Equipment Supplement: Acquisition of Confocal Scan to Upgrade Existing Fluorescence Microscope

Research Strategy Currently, I am in year 3 of my R35 project (Mechanisms and functions of Shoc2-transduced cellular signals) which elucidates the mechanisms by which Shoc2 modulates signal transmission of the ERK1/2 pathway and regulates early embryonic development. This proposed administrative supplement for additional equipment will pertain to the experiments proposed in the goals 2 in which we characterize the endocytic machinery involved in the spatial and temporal transmission of signals via the Shoc2 complex and the goal 3 where we determine the physiological roles of the Shoc2-ERK1/2 axis in embryonic development. Specifically, I am requesting funds to upgrade a Marianas™ wide-field fluorescence microscope that was purchased 12 years ago using start-up funds provided to me when I joined the University of Kentucky. This microscope has been an excellent resource and allowed to validate interactions within new components of the Shoc2 scaffolding complex and many other experiments that needed fluorescence microscopy. The SDC scanner will be easily integrated with the existing microscope to allow acquisition of high-speed confocal images in real- time. The capabilities of SDC scanner will give us access to a technology that will enhance and improve detection of fast endocytic events in live cells and will allow imaging zebrafish larvae. SDC scanner will solve the limitations of the current system such as speed, photobleaching, and phototoxicity, and simply not being able to image thick samples of zebrafish embryonic tissue. Designed to image a wide variety of samples, this upgrade of our imaging system will permit extended time-lapse imaging of live specimens (dramatically reducing photobleaching and phototoxicity), will reduce background noise due to light scattering (because there is no illumination outside the plane of focus), allow for imaging of thick samples, and will allow imaging of samples with rapid data acquisition without the need for oversampling that is not possible with a current microscope. SDC scanner acquires full-frame high-speed confocal images in real time and scans approximately 1,000 points of laser light across the field, simultaneously resulting in much faster image production. When this unit is combined with a sensitive camera that registers the signal from a quarter million or million pixels simultaneously, the quantum efficiency of light collection reaches 95%. This significant speed difference combined with the superior sensitivity of a high-end camera, makes spinning disk confocal microscopy essential for the research in our current R35 project that requires advanced imaging techniques (1). The SDC scanner would greatly enhance the goal 2 of this MIRA (R35) project to establish mechanisms by which endocytic trafficking modulates the transmission of signals via the Shoc2 complex. In these experiments, we aim to identify the endocytic machinery that is involved in the recruitment of the Shoc2 complex to endosomes and decipher the mechanisms by which cellular trafficking controls the spatial distribution of the Shoc2 complexes. Recently, we identified that proteins in the CCC-Retriever-WASH complex (CCDC22, CCDC93, COMMD1, and SNX27) that are involved in the trafficking of many critical cargo (2) interact with Shoc2. Next, we will determine whether the CCC-WASH complex facilitates retrieval of the Shoc2 module from late endosomes and whether Shoc2 is recycled to the PM to be “reactivated” to support another wave of ERK1/2 signaling. Moreover, we need to define more precisely the spatiotemporal patterns of the Shoc2 module during its remodeling, the distribution, and composition of the Shoc2-CCC-WASH complexes in cells, and their role inactivation of the ERK1/2 pathway as this new information will yield important insights into how the CCC-WASH complex affects the remodeling of the Shoc2 scaffolding module and will achieve a clear understanding of how Shoc2-mediated ERK1/2 signals impact the assembly and function of the CCC-WASH complex itself. he CCC complex has recently emerged as an endosome-associated assembly involved in the sorting of more than fifteen essential cargo (i.e. LDL receptor, integrin α5β1, Notch2) (3-6), and a significant body of literature demonstrates that CCC proteins play key roles in early development (7-10). Need for the requested instrument: The SDC microscope is the perfect equipment for carrying out our experiments that require imaging of rapidly occurring endocytic events in live cells. To trace and quantify the distribution of fluorescently labeled endogenous proteins in various cellular compartments, we generated CRISPR/Cas9-mediated knock-in cells in which Shoc2 is endogenously labeled with tagGFP2. Unfortunately, due to the low copy number of Shoc2 proteins in cells, detection of the endogenously labeled Shoc2 is very challenging and leaves us without the model to study trafficking of endogenous Shoc2. We had previously tried to image these cells using our current microscope and never achieved the needed resolution or sensitivity of signal. Moreover, our microscope does not have the acquisition capability required to image the fast trafficking events that occur on the millisecond range. Adding the SDC scanner with its minimal photobleaching, low toxicity, and incredibly fast image acquisition camera will make a perfect setup for our experiments. Upgrading our existing system will allow sufficient time on the instrument as we proceed to study the roles of different proteins within CCC-WASH complex in the distribution of the Shoc2 complexes, and the regulation of the ERK1/2 signals in cells. In addition to the experiments outlined above, the SDC scanner would greatly enhance our studies in determining the physiological roles of the Shoc2-ERK1/2 axis and Shoc2-ERK1/2 signals in Noonan syndrome with loose anagen hair (NSLH) where we utilize our novel Shoc2 CRISPR/Cas9 zebrafish knockout model (Goal 3). We have already identified a previously unrecognized role of Shoc2 in the development of neural crest-derived organs and hematopoiesis (11). Importantly, our Shoc2 mutants exhibit many of the clinical hallmarks associated with human NSLH, further emphasizing a critical role for the Shoc2 scaffold in embryogenesis and underscore the advantage of using zebrafish for dissecting the developmental contribution of the individual regulatory components of the ERK1/2 pathway. Our recent exciting discoveries demonstrated the robust effect of Shoc2 on the development of the lymphatic system. We found that shoc2 null zebrafish larvae develop severe trunk and eye edema and display nearly complete loss of critical lymphatic vessels: parachordal lines at 3 dpf (not shown), thoracic duct at 5 days post fertilization (dpf), highlighting the importance of Shoc2 during early embryonic development. The lymphatic vessel network plays vital roles in tissue fluid homeostasis, intestinal lipid absorption and immune response (1,12). Thus, given that lymphatic vessel malfunction is associated with NSLH as well as the pathogenesis of many diseases including lymphedema, fibrosis, inflammation, and metastatic tumor dissemination, deciphering the role of Shoc2 in lymphatics will contribute to our understanding of molecular mechanisms underlying a number of pathologies. Our goal is to obtain detailed information as to whether Shoc2 signals control 1) the specification of cells toward lymphatic fate, 2) the sprouting of lymphatic endothelia cell progenitors and/or 3) guide migration of lymphatic cells via a systematic analysis of lymphatic defects in Shoc2 null zebrafish and human primary cells. All of the studies described above are critically dependent on confocal microscopy, and zebrafish are uniquely amenable for this method due to their small size and optical clarity. Need for the requested instrument. In the next phase of our studies to determine how Shoc2 affects developmental lymphangiogenesis, we will utilize zebrafish transgenic lines in which vasculature and endothelial cells (ECs) are fluorescently labelled. We have already generated a shoc2 null, Tg(mrc1a:eGFP), Tg(kdrl:mCherry) double-transgenic zebrafish in which lymphatic ECs are EGFP- positive and arterial ECs are mCherry-positive (13,14). This and other transgenic lines will be used to define the role of Shoc2 in the formation and assembly of craniofacial and trunk lymphatic networks in development. We will also use these zebrafish lines to 1) identify whether Shoc2’s function in ECs is cell autonomous, 2) define if Shoc2 is critical for EC specification, and 3) establish if Shoc2 mediates proliferation in venous-derived lymphatic ECs. Methods will include transplantation assays of ECs expressing WT and mutant shoc2, analysis of progenitor sprouting using live-imaging as well as the analysis of the expression levels of markers of lymphatic EC fate (i.e. Prox1, LYVE1, Sox18, etc.) using in situ hybridization (15-17). The microscope equipped with confocal scanner would be an ideal instrument to carry out the studies described above more efficiently. Importantly, we currently do not have a confocal microscope to perform fast, long-term time-lapse imaging without photobleaching and photo-damaging the specimen. The SDC microscope with its low photobleaching and photodamage is essential for long-term developmental studies of various transgenic reporter zebrafish lines used in our experiments. Moreover, montage capabilities of SlideBook software that operates our current system will enable fluorescence imaging of whole embryos as the samples are positioned, oriented and uniformly illuminated and imaged with a sensitive camera that will greatly minimize experimental variability and the time required for data acquisition. The capabilities SDC microscope for the accurate imaging are of a critical importance for the reliable systemic characterization of mutant phenotypes. In summary, acquisition of the SDC scanner will fully support our growing scientific requirements will permit time-lapse live-cell imaging of rapid endocytic events and will allow us to analyze mutant phenotypes in living embryos and larvae. The SDC microscope would greatly benefit our studies, by providing much needed equipment to decipher Shoc2’s involvement in the biology of congenital disease and the regulation of lineage differentiation. Even though SDC microscopy has been commercially available for many years, researchers at the University of Kentucky have not yet had the opportunity to take advantage of this capability as this technology is currently not available at the University of Kentucky. Thus, acquisition of the SDC scanner will enable cutting-edge imaging analyses. In addition, this system will also support the research programs of Dr. Rodgers (grant xx) with whom we have standing active collaboration. Members of Rodgers lab are active users of the current system. Rigor and Reproducibility: Acquisition of the proposed instruments will improve the consistency and precision in examining individual larvae thereby improving scientific rigor of the proposed experiments. Analysis using the SDC microscope will make our methods of assessment significantly better, improve reproducibility of our proposed work. The significantly reduced time required to analyze clutches of larvae will allow us to perform more biological and technical replicates for our studies, which will positively impact our scientific rigor and reproducibility.