70 results on '"Srigokul Upadhyayula"'
Search Results
2. Origin of wiring specificity in an olfactory map revealed by neuron type–specific, time-lapse imaging of dendrite targeting
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Kenneth Kin Lam Wong, Tongchao Li, Tian-Ming Fu, Gaoxiang Liu, Cheng Lyu, Sayeh Kohani, Qijing Xie, David J Luginbuhl, Srigokul Upadhyayula, Eric Betzig, and Liqun Luo
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neural circuit assembly ,olfactory system ,projection neurons ,dendrite targeting ,neuronal remodeling ,time-lapse imaging ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
How does wiring specificity of neural maps emerge during development? Formation of the adult Drosophila olfactory glomerular map begins with the patterning of projection neuron (PN) dendrites at the early pupal stage. To better understand the origin of wiring specificity of this map, we created genetic tools to systematically characterize dendrite patterning across development at PN type–specific resolution. We find that PNs use lineage and birth order combinatorially to build the initial dendritic map. Specifically, birth order directs dendrite targeting in rotating and binary manners for PNs of the anterodorsal and lateral lineages, respectively. Two-photon– and adaptive optical lattice light-sheet microscope–based time-lapse imaging reveals that PN dendrites initiate active targeting with direction-dependent branch stabilization on the timescale of seconds. Moreover, PNs that are used in both the larval and adult olfactory circuits prune their larval-specific dendrites and re-extend new dendrites simultaneously to facilitate timely olfactory map organization. Our work highlights the power and necessity of type-specific neuronal access and time-lapse imaging in identifying wiring mechanisms that underlie complex patterns of functional neural maps.
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- 2023
- Full Text
- View/download PDF
3. Design and Validation of a Human Brain Endothelial Microvessel-on-a-Chip Open Microfluidic Model Enabling Advanced Optical Imaging
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Mootaz M. Salman, Graham Marsh, Ilja Kusters, Matthieu Delincé, Giuseppe Di Caprio, Srigokul Upadhyayula, Giovanni de Nola, Ronan Hunt, Kazuka G. Ohashi, Taylor Gray, Fumitaka Shimizu, Yasuteru Sano, Takashi Kanda, Birgit Obermeier, and Tom Kirchhausen
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blood-brain barrier (BBB) ,capillary ,microvessel ,shear stress ,microfluidics ,live cell imaging ,Biotechnology ,TP248.13-248.65 - Abstract
We describe here the design and implementation of an in vitro microvascular open model system using human brain microvascular endothelial cells. The design has several advantages over other traditional closed microfluidic platforms: (1) it enables controlled unidirectional flow of media at physiological rates to support vascular function, (2) it allows for very small volumes which makes the device ideal for studies involving biotherapeutics, (3) it is amenable for multiple high resolution imaging modalities such as transmission electron microscopy (TEM), 3D live fluorescence imaging using traditional spinning disk confocal microscopy, and advanced lattice light sheet microscopy (LLSM). Importantly, we miniaturized the design, so it can fit within the physical constraints of LLSM, with the objective to study physiology in live cells at subcellular level. We validated barrier function of our brain microvessel-on-a-chip by measuring permeability of fluorescent dextran and a human monoclonal antibody. One potential application is to investigate mechanisms of transcytosis across the brain microvessel-like barrier of fluorescently-tagged biologics, viruses or nanoparticles.
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- 2020
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4. Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling
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Roberta Pascolutti, Veronica Algisi, Alexia Conte, Andrea Raimondi, Mithun Pasham, Srigokul Upadhyayula, Raphael Gaudin, Tanja Maritzen, Elisa Barbieri, Giusi Caldieri, Chiara Tordonato, Stefano Confalonieri, Stefano Freddi, Maria Grazia Malabarba, Elena Maspero, Simona Polo, Carlo Tacchetti, Volker Haucke, Tom Kirchhausen, Pier Paolo Di Fiore, and Sara Sigismund
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Biology (General) ,QH301-705.5 - Abstract
Summary: Adaptor protein 2 (AP2) is a major constituent of clathrin-coated pits (CCPs). Whether it is essential for all forms of clathrin-mediated endocytosis (CME) in mammalian cells is an open issue. Here, we demonstrate, by live TIRF microscopy, the existence of a subclass of relatively short-lived CCPs lacking AP2 under physiological, unperturbed conditions. This subclass is retained in AP2-knockout cells and is able to support the internalization of epidermal growth factor receptor (EGFR) but not of transferrin receptor (TfR). The AP2-independent internalization mechanism relies on the endocytic adaptors eps15, eps15L1, and epsin1. The absence of AP2 impairs the recycling of the EGFR to the cell surface, thereby augmenting its degradation. Accordingly, under conditions of AP2 ablation, we detected dampening of EGFR-dependent AKT signaling and cell migration, arguing that distinct classes of CCPs could provide specialized functions in regulating EGFR recycling and signaling. : EGFR signaling controls different cell physiological processes, including proliferation and migration. Pascolutti et al. describe an additional layer of regulation of EGFR signaling, relying on the sequestration of receptors into molecularly distinct clathrin-coated vesicles that regulate receptor fate toward recycling versus degradation, with impact on the final cellular output. Keywords: EGFR, endocytosis, clathrin-coated pits, AP2, eps15, epsin, endocytic adaptors, signaling, receptor degradation, recycling, transcription
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- 2019
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5. Lamellar projections in the endolymphatic sac act as a relief valve to regulate inner ear pressure
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Ian A Swinburne, Kishore R Mosaliganti, Srigokul Upadhyayula, Tsung-Li Liu, David G C Hildebrand, Tony Y -C Tsai, Anzhi Chen, Ebaa Al-Obeidi, Anna K Fass, Samir Malhotra, Florian Engert, Jeff W Lichtman, Tomas Kirchhausen, Eric Betzig, and Sean G Megason
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inner ear ,pressure ,timelapse ,lamella ,valve ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The inner ear is a fluid-filled closed-epithelial structure whose function requires maintenance of an internal hydrostatic pressure and fluid composition. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear whose function is unclear. ES defects can cause distended ear tissue, a pathology often seen in hearing and balance disorders. Using live imaging of zebrafish larvae, we reveal that the ES undergoes cycles of slow pressure-driven inflation followed by rapid deflation. Absence of these cycles in lmx1bb mutants leads to distended ear tissue. Using serial-section electron microscopy and adaptive optics lattice light-sheet microscopy, we find a pressure relief valve in the ES comprised of partially separated apical junctions and dynamic overlapping basal lamellae that separate under pressure to release fluid. We propose that this lmx1-dependent pressure relief valve is required to maintain fluid homeostasis in the inner ear and other fluid-filled cavities.
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- 2018
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6. Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding
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Manuel Alonso Y Adell, Simona M Migliano, Srigokul Upadhyayula, Yury S Bykov, Simon Sprenger, Mehrshad Pakdel, Georg F Vogel, Gloria Jih, Wesley Skillern, Reza Behrouzi, Markus Babst, Oliver Schmidt, Michael W Hess, John AG Briggs, Tomas Kirchhausen, and David Teis
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ESCRT ,endosomes ,membrane budding ,lattice light sheet microscopy ,3D tracking ,electron tomography ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3–45 s lifetimes, the ESCRT-III assemblies accumulated 75–200 Snf7 and 15–50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.
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- 2017
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7. Seipin is required for converting nascent to mature lipid droplets
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Huajin Wang, Michel Becuwe, Benjamin E Housden, Chandramohan Chitraju, Ashley J Porras, Morven M Graham, Xinran N Liu, Abdou Rachid Thiam, David B Savage, Anil K Agarwal, Abhimanyu Garg, Maria-Jesus Olarte, Qingqing Lin, Florian Fröhlich, Hans Kristian Hannibal-Bach, Srigokul Upadhyayula, Norbert Perrimon, Tomas Kirchhausen, Christer S Ejsing, Tobias C Walther, and Robert V Farese Jr
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lipid droplet ,seipin ,organelle biogenesis ,LiveDrop ,endoplasmic reticulum ,lipid metabolism ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation—the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.
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- 2016
- Full Text
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8. Image-to-Image Regression with Distribution-Free Uncertainty Quantification and Applications in Imaging.
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Anastasios N. Angelopoulos, Amit Pal Singh Kohli, Stephen Bates, Michael I. Jordan, Jitendra Malik, Thayer Alshaabi, Srigokul Upadhyayula, and Yaniv Romano
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- 2022
9. Adaptive wavelet distillation from neural networks through interpretations.
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Wooseok Ha, Chandan Singh, François Lanusse, Srigokul Upadhyayula, and Bin Yu 0001
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- 2021
10. Author response: Origin of wiring specificity in an olfactory map revealed by neuron type–specific, time-lapse imaging of dendrite targeting
- Author
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Kenneth Kin Lam Wong, Tongchao Li, Tian-Ming Fu, Gaoxiang Liu, Cheng Lyu, Sayeh Kohani, Qijing Xie, David J Luginbuhl, Srigokul Upadhyayula, Eric Betzig, and Liqun Luo
- Published
- 2023
11. Characterization, comparison, and optimization of lattice light sheets
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Gaoxiang Liu, Xiongtao Ruan, Daniel E. Milkie, Frederik Görlitz, Matthew Mueller, Wilmene Hercule, Alison Kililea, Eric Betzig, and Srigokul Upadhyayula
- Subjects
Multidisciplinary ,Bioengineering - Abstract
Lattice light sheet microscopy excels at the non-invasive imaging of three-dimensional (3D) dynamic processes at high spatiotemporal resolution within cells and developing embryos. Recently, several papers have called into question the performance of lattice light sheets relative to the Gaussian sheets most common in light sheet microscopy. Here we undertake a comprehensive theoretical and experimental analysis of various forms of light sheet microscopy which both demonstrates and explains why lattice light sheets provide significant improvements in resolution and photobleaching reduction. The analysis provides a procedure to select the correct light sheet for a desired experiment and specifies the processing that maximizes the use of all fluorescence generated within the light sheet excitation envelope for optimal resolution while minimizing image artifacts and photodamage. Development of a new type of “harmonic balanced” lattice light sheet is shown to improve performance at all spatial frequencies within its 3D resolution limits and maintains this performance over lengthened propagation distances allowing for expanded fields of view.Significance StatementDespite its rapidly growing use, several misconceptions remain concerning the physics of image formation and its optimization in light sheet microscopy, particularly in high resolution variants tailored for subcellular imaging. These include the role of excitation sidelobes, the significance of out-of-focus fluorescence, the importance and optimization of deconvolution, and the perceived advantages of Gaussian beams. Here we attempt to shatter these misconceptions by showing that the professed tradeoffs between axial resolution and background haze, photobleaching rate, phototoxicity, and propensity for image artifacts do not exist for well-crafted lattice light sheets whose data is acquired and processed rigorously. The framework we provide should enable others to optimize light sheets and extract the most information at the lowest cost in their experiments.
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- 2023
12. Origin of wiring specificity in an olfactory map: dendrite targeting of projection neurons
- Author
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Kenneth Kin Lam Wong, Tongchao Li, Tian-Ming Fu, Gaoxiang Liu, Cheng Lyu, Sayeh Kohani, Qijing Xie, David J Luginbuhl, Srigokul Upadhyayula, Eric Betzig, and Liqun Luo
- Abstract
How does wiring specificity of neural maps emerge during development? Formation of the adultDrosophilaolfactory glomerular map begins with patterning of projection neuron (PN) dendrites at the early pupal stage. To better understand the origin of wiring specificity of this map, we created genetic tools to systematically characterize dendrite patterning across development at PN type–specific resolution. We find that PNs use lineage and birth order combinatorially to build the initial dendritic map. Specifically, birth order directs dendrite targeting in rotating and binary manners for PNs of the anterodorsal and lateral lineages, respectively. Two-photon– and adaptive optical lattice light-sheet microscope–based time-lapse imaging reveals that PN dendrites initiate active targeting with direction-dependent branch stabilization on the timescale of seconds. Moreover, PNs that are used in both the larval and adult olfactory circuits prune their larval-specific dendrites and re-extend new dendrites simultaneously to facilitate timely olfactory map organization. Our work highlights the power and necessity of type-specific neuronal access and time-lapse imaging in identifying wiring mechanisms that underlie complex patterns of functional neural maps.
- Published
- 2022
13. Antimicrobial properties of biodegradable magnesium for next generation ureteral stent applications.
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Jaclyn Y. Lock, Milan Draganov, Andrew Whall, Shan Dhillon, Srigokul Upadhyayula, and Valentine I. Vullev
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- 2012
- Full Text
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14. Dynamic staining of Bacillus endospores with Thioflavin T.
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Srigokul Upadhyayula, Samuel Lam, Alice Ha, Harbani K. Malik-Chaudhry, and Valentine I. Vullev
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- 2012
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15. A highly homogeneous polymer composed of tetrahedron-like monomers for high-isotropy expansion microscopy
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Edward S. Boyden, Kiryl D. Piatkevich, Chih-Chieh Jay Yu, Linyi Gao, Ruixuan Gao, Rachael L. Neve, James B. Munro, and Srigokul Upadhyayula
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Male ,Materials science ,Microscope ,Polymers ,Biomedical Engineering ,Bioengineering ,Mice, Transgenic ,02 engineering and technology ,Herpesvirus 1, Human ,010402 general chemistry ,01 natural sciences ,Article ,law.invention ,Polyethylene Glycols ,law ,Microscopy ,Homogeneity (physics) ,Image Processing, Computer-Assisted ,Animals ,Humans ,General Materials Science ,Electrical and Electronic Engineering ,Composite material ,Nanoscopic scale ,chemistry.chemical_classification ,Isotropy ,Virion ,Brain ,Hydrogels ,Polymer ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,Atomic and Molecular Physics, and Optics ,0104 chemical sciences ,HEK293 Cells ,chemistry ,Self-healing hydrogels ,Tetrahedron ,Click Chemistry ,Female ,0210 nano-technology ,HeLa Cells - Abstract
Expansion microscopy (ExM) physically magnifies biological specimens to enable nanoscale-resolution imaging using conventional microscopes. Current ExM methods permeate specimens with free-radical-chain-growth-polymerized polyacrylate hydrogels, whose network structure limits the local isotropy of expansion as well as the preservation of morphology and shape at the nanoscale. Here we report that ExM is possible using hydrogels that have a more homogeneous network structure, assembled via non-radical terminal linking of tetrahedral monomers. As with earlier forms of ExM, such ‘tetra-gel’-embedded specimens can be iteratively expanded for greater physical magnification. Iterative tetra-gel expansion of herpes simplex virus type 1 (HSV-1) virions by ~10× in linear dimension results in a median spatial error of 9.2 nm for localizing the viral envelope layer, rather than 14.3 nm from earlier versions of ExM. Moreover, tetra-gel-based expansion better preserves the virion spherical shape. Thus, tetra-gels may support ExM with reduced spatial errors and improved local isotropy, pointing the way towards single-biomolecule accuracy ExM. Expansion microscopy, or ExM, physically expands biological specimens upon embedding them in swellable polymeric hydrogels, allowing nanoscale imaging using conventional microscopes. This work presents a diamond lattice polymeric hydrogel synthesized via click chemistry, which achieves higher homogeneity than the ones currently in use, allowing a more accurate sample expansion.
- Published
- 2021
16. A serotonergic axon-cilium synapse drives nuclear signaling to alter chromatin accessibility
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Shu-Hsien Sheu, Srigokul Upadhyayula, Vincent Dupuy, Song Pang, Fei Deng, Jinxia Wan, Deepika Walpita, H. Amalia Pasolli, Justin Houser, Silvia Sanchez-Martinez, Sebastian E. Brauchi, Sambashiva Banala, Melanie Freeman, C. Shan Xu, Tom Kirchhausen, Harald F. Hess, Luke Lavis, Yulong Li, Séverine Chaumont-Dubel, David E. Clapham, Janelia Research Campus [Ashburn] (HHMI Janelia), Howard Hughes Medical Institute (HHMI), Harvard Medical School [Boston] (HMS), Boston Children's Hospital, University of California [Berkeley] (UC Berkeley), University of California (UC), Chan Zuckerberg BioHub [San Francisco, CA], Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Peking University [Beijing], Universidad Austral de Chile, ANR-17-CE16-0010,Sero6Dev,Réseau de signalisation associé au récepteur 5-HT6 et développement neuronal(2017), Guerineau, Nathalie C., and Réseau de signalisation associé au récepteur 5-HT6 et développement neuronal - - Sero6Dev2017 - ANR-17-CE16-0010 - AAPG2017 - VALID
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Cell Nucleus ,pyramidal neurons ,[SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology ,[SDV.NEU.NB] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology ,FIB-SEM ,Hippocampus ,General Biochemistry, Genetics and Molecular Biology ,Axons ,Chromatin ,fluorescence lifetime imaging ,serotonin ,primary cilia ,chromatin accessibility ,GPCR signaling ,nuclear actin ,Synapses ,histone modification ,Cilia ,Signal Transduction - Abstract
International audience; Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.
- Published
- 2022
17. A serotonergic axon-cilium synapse drives nuclear signaling to maintain chromatin accessibility
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David E. Clapham, Srigokul Upadhyayula, C. Shan Xu, Andrew L. Lemire, Deepika Walpita, Justin Houser, Luke D. Lavis, Lihua Wang, Vincent Dupuy, Silvia Sanchez-Martinez, Song Pang, Sebastian Brauchi, Shu-Hsien Sheu, Fei Deng, H. Amalia Pasolli, Yulong Li, Séverine Chaumont-Dubel, Sambashiva Banala, Tom Kirchhausen, Jin-Xia Wan, Melanie Freeman, and Harald F. Hess
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Synapse ,medicine.anatomical_structure ,nervous system ,Chemistry ,Cilium ,medicine ,Neuron ,Hippocampal formation ,Optogenetics ,Axon ,Serotonergic ,Neuroscience ,Chromatin - Abstract
SummaryChemical synapses between axons and dendrites mediate much of the brain’s intercellular communication. Here we describe a new kind of synapse – the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam – scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.
- Published
- 2021
18. Rescue of stalled clathrin-mediated endocytosis by asymmetric Arp2/3-mediated actin assembly
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David G. Drubin, Meiyan Jin, Srigokul Upadhyayula, Bowen Wang, Johannes Schöneberg, Cyna Shirazinejad, Amy Yan, and Ke Xu
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Chemistry ,Vesicle ,education ,Cell ,Endocytic cycle ,macromolecular substances ,Receptor-mediated endocytosis ,Endocytosis ,Membrane tension ,medicine.anatomical_structure ,medicine ,Biophysics ,Actin ,Vesicle scission - Abstract
Actin assembly facilitates vesicle formation in several trafficking pathways. Clathrin-mediated endocytosis (CME) shows elevated actin assembly dependence under high membrane tension. Why actin assembly at CME sites occurs heterogeneously even within the same cell, and how assembly forces are harnessed, are not fully understood. Here, endocytic dynamics, actin presence, and geometry of CME proteins from three different functional modules, were analyzed using three-dimensional (3D) super-resolution microscopy, live-cell imaging, and machine-learning-based computation. When hundreds of CME events were compared, sites with actin assembly showed a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. N-WASP is recruited to one side of CME sites where it is positioned to stimulate asymmetric actin assembly. We propose that asymmetric actin assembly rescues stalled CME sites by pulling vesicles into the cell much like a bottle opener pulls off a bottle cap.
- Published
- 2021
19. A proliferative to invasive switch is mediated by srGAP1 downregulation through the activation of TGF-β2 signaling
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Chandrani Mondal, Majo J. Gacha-Garay, Kathryn A. Larkin, Rebecca C. Adikes, Julie S. Di Martino, Chen-Chi Chien, Madison Fraser, Ireti Eni-aganga, Esperanza Agullo-Pascual, Katarzyna Cialowicz, Umut Ozbek, Alexandra Naba, Angelo Gaitas, Tian-Ming Fu, Srigokul Upadhyayula, Eric Betzig, David Q. Matus, Benjamin L. Martin, and Jose Javier Bravo-Cordero
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Mice ,Transforming Growth Factor beta2 ,Cell Line, Tumor ,Animals ,Down-Regulation ,General Biochemistry, Genetics and Molecular Biology ,Actins ,Zebrafish - Abstract
Many breast cancer (BC) patients suffer from complications of metastatic disease. To form metastases, cancer cells must become migratory and coordinate both invasive and proliferative programs at distant organs. Here, we identify srGAP1 as a regulator of a proliferative-to-invasive switch in BC cells. High-resolution light-sheet microscopy demonstrates that BC cells can form actin-rich protrusions during extravasation. srGAP1
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- 2021
20. A Proliferative to Invasive Switch is Mediated by srGAP1 Downregulation Through the Activation of TGFβ2 Signaling
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Julie Di Martino, Esperanza Agullo-Pascual, Benjamin L. Martin, Rebecca Adikes, Chen-Chi Chien, Angelo Gaitas, Srigokul Upadhyayula, Alexandra Naba, Ireti Eni-aganga, Jose Javier Bravo-Cordero, David Matus, Maria J. Gacha-Garay, Eric Betzig, Kathryn Larkin, Madison Fraser, Umut Ozbek, Chandrani Mondal, and Tian-Ming Fu
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education.field_of_study ,biology ,Population ,biology.organism_classification ,medicine.disease ,Phenotype ,Extravasation ,Metastasis ,Downregulation and upregulation ,Cancer cell ,Cancer research ,medicine ,Secretion ,education ,Zebrafish - Abstract
Many breast cancer (BC) patients suffer from complications of metastatic disease. In order to extravasate efficiently and form metastasis, cancer cells have to become migratory and coordinate both invasive and proliferative programs at distant organs. Here, we identify srGAP1 as a regulator of the proliferative-to-invasive switch in BC cells. Highresolution adaptive optics/light sheet microscopy demonstrates that BC cells form actinrich protrusions to contact and cross the endothelial layer during extravasation. srGAP1 low cells display a highly motile and invasive phenotype that facilitates their extravasation from blood vessels, as shown in zebrafish and mouse models, while attenuating tumor growth. Interestingly, a population of srGAP1low cells remain as solitary disseminated tumor cells (DTCs) in the lungs of mice bearing BC tumors. Furthermore, multiphoton imaging with BC cells expressing a Cdk2 biosensor show that srGAP1 low cells enter into a G0/G1 state and remain as single cells or small clusters in the lungs. Finally, RNAseq and secretome analyses reveal that srGAP1low cells have increased Smad2 activation and TGFβ2 secretion, resulting in an increase in both invasion and p27 levels to sustain quiescence. These findings describe a novel mechanism mediating the shift from a proliferative to an invasive phenotype in BC cells in vivo and identify srGAP1 as a mediator of this phenotypic switch through a TGFβ2 signaling axis.
- Published
- 2021
21. Adherens junction regulates cryptic lamellipodia formation for epithelial cell migration
- Author
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Masatoshi Takeichi, Srigokul Upadhyayula, Masayuki Ozawa, Tatsuo Shibata, Takaki Yamamoto, Yuko Mimori-Kiyosue, and Sylvain Hiver
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0303 health sciences ,Cadherin ,macromolecular substances ,Cell Biology ,Biology ,Epithelial cell migration ,Collective migration ,Cell biology ,Adherens junction ,03 medical and health sciences ,0302 clinical medicine ,Myosin ,Gene silencing ,biological phenomena, cell phenomena, and immunity ,Lamellipodium ,030217 neurology & neurosurgery ,Actin ,030304 developmental biology - Abstract
Collective migration of epithelial cells plays crucial roles in various biological processes such as cancer invasion. In migrating epithelial sheets, leader cells form lamellipodia to advance, and follower cells also form similar motile apparatus at cell–cell boundaries, which are called cryptic lamellipodia (c-lamellipodia). Using adenocarcinoma-derived epithelial cells, we investigated how c-lamellipodia form and found that they sporadically grew from around E-cadherin–based adherens junctions (AJs). WAVE and Arp2/3 complexes were localized along the AJs, and silencing them not only interfered with c-lamellipodia formation but also prevented follower cells from trailing the leaders. Disruption of AJs by removing αE-catenin resulted in uncontrolled c-lamellipodia growth, and this was brought about by myosin II activation and the resultant contraction of AJ-associated actomyosin cables. Additional observations indicated that c-lamellipodia tended to grow at mechanically weak sites of the junction. We conclude that AJs not only tie cells together but also support c-lamellipodia formation by recruiting actin regulators, enabling epithelial cells to undergo ordered collective migration.
- Published
- 2020
22. Anin-vitroBBB-on-a-chip open model of human blood-brain barrier enabling advanced optical imaging
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Tom Kirchhausen, Srigokul Upadhyayula, Giuseppe Di Caprio, Graham Marsh, Ilja Kusters, Ronan Hunt, Birgit Obermeier, Giovanni de Nola, Mootaz M. Salman, Kazuka G. Ohashi, Fumitaka Shimizu, Takashi Kanda, Matthieu Delince, and Yasuteru Sano
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Fluorescence-lifetime imaging microscopy ,medicine.anatomical_structure ,Materials science ,Transcytosis ,Confocal ,medicine ,Lattice light-sheet microscopy ,Blood–brain barrier ,Chip ,Microvessel ,Biomedical engineering ,Lumen (unit) - Abstract
We describe here the design and implementation of anin-vitroBBB-on-a-chip open model system capable of reconstituting the microenvironment of the blood brain barrier. This system allows controlled unidirectional flow of nutrients and biologicals on the lumen of the artificial microvessel. This BBB-on-a-chip is suitable for high resolution electron microscopy and it is amenable for quantitative 3D live fluorescence imaging using spinning confocal disk or lattice light sheet microscopy (LLSM) to follow, for example the transcytosis across the BBB-like barrier of fluorescently-tagged biological, viruses or nanoparticles.
- Published
- 2020
23. Adherens junction serves to generate cryptic lamellipodia required for collective migration of epithelial cells
- Author
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Masatoshi Takeichi, Srigokul Upadhyayula, Takaki Yamamoto, Yuko Mimori-Kiyosue, Tatsuo Shibata, Masayuki Ozawa, and Sylvain Hiver
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Adherens junction ,Contraction (grammar) ,animal structures ,Chemistry ,Myosin ,embryonic structures ,Gene silencing ,macromolecular substances ,Lamellipodium ,biological phenomena, cell phenomena, and immunity ,Actin ,Collective migration ,Cell biology - Abstract
Collective migration of epithelial cells plays crucial roles in various biological processes such as cancer invasion. In migrating epithelial sheets, leader cells form lamellipodia to advance, and follower cells also form similar motile apparatus at cell-cell boundaries, which are called cryptic lamellipodia (c-lamellipodia). Using adenocarcinoma-derived epithelial cells, we investigated how c-lamellipodia are generated, and found that they sporadically grew from Ecadherin-based adherens junctions (AJs). WAVE and Arp2/3 complexes were localized along the AJs, and silencing them not only interfered with c-lamellipodia formation but also prevented follower cells from trailing the leaders. Disruption of AJs by removing αE-catenin resulted in uncontrolled c-lamellipodia growth, and this was brought about by myosin II activation and the resultant contraction of AJ-associated actomyosin cables. Additional observations indicated that c-lamellipodia tended to grow at mechanically weak sites of the junction. We conclude that AJs not only tie cells together but also generate c-lamellipodia by recruiting actin regulators, enabling epithelial cells to undergo ordered collective migration.
- Published
- 2020
- Full Text
- View/download PDF
24. Dynamics of Auxilin 1 and GAK in clathrin-mediated traffic
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Minghe Ma, Raphael Gaudin, Iris Rapoport, Tom Kirchhausen, Eli Song, Wesley Skillern, Srigokul Upadhyayula, Kevin Bu, Ilja Kusters, Benjamin R Capraro, Kangmin He, and Song Dang
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animal structures ,Time Factors ,ATPase ,Auxilins ,Phosphatase ,Biophysics ,Coated vesicle ,macromolecular substances ,Auxilin ,Protein Serine-Threonine Kinases ,Phosphatidylinositols ,Clathrin ,Article ,chemistry.chemical_compound ,Chlorocebus aethiops ,Animals ,Humans ,Phosphatidylinositol ,Trafficking ,biology ,Vesicle ,HSC70 Heat-Shock Proteins ,Intracellular Signaling Peptides and Proteins ,Clathrin-Coated Vesicles ,Cell Biology ,Transport protein ,Protein Transport ,chemistry ,embryonic structures ,COS Cells ,biology.protein ,HeLa Cells ,Signal Transduction - Abstract
Coat disassembly, driven by the Hsc70 “uncoating ATPase” and mediated by auxilin, occurs within seconds after vesicle release. Using single-molecule imaging, He et al. find that auxilins are absent from assembling pits. Therefore, Hsc70 is not responsible for the clathrin exchange during pit formation., Clathrin-coated vesicles lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 “uncoating ATPase.” The J- and PTEN-like domain–containing proteins, auxilin 1 (Aux1) and auxilin 2 (GAK), recruit Hsc70. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin-mediated membrane scission has released the vesicle from the plasma membrane. These bursts contain a very small number of auxilins, and even four to six molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated pit assembly. We previously showed that clathrin-coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. The differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism., Graphical Abstract
- Published
- 2020
25. Inherited nuclear pore substructures template post-mitotic pore assembly
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Giuseppe Di Caprio, Tegy John Vadakkan, Kazuka G. Ohashi, Wesley Skillern, Song Dang, Srigokul Upadhyayula, Gustavo Scanavachi, Yi-ying Chou, Justin Houser, Tom Kirchhausen, Anwesha Sanyal, Kangmin He, and Alex J. B. Kreutzberger
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Cell division ,Nuclear Envelope ,Mitosis ,Biology ,Endoplasmic Reticulum ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Prophase ,otorhinolaryngologic diseases ,Humans ,Nuclear pore ,Interphase ,Molecular Biology ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,Endoplasmic reticulum ,Cell Cycle ,Cell Biology ,Cell cycle ,Cell biology ,Nuclear Pore Complex Proteins ,stomatognathic diseases ,Nuclear Pore ,Nucleoporin ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Nuclear envelope assembly during late mitosis includes rapid formation of several thousand complete nuclear pore complexes (NPCs). This efficient use of NPC components (nucleoporins or “NUPs”) is essential for ensuring immediate nucleocytoplasmic communication in each daughter cell. We show that octameric subassemblies of outer and inner nuclear pore rings remain intact in the mitotic endoplasmic reticulum (ER) after NPC disassembly during prophase. These “inherited” subassemblies then incorporate into NPCs during post-mitotic pore formation. We further show that the stable subassemblies persist through multiple rounds of cell division and the accompanying rounds of NPC mitotic disassembly and post-mitotic assembly. De novo formation of NPCs from newly synthesized NUPs during interphase will then have a distinct initiation mechanism. We postulate that a yet-to-be identified modification marks and “immortalizes” one or more components of the specific octameric outer- and inner-ring subcomplexes that then template post-mitotic NPC assembly during subsequent cell cycles.
- Published
- 2021
26. Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR–Cas9 genome editing
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Wataru Ebina, Tomas Kirchhausen, Suneet Agarwal, Srigokul Upadhyayula, Baris Boyraz, Paula Gutierrez-Martinez, Richard L. Frock, Rachita Yadav, Michael E. Talkowski, Anders Fasth, Frederick W. Alt, Bruna Paulsen, Pankaj Mandal, and Derrick J. Rossi
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0301 basic medicine ,DNA End-Joining Repair ,DNA Repair ,DNA repair ,Induced Pluripotent Stem Cells ,Biomedical Engineering ,Medicine (miscellaneous) ,Bioengineering ,Computational biology ,Biology ,Article ,Ectopic Gene Expression ,Homology directed repair ,03 medical and health sciences ,Genome editing ,Humans ,CRISPR ,DNA Breaks, Double-Stranded ,Gene ,Gene Editing ,Cas9 ,Recombinational DNA Repair ,Rad52 DNA Repair and Recombination Protein ,Computer Science Applications ,HEK293 Cells ,030104 developmental biology ,Ectopic expression ,CRISPR-Cas Systems ,Tumor Suppressor p53-Binding Protein 1 ,Biotechnology - Abstract
Gene disruption by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) is highly efficient and relies on the error-prone non-homologous end-joining pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than non-homologous end-joining in mammalian cells. Here, by testing whether manipulation of DNA repair factors improves HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative form of tumour protein p53-binding protein 1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of non-homologous end-joining-mediated double-strand break repair in the presence of these two factors is not suppressed and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.
- Published
- 2017
27. Miro1-mediated mitochondrial positioning shapes intracellular energy gradients required for cell migration
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Max-Hinderk Schuler, Wesley Skillern, Srigokul Upadhyayula, Tom Kirchhausen, Brian Cunniff, Agnieszka Lewandowska, Janet M. Shaw, and Giuseppe Di Caprio
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rho GTP-Binding Proteins ,0301 basic medicine ,Cytoplasm ,Cortical cytoskeleton ,Energy metabolism ,Mitochondrion ,Biology ,Microtubules ,Mitochondrial Proteins ,Mice ,03 medical and health sciences ,Adenosine Triphosphate ,Cell Movement ,Cell Adhesion ,Animals ,Cell adhesion ,Molecular Biology ,Cells, Cultured ,Cell migration ,Cell Biology ,Mitochondria ,Cell biology ,Adenosine Diphosphate ,030104 developmental biology ,Microscopy, Fluorescence ,Brief Reports ,Energy Metabolism ,Intracellular - Abstract
The ratio of ATP:ADP is highest at perinuclear sites, where mitochondria are dense, and dissipates toward the periphery. Miro1 positions mitochondria toward the cortical cytoskeleton. Deletion of Miro1 results in perinuclear clustering of mitochondria, altering intracellular ATP:ADP gradients, and impairs energy-expensive cell migratory processes., It has long been postulated, although never directly demonstrated, that mitochondria are strategically positioned in the cytoplasm to meet local requirements for energy production. Here we show that positioning of mitochondria in mouse embryonic fibroblasts (MEFs) determines the shape of intracellular energy gradients in living cells. Specifically, the ratio of ATP to ADP was highest at perinuclear areas of dense mitochondria and gradually decreased as more-peripheral sites were approached. Furthermore, the majority of mitochondria were positioned at the ventral surface of the cell, correlating with high ATP:ADP ratios close to the ventral membrane, which rapidly decreased toward the dorsal surface. We used cells deficient for the mitochondrial Rho-GTPase 1 (Miro1), an essential mediator of microtubule-based mitochondrial motility, to study how changes in mitochondrial positioning affect cytoplasmic energy distribution and cell migration, an energy-expensive process. The mitochondrial network in Miro1−/− MEFs was restricted to the perinuclear area, with few mitochondria present at the cell periphery. This change in mitochondrial distribution dramatically reduced the ratio of ATP to ADP at the cell cortex and disrupted events essential for cell movement, including actin dynamics, lamellipodia protrusion, and membrane ruffling. Cell adhesion status was also affected by changes in mitochondrial positioning; focal adhesion assembly and stability was decreased in Miro1−/− MEFs compared with Miro1+/+ MEFs. Consequently Miro1−/− MEFs migrated slower than control cells during both collective and single-cell migration. These data establish that Miro1-mediated mitochondrial positioning at the leading edge provides localized energy production that promotes cell migration by supporting membrane protrusion and focal adhesion stability.
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- 2017
28. A highly homogeneous expansion microscopy polymer composed of tetrahedron-like monomers
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Chih-Chieh Yu, Srigokul Upadhyayula, Rachael L. Neve, Kiryl D. Piatkevich, Linyi Gao, Ruixuan Gao, and Edward S. Boyden
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chemistry.chemical_classification ,0303 health sciences ,Microscope ,Materials science ,Biomolecule ,Resolution (electron density) ,Polymer ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences ,law.invention ,Sphericity ,03 medical and health sciences ,chemistry ,law ,Chemical physics ,Microscopy ,Self-healing hydrogels ,Tetrahedron ,030304 developmental biology - Abstract
Expansion microscopy (ExM) physically magnifies biological specimens to enable nanoscale-resolution imaging on conventional microscopes. Current ExM methods permeate biological specimens with free radical-polymerized polyacrylate hydrogels, whose network structure limits the microscopy resolution enabled by ExM. Here we report that ExM is possible using hydrogels with more homogeneous network structure, assembled via non-radical terminal linking of monomers of tetrahedral shape. As with earlier forms of ExM, such “tetra-gel”-embedded specimens can be iteratively expanded for greater physical magnification. Iterative tetra-gel expansion of herpes simplex virus type 1 (HSV-1) virions by ~10x in linear dimension results in a viral envelope deviation from sphericity of 9.2 nm, rather than the 14.3 nm enabled by free radical-polymerized hydrogels used in earlier versions of ExM. Thus, tetra-gel polymer chemistry may support new forms of ExM imaging that introduce fewer spatial errors than earlier versions, and raise the question of whether single biomolecule precision may be achievable.
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- 2019
29. Dynamics of Auxilin1 and GAK in clathrin-mediated traffic
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Wesley Skillern, Raphael Gaudin, Minghe Ma, Kangmin He, Srigokul Upadhyayula, Benjamin R Capraro, Kevin Bu, Iris Rapoport, Ilja Kusters, Tom Kirchhausen, Eli Song, and Song Dang
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0303 health sciences ,biology ,Chemistry ,Kinase ,Vesicle ,Phosphatase ,Coated vesicle ,Clathrin ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein kinase domain ,biology.protein ,Phosphatidylinositol ,030217 neurology & neurosurgery ,030304 developmental biology ,Dynamin - Abstract
Clathrin coated vesicles formed at the plasma membrane lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 “uncoating ATPase”. The J-domain containing proteins, auxilin1 (Aux1) and auxilin2/cyclin-G dependent kinase (GAK), recruit Hsc70. Aux1 and GAK are closely related homologs, each with a phosphatase- and tensin-like (PTEN-like) domain, a clathrin-binding region, and a C-terminal J-domain; GAK has an additional, N-terminal Ser/Thr kinase domain. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin mediated membrane scission has released the vesicle from the plasma membrane. We show here that these bursts represent recruitment of a very small number of auxilins such that even 4-6 molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated-pit assembly. We have also shown previously that clathrin coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. We now show that differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism.
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- 2019
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30. Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling
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Maria Grazia Malabarba, Pier Paolo Di Fiore, Roberta Pascolutti, Raphael Gaudin, Stefano Freddi, Sara Sigismund, Tanja Maritzen, Srigokul Upadhyayula, Alexia Conte, Elena Maspero, Volker Haucke, Simona Polo, Chiara Tordonato, Veronica Algisi, Mithun Pasham, Stefano Confalonieri, Andrea Raimondi, Elisa Barbieri, Tom Kirchhausen, Carlo Tacchetti, Giusi Caldieri, Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1), San Raffaele Scientific Institute, Vita-Salute San Raffaele University and Center for Translational Genomics and Bioinformatics, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), European Institute of Oncology [Milan] (ESMO), Department of Experimental Oncology, Departimenta di Medicina Sperimentale, University of Genoa (UNIGE), Leibniz-Institut für Molekulare Pharmakologie, Boston Children's Hospital, Pascolutti, R and, Algisi, V, Conte, A, Raimondi, A, Pasham, M, Upadhyayula, S, Gaudin, R, Maritzen, T, Barbieri, E, Caldieri, G, Tordonato, C, Confalonieri, S, Freddi, S, Malabarba, Mg, Maspero, E, Polo, S, Tacchetti, C, Haucke, V, Kirchhausen, T, Di Fiore, Pp, and Sigismund, S
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0301 basic medicine ,Epsin ,[SDV]Life Sciences [q-bio] ,Endocytic cycle ,recycling ,eps15 ,Mice ,0302 clinical medicine ,Cell Movement ,epsin ,clathrin-coated pits ,RNA, Small Interfering ,Internalization ,lcsh:QH301-705.5 ,media_common ,Gene Editing ,biology ,Chemistry ,Signal transducing adaptor protein ,food and beverages ,Cell migration ,Clathrin-Coated Vesicles ,Cell biology ,ErbB Receptors ,RNA Interference ,signaling ,transcription ,receptor degradation ,Signal Transduction ,Transcriptional Activation ,media_common.quotation_subject ,EGFR ,Adaptor Protein Complex 2 ,Transferrin receptor ,Endocytosis ,Clathrin ,General Biochemistry, Genetics and Molecular Biology ,Article ,Cell Line ,03 medical and health sciences ,endocytic adaptors ,endocytosis ,Animals ,Humans ,AP2 ,Adaptor Proteins, Signal Transducing ,Epidermal Growth Factor ,Adaptor Proteins, Vesicular Transport ,030104 developmental biology ,lcsh:Biology (General) ,Microscopy, Fluorescence ,biology.protein ,Proto-Oncogene Proteins c-akt ,030217 neurology & neurosurgery ,HeLa Cells - Abstract
Summary Adaptor protein 2 (AP2) is a major constituent of clathrin-coated pits (CCPs). Whether it is essential for all forms of clathrin-mediated endocytosis (CME) in mammalian cells is an open issue. Here, we demonstrate, by live TIRF microscopy, the existence of a subclass of relatively short-lived CCPs lacking AP2 under physiological, unperturbed conditions. This subclass is retained in AP2-knockout cells and is able to support the internalization of epidermal growth factor receptor (EGFR) but not of transferrin receptor (TfR). The AP2-independent internalization mechanism relies on the endocytic adaptors eps15, eps15L1, and epsin1. The absence of AP2 impairs the recycling of the EGFR to the cell surface, thereby augmenting its degradation. Accordingly, under conditions of AP2 ablation, we detected dampening of EGFR-dependent AKT signaling and cell migration, arguing that distinct classes of CCPs could provide specialized functions in regulating EGFR recycling and signaling., Graphical Abstract, Highlights • Distinct classes of CCPs exist, molecularly defined by the presence or lack of AP2 • The AP2-negative CCPs support the internalization of EGFR but not of TfR • The AP2-negative CCPs rely on the endocytic adaptors eps15/eps15L1 and epsin1 • The two classes of CCPs determine distinct EGFR fates and signaling outputs, EGFR signaling controls different cell physiological processes, including proliferation and migration. Pascolutti et al. describe an additional layer of regulation of EGFR signaling, relying on the sequestration of receptors into molecularly distinct clathrin-coated vesicles that regulate receptor fate toward recycling versus degradation, with impact on the final cellular output.
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- 2019
31. Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution
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Eric Betzig, Igor Pisarev, Srigokul Upadhyayula, Shu-Hsien Sheu, Yongxin Zhao, Austin R. Graves, Christopher T. Zugates, Sean G. Megason, John A. Bogovic, Harald F. Hess, Adam W. Hantman, Song Pang, C. Shan Xu, Susan Tappan, Kishore R. Mosaliganti, Yoshinori Aso, Ved P. Singh, Shoh Asano, Jennifer Lippincott-Schwartz, Carolyn Ott, Gerald M. Rubin, H. Amalia Pasolli, Jennifer Colonell, Edward S. Boyden, Daniel E. Milkie, Grace H. Huynh, Tom Kirchhausen, Stephan Saalfeld, Alfredo Rodriguez, Tsung-Li Liu, and Ruixuan Gao
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Male ,0301 basic medicine ,Dendritic spine ,Materials science ,Dendritic Spines ,Confocal ,Mice, Transgenic ,Neuroimaging ,Kidney ,Imaging phantom ,Mice ,03 medical and health sciences ,Imaging, Three-Dimensional ,0302 clinical medicine ,Microscopy ,Image Processing, Computer-Assisted ,Fluorescence microscope ,medicine ,Biological neural network ,Animals ,Humans ,Nanotechnology ,Multidisciplinary ,Phantoms, Imaging ,Optical Imaging ,Brain ,Somatosensory Cortex ,Photobleaching ,Axons ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Synapses ,Biophysics ,Drosophila ,Female ,Cortical column ,030217 neurology & neurosurgery - Abstract
INTRODUCTION Neural circuits across the brain are composed of structures spanning seven orders of magnitude in size that are assembled from thousands of distinct protein types. Electron microscopy has imaged densely labeled brain tissue at nanometer-level resolution over near-millimeter-level dimensions but lacks the contrast to distinguish specific proteins and the speed to readily image multiple specimens. Conversely, confocal fluorescence microscopy offers molecular contrast but has insufficient resolution for dense neural tracing or the precise localization of specific molecular players within submicrometer-sized structures. Last, superresolution fluorescence microscopy bleaches fluorophores too quickly for large-volume imaging and also lacks the speed for effective brain-wide or cortex-wide imaging of multiple specimens. RATIONALE We combined two imaging technologies to address these issues. Expansion microscopy (ExM) creates an expanded, optically clear phantom of a fluorescent specimen that retains its original relative distribution of fluorescent tags. Lattice light-sheet microscopy (LLSM) then images this phantom in three dimensions with minimal photobleaching at speeds sufficient to image the entire Drosophila brain or across the width of the mouse cortex in ∼2 to 3 days, with multiple markers at an effective resolution of ∼60 by 60 by 90 nm for 4× expansion. RESULTS We applied expansion/LLSM (ExLLSM) to study a variety of subcellular structures in the brain. In the mouse cortex, we quantified the volume of organelles, measured morphological parameters of ~1500 dendritic spines, determined the variation of distances between pre- and postsynaptic proteins, observed large differences in postsynaptic expression at adjacent pyramidal neurons, and studied both the azimuthal asymmetry and layer-specific longitudinal variation of axonal myelination. In Drosophila , we traced the axonal branches of olfactory projection neurons across one hemisphere and studied the stereotypy of their boutons at the calyx and lateral horn across five animals. We also imaged all dopaminergic neurons (DANs) across the brain of another specimen, visualized DAN morphologies in all major brain regions, and traced a cluster of eight DANs to their termini to determine their respective cell types. In the same specimen, we also determined the number of presynaptic active zones (AZs) across the brain and the local density of all AZs and DAN-associated AZs in each brain region. CONCLUSION With its high speed, nanometric resolution, and ability to leverage genetically targeted, cell type–specific, and protein-specific fluorescence labeling, ExLLSM fills a valuable niche between the high throughput of conventional optical pipelines of neural anatomy and the ultrahigh resolution of corresponding EM pipelines. Assuming the development of fully validated, brain-wide isotropic expansion at 10× or beyond and sufficiently dense labeling, ExLLSM may enable brainwide comparisons of even densely innervated neural circuits across multiple specimens with protein-specific contrast at 25-nm resolution or better.
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- 2019
32. Cortical Column and Whole Brain Imaging of Neural Circuits with Molecular Contrast and Nanoscale Resolution
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Kishore R. Mosaliganti, Tom Kirchhausen, Adam W. Hantman, Eric Betzig, Yongxin Zhao, Grace H. Huynh, Stephan Saalfeld, Igor Pisarev, Daniel E. Milkie, Srigokul Upadhyayula, Ved P. Singh, Alfredo Rodriguez, Christopher T. Zugates, Edward S. Boyden, Susan Tappan, John A. Bogovic, Austin R. Graves, Gerald M. Rubin, Shoh Asano, Jennifer Lippincott-Schwartz, Yoshinori Aso, Jennifer Colonell, Sean G. Megason, Carolyn Ott, Tsung-Li Liu, and Ruixuan Gao
- Subjects
0303 health sciences ,Dendritic spine ,Chemistry ,Resolution (electron density) ,Lattice light-sheet microscopy ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,Microscopy ,medicine ,Neuropil ,Biological neural network ,Cortical column ,Neuroscience ,Neural development ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Optical and electron microscopy have made tremendous inroads in understanding the complexity of the brain, but the former offers insufficient resolution to reveal subcellular details and the latter lacks the throughput and molecular contrast to visualize specific molecular constituents over mm-scale or larger dimensions. We combined expansion microscopy and lattice light sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entireDrosophilabrain, including synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly neuropil domain. The technology should enable statistically rich, large scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.One Sentence SummaryCombined expansion and lattice light sheet microscopy enables high speed, nanoscale molecular imaging of neural circuits over large volumes.
- Published
- 2018
33. Author response: Lamellar projections in the endolymphatic sac act as a relief valve to regulate inner ear pressure
- Author
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David G. C. Hildebrand, Samir Malhotra, Ian A. Swinburne, Srigokul Upadhyayula, Kishore R. Mosaliganti, Florian Engert, Tony Y.-C. Tsai, Anzhi Chen, Sean G. Megason, Tomas Kirchhausen, Tsung-Li Liu, Anna K. Fass, Eric Betzig, Ebaa Al-Obeidi, and Jeff W. Lichtman
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medicine.anatomical_structure ,business.industry ,Medicine ,Lamellar structure ,Inner ear ,Anatomy ,Relief valve ,business ,Endolymphatic sac - Published
- 2018
34. Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms
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Abraham Q. Kohrman, Kishore R. Mosaliganti, Kai Wang, Srigokul Upadhyayula, Hanako Yashiro, David Q. Matus, Elliot M. Meyerowitz, Yuan Ruan, Tom W. Hiscock, Brian Cunniff, Dirk Hockemeyer, David G. Drubin, Minoru Koyama, Eric Betzig, Zach M. Collins, Ryan Forster, Ved P. Singh, Daniel E. Milkie, Ian A. Swinburne, Daphné Dambournet, Benjamin L. Martin, Tsung-Li Liu, Sean G. Megason, Steffen Scholpp, Tom Kirchhausen, Jamien Shea, and Taylor N Medwig
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0301 basic medicine ,General Science & Technology ,1.1 Normal biological development and functioning ,Cell ,Mitosis ,Bioengineering ,Lattice light-sheet microscopy ,Endocytosis ,Eye ,Article ,Imaging ,03 medical and health sciences ,0302 clinical medicine ,Imaging, Three-Dimensional ,Single-cell analysis ,Underpinning research ,Cell Movement ,Organelle ,Genetics ,medicine ,Animals ,Humans ,Zebrafish ,Organelles ,Microscopy ,Multidisciplinary ,biology ,biology.organism_classification ,Phenotype ,Cell biology ,Multicellular organism ,030104 developmental biology ,medicine.anatomical_structure ,Three-Dimensional ,Cancer cell ,Biomedical Imaging ,Generic health relevance ,Single-Cell Analysis ,030217 neurology & neurosurgery - Abstract
True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.One Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.
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- 2018
35. Lamellar projections in the endolymphatic sac act as a relief valve to regulate inner ear pressure
- Author
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Anna K. Fass, Kishore R. Mosaliganti, Jeff W. Lichtman, David G. C. Hildebrand, Srigokul Upadhyayula, Florian Engert, Tony Y.-C. Tsai, Eric Betzig, Ebaa Al-Obeidi, Tom Kirchhausen, Samir Malhotra, Anzhi Chen, Sean G. Megason, Tsung-Li Liu, and Ian A. Swinburne
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0301 basic medicine ,inner ear ,Male ,Embryo, Nonmammalian ,Hydrostatic pressure ,timelapse ,Gene Expression ,Animals, Genetically Modified ,pressure ,Hearing ,Homeostasis ,Biology (General) ,In Situ Hybridization, Fluorescence ,Zebrafish ,lamella ,General Neuroscience ,General Medicine ,Anatomy ,medicine.anatomical_structure ,Larva ,Medicine ,Female ,Relief valve ,valve ,Human ear ,QH301-705.5 ,Science ,Biology ,Time-Lapse Imaging ,General Biochemistry, Genetics and Molecular Biology ,Endolymphatic sac ,03 medical and health sciences ,medicine ,otorhinolaryngologic diseases ,Hydrostatic Pressure ,Animals ,Inner ear ,General Immunology and Microbiology ,Balance disorders ,Zebrafish Proteins ,Equilibrioception ,Microscopy, Electron ,030104 developmental biology ,Mutation ,Zebrafish embryo ,sense organs ,Endolymphatic Sac ,Transcription Factors - Abstract
The most internal part of the human ear, the inner ear, is essential for us to hear and have a sense of balance. It is formed by a complex series of connected cavities filled by a liquid. When sound waves and changes in the position of the body make this liquid move, specialized ‘hair’ cells can detect these subtle movements; neurons then relay this information to the brain where it is decoded and interpreted. For the inner ear to work properly, the body needs to finely regulate the pressure created by the liquid inside the cavities. For example, people with unstable pressure in their ears can experience deafness or problems with balance. A structure known as the endolymphatic sac, which is a balloon-like chamber connected to the rest of the inner ear by a thin tube, helps with this regulation. However, scientists are still unsure about how exactly the sac performs its role. One problem is that the inner ear is difficult to study because it is encased in one of the densest bones in the body. Many other animals also have inner ears, from fish to birds and mammals. Here, Swinburne et al. examine the inner ear of zebrafish embryos because, in this fish, the ear starts working before the bones around it form; the structure is therefore accessible for injections and microscopy. Experiments show that when the pressure in the inner ear rises, the endolymphatic sac slowly fills up with the ear liquid, and then it rapidly deflates. Fish with mutations that stop the sac from deflating have overinflated sacs, which is a symptom also found in certain patients with hearing and balance disorders. Looking into the details of these inflation-deflation cycles, Swinburne et al. found that the cells that form the sac have gaps between them, unlike a normal sheet of cells. A flap covers these gaps to keep the liquid in, but under pressure, the flap opens and the liquid can escape. These results show that the endolymphatic sac works as a pressure relief valve for the inner ear. Ultimately, understanding how pressure is regulated in the ear could help patients with inner ear disorders. It could also serve as a template to investigate how eyes, kidneys and the brain, which all have liquid-filled cavities, control their internal pressure.
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- 2018
36. Reconstitution of Clathrin Coat Disassembly for Fluorescence Microscopy and Single-Molecule Analysis
- Author
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Iris Rapoport, Till Böcking, Srigokul Upadhyayula, Tom Kirchhausen, and Benjamin R Capraro
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0301 basic medicine ,animal structures ,Total internal reflection fluorescence microscope ,biology ,Chemistry ,Vesicle ,Coated vesicle ,Auxilin ,Clathrin ,Article ,03 medical and health sciences ,030104 developmental biology ,Clathrin coat disassembly ,Biophysics ,Fluorescence microscope ,biology.protein ,Molecule - Abstract
The disassembly of the clathrin lattice surrounding coated vesicles is the obligatory last step in their life cycle. It is mediated by the coordinated recruitment of auxilin and Hsc70, an ATP-driven molecular clamp. Here, we describe the preparation of reagents and the single-particle fluorescence microscopy imaging assay in which we visualize directly the Hsc70-driven uncoating of synthetic clathrin coats or clathrin-coated vesicles.
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- 2018
37. Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic
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Benjamin R Capraro, Wesley Skillern, Weiming Wang, Raphael Gaudin, Tom Kirchhausen, Eli Song, Srigokul Upadhyayula, Song Dang, Minghe Ma, Kangmin He, Robert Marsland, Nanjing Institute of Geology and Palaeontology (NANJING INSTITUTE OF GEOLOGY AND PALAEONTOLOGY), Nanjing Institute of Geology and Palaeontology, Department of Cell Biology, Harvard Medical School, and Boston Children's Hospital
- Subjects
Phosphatidylinositol 4,5-Diphosphate ,0301 basic medicine ,Endosome ,[SDV]Life Sciences [q-bio] ,Auxilins ,Endocytic cycle ,Endosomes ,Phosphatidylinositols ,Endocytosis ,Clathrin ,Article ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,Phosphatidylinositol Phosphates ,Chlorocebus aethiops ,Animals ,Humans ,Phosphatidylinositol ,Phosphorylation ,ComputingMilieux_MISCELLANEOUS ,rab5 GTP-Binding Proteins ,Multidisciplinary ,biology ,Chemistry ,Vesicle ,Cell Membrane ,Phosphotransferases ,Clathrin-Coated Vesicles ,Coated Pits, Cell-Membrane ,Phosphoric Monoester Hydrolases ,Transport protein ,030104 developmental biology ,Membrane ,COS Cells ,biology.protein ,Biophysics - Abstract
Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes1,2. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphateand phosphatidylinositol-4-phosphate mark distinct endosomal compartments2,3. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe ‘coincidence-detecting’ sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate.Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.
- Published
- 2017
38. Photoinduced dynamics of a cyanine dye: parallel pathways of non-radiative deactivation involving multiple excited-state twisted transients
- Author
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Srigokul Upadhyayula, Vicente Nuñez, Eli M. Espinoza, Jillian M. Larsen, Duoduo Bao, Dewen Shi, Jenny T. Mac, Bahman Anvari, and Valentine I. Vullev
- Subjects
Chemistry ,010405 organic chemistry ,Chemical Sciences ,General Chemistry ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences ,3. Good health - Abstract
Cyanine dyes are broadly used for fluorescence imaging and other photonic applications. 3,3'-Diethylthiacyanine (THIA) is a cyanine dye composed of two identical aromatic heterocyclic moieties linked with a single methine, -CH 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 . The torsional degrees of freedom around the methine bonds provide routes for non-radiative decay, responsible for the inherently low fluorescence quantum yields. Using transient absorption spectroscopy, we determined that upon photoexcitation, the excited state relaxes along two parallel pathways producing three excited-state transients that undergo internal conversion to the ground state. The media viscosity impedes the molecular modes of ring rotation and preferentially affects one of the pathways of non-radiative decay, exerting a dominant effect on the emission properties of THIA. Concurrently, the polarity affects the energy of the transients involved in the decay pathways and further modulates the kinetics of non-radiative deactivation.
- Published
- 2015
39. Lamellar junctions in the endolymphatic sac act as a relief valve to regulate inner ear pressure
- Author
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Eric Betzig, Anna K. Fass, Srigokul Upadhyayula, Kishore R. Mosaliganti, Tom Kirchhausen, Tony Y.-C. Tsai, Florian Engert, David G. C. Hildebrand, Jeff W. Lichtman, Samir Malhotra, Ebaa Al-Obeidi, Tsung-Li Liu, Anzhi Chen, Sean G. Megason, and Ian A. Swinburne
- Subjects
0303 health sciences ,Chemistry ,Hydrostatic pressure ,Balance disorders ,Anatomy ,Endolymphatic sac ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,Ion homeostasis ,Live cell imaging ,medicine ,Lamellar structure ,Inner ear ,sense organs ,Relief valve ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
The inner ear is a fluid-filled closed-epithelial structure whose normal function requires maintenance of an internal hydrostatic pressure and fluid composition by unknown mechanisms. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear. ES defects can cause distended ear tissue, a pathology often seen in hearing and balance disorders. Using live imaging of zebrafish larvae, we reveal that the ES undergoes cycles of slow pressure-driven inflation followed by rapid deflation every 1-3 hours. Using serial-section electron microscopy and adaptive optics lattice light-sheet microscopy, we find a pressure relief valve in the ES comprised of thin overlapping basal lamellae that dynamically extend over neighboring cells before rupturing under pressure leading to ES collapse. The unexpected discovery of a physical relief valve in the ear emphasizes the need for further study into how organs control fluid pressure, volume, flow, and ion homeostasis in development and disease.
- Published
- 2017
40. Single-Particle Detection of Transcription following Rotavirus Entry
- Author
-
Srigokul Upadhyayula, Stephen C. Harrison, and Eric N. Salgado
- Subjects
0301 basic medicine ,Immunology ,Cell ,Biology ,virus entry ,medicine.disease_cause ,Microbiology ,fluorescence microscopy ,Virus ,03 medical and health sciences ,Viral entry ,Transcription (biology) ,Virology ,Rotavirus ,medicine ,Spotlight ,RNA synthesis ,030102 biochemistry & molecular biology ,RNA ,Virus-Cell Interactions ,Cytosol ,030104 developmental biology ,medicine.anatomical_structure ,rhesus rotavirus ,Insect Science ,Biophysics ,Intracellular - Abstract
Infectious rotavirus particles are triple-layered, icosahedral assemblies. The outer layer proteins, VP4 (cleaved to VP8* and VP5*) and VP7, surround a transcriptionally competent, double-layer particle (DLP), which they deliver into the cytosol. During entry of rhesus rotavirus, VP8* interacts with cell surface gangliosides, allowing engulfment into a membrane vesicle by a clathrin-independent process. Escape into the cytosol and outer-layer shedding depend on interaction of a hydrophobic surface on VP5* with the membrane bilayer and on a large-scale conformational change. We report here experiments that detect the fate of released DLPs and their efficiency in initiating RNA synthesis. By replacing the outer layer with fluorescently tagged, recombinant proteins and also tagging the DLP, we distinguished particles that have lost their outer layer and entered the cytosol (uncoated) from those still within membrane vesicles. We used fluorescent in situ hybridization with probes for nascent transcripts to determine how soon after uncoating transcription began and what fraction of the uncoated particles were active in initiating RNA synthesis. We detected RNA synthesis by uncoated particles as early as 15 min after adding virus. The uncoating efficiency was 20 to 50%; of the uncoated particles, about 10 to 15% synthesized detectable RNA. In the format of our experiments, about 10% of the added particles attached to the cell surface, giving an overall ratio of added particles to RNA-synthesizing particles of between 250:1 and 500:1, in good agreement with the ratio of particles to focus-forming units determined by infectivity assays. Thus, RNA synthesis by even a single, uncoated particle can initiate infection in a cell. IMPORTANCE The pathways by which a virus enters a cell transform its packaged genome into an active one. Contemporary fluorescence microscopy can detect individual virus particles as they enter cells, allowing us to map their multistep entry pathways. Rotaviruses, like most viruses that lack membranes of their own, disrupt or perforate the intracellular, membrane-enclosed compartment into which they become engulfed following attachment to a cell surface, in order to gain access to the cell interior. The properties of rotavirus particles make it possible to determine molecular mechanisms for these entry steps. In the work described here, we have asked the following question: what fraction of the rotavirus particles that penetrate into the cell make new viral RNA? We find that of the cell-attached particles, between 20 and 50% ultimately penetrate, and of these, about 10% make RNA. RNA synthesis by even a single virus particle can initiate a productive infection.
- Published
- 2017
41. Degradation and antibacterial properties of magnesium alloys in artificial urine for potential resorbable ureteral stent applications
- Author
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Vicente Nuñez, Valentine I. Vullev, Jaclyn Y. Lock, Srigokul Upadhyayula, Huinan Liu, Andrew Whall, and Eric Wyatt
- Subjects
Medical device ,Materials science ,Magnesium ,medicine.medical_treatment ,Metals and Alloys ,Biomedical Engineering ,Stent ,chemistry.chemical_element ,Ureteral stents ,equipment and supplies ,Biomaterials ,surgical procedures, operative ,Artificial urine ,chemistry ,Ceramics and Composites ,medicine ,Degradation (geology) ,Magnesium ion ,Biomedical engineering ,Bacterial colony - Abstract
This article presents an investigation on the effectiveness of magnesium and its alloys as a novel class of antibacterial and biodegradable materials for ureteral stent applications. Magnesium is a lightweight and biodegradable metallic material with beneficial properties for use in medical devices. Ureteral stent is one such example of a medical device that is widely used to treat ureteral canal blockages clinically. The bacterial colony formation coupled with the encrustation on the stent surface from extended use often leads to clinical complications and contributes to the failure of indwelling medical devices. We demonstrated that magnesium alloys decreased Escherichia coli viability and reduced the colony forming units over a 3-day incubation period in an artificial urine (AU) solution when compared with currently used commercial polyurethane stent. Moreover, the magnesium degradation resulted in alkaline pH and increased magnesium ion concentration in the AU solution. The antibacterial and degradation properties support the potential use of magnesium-based materials for next-generation ureteral stents. Further studies are needed for clinical translation of biodegradable metallic ureteral stents.
- Published
- 2013
42. Photoinduced Electron Transfer Between Pyridine Coated Cadmium Selenide Quantum Dots and Single Sheet Graphene
- Author
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Srigokul Upadhyayula, Jennifer Reiber Kyle, Shirui Guo, Mihrimah Ozkan, Ali Bilge Guvenc, Cengiz S. Ozkan, Hamed Hosseinibay, Wei Wang, Krassimir N. Bozhilov, Duoduo Bao, and Valentine I. Vullev
- Subjects
Materials science ,Cadmium selenide ,Graphene ,business.industry ,Nanotechnology ,Condensed Matter Physics ,Photoinduced electron transfer ,Electronic, Optical and Magnetic Materials ,law.invention ,Nanomaterials ,Biomaterials ,chemistry.chemical_compound ,Nanoelectronics ,chemistry ,law ,Quantum dot ,Electrochemistry ,Optoelectronics ,Bilayer graphene ,business ,Graphene nanoribbons - Abstract
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Interest in graphene as a two-dimensional quantum-well material for energy applications and nanoelectronics has increased exponentially in the last few years. The recent advances in large-area single-sheet fabrication of pristine graphene have opened unexplored avenues for expanding from nanoto meso-scale applications. The relatively low level of absorptivity and the short lifetimes of excitons of single-sheet graphene suggest that it needs to be coup led with light sensitizers in order to explore its feasibility for photonic applications, such as solar-energy conversion. Red-emitting CdSe quantum dots are employed for photosensitizing single-sheet graphene with areas of several square centimeters. Pyridine coating of the quantum dots not only enhances their adhesion to the graphene surface, but also provides good electronic coup ling between the CdSe and the two-dimensional carbon allotrope. Illumination of the quantum dots led to injection of n-carrier in the graphene phase. Time-resolved spectroscopy reveals three modes of photoinduced electron transfer between the quantum dots and the graphene occurring in the femtosecond and picosecond time-domains. Transient absorption spectra provide evidence for photoinduced hole-shift from the CdSe to the pyridine ligands, thereby polarizing the surface of the quantum dots. That is, photoinduced electrical polarization, which favors the simultaneous electron transfer from the CdSe to the graphene phase. These mechanistic insights into the photoinduced interfacial charge transfer have a promising potential to serve as guidelines for the design and development of composites of graphene and inorganic nanomaterials for solar-energy conversion applications.
- Published
- 2013
43. Microfluidic Space-Domain Time-Resolved Emission Spectroscopy of Terbium(III) and Europium(III) Chelates with Pyridine-2,6-Dicarboxylate
- Author
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Brent Millare, Srigokul Upadhyayula, Hong Xu, Prashanthi Vandrangi, Jillian M. Larsen, Valentine I. Vullev, Sharad Gupta, Sanghoon Shin, Adam Lin, Ali Hadian, Vicente Nuñez, and Georgi Georgiev
- Subjects
Time Factors ,Microchannel ,Chemistry ,Analytical chemistry ,chemistry.chemical_element ,Terbium ,Laminar flow ,Microfluidic Analytical Techniques ,Analytical Chemistry ,Ion ,Physics::Fluid Dynamics ,Photoexcitation ,Europium ,Microscopy, Fluorescence ,Emission spectrum ,Picolinic Acids ,Spectroscopy ,Chelating Agents - Abstract
This article describes the utilization of laminar microflows for time-resolved emission measurements with steady-state excitation and detection. Passing a laminar flow through a short illuminated section of a microchannel provided a means for pulsed-like photoexcitation of the moieties carried by the fluid. Imaging the microchannel flows carrying thus photoexcited chelates of lanthanide ions allowed us to extract their excited-state lifetimes from the spatial distribution of the changes in the emission intensity. The lifetime values obtained using this space-domain approach agreed well with the lifetimes from time-domain measurements. This validated space-domain microfluidic approach reveals a means for miniaturization of time-resolved emission spectroscopy.
- Published
- 2013
44. Anthranilamides as Bioinspired Molecular Electrets: Experimental Evidence for a Permanent Ground-State Electric Dipole Moment
- Author
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Duoduo Bao, Srigokul Upadhyayula, Bing Xia, Valentine I. Vullev, and Guilford Jones
- Subjects
Magnetic Resonance Spectroscopy ,Chemistry ,Static Electricity ,Organic Chemistry ,Proteins ,Nanotechnology ,Dielectric ,Electric dipole moment ,Dipole ,Magnet ,Static electricity ,Energy transformation ,ortho-Aminobenzoates ,Electret ,Ground state - Abstract
As electrostatic equivalents of magnets, organic electrets offer unparalleled properties for impacting energy conversion and electronic applications. While biological systems have evolved to efficiently utilize protein α-helices as molecular electrets, the synthetic counterparts of these conjugates still remain largely unexplored. This paper describes a study of the electronic properties of anthranilamide oligomers, which proved to be electrets based on their intrinsic dipole moments as evident from their spectral and dielectric properties. NMR studies provided the means for estimating the direction of the intrinsic electric dipoles of these conjugates. This study sets the foundation for the development of a class of organic materials that are de novo designed from biomolecular motifs and possess unexplored electronic properties.
- Published
- 2013
45. Seipin is required for converting nascent to mature lipid droplets
- Author
-
Qingqing Lin, Maria Jesus Olarte, Tobias C. Walther, Morven Graham, Srigokul Upadhyayula, Michel Becuwe, Christer S. Ejsing, Ashley J. Porras, David B. Savage, Xinran Liu, Norbert Perrimon, Abdou Rachid Thiam, Abhimanyu Garg, Chandramohan Chitraju, Huajin Wang, Tomas Kirchhausen, Anil K. Agarwal, Florian Fröhlich, Robert V. Farese, Hans Kristian Hannibal-Bach, Benjamin E. Housden, Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Department of Chemistry, Pennsylvania State University (Penn State), Penn State System-Penn State System, Laboratoire de Physique Statistique de l'ENS (LPS), Fédération de recherche du Département de physique de l'Ecole Normale Supérieure - ENS Paris (FRDPENS), Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris)-Université Paris Diderot - Paris 7 (UPD7)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Medicine Center Dallas, Department Medicine, University of Texas Southwestern Medical Center (UTSW), Yale University School of Medicine, Dept of Genetics, Harvard Medical School, Harvard Medical School [Boston] (HMS), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,endocrine system ,QH301-705.5 ,Science ,[SDV]Life Sciences [q-bio] ,lipid droplet ,Economic shortage ,Biology ,complex mixtures ,General Biochemistry, Genetics and Molecular Biology ,Seipin ,03 medical and health sciences ,Lipid droplet ,cell biology ,lipid metabolism ,human ,Biology (General) ,LiveDrop ,General Immunology and Microbiology ,D. melanogaster ,General Neuroscience ,Endoplasmic reticulum ,technology, industry, and agriculture ,A protein ,Lipid metabolism ,General Medicine ,eye diseases ,Cell biology ,endoplasmic reticulum ,030104 developmental biology ,seipin ,Large lipid droplets ,Medicine ,Organelle biogenesis ,organelle biogenesis ,Research Article - Abstract
How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation—the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs. DOI: http://dx.doi.org/10.7554/eLife.16582.001, eLife digest Living organisms often store energy in the form of fat molecules called triglycerides. Enzymes in a compartment of the cell called the endoplasmic reticulum catalyze the chemical reactions needed to make these triglycerides. The cell then stores the triglycerides in a different structure called the lipid droplet. Lipid droplets form from the endoplasmic reticulum in an organized manner, but little is known about the cellular machinery that gives rise to lipid droplets. A protein called seipin is thought to be involved in lipid droplet formation. Seipin resides in the endoplasmic reticulum and a shortage of this protein in cells leads to abnormal lipid droplets – that is, cells often have lots of tiny lipid droplets or a few giant ones. People who lack seipin lose much of their fat tissue and instead store fat in the wrong places, such as the liver. Now, Wang et al. have studied the seipin protein in insect and human cells grown in the laboratory. The experiments confirmed that cells that lack the seipin protein form lots of tiny dot-like structures containing triglycerides that fail to grow into normal-sized lipid droplets. These lipid droplets have different proteins on their surface, which may impair their ability to store fat. Wang et al. also discovered that in normal cells, the seipin protein is found at distinct spots in the endoplasmic reticulum. This distribution appears to allow seipin to come into contact with the small, newly formed lipid droplets and enable them to grow. Together these findings suggest that the seipin protein could form part of a molecular machine that allows more triglycerides to be added into newly formed lipid droplets causing the droplets to grow as normal. When seipin is not present the newly formed lipid droplets initially become stuck in a smaller form. As a consequence, a few of these tiny droplets later enter a different cellular pathway of lipid droplet expansion, which turns them into abnormally large lipid droplets. Future challenges will be to determine precisely how seipin enables newly formed lipid droplets to grow. It will also be important to confirm whether seipin works with other proteins as part of a molecular machine and, if so, to investigate how these proteins affect the formation and growth of lipid droplets. DOI: http://dx.doi.org/10.7554/eLife.16582.002
- Published
- 2016
46. Author response: Seipin is required for converting nascent to mature lipid droplets
- Author
-
Norbert Perrimon, Maria-Jesus Olarte, Hans Kristian Hannibal-Bach, Srigokul Upadhyayula, Benjamin E. Housden, Florian Fröhlich, David B. Savage, Abhimanyu Garg, Tobias C. Walther, Chandramohan Chitraju, Qingqing Lin, Christer S. Ejsing, Abdou Rachid Thiam, Michel Becuwe, Huajin Wang, Xinran Liu, Tomas Kirchhausen, Robert V. Farese, Morven Graham, Ashley J. Porras, and Anil K. Agarwal
- Subjects
Chemistry ,Lipid droplet ,Seipin ,Cell biology - Published
- 2016
47. Coatings of Polyethylene Glycol for Suppressing Adhesion between Solid Microspheres and Flat Surfaces
- Author
-
Jeremy Stubbs, Pratima Nallagatla, Baharak Bahmani, Timothy Quinata, Srigokul Upadhyayula, Pamela Jreij, Sharad Gupta, Kliment Bozhilov, Valentine I. Vullev, Noah R. Johnson, and Stephen Bishop
- Subjects
chemistry.chemical_classification ,Materials science ,Surface Properties ,Surfaces and Interfaces ,Polyethylene glycol ,Polymer ,Adhesion ,engineering.material ,Condensed Matter Physics ,Microspheres ,Polyethylene Glycols ,chemistry.chemical_compound ,Coating ,chemistry ,Chemical engineering ,Polymer chemistry ,PEG ratio ,Microscopy ,Microscopy, Electron, Scanning ,Electrochemistry ,engineering ,Surface modification ,General Materials Science ,Spectroscopy ,Superparamagnetism - Abstract
This article describes the development and the examination of surface coatings that suppress the adhesion between glass surfaces and polymer microspheres. Superparamagnetic doping allowed for exerting magnetic forces on the microbeads. The carboxyl functionalization of the polymer provided the means for coating the beads with polyethylene glycol (PEG) with different molecular weight. Under gravitational force, the microbeads settled on glass surfaces with similar polymer coatings. We examined the efficacy of removing the beads from the glass surfaces by applying a pulling force of ~1.2 pN. The percent beads remaining on the surface after applying the pulling force for approximately 5 s served as an indication of the adhesion propensity. Coating of PEG with molecular weight ranging between 3 and 10 kDa was essential for suppressing the adhesion. For the particular substrates, surface chemistry and aqueous media we used, coatings of 5 kDa manifested optimal suppression of adhesion: that is, only 3% of the microbeads remained on the surface after applying the pulling magnetic force. When either the glass or the beads were not PEGylated, the adhesion between them was substantial. Addition of a noncharged surfactant, TWEEN, above its critical micelle concentrations (CMCs) suppressed the adhesion between noncoated substrates. The extent of this surfactant-induced improvement of the adhesion suppression, however, did not exceed the quality of preventing the adhesion that we attained by PEGylating both substrates. In addition, the use of surfactants did not significantly improve the suppression of bead-surface adhesion when both substrates were PEGylated. These findings suggest that such surfactant additives tend to be redundant and that covalently grafted coatings of PEGs with selected chain lengths provide sufficient suppression of nonspecific interfacial interactions.
- Published
- 2012
48. Amyloid Histology Stain for Rapid Bacterial Endospore Imaging
- Author
-
Srigokul Upadhyayula, Bing Xia, Vicente Nuñez, Mohammad Sarshar, Harbani Kaur Malik, Pavel Landsman, Bahman Anvari, Jingqiu Hu, Samuel Lam, Valentine I. Vullev, Guilford Jones, and Sharad Gupta
- Subjects
Spores, Bacterial ,Microbiology (medical) ,Bacteriological Techniques ,Endospore coat ,Endospore staining ,Staining and Labeling ,biology ,Bacillus ,Exosporium ,Bacteriology ,biology.organism_classification ,Endospore ,Staining ,Geobacillus stearothermophilus ,Thiazoles ,chemistry.chemical_compound ,Bacterial Proteins ,Microscopy, Fluorescence ,chemistry ,Biochemistry ,Image Processing, Computer-Assisted ,Thioflavin ,Benzothiazoles ,Amyloid (mycology) - Abstract
Bacterial endospores are some of the most resilient forms of life known to us, with their persistent survival capability resulting from a complex and effective structural organization. The outer membrane of endospores is surrounded by the densely packed endospore coat and exosporium, containing amyloid or amyloid-like proteins. In fact, it is the impenetrable composition of the endospore coat and the exosporium that makes staining methodologies for endospore detection complex and challenging. Therefore, a plausible strategy for facile and expedient staining would be to target components of the protective surface layers of the endospores. Instead of targeting endogenous markers encapsulated in the spores, here we demonstrated staining of these dormant life entities that targets the amyloid domains, i.e., the very surface components that make the coats of these species impenetrable. Using an amyloid staining dye, thioflavin T (ThT), we examined this strategy. A short incubation of bacillus endospore suspensions with ThT, under ambient conditions, resulted in (i) an enhancement of the fluorescence of ThT and (ii) the accumulation of ThT in the endospores, affording fluorescence images with excellent contrast ratios. Fluorescence images revealed that ThT tends to accumulate in the surface regions of the endospores. The observed fluorescence enhancement and dye accumulation, coupled with the sensitivity of emission techniques, provide an effective and rapid means of staining endospores without the inconvenience of pre- or posttreatment of samples.
- Published
- 2011
49. Dependence of the quality of adhesion between poly(dimethylsiloxane) and glass surfaces on the composition of the oxidizing plasma
- Author
-
Vicente Nuñez, Srigokul Upadhyayula, Adam Lin, Brent Millare, Hong Xu, Kenny Chau, and Valentine I. Vullev
- Subjects
Materials science ,Fabrication ,Polydimethylsiloxane ,technology, industry, and agriculture ,macromolecular substances ,02 engineering and technology ,Plasma ,Adhesion ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,Elastomer ,01 natural sciences ,0104 chemical sciences ,Electronic, Optical and Magnetic Materials ,chemistry.chemical_compound ,chemistry ,Chemical engineering ,Oxidizing agent ,Polymer chemistry ,Materials Chemistry ,Capacitively coupled plasma ,0210 nano-technology ,Microfabrication - Abstract
Controlled surface oxidation of polydimethylsiloxane (PDMS) is essential for permanent adhesion between device components composed of this elastomer. The permanent adhesion between such microdevice components results from covalent crosslinking across the interfaces between PDMS and other silica-based materials, such as glass, quartz, and PDMS. Optimal duration and conditions of oxidation, attained via treatments with oxygen-containing plasma, are crucial for microfabrication procedures with quantitative yields. While insufficient PDMS oxidation does not provide high enough surface density of siloxyl groups for cross-interface linking, overoxidation of PDMS yields rough silica surface layers that prevent the adhesion between flat substrates. Ideally, for a set of plasma conditions, the range of treatment durations producing permanent adhesion should be as broad as possible: i.e., the surface oxidation of PDMS sufficient for irreversible binding has to complete significantly before the effects of overoxidation become apparent. Such a requirement assures that relatively small fluctuations in the treatment conditions will not result in over- or under-oxidation and, hence, will not compromise the yields of the fabrication procedures. We examined the dependence of the quality of adhesion (QA) between plasma-treated PDMS and glass substrates on the composition of the oxygen-containing plasma and on the radio frequency (RF) of the plasma generator. We observed that plasma generated at megahertz RF provided superior conditions than kilohertz RF. Concurrently, an increase in the oxygen content of binary gas mixtures, used for the plasma, broadened the treatment durations that afford superior QA.
- Published
- 2010
50. Correction: Photoinduced dynamics of a cyanine dye: parallel pathways of non-radiative deactivation involving multiple excited-state twisted transients
- Author
-
Srigokul Upadhyayula, Dewen Shi, Vicente Nuñez, Bahman Anvari, Eli M. Espinoza, Jillian M. Larsen, Jenny T. Mac, Duoduo Bao, and Valentine I. Vullev
- Subjects
General Chemistry ,Internal conversion (chemistry) ,Photochemistry ,Fluorescence ,Photoexcitation ,chemistry.chemical_compound ,Chemistry ,chemistry ,Excited state ,Ultrafast laser spectroscopy ,Chemical Sciences ,Cyanine ,Spectroscopy ,Ground state - Abstract
A photoexcited cyanine dye deactivates via multiple non-radiative pathways, only one of which is principally responsible for quenching its fluorescence., Cyanine dyes are broadly used for fluorescence imaging and other photonic applications. 3,3′-Diethylthiacyanine (THIA) is a cyanine dye composed of two identical aromatic heterocyclic moieties linked with a single methine, –CH 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 . The torsional degrees of freedom around the methine bonds provide routes for non-radiative decay, responsible for the inherently low fluorescence quantum yields. Using transient absorption spectroscopy, we determined that upon photoexcitation, the excited state relaxes along two parallel pathways producing three excited-state transients that undergo internal conversion to the ground state. The media viscosity impedes the molecular modes of ring rotation and preferentially affects one of the pathways of non-radiative decay, exerting a dominant effect on the emission properties of THIA. Concurrently, the polarity affects the energy of the transients involved in the decay pathways and further modulates the kinetics of non-radiative deactivation.
- Published
- 2015
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