2665-Pos Board B681 Super-Resolution Imaging of DNA Replisome Dynamics in Live Bacillus subtilis Yilai Li 1 , Jeremy W. Schroeder 2 , Yi Liao 3 , Ziyuan Chen 1 , Lyle A. Simmons 2 , Julie S. Biteen 3 . 1 Biophysics, University of Michigan, Ann Arbor, MI, USA, 2 Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, USA, 3 Chemistry, University of Michigan, Ann Arbor, MI, USA. DNA replication happens in all living organisms and assures that the genome is accurately copied and maintained. The replisome is the molecular machine in cells that replicates DNA, and this protein assembly includes DNA poly- merases which directly synthesize DNA by adding nucleotides. Although the bacterial replisome has been studied extensively in vitro, single-molecule microscopy is now providing a new perspective on the dynamics and archi- tecture of replisome components in vivo. Here we study the architecture and dynamics of several highly conserved replisome components in vivo in the model organism Bacillus subtilis. Photoactivated localization microscopy (PALM) and single-molecule tracking enable us to localize and track every single protein molecule with a resolution of 20 - 40 nm. In our study, we investigate the dynamics of a number of replisome components under different conditions, including the replicative DNA polymerases PolC and DnaE and the b-clamp loader DnaX. We study quantitatively the real-time behavior of different replisome components during the DNA synthesis pro- cess. Surprisingly, our investigations have revealed that all of these repli- some components are highly dynamic and exchange more rapidly than previously expected, and we characterize the molecular scale distribution of each replisome component as well as responses to cellular mutations and external stimuli with a combination of single-molecule tracking, time- lapse imaging, and spatiotemporal image correlation spectroscopy. Overall, these new insights into DNA replication indicate that the activities of bacte- rial replisomal proteins may be regulated in cells by coordinating and modu- lating the dynamics of protein recruitment, binding, and unbinding at the site of DNA synthesis. 2666-Pos Board B682 3D Architectural Reconstruction of Mammalian Centriole Distal Appendages using Superresolution Microscopy T Tony Yang 1 , Weng Man Chong 1 , Zhengmin Chen 1 , Meng-Fu Bryan Tsou 2 , Jung-Chi Liao 1 . 1 Academia Sinica, Taipei, Taiwan, 2 Memorial Sloan-Kettering Cancer Center, New York, NY, USA. The primary cilium is an essential microdomain of cells grown upon the mother centriole/basal body serving multiple sensory roles (e.g., smell, light, force) and mediating multiple signaling activities (e.g., hedgehog, Wnt, cAMP). Distal appendages (DAPs) are nanoscale, pinwheel-like structures protruding from the distal end of the centriole that mediate membrane dock- ing during ciliogenesis, marking the cilia base around the ciliary gate. Despite their functional importance, the detailed architecture of DAPs re- mains largely unknown. Here, we determined a superresolved multiplex of 16 centriole-distal-end components. Surprisingly, rather than pinwheels, intact DAPs exhibit a cone-shaped architecture with components filling the space between each pinwheel blade, a new structural element we termed the distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1, and CEP164 form the backbone of pinwheel blades, with CEP83 confined at the root and CEP164 extending to the tip near the membrane-docking site. By contrast, FBF1 marks the distal end of the DAM near the ciliary membrane. Strikingly, unlike CEP164 which is essential for ciliogenesis, FBF1 is required for ciliary gating of transmembrane proteins, revealing DAPs as an essential component of the ciliary gate. Our findings redefine both the structure and function of DAPs. Funding sources: Ministry of Science and Technology, Taiwan (Grant No. 103- 2112-M-001-039-MY3), Academia Sinica Career Development Award. 2667-Pos Board B683 In Situ Imaging of Spatial Organization of Accessible Chromatin at the Nanoscale with ATAC-see and Single-Molecule Super-Resolution Fluorescence Microscopy Maurice Y. Lee 1,2 , Xingqi Chen 3 , Anna-Karin Gustavsson 2 , Howard Y. Chang 3 , W.E. Moerner 2 . 1 Biophysics, Stanford University, Stanford, CA, USA, 2 Chemistry, Stanford University, Stanford, CA, USA, 3 Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, USA. The spatial organization of accessible chromatin in the nucleus tightly regu- lates DNA replication, DNA repair, and gene regulation. Recently, a new biochemical labeling technology for the sequencing and imaging of accessible chromatin was developed. This method, called assay of transposase- accessible chromatin with visualization (ATAC-see), uses the hyperactive Tn5 transposase to covalently insert fluorescently-labeled DNA sequencing adapters directly into the accessible genome. Here, we combined ATAC- see with our latest super-resolution fluorescence microscopy methods, which use long axial range point spread functions for fiducial localization, to chart out the 2D map of how accessible chromatin is spatially organized in the HeLa cell nucleus at the nanoscale. In addition, with multi-color super-reso- lution imaging, this map of accessible chromatin can be cross-referenced with the maps of other landmark proteins to gain a better understanding of how the many different players work together in concert to regulate gene expression in the human nucleus. 2668-Pos Board B684 Quantitative Super-Resolution Microscopy of Proteins at the Synaptic Level Silvia Scalisi 1,2 , Andrea Barberis 3 , Enrica Maria Petrini 3 , Alberto Diaspro 1,2 , Francesca Cella Zanacchi 1 . 1 Nanoscopy, Istituto Italiano Tecnologia, Genova, Italy, 2 Physics, University of Genoa, Genova, Italy, 3 Neuroscience and Brain Technologies, Istituto Italiano Tecnologia, Genova, Italy. Single-molecule localization (SML) techniques provide a powerful tool to answer biological questions requiring the observation of subcellular structures at the nanoscale. Quantitative single-molecule analysis allows quantifying the number and distribution of molecules in several biological systems beyond the diffraction limit [1]. In the last few years, many computational methods employ- ing clustering analysis algorithms have been developed to extract quantitative information from SML data sets. In neuroscience, quantitative SML has been applied to reveal density and spatial organization of synaptic proteins [2]. Recently, it has been reported that under plasticity conditions, chemically induced by long term potentiation (iLTP) of inhibitory synapses, GABAA receptors are immobilized and confined at synapses in cultured hippocam- pal neurons. iLTP expression relies on the recruitment and accumulation of the scaffold protein gephyrin at synaptic areas [3], thereby enhancing the clustering of synaptic GABAA receptors and potentiating GABAergic synaptic currents [4]. In our work we exploit super-resolution approaches (STORM) combined with clustering analysis to study the nanoscale distribution of the inhibitory postsynaptic scaffold, in particular to count GABAA receptors in close proximity to gephyrin nanodomains during iLTP. Furthermore we applied the quantitative SML approaches to study a neuronal protein complex (formed by Protocadherin19, Negr1, FGFR2 and NCAM) involved in neurodevelopmental autism spectrum disorder [5]. We used quan- titative STORM to highlight the role of Protocadherin19 in relation to the dis- tribution of the other proteins forming the complex. References: [1] Deschout H., Nature Methods. (2014), 11, 253. [2] Specht C.G., Neuron. (2013), 79, 308. [3] Pennacchietti F., Journal of Neuuroscience. (2017) 37, 1747. [4] Petrini E.M., Nature Communication. (2014) 5, 3921. [5] Pischedda F., Molecular & Cellular Proteomics (2014), 13, 733. 2669-Pos Board B685 Studying Protein Dynamics and Organization in Live Cell Membranes by Imaging FCS and SOFI/SRRF Analyses Xue Wen Ng 1,2 , George Barbastathis 2,3 , Thorsten Wohland 1,4 . 1 NUS Centre for Bioimaging Sciences and Department of Chemistry, National University of Singapore, Singapore, Singapore, 2 Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore, Singapore, 3 Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA, 4 Department of Biological Sciences, National University of Singapore, Singapore, Singapore. Super-resolution imaging based on localization of sparsely distributed fluores- cence image series has been instrumental over the past decade in understanding biological systems at superior spatial resolution. However, their applications are often limited to fixed cells due to difficulties in localization of mobile particles. Recent computational super-resolution techniques, namely super- resolution optical fluctuation imaging (SOFI) and super-resolution radial fluctuations (SRRF), had shown great promise to extract high spatial resolution images from living systems. SOFI performs higher-order temporal correlation analysis on the fluorescence fluctuations of fluorophores that are able to stochastically switch between a bright and dark state independently while SRRF conducts a spatial analysis on a diffraction-limited image stack to Tuesday, February 20, 2018 539a