Bacterial Type 3 Secretion Systems: High-Throughput 3D Single-Molecule Tracking of Sorting Platform Proteins in Live Cells

Sunday, February 12, 2017 728-Pos Board B493 Diffusion of DNA-Binding Species in Thenucleus: A Transient Anomalous Subdiffusion Model Michael J. Saxton. Biochemistry & Molec Med, University of California, Davis, CA, USA. Recent single-particle tracking experiments have measured the distribution of dwell times of DNA-binding species diffusing in living cells: CRISPR-Cas9, TetR, and LacI [Knight, Science 2015; Normanno, Nat Commun 2015; Caccianini, Faraday Disc 2015]. The observed distribution, a truncated power law, implies transient anomalous subdiffusion, in which diffusion is anomalous at short times (mean-square displacement proportional to t^a, a < 1) and normal at long times (MSD proportional to t) [Saxton, Biophys J 66 (1994), 70 (1996), 92 (2007)]. Monte Carlo simulations are used to characterize the time-dependent diffusion coefficient D(t) in terms of the exponent a, the crossover time, D(0), and D(N), and these quantities in terms of the dwell time distribution. The simplest interpretation is that the dwell time is an actual binding time to DNA. One alternative interpretation is that the dwell time is the period of 1D diffusion on DNA in the standard model combining 1D and 3D search. The model has several implications for cell biophysics. (1) The initial anomalous regime represents the search of the DNA-binding species for its target DNA sequence. (2) Non-target DNA sites have a significant effect on search kinetics. False positives in bioinformatic searches are potentially rate-determining in vivo. For simple binding, the search would be speeded if false-positive sequences were eliminated from the genome. (3) Both binding and obstruction affect diffusion. The proper controls for obstruction are GFP as a calibration standard among laboratories and cell types, and the DNA-binding species with the binding site inactivated. (4) Overexpression of the DNA binding species reduces anomalous subdiffusion because the deepest binding sites are occupied and unavailable. (5) The model provides a coarse-grained phenomenological description of diffusion of a DNA-binding species, useful in larger-scale modeling of kinetics and FRAP. (Supported in part by NIH grant GM038133). 729-Pos Board B494 Spatial Dynamics of SIRT1 Relates to Metabolic Transitions in the Cell Nucleus Suman Ranjit1, Lorena Aguilar-Arnal2,3, Chiara Stringari1,4, Paolo Sassone-Corsi2, Enrico Gratton1. 1 Biomedical Engineering, University of California Irvine, Irvine, CA, USA, 2 Center for Epigenetics and Metabolism, University of California Irvine, Irvine, CA, USA, 3Institute for Biomedical Research, UNAM, Mexico, Mexico, 4Laboratory for Optics and Biosciences, Ecole Polytechnique, Paris, France. SIRT1 is a NADþ -dependent deacetylase functioning as metabolic sensor of cellular energy and it adapts different biochemical pathways to the changes in the environment. SIRT1 substrates include histones and proteins related to enhancement of mitochondrial and antioxidant protection. Fluctuations in intracellular NADþ levels regulate SIRT1 activity, yet the exact pathway SIRT1 enzymatic activity impacts NADþ levels and its intracellular distribution remains unclear. Here, we demonstrate that SIRT1 determines the nuclear organization of protein bound NADH. Using multiphoton microscopy in live cells, we show that free and bound NADH are compartmentalized inside of the nucleus, and its subnuclear distribution depends on SIRT1. Importantly, SIRT6, a chromatin-bound deacetylase of the same class does not influence NADH nuclear localization. In addition, using fluorescence fluctuation spectroscopy, especially phasorFCS in single living cells, we reveal that NADþ metabolism in the nucleus is linked to subnuclear dynamics of active SIRT1. SIRT1 diffuses faster on the periphery of nucleus and the diffusion is slower in the center. Comparison of results from phasorFCS and autofluorescence FLIM divulge a relationship between NADþ metabolism, NADH distribution and SIRT1 activity in the nucleus of live cells, and leads off to decipher links between nuclear organization and metabolism. 730-Pos Board B495 Spatiotemporal Fluctuation Analysis: A Powerful Tool for the Future Nanoscopy of Dynamic Molecular Processes Francesco Cardarelli1, Enrico Gratton2, Fabio Beltram3, Carmine Di Rienzo3. 1 Nanomedicine, Center for Nanotechnology Innovation at NEST, Istituto Italiano di Tecnologia, Pisa, Italy, 2Department of Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California at Irvine, California, US, Irvine, CA, USA, 3Center for Nanotechnology Innovation at NEST, Istituto Italiano di Tecnologia; NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Pisa, Italy. A major challenge of present and future biophysics is to quantitatively study how biomolecules dynamically fulfill their physiological role in living cells, tis-

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sues, or entire organisms. In the last few decades, new experimental methodologies were introduced that are able to unveil details on a length scale that is a tiny fraction of the wavelength of light, thus moving spatial resolution far beyond the diffraction limit set by Ernst Abbe’s equation. Still, the enormous wealth of information available today from optical microscopy measurements on living samples is often underexploited. We argue that spatiotemporal fluorescence correlation spectroscopy (spFCS) can enhance the performances of current nanoscopy methods and provide further insight into dynamic molecular processes of high biological relevance (Di Rienzo, C. et al. Biophys J, 111, 679685, 2016). We present exemplary biological applications of spFCS to measure dynamic molecular parameters well below the diffraction limit in a standard optical setup, including the measurement of the nanoscale displacement of GFP in the cell cytoplasm (Di Rienzo, C. et al. Nat comm 5, n.5891, 2014), and of Transferrin Receptor-GFP (TfR-GFP) sub-diffraction confinement on the plasma membrane of live cells (Di Rienzo, C. et al. PNAS, 110 (30), 12307-12312, 2013). Also we discuss how standard super-resolution methods, which are intrinsically endowed with high static spatial resolution properties, can take advantage of the resolution improvements that are accessible through spFCS to describe dynamical molecular processes. Finally, we argue that by using spFCS we can definitely integrate the arsenal of methods at our disposal to investigate living matter at the nanoscale. 731-Pos Board B496 Bacterial Type 3 Secretion Systems: High-Throughput 3D Single-Molecule Tracking of Sorting Platform Proteins in Live Cells Julian Rocha1, Andreas Diepold2, Judith P. Armitage2, Andreas Gahlmann3. 1 Department of Chemistry, University of Virginia, Charlottesville, VA, USA, 2 Department of Biochemistry, University of Oxford, Oxford, United Kingdom, 3Departments of Chemistry and Molecular Physiology & Biological Physics, University of Virginia, Charlottesville, VA, USA. Bacterial secretion systems are large biomolecular assemblies that rely on static and transient interactions between individual molecular subunits. A central example is the Type 3 Secretion System (T3SS) which consists of both the static membrane-embedded needle complex and the much more dynamic cytoplasmic sorting platform. Single-subunit turnover in the sorting platform and the resulting structural heterogeneity have made it challenging to decipher the molecular-level mechanism of Type 3 secretion. Live-cell single-molecule super-resolution microscopy is ideally suited to measure spatial locations and trajectories of individual molecular subunits with nanoscale precision. Extracting meaningful biological results, however, requires characterizing the entire distribution of molecular behaviors, which in turn, necessitates a large number of individual measurements. Here, we apply high-throughput aberration-corrected 3D single-molecule localization microscopy to quantitatively measure the diffusion behaviors of over 100,000 individual T3SS sorting platform proteins. The single-molecule trajectories reveal multiple diffusive populations in the bacterial cytoplasm suggesting the pre-formation of functionally important higher-order molecular complexes. By providing information on the spatiotemporal regulation of protein function in living cells, our results complement recent structural and biochemical findings that the cytoplasmic T3SS sorting platforms contain large pod-like structures and that cytoplasmic C-ring proteins may pre-assemble into oligomeric complexes prior to binding to the T3SS sorting platforms. 732-Pos Board B497 Molecular Tattoo: Subcellular Confinement of Drug Effects In Vivo with Two-Photon Microscopy ´ . Rauscher2, La´szlo´ Ve´gner1, Bogla´rka Va´rkuti1, Miklo´s Ke´piro´1, Anna A ´ da´m I. Horva´th1, ´ ron Zsigmond1, Vanda Imrich1, Szilvia Ra´ti1, A A Ma´te´ Varga3, Miklo´s S. Kellermayer4, Malnasi-Csizmadia Andras1. 1 Department of Biochemistry, MTA-ELTE Molecular Biophysics Research Group, Budapest, Hungary, 2Printnet Ltd., Budapest, Hungary, 3Department of Genetics, Eo¨tvo¨s University, Budapest, Hungary, 4Department of Biophysics and Radiation Biology, MTA-SE Molecular Biophysics Research Group, Budapest, Hungary. Technological resources for sustained local control of molecular effects within organs, cells, or subcellular regions are currently unavailable, even though such technologies would be pivotal for unveiling the molecular actions underlying collective mechanisms of neuronal networks, signaling systems, complex machineries, and organism development. We present a novel optopharmacological technology named molecular tattooing, which combines photoaffinity labeling with two-photon microscopy.Moleculartattooing covalently attaches a photoreactive bioactive compound to its target by two-photon irradiation without any systemic effects outside the targeted area, thereby achieving subfemtoliter,long-term confinement of target-specific effects in vivo. As we demonstrated in melanoma cells and zebrafish embryos, molecular tattooing is