Grant-in-Aid of Research, Artistry and Scholarship Live cell imaging system for molecular trafficking and interaction studies PI: Do-Hyung Kim, Biochemistry, Molecular Biology and Biophysics, UMTC Abstract: High-resolution analysis of cellular structures and molecule processes in living

High-resolution analysis of cellular structures and molecule processes in living cells has been increasingly important in the current biological research. Many laboratories in the Molecular and Cellular Biology building carry out research relying on high-resolution analysis of cellular processes such as protein-protein/protein-DNA interaction, protein localization, and HIV/virus/membrane trafficking, and have had increasing demands on high-resolution live cell imaging systems. To that end, several laboratories have joined forces to identify the best system for our requirement and found the DeltaVision personalDV Live-Cell Imaging System as the best fit among 6 demonstrated systems. The instrument was determined not only based on its performance but also recommendation by cell and molecular biology experts. The DeltaVision would be a unique system of this kind in the University and therefore its installation would help researchers on the campus to perform fundamental researches of live cell analysis that can bring up new ideas and discoveries. Principal Investigator/Program Director: Kim, Do-Hyung Page 1 Live cell imaging system for molecular trafficking and interaction studies 1. Category Justification: This grant proposal (category #2) is to request funds for installation of the DeltaVision fluorescence microscope workstation, a high-resolution live cell imaging system, which will be shared by research laboratories in the Molecular and Cellular Biology building. 2. Present Status of Knowledge: Important biological discoveries are often driven by investigation of cellular and molecular events in living cells. Despite an increasing demand on live cell imaging, our University has not provided an appropriate facility for this direction. Given the circumstances, several laboratories in the BMBB department have joined forces to purchase the DeltaVision live-cell imaging system. After carefully considering the options between 6 demonstrated systems, we have concluded that the DeltaVision is the best fit for our needs. We have also consulted several cell biologists, including Drs. Tom Hope at Northwestern and Ulrich Mueller at the Scripps Research Institute. The DeltaVision system utilizes optical and computational techniques to quantitatively resolve localization in 3D at high resolution. In addition, the deconvolution microscopy is particularly well-suited to imaging live cells because acquisition is rapid and sensitive, thereby minimizing damaging phototoxicity. The microscope is highly photon efficient, up to hundreds of times more efficient than confocal systems, which enables it to outperform other live-cell imaging systems. In contrast to other systems, the Deltavision’s sophisticated constrained iterative algorithm enhances the image contrast while actually increasing the numerical accuracy of the image, the algorithm that has been quantitatively verified to accurately represent the original 3D object (Swedlow et al., 2002). The DeltaVision has strong features in the aspect of throughput analysis. Its nano-positioning technology allows for multiplexing multiple field-of-view collected to be virtually stationary although many different locations may have been visited between timepoints even at high resolutions. Thus, the system allows for increasing sample size and the number of experiments that can be run in parallel thus achieving high spatial resolution and large sample sizes. The DeltaVision provides quantitative data of images, enables advanced photokinetic experiments [FRAP, photo-activation, FRET] through the use of the optical Quantifiable laser module. The system is even considered superior to the laserscanning confocal microscopes at the Biomedical image processing lab to obtain high-resolution, quantitative fixed cell images. 3. Plan of Work: The Deltavision workstation will be used mainly by several investigators in BMBB (please see support letters) all working in the Molecular and Cellular Biology building. The main user group includes Ervasti, Harris, Sobeck, and Kim laboratories. Minor user group includes Bernlohr, Bielinsky, Potter, and Towle. Potential other user group includes Michel Sanders and Eric Hendrickson labs. The research plans of the user groups to use the DeltaVision are summarized below. Do-Hyung Kim lab (PI): My lab studies cellular signaling processes that regulate cell growth, survival, and metabolism in response to nutrient and growth factor signals (Kim et al., 2002, 2003; Vander Haar et al., 2007; Woo et al., 2007) (MMF, NIH, ADA, and DOD grants). My lab research highly depends on analyzing protein-protein interactions and membrane trafficking. The DeltaVision will benefit our two specific research topics detailed below. (1) Autophagy (cellular self-eating), a nutrient-regulated process through which faulty cell components and organells are degraded during stress or starvation, has a growing number of links to a variety of human disease and physiology including cancer, aging, neurodegeneration, and microbial infection (Cuervo et al., 2005; Ravikumar et al., 2004; Mizushima et al., 2008). Despite such wide spread appreciation for autophagy, the molecular mechanism underlying the Principal Investigator/Program Director: Kim, Do-Hyung Page 2 induction of autophagy remains unclear. Our recent study has revealed a key process of autophagy induction involving protein kinases mTOR and ULK1. In normal growth conditions, mTOR inhibits ULK1. When cells are deprived of nutrients, mTOR activity is reduced and no longer inhibits ULK1. Then, activated ULK1 triggers formation of autophagosomes, the membrane structures whose origins are unclear. It is crucial to analyze the autophagosome formation by spatial and temporal visualization of the processes in order to understand their origins and mechanisms of formation. We will utilize the DeltaVision to visualize protein localizational changes and membrane structural alteration during the autophagosome formation. This new approach will be important for us to renew our NIH R01 DK072004 grant. (2) Cellular responses to nutrient starvation involve changes in the DNA chromosomal structure and DNA-damage response pathway according to our recent study (manuscript in preparation). In particular, we found that double strand breaks occur and the proteins involved in DNA repair are recruited to the site of the DNA breaks upon nutrient deprivation. We are interested in understanding how DNA-damage-recognizing proteins such as p53bp1, H2AX, and Ku70/86 are recruited to the damaged sites in live cells upon nutrient starvation using the Deltavision system. The visualization of the live processes of the protein recruitment to DNA damage sites has never been examined. Therefore, our research will provide unique, novel insight into the mechanism of the recognition of the damaged DNA regions. This study, so far having been supported by the NIH R21 and DOD grants, will help us to obtain important data that will surely strengthen a resubmission of an NIH R01 grant next year. James Ervasti lab (Co-PI): While my laboratory is primarily focused on the molecular pathogenesis of muscular dystrophy (NIH grants AR042423 and AR 049899, MDA, Nash Avery Foundation and Charley’s Fund), we have generated several animal models that uniquely enable us to elucidate the role of cytoplasmic actin isoforms in the establishment and maintenance of polarized morphologies important to nervous system development and function. In particular, we have generated lines of mice that allow us to conditionally knockout the Actb and Actg1 alleles in any tissue (Sonnemann et al., 2006 Dev. Cell) and we are actively assessing how ablation of these important cytoskeletal elements affects neuronal morphology and migration as well as cochlear hair cell morphology and hearing. Importantly, our new studies have great potential to elucidate the pathomechanisms of progressive deafness and spinal muscular atrophy in humans. We are aggressively pursuing new extramural funding for this project but immediate access to the Deltavision system will allow us to acquire key preliminary data, which will enhance our chances of funding. The eukaryotic actin cytoskeleton is crucial for virtually all aspects of cell structure and function. The actin cytoskeleton is composed of six different actin isoforms including four muscle-specific isoforms and two ubiquitously expressed cytoplasmic isoforms, βcytoand γcytoactin. We have generated two new lines of mice in which either βcytoor γcyto-actin can be ablated in any tissue. We recently showed that muscle-specific γcyto-actin knockout mice present with a novel centronuclear myopathy (Sonneman et al., 2006 Dev. Cell) while ablation of γcyto-actin in hair cells results in a progressive deafness phenotype modeling that presented by humans with mutant γcyto-actin (Belyantseva et al., in preparation). Finally, preliminary studies of muscle-specific βcyto-actin knockout mice revealed a myopathy that may inform on the poorly understood pathomechansim of spinal muscular atrophy. To further elucidate the mechanism(s) by which βcytoand γcyto-actins function, we require access to high-resolution imaging technology so that we can spatially and temporally localize proteins in living cells. The Deltavision system will advance our investigation of actins in Spinal Muscle Atrophy and deafness by allowing us to dynamically image live βcytoor γcyto-actin knockout neurons to identify defects in motility and growth cone guidance and also provide optimal high-resolution imaging of cochlear hair cells, which are key to our mechanistic studies of progressive deafness associated actinopathy. While we are aggressively pursuing extramural funding for these new research Principal Investigator/Program Director: Kim, Do-Hyung Page 3 directions, our chances for success will be greatly improved with immediate access to the Deltavision so that we can acquire preliminary data showing feasibility of our