Bright and Photostable Fluorophores for Advanced Fluorescence Microscopy

Sunday, February 12, 2017 the viral transport from both the cargo and the ‘‘gate’’ (NPC) level in an unprecedented manner. 108-Plat Development of a Simultaneous Six-Color Fluorescence Microscope with Single-Molecule Sensitivity Jingyu Wang, Jamie Barnett, Luke Springall, Neil M. Kad. School of Biosciences, University of Kent, Canterbury, United Kingdom. Prokaryotic Nucleotide excision repair (NER) is a complex mechanism involving six proteins: UvrA, UvrB, UvrC, UvrD, DNA polymerase 1 and DNA ligase. To holistically study the kinetics of NER, multiple labelling and imaging channels are required to differentiate the parties involved in the damage search and repair activities. Using Quantum dot (Qdot)-conjugated UvrA, UvrB, UvrC, UvrD, DNA polymerase 1 and a Qdot labelled lesion we aim to detect and determine the significance of all the complexes that contribute to repair. For this purpose, we have adapted an existing fluorescence microscope into a multi-color simultaneous fluorescence microscope by spectrally separating 6 emission channels based on available Qdots: 525 nm, 565 nm, 585 nm, 625 nm, 655 nm and 705 nm. These six channels were created using parallel optical splitting devices distributed into two paths and imaged on two identical state-of-the-art Hamamatsu, ORCA-Flash4.0 V2 scientific CMOS cameras. Each low noise, high QE (>80%) camera sensor has 2048 x 2048 pixels, therefore every spectral channel has 682 x 2048 pixels; sufficiently large to image elongated DNA tightropes. Although the Qdots can be illuminated by a single 488 nm source, the fluorescence microscope also uses four excitation lasers: 405 nm, 488 nm, 561 nm and 637 nm combined into a single path. This allows for numerous combinations of channels to be excited and separately modulated using an Arduino-based control element linking the cameras to the lasers. Using this system, we present data obtained on the heterogeneity of the complexes formed during NER. 109-Plat Intracellular Delivery of Membrane Impermeable Photostable Fluorescent Probes into Living Cells for Super-Resolution Microscopy Yuji Ishitsuka1, Kai Wen Teng1, Pin Ren1, Yeoan Youn1, Xiang Deng2, Pinghua Ge1, Andrew Belmont2, Paul R. Selvin1. 1 Department of Physics, University of Illinois Urbana-Champaign, Urbana, IL, USA, 2Department of Cell and Developmental Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA. Specific labeling of the cellular target with fluorophore is one of the fundamental requirement in all fluorescence imaging techniques including single molecule and super-resolution microscopy techniques. While extracellular targets may be tagged with virtually any kind of probes, intracellular labeling of living cells is limited to the use of fluorescent proteins and limited selection of membrane permeable dyes. Here we show that pore forming proteins can be used to temporarily permeabilize the cells and allow delivery of various fluorescent probes, ranging from organic dyes (<1 kDa) to fluorescent immunoglobulin antibody (~150 kDa), for specific labeling of intracellular targets for live fluorescence cell imaging. We demonstrate that permeabilized cells can efficiently be recovered to carry on normal cellular processes shown by nuclear translocation of nanobody-labeled p65 proteins in response to chemical stimulation. One of the most photostable but membrane impermeable organic fluorophore, Atto 647N, has been delivered into cells to label actin fibers and kinesin dimers and to perform super-resolution fluorescence microscopy imaging dSTORM and single molecule tracking, respectively. This technique opens up a large number of stable, but otherwise membrane impermeable fluorescence labeling probes that are available for investigating transfected and endogenous intracellular targets. 110-Plat Bright and Photostable Fluorophores for Advanced Fluorescence Microscopy Qinsi Zheng1, Jonathan B. Grimm1, Anand K. Muthusamy1, Robert H. Singer1,2, Luke D. Lavis1. 1 Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA, 2Albert Einstein College of Medicine, New York, NY, USA. Advanced fluorescence microscopy, including single-molecule and superresolution imaging, demands bright and photostable fluorophores. We have recently reported a general approach to improve fluorophores brightness in living cells by substituting the N,N-dimethylamino groups found in classic dyes with four-membered azetidine rings (Nature Methods 12, 244250 (2015)). In an unpublished work we have synthesized new derivatives containing substituents on the azetidine ring. Using this approach we were able to fine tune the wavelength and fluorogenecity of the fluorophore without affecting brightness. Here, we report that several of these novel substituted-azetidine fluorophores, as well as the substituted-xanthene ones, exhibit substantial

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improvements in photostability in living cells. These fluorophores enable robust multi-color, wash-free imaging with a large photon budget. We are investigating their phototoxicity and mechanism in order to maximize the photostability, to generalize this approach to different fluorophores, and to apply these fluorophores to diverse biological settings, including living cells, tissues, and animals. We freely share our fluorophores with the academic community. This work is supported by HHMI and NIH (U01 EB 021236).

Platform: Membrane Protein Structures I 111-Plat Structure Inhibition and Regulation of a Two-Pore Channel TPC1 Alexander F. Kintzer, Robert M. Stroud. Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA. Membrane transport serves vital functions in the cell, providing nutrients for growth, relaying electrical signals, evading pathogens, and maintaining homeostasis. Voltage- and ligand-gated ion channels propagate cellular electrical signals by coupling changes in membrane potential and the binding of molecules to channel opening and selective passage of ions through the membrane barrier. Two-pore channels (TPCs) are intracellular ion channels that integrate changes in membrane potential, second messengers, and phosphorylation to control endolysosomal trafficking, autophagy, cellular ion and amino acid homeostasis, and ultra-long action potential-like signals. They broadly impact human diseases related to trafficking including filoviral infections, Parkinson’s disease, obesity, fatty liver disease, and Alzheimer’s disease. The response of TPCs to multiple cellular inputs suggests a multistate gating mechanism. Nevertheless, the mechanisms that govern cycles of activation and deactivation or ‘gating’ in the channel remain poorly understood. To understand the bases for ion permeation, channel activation, the location of voltage-sensing domains and regulatory ion-binding sites, and phosphoregulation, we determined the crystal structure of TPC1 from ˚ resolution. This reveals for the first time Arabidopsis thaliana to 2.87A how TPC channels assemble as ‘quasi-tetramers’ from two non-equivalent tandem pore-forming subunits. We determined sites of phosphorylation in the N-terminal and C-terminal domains that are positioned to allosterically modulate channel activation by cytoplasmic calcium. One of the two voltage sensing domains (VSD2) encodes voltage sensitivity and inhibition by luminal calcium locks VSD2 in a ‘resting’ conformation, distinct from the activated VSDs observed in structures of other voltage-gated ion channels. The structure shows how potent pharmacophore trans-Ned-19 allosterically acts to inhibit channel opening. In animals, trans-Ned-19 prevents infection by Ebola virus and Filoviruses by blocking fusion of the viral and endolysosomal membranes, thereby preventing delivery of their RNA into the host cytoplasm. The structure of TPC1 paves the way for understanding the complex function of these channels and may aid the development of antiviral compounds. 112-Plat Novel Mechanism of Gating in the TrkH-TrkA Complex Hanzhi Zhang1, Zhao Wang1,2, Mingqiang Rong1,3, Yaping Pan1, Wah Chiu1,2, Ming Zhou1. 1 Baylor College of Medicine, Houston, TX, USA, 2National Center for Macromolecular Imaging, Houston, TX, USA, 3Kunming Institute of Zoology, China Academic of Science, Kunming, China. The superfamily of Kþ transporters (SKT) is ubiquitous in bacteria, fungi and plants. SKT proteins are required for survival of bacteria in low Kþ conditions and are involved in salt regulation in fungi and plants. Bacterial SKTs have two components, a membrane embedded protein that resembles an ion channel and a cytosolic protein that regulates channel gating [1]. Crystal structures of two bacterial SKT systems were reported recently [2,3]. In both structures, the membrane embedded component forms a homodimer onto which a homotetrameric ring of the cytosolic protein docks. Single-channel activities of one of the complexes, the TrkH (membrane embedded) -TrkA (cytosolic) complex, were recorded and analyzed: ATP or ATP analogs such as AMPPNP activates the channel while ADP closes it [2]. The structure of the TrkH-TrkA complex is likely in a closed conformation because it was crystallized in the presence of NADH, a ligand that does not activates the channel. In order to understand how ATP or its analogs induces channel opening, we solved the structure of ˚ by x-ray crysthe TrkH-TrkA complex in the presence of AMPPNP to 3.29 A tallography. When compared to the previous structures, the new structure shows that each TrkA protomer binds to two AMPPNP molecules, and that the TrkA tetramer assumes an elongated conformation that likely induces a change in the TrkH. Conformational changes in the TrkH involve significant changes in the dimer interface and are different from any other channels of