Collective Live-Cell Superresolved Traces Reveal Nonaxonomal Dynamics of Intraflagellar Transport Particles at the Ciliary Base

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Tuesday, February 14, 2017

conductance, that is perceived by the nervous system, and we believe this translation occur through the action of mechanosensitive ion channels. Recently it has been demonstrated that eukaryotic mechanosensitive channels, just like their prokaryotic counterparts, directly sense force changes in the lipid bilayers, so now the important question is: how does membrane modulation couple to channel activity? To address this question we use purified mechanosensitive TREK-2 channels, which we reconstitute into planar lipid patches at a concentration that allow us to study channel activity at the single molecule level. We find that the reconstituted channels have a random orientation, and that we can clearly distinguish between the two possible orientations by analyzing electrophysiological characteristics as conductance and open dwell times. To investigate the sensitivity of the channels to mechanical modulation, we apply different degrees of negative or positive pressure to the patches, and analyze how such stimulations affect the activity of differently oriented channels. With this approach we can demonstrate that mechanical activation of TREK2 depends on the direction of the pressure pulse, i.e. one orientation is activated only by negative pressure while the other orientation is activated only by positive pressure. We carefully suggest that these results demonstrate that TREK-2 channels are sensitive to the direction of membrane curvature. Furthermore we are in the process of deducing a kinetic model that describes TREK-2 activation by pressure.

Platform: Optical Microscopy and SuperResolution Imaging: Applications to Cellular Molecules 1533-Plat Illuminating Bacterial Electrophysiology Giancarlo N. Bruni, Benjamin Dodd, Joel Kralj. Biofrontiers Institute, MCDB, University of Colorado Boulder, Boulder, CO, USA. The revelation that bacteria undergo rapid voltage transients sparked a potential union between the fields of microbiology and electrophysiology. Voltage transients in biology are canonically studied in neuronal and cardiac contexts, where a voltage depolarization induces calcium fluctuations. Using genetically encoded voltage and calcium indicators, we sought to uncover a physiological role for voltage depolarizations in E. coli. We found bacteria are similar to electrically excitable eukaryotic cells; voltage depolarization induces calcium influx, hinting at a potential signaling mechanism. Surprisingly, we found that changing the mechanical environment can trigger calcium influx and also alters gene expression. We hypothesize that, similar to sensory neurons, E. coli mechanosensation is mediated by a voltage-gated calcium channel. Further, we hypothesize that the calcium dynamics we observe mediate the correlated gene expression changes, akin to eukaryotic cells. Understanding how bacteria utilize voltage fluctuations to enact physiological change could lead to the development of novel antibiotics, and potentially give clues as to the origins of voltage as a signal in biology. 1534-Plat Understanding the Asymmetric MipZ Gradient in Caulobacter Crescentus Matthew D. Stilwell, Nikolai P. Radzinski, James C. Weisshaar, Douglas B. Weibel. University of Wisconsin-Madison, Madison, WI, USA. Caulobacter crescentus is a freshwater a-proteobacterium that divides into two asymmetric daughter cells. These curved, rod-shaped cells differ in their chromosome replication fates and in their cell length at division. C. crescentus divides into a replication-competent ‘stalked’ cell and a smaller, replicationquiescent ‘swarmer’ cell. The site of cell division is dictated by the polymerization of the eukaryotic tubulin homologue, FtsZ. MipZ is an essential ATPase and inhibitor of FtsZ polymerization. MipZ forms an intracellular bipolar gradient that directs FtsZ to the lowest concentration of MipZ, which is found slightly towards the new pole as opposed to the exact mid-cell. This localization bias leads to two differently sized daughter cells. To better understand how MipZ directs FtsZ to this off-center location, we employed singleparticle tracking photoactivated localization microscopy (sptPALM) to characterize the diffusive states and transition state kinetics of MipZ. We then used deterministic and stochastic simulations with these biophysical parameters to recreate the asymmetric MipZ gradient in silico. These simulations produce a distribution of cell lengths after cell division similar to that observed in vivo. These studies provide more insight into the asymmetric division of C. crescentus, but further questions remain regarding the nature of the asymmetric division.

1535-Plat A Novel DNA Binding Mode of H-NS Drives Chromosome Compaction and Gene Silencing in Single Bacterial Cells Linda J. Kenney. Mechanobiology Institute, National University of Singapore, Singapore, Singapore. Nucleoid-associated proteins (NAPS) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes acquired by horizontal gene transfer. We used genetics, single-particle tracking, super-resolution fluorescence microscopy, atomic force microscopy and molecular dynamics simulations to examine H-NS/DNA interactions in single bacterial cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization domain and the C-terminal DNA binding domain. Amino acids in the linker stabilize initial H-NS/DNA binding, facilitating polymerization of H-NS along DNA. In the absence of linker contacts, single particle tracking experiments indicated that H-NS binding was significantly reduced and in PALM images, the chromosome was de-condensed. In contrast to previous reports, H-NS was not localized to two distinct foci, rather it was scattered all around the nucleoid. Amino acids in the linker make DNA contacts that are required for both gene silencing and chromosome compaction, linking these two important functions. Supported by NIH-R21123640, VAIBX000372 and an RCE in Mechanobiology, NUS from the Ministry of Education, Singapore. 1536-Plat Investigating RNAP Search Dynamics in Live E. Coli Cells using Single Molecule and Statistical Methods Kelsey Bettridge1, Chris Bohrer1,2, Jie Xiao1. 1 Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD, USA, 2Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA. Transcription is an essential component of gene regulation and one of the most highly regulated processes in the cell. Because bacteria lack a nuclear envelope, genes transcribed by RNA polymerase (RNAP), the key enzyme of transcription, are essentially immediately translated into protein, leaving the kinetics of transcription significantly influencing the resulting protein expression timing and levels. However, while the kinetics of transcription is greatly studied in vitro, the in vivo dynamics of RNAP, including the typical search time and transcription time, are difficult to obtain. Our lab takes advantage of the sensitivity and fast time scale of single-molecule tracking to determine the kinetics of RNAP binding and transcription in live E. coli cells. We used a strain of E. coli in which the b’ subunit (rpoC) of RNAP is tagged with a photoactivatable fluorescent protein, PAmCherry, replacing the endogenous chromosomal copy. We discovered there are three states of RNAP with distinct diffusion coefficients. Using genetic manipulations and drug treatments, we were able to assign each diffusion state to a particular function of RNAP, i.e. RNAP that is bound to the DNA, RNAP that is undergoing rapid association and dissociation from the nucleoid, and freely diffusing RNAP. Further, using a Markov chain Monte Carlo algorithm, we were able to form a hidden Markov model of our data to parse out the dynamics of RNAP switching states, which enabled us to parse out a complete dynamical model of RNAP in living cells. We obtainined a wide variety of important parameters, including the non-specific dwell time, the typical promoter search time, and the typical transcription time for an average gene. These parameters will be useful for future gene regulations studies of all kinds, as well as to help understand general protein-nucleic acid interactions and dynamics within the crowded cytoplasm of a live cell. 1537-Plat Collective Live-Cell Superresolved Traces Reveal Nonaxonomal Dynamics of Intraflagellar Transport Particles at the Ciliary Base Tony Yang, Nguyet Thi, Minh Tran, Weng Man Chong, Jung-Chi Liao. Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan. The primary cilium is an essential organelle responsible for multiple sensory and signaling activities. Ciliogenesis is achieved by delivery of precursors such as tubulins along the axoneme through intraflagellar transport (IFT), which is mediated by molecular motors and various IFT particles. Distal appendages (DAPs) are known to serve as the recruiting site of IFT proteins. During ciliogenesis, IFT proteins must go through several different zones in cilia. One of missing links of the IFT dynamics is how IFT particles move between the DAPs and the ciliary axoneme. The major obstacle comes from the tiny volume surrounding the DAPs and TZ and the high density of IFT particles in this region, which is far smaller than the diffraction-limited

Tuesday, February 14, 2017 spot. Here we performed live-cell sptPALM-based superresolution tracking of short trajectories to demonstrate IFT particle dynamics at the ciliary base with the optimization of particle density and trajectory duration suitable for IFT motion speed. Our results revealed the DAPs and TZ accommodate not only axonemal but also transverse IFT88 movement. IFT particles move slower at the base than in the ciliary compartment. Moreover, diffusion analysis revealed that IFT particle movement was confined at the distal TZ while superdiffusive at the proximal TZ. This heterogeneous diffusion characteristics was likely attributed to a complex organization at the DAPs, spatially partitioned into some obstructed regions and some unhindered areas. Together, our live-cell superresolution studies revealed that IFT proteins adopt location-dependent stochastic paths in different regions of the ciliary base, with newly reported dynamic characteristics of IFT particles to shed light on the mechanisms of IFT particle traffic and gating facilitating ciliogenesis. 1538-Plat Real-Time Subcellular Localization Reveals Hidden Intraflagellar Transport Mechanisms Anthony Kovacs1, Jonathan Kessler1, Je-Luen Li2, HuaWen Lin3, Susan Dutcher1, Yan Mei Wang4,5. 1 Physics, Washington University in St. Louis, St. Louis, MO, USA, 2D. E. Shaw Research, New York, NY, USA, 3Genetics, Washington University in St. Louis, St. Louis, MO, USA, 4Physics, Princeton University, Princeton, NJ, USA, 5Physics, Washington University In St. Louis, St. Louis, NJ, USA. Subcellular localization, a process that determines the location of a protein at a specific time during its trajectory in cell, is essential for understanding protein function and mechanisms. Current localization techniques such as CLEM (correlative light-electron microscopy) and colocalization measurements, however, compromise on accuracy and/or precision due to their limited temporal and/or spatial resolutions. CLEM does not localize proteins at the time of imaging; rather a delayed time of at least seconds due to cell fixation. During this time, the proteins could have relocated. Colocalization measurements have a spatial resolution of at best 10 nm, rendering adjacent organelles/structures not differentiable. Here we report a real time subcellular localization method (Reticello) by using single-particle tracking measurements that can differentiate interfacing organelles/structures in live cells with millisecond resolution. The underlying principle of this method is as follows: on or in different organelles/structures in live cells, a protein is confined to different geometrical spaces or surfaces and therefore exhibit different movement patterns that can be predicted and tested. We demonstrate the Reticello method by revealing transient and unknown BBSome and kinesin-2 motor localizations in the 250 nm-wide C. reinhardtii flagella, differentiating among four interfacing structures. At the flagellar tip, these proteins reorganize by free diffusion for a mean of 2.2 sec on the membrane and 1.3 sec on the microtubules, respectively. After the reorganization, kinesin-2 motors perform retrograde travel by diffusion in the flagellar matrix. 1539-Plat Spatial Organization of Nuclear Structures by Dual Colour Super-Resolution Microscopy Maria J. Sarmento1, Lorenzo Scipioni1,2, Melody Di Bona1,3, Mario Faretta4, Laura Furia4, Gaetano I. Dellino4, Pier G. Pelicci4, Paolo Bianchini1,5, Alberto Diaspro1,3, Luca Lanzano`1. 1 Nanoscopy, Istituto Italiano di Tecnologia, Genoa, Italy, 2Department of Informatics, Bioengineering, Robotics and Systems Engineering, University of Genoa, Genoa, Italy, 3Department of Physics, University of Genoa, Genoa, Italy, 4Department of Experimental Oncology, European Institute of Oncology, Milan, Italy, 5Nikon Imaging Center, Istituto Italiano di Tecnologia, Genoa, Italy. The overall function of DNA in cell nuclei has long been related with different levels of chromatin organization. In fact, besides its primary sequence, proper chromatin structure and dynamics are required for healthy cell proliferation and maintenance. Errors in vital nuclear functions such as DNA replication and transcription, and the consequent DNA damage, have frequently been identified as the source of genomic instability responsible for numerous diseases, e.g., cancer development. Recently, new in vitro approaches have been developed that allow the genome mapping of these nuclear processes and the identification of substantial differences between health and disease. However, all genomic processes have also an underlying spatio-temporal organization within the cell nucleus that goes well beyond the pure linear sequence. Here, our goal was to detect and visualize different nuclear structures directly within the nuclei of intact cells. Upon immunostaining, the

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nanoscale spatial arrangement of these processes was then assessed by dual colour Stimulated Emission Depletion (STED) microscopy. More specifically, we used a novel approach recently introduced by our group (Separation of Photons by Lifetime Tuning, SPLIT)1 to achieve the nanoscale resolution required to image subnuclear structures. In this approach, the use of the phasor plot to represent the emission dynamics of both fluorophores allows for the proper spatial tuning of each signal dynamics as a function of their respective positions within the detection volume. In this way, we are able to increase the spatial resolution of two fluorophore signals simultaneously beyond the diffraction limit, ultimately leading to the proper spatial mapping of different structures within the cell nucleus at the nanoscale. [1] Lanzano` L. et al. (2015) Nat. Commun. 6: 6701-9 [Work partly funded by AIRC-Cariplo TRIDEO #17215] 1540-Plat Time-Resolved Single Cell, Sub-Cellular Compartmentalized Proteomics, Combining Precise Microfluidics, Deconvolution and Ultrasensitive SingleMolecule Microscopy Adam J.M. Wollman1, Sviatlana Shashkova2, Niek Welkenhuysen3, Erik G. Hedlund1, Stefan Hohmann3, Mark C. Leake1. 1 Physics and Biology, University of York, York, United Kingdom, 2 Department of Chemistry & Molecular Biology, University of Gothenburg, Gothenburg, Sweden, 3Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden. Optical microscopy is emerging as a powerful tool in quantified in vivo proteomics, allowing protein copy number determination in live cells and even sub-cellular compartments, but to date without dynamic information. Here, we have combined a novel microfluidics technology, Fluicell(1) with cell tracking and deconvolution software(2), to create a new high-throughput dynamic proteomics platform, capable of quantifying copy number changes in sub-cellular compartments in live cells in response to extracellular microenvironmental changes. We used a canonical signal transduction pathway in eukaryotic budding yeast cells as a test system to observe the localisation changes of the Mig1 transcription factor between nucleus and cytoplasm in response to extra-cellular glucose concentration changes. Combined with single-molecule Slimfield measurements of the dynamics of individual Mig1 molecular clusters, we find that clusters of Mig1 molecules translocate across the nuclear envelope and bind target genes, enabling a tuneable response to the glucose concentration signal. By combining microfluidics control of the input signal, with molecular quantification of the output on a single cell level, this new platform generates novel insight into the precise mechanisms of cellular processes. 1. A. Ainla, G. D. Jeffries, R. Brune, O. Orwar, A. Jesorka, A multifunctional pipette. Lab Chip. 12, 1255-1261 (2012). 2. A. J. M. Wollman, M. C. Leake, Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time. Faraday Discuss.184, 401-24 (2015).

Platform: Protein-Nucleic Acid Interactions II 1541-Plat Genomic RNA Binding Promotes Retroviral Gag Protein Interactions in an Assembly-Competent Conformation Leading to Selective Genome Packaging Ioulia Rouzina1, Shuohui Liu1, Erik D. Olson1, Tiffiny Rye-McCurdy1, Christiana Binkley1, Joshua-Paolo Reyes1, Leslie J. Parent2, Karin Musier-Forsyth1. 1 OSU, Columbus, OH, USA, 2Penn State College of Medicine, Hershey, PA, USA. In HIV-1 infected cells, full-length viral genomic RNA (gRNA) is selectively packaged by the HIV-1 Gag protein despite a vast excess of spliced viral and host RNAs. The mechanism of this selective packaging is incompletely understood but a region of gRNA known as ‘‘Psi’’ mediates specific Gag interaction. HIV-1 Gag binds Psi and non-Psi RNA with similar affinity under physiological salt concentration (~150 mM NaCl), but the salt dependence of these two binding events differs dramatically (Webb, JA, et al, RNA 2013). HIV-1 Gag binds Psi RNA with a strong non-electrostatic binding component and a small effective charge (Zeff ~ 5). In contrast, HIV-1 Gag binds non-Psi RNA with a very weak non-electrostatic binding component and a larger effective charge (Zeff ~ 9). In this work, we use salt-titration binding assays to study the effect of various Gag and Psi RNA mutations on the Psi and non-Psi binding interactions of HIV-1 and Rous sarcoma virus Gag proteins. Our findings are consistent with a model in which Gag binding to cognate Psi RNA shifts the