James E. Bear, Ph.D. - US grants

protein protein interaction; cytoskeletal proteins; guanosinetriphosphatases; protein structure function; actins; cell motility; protein biosynthesis; yeast two hybrid system; microinjections; laboratory mouse; genetically modified animals; tissue /cell culture;

[unreadable] DESCRIPTION (provided by applicant): This is a proposal to upgrade our one- and multi-photon laser scanning microscope system that is based on our Zeiss 510 NLO system. The upgrade consists of 1) the Chameleon, an easily use femtosecond pulse Ti:Sapphire laser to replace our aging and difficult to use Ar pumped Mira laser; 2) Zeiss Physiology software for the 510 system that allows more sophisticated image processing and analysis; 3) workstation software to duplicate the 510 analysis functionality on standalone computers so that our heavily used 510 system can be used for collecting data. Our Zeiss 510 system is in a heavily used multi-user facility that provides image acquisition and analysis for a wide variety of one- and multi-photon excitation projects. These upgrades are sought 1) to improve the reliability and ease of use of multi-photon excitation so that an average user can work independently; 2) greatly increase the speed of changing wavelengths of multi-photon excitation to allow novel experimental approaches; 3) provide increased software functionality on the 510 system; and 4) to make analysis functions available "off-line" so that the 510 system can be dedicated to data acquisition. Our facility includes two other laser scanning confocal microscopes, a Zeiss 410 and a BioRad 600, and a spinning disk confocal microscope, from Atto Instruments. Our microscope systems are used for a variety of projects by investigators from several laboratories. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cell migration is essential for many physiological processes such as wound healing, immune response and morphogenesis, and plays a significant role in pathological processes such as cancer metastasis. In order for cells to migrate, they must actively and coordinately remodel their actin cytoskeleton on a time scale of seconds. The control of actin dynamics requires dozens of proteins, but two critical components are the Arp2/3 complex that nucleates new filaments in branched arrays and ADF/Cofilin proteins that promote actin filament turnover by severing and enhanced depolymerization. It has long been suspected that these two factors function in a coordinated manner, but the mechanism of this coordination was unknown. Recent data from our lab indicate that Coronins, highly conserved WD-repeat proteins, have a unique function as coordinators of Arp2/3 and Cofilin activity at the leading edge of motile cells. This proposal seeks to elucidate the mechanisms of this coordination and determine how this activity contributes to cell migration and cancer metastasis. To achieve this, we propose four specific aims: 1) The regulation of Arp2/3 function by Coronin 1B;2) The regulation of Cofilin activity by Coronin 1B via the Slingshot phosphatases;3) The spatial and temporal control of Coronin 1B activity by Ser2 phosphorylation;and 4) The comparative analysis of Coronin 1B and Coronin 1C regulation and function.

DESCRIPTION (provided by applicant): Cells in a variety of contexts migrate towards soluble chemical cues in a process known as chemotaxis. Despite nearly a century of study, the mechanistic underpinnings of chemotaxis remain incompletely understood. Spatial gradients of growth factors direct the movements of mesenchymal cells in tissues to coordinate and accelerate physiologically important processes such as wound healing, and mesenchymal chemotaxis has been implicated in pathological conditions such as cardiovascular and fibrotic diseases. Yet, the vast majority of chemotaxis studies have focused on leukocytes and other fast-moving, amoeboid cells. Mesenchymal chemotaxis has been prohibitively difficult to study, because it requires maintenance of stable gradients for many hours. Traditional methods such as transwell assays provide little or no dynamic information and poorly discriminate effects on the efficiency of motility from actual directional sensing. To overcome these technical limitations we recently established a microfluidic chemotaxis assay that allows direct observation of mesenchymal cells in stable, linear gradients over many hours, allowing both single-cell tracking and high-resolution live-cell imaging approaches such as TIRF microscopy. Our preliminary data indicate that the growth factor receptor, PDGF-R controls mesenchymal chemotaxis by a PLC > PKC > Myosin II pathway and requires the coordination of signaling events and cytoskeletal organization. We propose to elucidate the mechanisms of mesenchymal chemotaxis by 1) Dissecting the spatio- temporal nature of chemotactic signaling in mesenchymal cells 2) Understanding the dynamic organization of the cytoskeleton during chemotaxis in this cell type and 3) Delineating the coordination of signaling and cytoskeletal events that lead to complex chemotactic behaviors such as re-orientation to new cues and chemotaxis in 3D environments. These studies will directly contribute to our understanding the physiological basis of disease states such tumor metastasis, fibrosis and cardiovascular disease, as well as our understanding of physiological processes such as wound healing.

DESCRIPTION (provided by applicant): The proper regulation of actin dynamics is essential for many biological processes such as wound healing, immune response and morphogenesis, and plays a significant role in pathological processes such as cancer metastasis and cardiovascular disease, the two leading causes of death in the developed world. Despite its widespread involvement in normal physiology and disease, efforts to target actin dynamics for therapeutic purposes are at an early stage and clearly require a deeper understanding of the processes involved. The Arp2/3 complex is a critical player in actin dynamics that generates branched actin arrays which are thought to be important for many cellular processes including cell migration, phagocytosis and cell adhesion. Using cells derived from a conditional Arp2/3 knockout mouse (Arpc2 gene), we propose to address several important questions for the field of actin dynamics: 1) How are Arp2/3-branched actin networks disassembled and dynamically turned over in cells? We have developed a new optogenetic method to control Arp2/3 function in cells with light that will allow us to dissect the de-branching pathway. 2) Is Arp2/3 required for actin-dependent processes such as directed migration, phagocytosis and cell-cell junction establishment? This will be addressed using a clean, genetic deletion approach in primary cells both ex vivo using live-cell imaging approaches and in vivo using multiphoton intravital imaging. 3) How do cells coordinate Arp2/3 and non-Arp2/3 actin pathways to produce optimal actin dynamics? Using our Arp2/3- deficient cells, we will interrogate the Arp2/3-independent pathways that partially compensate for its loss and study how Arp2/3-dependent and -independent pathway act in a coordinated pathway to produce optimal actin dynamics in cells.

ABSTRACT-Microscopy (MICRO) Shared Resource The Microscopy Shared Resource (MICRO) is a CCSG basic shared resource that combines the expertise and equipment from three different imaging modalities: light microscopy, intravital microscopy and electron microscopy. This combination gives LCCC members access to outstanding imaging capabilities. Visualization of the process of oncogenesis from tumors in whole organs, to cancer cells invading normal tissues, to transformed cells growing in culture and finally visualization of the macromolecules which lie at the heart of transformation provides a critical means of understanding, manipulating, and ultimately combating cancer. Having a broad range of imaging tools immediately available to the cancer research community at UNC within the LCCC is essential if the diverse research of the many cutting edge studies are to progress rapidly toward their goals in cancer research. This requires specialized techniques spanning a range of visualization that encompasses light and electron microscopy and dimensions of millimeters to nanometers. To provide this technology to the LCCC members, three highly specialized laboratories work together and each has an excellent history of accomplishments. The three components of the LCCC Microscopy SR consist of: The Microscopy Services Laboratory (MSL) which stands as the main supplier of widefield, confocal, and live cell imaging plus image analysis and morphometry. The Intravital Imaging SR provides a unique set of microscopes and expertise for imaging cells, tissues and organs within a living animal. The Electron Microscopy SR provides specialized imaging techniques for macromolecular imaging that have been developed by the SR director and are used world wide. Taken together, these three SRs provide a powerful tool for our cancer researchers. Future directions include expansion of light microscopy into the realm of super-resolution imaging and establishment of new methods for tagging specific proteins such that they can be localized in cells by high resolution thin sectioning.

Abstract: Cell motility is arguably the oldest problem in cell biology as the movement of cells was one of the first things noted when the microscope was developed in the 17th century. The last sixty years of work revealed that the molecular underpinnings of motility involve the active control of the cytoskeleton. However, many questions remain unanswered. While we have identified many of the components of the cytoskeleton and know a great deal about their biochemical and structural characteristics, we lack a systematic understanding of how the parts interact to produce coordinated cytoskeletal function such as during cell migration. Perhaps the most important problem in cell motility is understanding how cells perceive various cues in their environment and convert this information into a directed migration response. A deeper understanding of these two inter-related problems will inform higher order biological processes such as embryogenesis, immune response and wound healing, as well as diseases such as cancer metastasis. In 2012, our published a watershed paper for our research program that has shaped the course of our work ever since (Wu et al, Cell). In this paper, we discovered that mammalian fibroblasts could be nearly completely depleted of Arp2/3 by RNAi without significantly compromising viability. This allowed us to go the heart of the problem and study the function of the Arp2/3 complex directly. Using these cells and microfluidic devices to produce long-lasting gradients, we made the surprising observation that cells did not need the Arp2/3 complex for chemotaxis, but absolutely required it for haptotaxis. Inspired by our success with RNAi, we shifted to a conditional knockout mouse model where the gene encoding the critical Arpc2 (p34) subunit of Arp2/3 could be deleted on command. Using these tools, we have pursued two goals: 1) Investigate how cells can build an actin cytoskeleton without the Arp2/3 complex (funded by RO1 GM111557) and 2) Interrogate the molecular mechanisms of directed cell migration of mesenchymal cell and other cell types (funded by RO1 GM110155). Building on our progress in the last five years, we propose to extend our work in these two areas by defining the roles of Arp2/3 and non-Arp2/3 actin networks in contributing to cell behaviors such as directed migration towards chemical (chemotaxis), substrate- bound (haptotaxis) and mechanical (durotaxis) cues.

ABSTRACT: CANCER CELL BIOLOGY PROGRAM The overarching goals of the Cancer Cell Biology (CCB) Program are: (i) to understand, at the molecular and cellular levels, mechanisms underlying tumor initiation, progression, metastasis and resistance to therapeutic treatment, and (ii) to identify and validate new targets for cancer therapy. Insight derived from these studies, when integrated with research and development from other programs, will provide targets and guidance for the development of strategies for therapeutic intervention of cancer. Toward these two goals, the Program faculty investigate various aspects of cancer cell biology, including growth factors and receptors; angiogenesis and vascular biology; apoptosis; cell cycle regulation; chromatin biochemistry and transcriptional regulation; cell microstructure and function; DNA replication and repair; metabolism; regulatory RNA; and signal transduction. Led by two co-leaders with complementary expertise, Yue Xiong and James Bear, the program organizes these different areas into four major research aims: (1) Cell Cycle, (2) Cell Signaling, (3) Cell Movement and Organization, and (4) Chromatin Biology. The major emphasis of the Program is to foster integrated research that spans these inter-related themes, enhancing the research and translational capabilities of program investigators through the establishment, expansion and utilization of appropriate core facilities, and promoting interactions with investigators from other LCCC basic, clinical and population sciences programs. CCB has made concerted and focused efforts to improve translational output, by fostering inter-programmatic collaboration directed towards translation of basic science discoveries, engaging in entrepreneurship and brining small molecule inhibitors to pre-clinical and clinical trials. The research of CCB addresses fundamental biology that applies to all cancers but has a particular impact on cancers relevant to our catchment area such as multiple myeloma, lung cancer and melanoma. In addition, members of CCB have been active in community outreach and engagement activities such as advising state legislators on e-cigarette regulation. Finally, the members of CCB are fully committed to education, training and mentoring at levels ranging from middle schoolers through junior faculty at our own institution. The Cancer Cell Biology Program consists of 43 members who are associated with 12 basic science and 3 clinical departments at UNC-Chapel Hill and affiliated institutions. During the last funding period, program members published 740 cancer-related articles. Of these, 23% were inter-programmatic and 10% were intra- programmatic (31% collaborative). In 2019, our program members held grants totaling $18.2M (direct cost) in cancer-relevant extramural funding, including $3.5M (direct costs) from the NCI and $13.6M other peer.