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900 results on '"Rothman, James E."'

1. Biallelic NAA60 variants with impaired N-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications

Author

Chelban, Viorica, Aksnes, Henriette, Maroofian, Reza, LaMonica, Lauren C., Seabra, Luis, Siggervåg, Anette, Devic, Perrine, Shamseldin, Hanan E., Vandrovcova, Jana, Murphy, David, Richard, Anne-Claire, Quenez, Olivier, Bonnevalle, Antoine, Zanetti, M. Natalia, Kaiyrzhanov, Rauan, Salpietro, Vincenzo, Efthymiou, Stephanie, Schottlaender, Lucia V., Morsy, Heba, Scardamaglia, Annarita, Tariq, Ambreen, Pagnamenta, Alistair T., Pennavaria, Ajia, Krogstad, Liv S., Bekkelund, Åse K., Caiella, Alessia, Glomnes, Nina, Brønstad, Kirsten M., Tury, Sandrine, Moreno De Luca, Andrés, Boland-Auge, Anne, Olaso, Robert, Deleuze, Jean-François, Anheim, Mathieu, Cretin, Benjamin, Vona, Barbara, Alajlan, Fahad, Abdulwahab, Firdous, Battini, Jean-Luc, İpek, Rojan, Bauer, Peter, Zifarelli, Giovanni, Gungor, Serdal, Kurul, Semra Hiz, Lochmuller, Hanns, Da’as, Sahar I., Fakhro, Khalid A., Gómez-Pascual, Alicia, Botía, Juan A., Wood, Nicholas W., Horvath, Rita, Ernst, Andreas M., Rothman, James E., McEntagart, Meriel, Crow, Yanick J., Alkuraya, Fowzan S., Nicolas, Gaël, Arnesen, Thomas, and Houlden, Henry

Published
2024
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3. Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution

Author

Hao, Xiang, Allgeyer, Edward S, Lee, Dong-Ryoung, Antonello, Jacopo, Watters, Katherine, Gerdes, Julianne A, Schroeder, Lena K, Bottanelli, Francesca, Zhao, Jiaxi, Kidd, Phylicia, Lessard, Mark D, Rothman, James E, Cooley, Lynn, Biederer, Thomas, Booth, Martin J, and Bewersdorf, Joerg

Subjects
Biological Sciences, Bioengineering, Imaging, Three-Dimensional, Microscopy, Fluorescence, Nanotechnology, Optics and Photonics, Signal-To-Noise Ratio, Technology, Medical and Health Sciences, Developmental Biology, Biological sciences
Abstract

Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.

Published
2021

4. Snare machinery is optimized for ultrafast fusion

Author

Manca, Fabio, Pincet, Frederic, Truskinovsky, Lev, Rothman, James E, Foret, Lionel, and Caruel, Matthieu

Subjects
Physics - Biological Physics
Abstract

SNARE proteins zipper to form SNAREpins that power vesicle fusion with target membranes in a variety of biological processes. A single SNAREpin takes about 1 second to fuse two bilayers, yet a handful can ensure release of neurotransmitters from synaptic vesicles much faster, in a 10th of a millisecond. We propose that, similar to the case of muscle myosins, the ultrafast fusion results from cooperative action of many SNAREpins. The coupling originates from mechanical interactions induced by confining scaffolds. Each SNAREpin is known to have enough energy to overcome the fusion barrier of 25-35 kB T, however, the fusion barrier only becomes relevant when the SNAREpins are nearly completely zippered and from this state each SNAREpin can deliver only a small fraction of this energy as mechanical work. Therefore they have to act cooperatively and we show that at least 3 of them are needed to ensure fusion in less than a millisecond. However, to reach the pre-fusion state collectively, starting from the experimentally observed half-zippered metastable state, the SNAREpins have to mechanically synchronize which takes exponentially longer time as the number of SNAREpins increases. Incorporating this somewhat counter-intuitive idea in a simple coarse grained model results in the novel prediction that there should be an optimum number of SNAREpins for sub-ms fusion: 3-6 over a wide range of parameters. Interestingly, in situ cryo-electron microscope tomography has very recently shown that exactly six SNAREpins participate in the fusion of each synaptic vesicle. This number is in the range predicted by our theory.

Published
2019
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5. The golgin family exhibits a propensity to form condensates in living cells

Author

Ziltener, Pascal, Rebane, Aleksander A, Graham, Morven, Ernst, Andreas M, and Rothman, James E

Subjects
Generic health relevance, Autoantigens, Cell Survival, Golgi Apparatus, Golgi Matrix Proteins, HeLa Cells, Humans, Time-Lapse Imaging, trans-Golgi Network, Hela Cells, Medicinal and Biomolecular Chemistry, Biochemistry and Cell Biology, Evolutionary Biology, Biochemistry & Molecular Biology
Abstract

The Golgi is surrounded by a ribosome-excluding matrix. Recently, we reported that the cis-Golgi-localized golgin GM130 can phase-separate to form dynamic, liquid-like condensates in vitro and in vivo. Here, we show that the overexpression of each of the remaining cis (golgin160, GMAP210)- and trans (golgin97, golgin245, GCC88, GCC185)-golgins results in novel protein condensates. Focused ion beam scanning electron microscopy (FIB-SEM) images of GM130 condensates reveal a complex internal organization with branching aqueous channels. Pairs of golgins overexpressed in the same cell form distinct juxtaposed condensates. These findings support the hypothesis that, in addition to their established roles as vesicle tethers, phase separation may be a common feature of the golgin family that contributes to Golgi organization.

Published
2020

6. Labeling Strategies Matter for Super-Resolution Microscopy: A Comparison between HaloTags and SNAP-tags

Author

Erdmann, Roman S, Baguley, Stephanie Wood, Richens, Jennifer H, Wissner, Rebecca F, Xi, Zhiqun, Allgeyer, Edward S, Zhong, Sheng, Thompson, Alexander D, Lowe, Nicholas, Butler, Richard, Bewersdorf, Joerg, Rothman, James E, St Johnston, Daniel, Schepartz, Alanna, and Toomre, Derek

Subjects
Biochemistry and Cell Biology, Biological Sciences, Animals, Drosophila, Fluorescent Dyes, Green Fluorescent Proteins, HeLa Cells, Humans, Microscopy, Confocal, Microscopy, Fluorescence, Proteins, Recombinant Fusion Proteins, Rhodamines, Staining and Labeling, Hela Cells, HaloTag, SNAP-tag, STED, fluorophores, live-cell imaging, microscopy, nanoscopy, self-labeling proteins, super-resolution microscopy, Biochemistry and cell biology, Medicinal and biomolecular chemistry
Abstract

Super-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. Organic fluorophores may help here as they can yield more photons-by orders of magnitude-than fluorescent proteins. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAP-tags being the most common. However, how these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. Herein, we compare the two labeling approaches in confocal and STED imaging using various proteins and two model systems. Strikingly, we find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging.

Published
2019

11. Long time-lapse nanoscopy with spontaneously blinking membrane probes

Author

Takakura, Hideo, Zhang, Yongdeng, Erdmann, Roman S, Thompson, Alexander D, Lin, Yu, McNellis, Brian, Rivera-Molina, Felix, Uno, Shin-nosuke, Kamiya, Mako, Urano, Yasuteru, Rothman, James E, Bewersdorf, Joerg, Schepartz, Alanna, and Toomre, Derek

Subjects
Biochemistry and Cell Biology, Physical Sciences, Biological Sciences, Cellular Structures, Fluorescent Dyes, HeLa Cells, Humans, Microscopy, Fluorescence, Nanotechnology, Hela Cells
Abstract

Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resolution images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.

Published
2017

12. A novel physiological role for ARF1 in the formation of bidirectional tubules from the Golgi

Author

Bottanelli, Francesca, Kilian, Nicole, Ernst, Andreas M, Rivera-Molina, Felix, Schroeder, Lena K, Kromann, Emil B, Lessard, Mark D, Erdmann, Roman S, Schepartz, Alanna, Baddeley, David, Bewersdorf, Joerg, Toomre, Derek, and Rothman, James E

Subjects
Biochemistry and Cell Biology, Biological Sciences, Genetics, Biotechnology, ADP-Ribosylation Factor 1, COP-Coated Vesicles, Clathrin, Coat Protein Complex I, GTP Phosphohydrolases, Golgi Apparatus, Guanosine Triphosphate, HeLa Cells, Humans, Hydrolysis, Intracellular Membranes, Hela Cells, Medical and Health Sciences, Developmental Biology, Biochemistry and cell biology
Abstract

Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Besides its well-established role in generating COPI vesicles, we find that ARF1 is also involved in the formation of long (∼3 µm), thin (∼110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins coatomer (COPI) and clathrin. Instead, they contain ARF1 along their entire length at a density estimated to be in the range of close packing. Experiments using a mutant form of ARF1 affecting GTP hydrolysis suggest that ARF1[GTP] is functionally required for the tubules to form. Dynamic confocal and stimulated emission depletion imaging shows that ARF1-rich tubular compartments fall into two distinct classes containing 1) anterograde cargoes and clathrin clusters or 2) retrograde cargoes and coatomer clusters.

Published
2017

13. Ultra-High Resolution 3D Imaging of Whole Cells.

Author

Huang, Fang, Sirinakis, George, Allgeyer, Edward S, Schroeder, Lena K, Duim, Whitney C, Kromann, Emil B, Phan, Thomy, Rivera-Molina, Felix E, Myers, Jordan R, Irnov, Irnov, Lessard, Mark, Zhang, Yongdeng, Handel, Mary Ann, Jacobs-Wagner, Christine, Lusk, C Patrick, Rothman, James E, Toomre, Derek, Booth, Martin J, and Bewersdorf, Joerg

Subjects
Spermatocytes, Synaptonemal Complex, COP-Coated Vesicles, Golgi Apparatus, Animals, Mice, Bacteriophages, Microscopy, Fluorescence, Cytological Techniques, Male, Single Molecule Imaging, Bioengineering, Underpinning research, 1.1 Normal biological development and functioning, 1.5 Resources and infrastructure (underpinning), Generic health relevance, Biological Sciences, Medical and Health Sciences, Developmental Biology
Abstract

Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

Published
2016

14. Two-colour live-cell nanoscale imaging of intracellular targets.

Author

Bottanelli, Francesca, Kromann, Emil B, Allgeyer, Edward S, Erdmann, Roman S, Wood Baguley, Stephanie, Sirinakis, George, Schepartz, Alanna, Baddeley, David, Toomre, Derek K, Rothman, James E, and Bewersdorf, Joerg

Subjects
COS Cells, Hela Cells, Animals, Cercopithecus aethiops, Humans, Rhodamines, Luminescent Proteins, Microscopy, Fluorescence, Nanotechnology, Heterocyclic Compounds, 4 or More Rings, Chlorocebus aethiops, HeLa Cells, Microscopy, Fluorescence, Heterocyclic Compounds, or More Rings
Abstract

Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution.

Published
2016

20. Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery

Author

Kalyana Sundaram, R. Venkat, primary, Chatterjee, Atrouli, additional, Bera, Manindra, additional, Grushin, Kirill, additional, Panda, Aniruddha, additional, Li, Feng, additional, Coleman, Jeff, additional, Lee, Seong, additional, Ramakrishnan, Sathish, additional, Ernst, Andreas M., additional, Gupta, Kallol, additional, Rothman, James E., additional, and Krishnakumar, Shyam S., additional

Published
2023
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29. Two successive oligomeric Munc13 assemblies scaffold vesicle docking and SNARE assembly to support neurotransmitter release

Author

Bera, Manindra, primary, Grushin, Kirill, additional, Sundaram, R Venkat Kalyana, additional, Shahanoor, Ziasmin, additional, Chatterjee, Atrouli, additional, Radhakrishnan, Abhijith, additional, Lee, Seong, additional, Padmanarayana, Murugesh, additional, Coleman, Jeff, additional, Pincet, Frédéric, additional, Rothman, James E, additional, and Dittman, Jeremy S, additional

Published
2023
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30. Novel Roles for Diacylglycerol in Synaptic Vesicle Priming and Release Revealed by Complete Reconstitution of Core Protein Machinery

Author

Sundaram, R Venkat Kalyana, primary, Chatterjee, Atrouli, additional, Bera, Manindra, additional, Grushin, Kirill, additional, Panda, Aniruddha, additional, Li, Feng, additional, Coleman, Jeff, additional, Lee, Seong, additional, Ramakrishnan, Sathish, additional, Ernst, Andreas M., additional, Gupta, Kallol, additional, Rothman, James E., additional, and Krishnakumar, Shyam S., additional

Published
2023
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44. Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer

Author

Panda, Aniruddha, primary, Giska, Fabian, additional, Duncan, Anna L., additional, Welch, Alexander J., additional, Brown, Caroline, additional, McAllister, Rachel, additional, Hariharan, Parameswaran, additional, Goder, Jean N. D., additional, Coleman, Jeff, additional, Ramakrishnan, Sathish, additional, Pincet, Frédéric, additional, Guan, Lan, additional, Krishnakumar, Shyam, additional, Rothman, James E., additional, and Gupta, Kallol, additional

Published
2023
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