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1366 Janelia Publications

Showing 61-70 of 1366 results
03/07/18 | cisTEM, User-friendly software for single-particle image processing.
Grant T, Rohou A, Grigorieff N
eLife. 2018 Mar 07;7:. doi: 10.7554/eLife.35383

We have developed new open-source software calledTEM (computational imaging system for transmission electron microscopy) for the processing of data for high-resolution electron cryo-microscopy and single-particle averaging.TEM features a graphical user interface that is used to submit jobs, monitor their progress, and display results. It implements a full processing pipeline including movie processing, image defocus determination, automatic particle picking, 2D classification, ab-initio 3D map generation from random parameters, 3D classification, and high-resolution refinement and reconstruction. Some of these steps implement newly-developed algorithms; others were adapted from previously published algorithms. The software is optimized to enable processing of typical datasets (2000 micrographs, 200k - 300k particles) on a high-end, CPU-based workstation in half a day or less, comparable to GPU-accelerated processing. Jobs can also be scheduled on large computer clusters using flexible run profiles that can be adapted for most computing environments.TEM is available for download from cistem.org.

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03/01/18 | From electron crystallography of 2D crystals to MicroED of 3D crystals.
Martynowycz MW, Gonen T
Current Opinion in Colloid & Interface Science . 2018 Mar;34:9-16. doi: 10.1016/j.cocis.2018.01.010

Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.

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02/26/18 | A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.
Piatkevich KD, Jung EE, Straub C, Linghu C, Park D, Suk H, Hochbaum DR, Goodwin D, Pnevmatikakis E, Pak N, Kawashima T, Yang C, Rhoades JL, Shemesh O, Asano S, Yoon Y, Freifeld L, Saulnier JL, Riegler C, Engert F, Hughes T, Drobizhev M, Szabo B, Ahrens MB, Flavell SW, Sabatini BL, Boyden ES
Nature Chemical Biology. 2018 Feb 26:. doi: 10.1038/s41589-018-0004-9

We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

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02/24/18 | Integrative whole-brain neuroscience in larval zebrafish.
Vanwalleghem GC, Ahrens MB, Scott EK
Current Opinion in Neurobiology. 2018 Feb 24;50:136-145. doi: 10.1016/j.conb.2018.02.004

Due to their small size and transparency, zebrafish larvae are amenable to a range of fluorescence microscopy techniques. With the development of sensitive genetically encoded calcium indicators, this has extended to the whole-brain imaging of neural activity with cellular resolution. This technique has been used to study brain-wide population dynamics accompanying sensory processing and sensorimotor transformations, and has spurred the development of innovative closed-loop behavioral paradigms in which stimulus-response relationships can be studied. More recently, microscopes have been developed that allow whole-brain calcium imaging in freely swimming and behaving larvae. In this review, we highlight the technologies underlying whole-brain functional imaging in zebrafish, provide examples of the sensory and motor processes that have been studied with this technique, and discuss the need to merge data from whole-brain functional imaging studies with neurochemical and anatomical information to develop holistic models of functional neural circuits.

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02/23/18 | BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.
McArthur K, Whitehead LW, Heddleston JM, Li L, Padman BS, Oorschot V, Geoghegan ND, Chappaz S, Davidson S, San Chin H, Lane RM, Dramicanin M, Saunders TL, Sugiana C, Lessene R, Osellame LD, Chew T, Dewson G, Lazarou M, Ramm G, Lessene G, Ryan MT, Rogers KL, van Delft MF, Kile BT
Science (New York, N.Y.). 2018 02 23;359(6378):. doi: 10.1126/science.aao6047

Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that induce mitochondrial outer membrane permeabilization, leading to cytochrome c release and activation of apoptotic caspases. In the absence of active caspases, mitochondrial DNA (mtDNA) triggers the innate immune cGAS/STING pathway, causing dying cells to secrete type I interferon. How cGAS gains access to mtDNA remains unclear. We used live-cell lattice light-sheet microscopy to examine the mitochondrial network in mouse embryonic fibroblasts. We found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner mitochondrial membrane to herniate into the cytosol, carrying with it mitochondrial matrix components, including the mitochondrial genome. Apoptotic caspases did not prevent herniation but dismantled the dying cell to suppress mtDNA-induced innate immune signaling.

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02/20/18 | VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis.
Ott C, Nachmias D, Adar S, Jarnik M, Sherman S, Birnbaum RY, Lippincott-Schwartz J, Elia N
Scientific Reports. 2018 Feb 20;8(1):3353. doi: 10.1038/s41598-018-21491-x

The hexameric AAA ATPase VPS4 facilitates ESCRT III filament disassembly on diverse intracellular membranes. ESCRT III components and VPS4 have been localized to the ciliary transition zone and spindle poles and reported to affect centrosome duplication and spindle pole stability. How the canonical ESCRT pathway could mediate these events is unclear. We studied the association of VPS4 with centrosomes and found that GFP-VPS4 was a dynamic component of both mother and daughter centrioles. A mutant, VPS4, which can't hydrolyze ATP, was less dynamic and accumulated at centrosomes. Centrosome localization of the VPS4mutant, caused reduced γ-tubulin levels at centrosomes and consequently decreased microtubule growth and altered centrosome positioning. In addition, preventing VPS4 ATP hydrolysis nearly eliminated centriolar satellites and paused ciliogensis after formation of the ciliary vesicle. Zebrafish embryos injected with GFP-VPS4mRNA were less viable, exhibited developmental defects and had fewer cilia in Kupffer's vesicle. Surprisingly, ESCRT III proteins seldom localized to centrosomes and their depletion did not lead to these phenotypes. Our data support an ESCRT III-independent function for VPS4 at the centrosome and reveal that this evolutionary conserved AAA ATPase influences diverse centrosome functions and, as a result, global cellular architecture and development.

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02/19/18 | Single excitatory axons form clustered synapses onto CA1 pyramidal cell dendrites
Bloss EB, Cembrowski MS, Karsh B, Colonell J, Fetter RD, Spruston N
Nature Neuroscience. 2018 Mar;21(3):353-63. doi: 10.1038/s41593-018-0084-6

CA1 pyramidal neurons are a major output of the hippocampus and encode features of experience that constitute episodic memories. Feature-selective firing of these neurons results from the dendritic integration of inputs from multiple brain regions. While it is known that synchronous activation of spatially clustered inputs can contribute to firing through the generation of dendritic spikes, there is no established mechanism for spatiotemporal synaptic clustering. Here we show that single presynaptic axons form multiple, spatially clustered inputs onto the distal, but not proximal, dendrites of CA1 pyramidal neurons. These compound connections exhibit ultrastructural features indicative of strong synapses and occur much more commonly in entorhinal than in thalamic afferents. Computational simulations revealed that compound connections depolarize dendrites in a biophysically efficient manner, owing to their inherent spatiotemporal clustering. Our results suggest that distinct afferent projections use different connectivity motifs that differentially contribute to dendritic integration.

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02/16/18 | Physical basis of amyloid fibril polymorphism.
Close W, Neumann M, Schmidt A, Hora M, Annamalai K, Schmidt M, Reif B, Schmidt V, Grigorieff N, Fändrich M
Nature Communications. 2018 Feb 16;9(1):699. doi: 10.1038/s41467-018-03164-5

Polymorphism is a key feature of amyloid fibril structures but it remains challenging to explain these variations for a particular sample. Here, we report electron cryomicroscopy-based reconstructions from different fibril morphologies formed by a peptide fragment from an amyloidogenic immunoglobulin light chain. The observed fibril morphologies vary in the number and cross-sectional arrangement of a structurally conserved building block. A comparison with the theoretically possible constellations reveals the experimentally observed spectrum of fibril morphologies to be governed by opposing sets of forces that primarily arise from the β-sheet twist, as well as peptide-peptide interactions within the fibril cross-section. Our results provide a framework for rationalizing and predicting the structure and polymorphism of cross-β fibrils, and suggest that a small number of physical parameters control the observed fibril architectures.

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02/14/18 | Synthetic and genetically encoded fluorescent neural activity indicators.
Deo C, Lavis LD
Current Opinion in Neurobiology. 2018 Feb 14;50:101-108. doi: 10.1016/j.conb.2018.01.003

The ultimate goal of neuroscience is to relate the complex activity of cells and cell-networks to behavior and cognition. This requires tools and techniques to visualize neuronal activity. Fluorescence microscopy is an ideal tool to measure activity of cells in the brain due to the high sensitivity of the technique and the growing portfolio of optical hardware and fluorescent sensors. Here, we give a chemist's perspective on the recent progress of fluorescent activity indicators that enable the measurement of cellular events in the living brain. We discuss advances in both chemical and genetically encoded sensors and look forward to hybrid indicators, which incorporate synthetic organic dyes into genetically encoded protein constructs.

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02/13/18 | Measuring integrin conformational change on the cell surface with super-resolution microscopy.
Moore TI, Aaron J, Chew T, Springer TA
Cell Reports. 2018 Feb 13;22(7):1903-1912. doi: 10.1016/j.celrep.2018.01.062

We use super-resolution interferometric photoactivation and localization microscopy (iPALM) and a constrained photoactivatable fluorescent protein integrin fusion to measure the displacement of the head of integrin lymphocyte function-associated 1 (LFA-1) resulting from integrin conformational change on the cell surface. We demonstrate that the distance of the LFA-1 head increases substantially between basal and ligand-engaged conformations, which can only be explained at the molecular level by integrin extension. We further demonstrate that one class of integrin antagonist maintains the bent conformation, while another antagonist class induces extension. Our molecular scale measurements on cell-surface LFA-1 are in excellent agreement with distances derived from crystallographic and electron microscopy structures of bent and extended integrins. Our distance measurements are also in excellent agreement with a previous model of LFA-1 bound to ICAM-1 derived from the orientation of LFA-1 on the cell surface measured using fluorescence polarization microscopy.

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