Stephen Rogers, Ph. D. - US grants

Microtubules (Mts) are polymers of the cytoskeleton that serve to organize the cytoplasm and to act as tracks for the delivery of cargo by motor proteins. In order to perform these functions, the ends of Mts sometimes need to form stable linkages with targets, such as organelles or cortical domains. These linkages are thought to form between MT plus end-associated proteins and target-bound 'receptors.' The EB1 family of proteins is believed to be one such MT-bound protein. We propose to study the cellular functions of EB1 in Drosophila and to understand the molecular mechanisms that target it to MT plus ends by pursuing three specific aims. In the first, we will inhibit the function of EB1 in cultured cells using RNAi and use immunofluorescence- microscopy to assess the role EB1 plays in MT organization during interphase and mitosis. We will also employ real-time confocal microscopy to observe the role of inhibitory antibodies on the mitotic spindles of living fly embryos. In aim two, we will reconstitute EB1 targeting to the plus ends of MTs using in vitro assays and study the interactions between tubulin and EB1 using biochemical techniques, as well as atomic force microscopy. In the third, we will identify EB1- interacting proteins using biochemical approaches, such as affinity chromatography and immunoprecipitation, and by using two-hybrid interaction to identify EB1Q effector proteins and candidate EB1 receptors. Collectively, these studies will allow us to assign cellular functions to EB1 and to determine how this protein interacts with the MT cytoskeleton.

[unreadable] DESCRIPTION (provided by applicant): The ability of cells to alter their shape is critical to the ontogeny of most organisms. Within tissues, for example, changes in cellular morphology drive tissue remodeling during morphogenesis and are essential for wound repair. At the level of the individual cell, cycles of shape change allow some cell types to migrate during embryogenesis, immune function, and (more insidiously) during metastasis. Cellular morphology is dictated by the cytoskeleton - the network of actin filaments and microtubules. The long-term goal of this project is to understand the principles and mechanisms underlying cellular morphology at the molecular level by studying the pathways that regulate and integrate cytoskeletal dynamics. Importantly, the networks of actin and microtubules do not act in isolation, rather there is an unprecedented degree of cross-talk, both regulatory interactions and mechanical interactions. Microtubule plus end-tracking proteins (or +TIPs) are a class of molecules that selectively localize to the tips of growing and shrinking microtubules. Since their discovery in 1999, +TIPs have been implicated in almost every microtubule-dependent cellular function including regulation of microtubule dynamic instability, organelle and chromosomal transport, assembly of the mitotic spindle, establishment of cellular polarity, and cell migration. In this proposal, we focus on +TIPs with a particular emphasis on actin-microtubule cross-talk as this represents a relatively unexplored functional interface between the two cytoskeletal networks. Our core hypothesis is that microtubule plus ends are dynamic platforms that deliver information to cortical regulatory networks governing cell shape and also act as sites of structural integration between actin and microtubules. We will test these ideas using novel assays we have developed with cultured Drosophila cell lines as this model system is amenable to high-resolution light microscopy, biochemical analyses, and gene inhibition using RNAi. The results of these studies will contribute to a basic understanding about the network of cellular components that mediate changes in cellular shape during processes such as morphogenesis and cell migration. The goal of this proposal is to understand the mechanistic basis of cellular morphogenesis and motility. The proper execution of cellular shape changes is essential for embryonic development - if they are not synchronized during development, or fail to occur at all, this can result in congenital birth defects. Like wise, cellular motility underlies processes such as wound healing and immune response. Improper cell motility is also an underlying cause of atherosclerosis, inflammation, and metastasis. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The microtubule cytoskeleton is a dynamic scaffold used to facilitate polarized intracellular transport, cell migration and formation of the mitotic spindle. Microtubules are polymers of a[unreadable]-tubulin heterodimers. The microtubule has inherent dynamics regulated by GTP hydrolysis in [unreadable]-tubulin's exchangeable nucleotide site. A host of microtubule associated proteins regulate the polymer's dynamics both spatially and temporally. Defects in microtubule dynamics result in a wide spectrum of diseases including, but not limited to neurodegeneration, spastic paraplegia and aneuploidy. TOG domains are tubulin binding domains found in two conserved protein families that regulate microtubule dynamics, defined by members XMAP215 and CLASP. Across both families, TOG domains are found arrayed;however XMAP215 and CLASP differentially regulate microtubule dynamics, promoting polymerization and pause respectively. The mechanism TOG domains use to bind tubulin and the role arrayed TOG domains play to modulate microtubule dynamics remains to be determined. This proposal develops the hypothesis that arrayed TOG domains provide multiple tubulin/microtubule binding sites and this multivalent architecture coupled with TOG class-specific determinants is central to the mechanism by which XMAP215 and CLASP differentially regulate microtubule dynamics. Three series of experiments examine the structure and function of TOG domains to determine a multi- resolution model for arrayed TOG mechanism. The first objective is to define, at atomic resolution, the structure and unique features of TOG domain classes across the XMAP215 and CLASP protein families using X-ray crystallography. The second objective is to ascertain the tubulin and microtubule binding capacity of individual and arrayed TOG domains and map tubulin binding determinants. This examination will use in vitro tubulin and microtubule binding assays as well as a F"rster resonance energy transfer assay to generate a model of the TOG-tubulin complex. The third objective is to determine the mechanism arrayed TOG domains use to modulate microtubule dynamics in vivo. This study will use live cell fluorescence imaging to monitor microtubule dynamics when the wild-type TOG protein has been depleted and replaced with a fluorescently- labeled truncated, mutant or chimeric construct. The long term objectives of this investigation are to determine at the atomic level, the mechanism arrayed TOG domains employ to modulate microtubule dynamics individually and in concert with other microtubule associated proteins. A fundamental understanding of TOG domain mechanism and how this is utilized in different protein families will enhance our understanding of microtubule dynamics and the role it plays in human health and disease including various neuropathies, ciliopathies, aneuploidy and cancer. PUBLIC HEALTH RELEVANCE: The microtubule cytoskeleton is a dynamic, polarized cellular scaffold that facilitates intracellular transport, ciliogenesis, cell migration and mitosis;all processes that rely on the regulated dynamic instability of the microtubule polymer. Defects in microtubule dynamics manifest in a range of disorders that effect human health including spastic paraplegia, lissencephaly, ciliopathies and aneuploidy;at the same time, microtubule dynamics is a key target for chemotherapeutics as inhibition of microtubule dynamics, and thus mitosis, is a means to prevent the rapid cell division associated with cancer. The proposed research aims to examine the molecular mechanism of TOG domain-containing proteins and the role they play in modulating microtubule dynamics;the research will impact public health by establishing a fundamental mechanistic framework under which healthy microtubule dynamics operates, from which aberrant manifestations can be properly examined and therapeutic strategies developed.

DESCRIPTION (provided by applicant): One of the key problems in developmental neuroscience is understanding how different types of neurons respond to external signals to establish synapses with their appropriate targets and to generate their specific axonal and dendritic architecture. The cytoskeletal network of actin filaments and microtubules acts as a scaffolding to allow navigating growth cones to alter their trajectories in response to attractive and repulsive guidance cues, to define the morphology of mature neurons, and to remodel in response to injury. For all of these processes to proceed normally, neurons must coordinate the dynamics of actin filaments and microtubules in a highly regulated manner. The goal of this proposal is to understand how neuronal cytoskeletal dynamics are regulated by studying the Drosophila spectraplakin Short stop (Shot). Flies lacking Shot exhibit axon outgrowth defects and CNS malformation. Spectraplakins are conserved among animal lineages and their mutation causes sensory neuron degeneration and developmental brain malformations in mice and neurodegenerative disease in humans. At the cellular level, spectraplakins act as cross-linkers to physically bridge actin and microtubules. Despite these essential functions in the nervous system, we know very little about how spectraplakins are regulated at the molecular level. In this proposal, we will use Shot as a paradigm to ask how spectraplakins are regulated using cutting-edge single molecule visualization techniques. 1) We will establish an assay for visualizing Shot actin-microtubule cross-linking at single molecule resolution. 2) We will test the hypothesis that Shot is regulated by an intramolecular inhibition mechanism. Given the high degree of conservation among members of this family of proteins, we anticipate that these regulatory mechanisms will be conserved with vertebrate spectraplakins, as well.

DESCRIPTION (provided by applicant): Heterotrimeric G proteins are important bi-molecular switches that transmit signals from a diverse array of membrane receptors for neurotransmitters, hormones and extracellular morphogens to activate downstream intracellular signaling pathways. Members of the G?12/13 family are particularly important during embryonic development as they are essential for the development of the circulatory system and are required for embryonic angiogenesis. The goal of this proposal is to understand how Ric-8 participates in signaling in the G?12/13 pathway. We will use the Drosophila gastrulation pathway as a model system to study Ric-8 to take advantage of the powerful cell biological tools and rich understanding of the G?12/13 signaling pathway in flies. In this proposal we will 1) Conduct a structure-function study of Ric-8 within the context of G?12/13 signaling; and 2) Identify novel proteins that are required for regulating Ric-8 activity in Drosophila. Given the hih degree of conservation of these pathways between flies and humans, these studies will provide understanding of the regulation of G?12/13 protein signaling during vascular development and will identify novel molecules with potential therapeutic importance in the regulation of angiogenesis.

This project will improve the security, preservation, and accessibility of the Carnegie Museum of Natural History Herpetological Collection, a world-class resource of over 235,000 specimens. This collection includes large historical holdings from areas that have undergone drastic environmental change or may face important change in the future. Recent research citing the collection exemplifies how new and unpredicted scientific paradigms, environmental issues, and technological advances require the existence of extensive and accessible historical scientific collections. Genetics, molecular phylogenetics, museomics, biomechanics, global change, emerging diseases, and invasive species are research areas for which specimens from this collection have recently been used. Based on recommendations from recent assessments that identified a number of urgent challenges affecting the collection and its related data, Carnegie Museum of Natural History has developed an extensive plan to preserve and increase access to the collection, ensuring that this important resource is sustained and able to support research for generations to come.

Ranking tenth among US collections of its kind, the Herpetology Collection at Carnegie Museum of Natural History spans more than 100 years of scientific collecting in 170 countries. It includes 148 holotypes and 2,007 paratypes (specimens of special scientific and historic significance used in the original species descriptions), specimens of six extinct and 78 critically endangered species, and one of the world's largest turtle collections. Through this award, Carnegie Museum of Natural History will: a) optimize storage space to improve curation and access and mitigate preservation risks due to overcrowding and suboptimal containers; b) digitize vouchered archival data related to specific specimens, providing vital context for the collection; c) accession and catalog ca. 12,000 specimens, georeference locality data, and make data and images of holotypes and paratypes digitally accessible; d) update taxonomy throughout the Section and reorganize the collection accordingly; e) enhance the public's appreciation of the value of collections and research by developing new collections-based programs and exhibits. Ensuring the long-term preservation and availability of the Section's specimens and related data is key for supporting future research efforts. Likewise, digitizing important archival records will expand the relevance of the collection for studies in ecology, systematics, conservation, and the history of natural history collections. Updating taxonomy and georeferencing additional records will enhance the precision of biodiversity data, improving usefulness to the global research community. In addition, collaborative planning will lead to the implementation of new educational programming and two new museum exhibits designed to enhance the public's appreciation of the value of collections for research and increase understanding of the collection's importance in the public's day-to-day life. Project results are available on line (www.carnegiemnh.org/projects/alcohol-house), and data will be shared and made available through iDigBio (www.idigbio.org), VertNet, and the Global Biodiversity Information Facility (GBIF).

This REU Site award to The University of North Carolina located in Chapel Hill, NC, will support the training of 10 students for 10 weeks during the summers of 2016 - 2018.

The REU Program in Molecular Biosciences at UNC-Chapel Hill provides students the opportunity to carry out independent research projects under the guidance of faculty mentors in biochemistry, microbiology, cell biology, genetics, and structural biology. Students work side-by-side with graduate students, postdoctoral fellows and faculty who serve as role models and mentors. Participants discover how modern biological research is formulated, carried out, and reported in an environment that stresses collaboration and interdisciplinary approaches to problem solving. Students acquire skills critical to success in research and teaching careers and to gain entry to competitive graduate programs. Making informed career choices is a major theme of the Program; participants hear about careers and lifestyles in forums with scientists inside and outside of academia and with current graduate students and post-doctoral fellows at UNC. The journal club and seminar program expose students to emerging areas of research and provide insight into the scientific method. The program culminates with all students presenting their work at an annual Summer Research Symposium.

It is anticipated that a total of 30 students, primarily from schools with limited research opportunities, will be trained in the program. Preference is given to students with little prior independent research experience, however all talented undergraduates with a genuine desire to pursue a career in biological research and teaching are encouraged to apply. First generation college students and students from groups under-represented in the sciences are strongly encouraged to apply.

A common web-based assessment tool used by all REU programs funded by the Division of Biological Infrastructure (Directorate for Biological Sciences) will be used to determine the effectiveness of the training program. Students will be tracked after the program in order to determine student career paths. Students will be asked to respond to an automatic email sent via the NSF reporting system. Program information and application instructions are available at http://www.med.unc.edu/oge/stad/sure . For questions contact the PI (Dr. Tom Kawula at kawula@med.unc.edu).