the protein level. This discovery challenges current understanding of the mechanisms of molecular motor regulation and transport, and also likely cytokinesis. If successful, the knowledge generated from this research will transform current understanding about the regulation of molecular motors and cell division in all aspects of human and organismal biology.