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4106 Publications

Showing 2151-2160 of 4106 results
Gonen Lab
10/01/09 | Lipid-protein interactions probed by electron crystallography.
Reichow SL, Gonen T
Current Opinion in Structural Biology. 2009 Oct;19(5):560-5. doi: 10.1016/j.sbi.2009.07.012

Electron crystallography is arguably the only electron cryomicroscopy (cryoEM) technique able to deliver an atomic-resolution structure of membrane proteins embedded in the lipid bilayer. In the electron crystallographic structures of the light driven ion pump, bacteriorhodopsin, and the water channel, aquaporin-0, sufficiently high resolution was obtained and both lipid and protein were visualized, modeled, and described in detail. An extensive network of lipid-protein interactions mimicking native membranes is established and maintained in two-dimensional (2D) crystalline vesicles used for structural analysis by electron crystallography. Lipids are tightly integrated into the protein’s architecture where they can affect the function, structure, quaternary assembly, and the stability of the membrane protein.

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01/01/10 | Lipids and cholesterol as regulators of traffic in the endomembrane system.
Lippincott-Schwartz J, Phair RD
Annual review of biophysics. 2010;39:559-78. doi: 10.1146/annurev.biophys.093008.131357

The endomembrane system of eukaryotic cells uses membrane-enclosed carriers to move diverse macromolecules among different membrane-bound compartments, a requirement for cells to secrete and take up molecules from their environment. Two recycling pathways-biosynthetic and endocytic, each with specific lipid components-make up this system, with the Golgi apparatus mediating transport between the two. Here, we integrate lipid-based mechanisms into the description of this system. A partitioning model of the Golgi apparatus is discussed as a working hypothesis to explain how membrane lipids and proteins that are segregated based on lateral lipid partitioning support the unique composition of the biosynthetic and endocytic recycling pathways in the face of constant trafficking of molecular constituents. We further discuss how computational modeling can allow for interpretation of experimental findings and provide mechanistic insight into these important cellular pathways.

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06/07/21 | Live and Let Dye.
Lavis LD
Biochemistry. 2021 Jun 07:. doi: 10.1021/acs.biochem.1c00299

The measurement of ion concentrations and fluxes inside living cells is key to understanding cellular physiology. Fluorescent indicators that can infiltrate and provide intel on the cellular environment are critical tools for biological research. Developing these molecular informants began with the seminal work of Racker and colleagues ( (1979) 18, 2210), who demonstrated the passive loading of fluorescein in living cells to measure changes in intracellular pH. This work continues, employing a mix of old and new tradecraft to create innovative agents for monitoring ions inside living systems.

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12/30/16 | Live cell single molecule-guided Bayesian localization super resolution microscopy.
Xu F, Zhang M, He W, Han R, Xue F, Liu Z, Zhang F, Lippincott-Schwartz J, Xu P
Cell Research. 2016 Dec 30:. doi: 10.1038/cr.2015.160
03/01/14 | Live imaging and quantitative analysis of gastrulation in mouse embryos using light-sheet microscopy and 3D tracking tools.
Ichikawa T, Nakazato K, Keller PJ, Kajiura-Kobayashi H, Stelzer EH, Mochizuki A, Nonaka S
Nature Protocols. 2014 Mar;9(3):575-85. doi: 10.1038/nprot.2014.035

This protocol describes how to observe gastrulation in living mouse embryos by using light-sheet microscopy and computational tools to analyze the resulting image data at the single-cell level. We describe a series of techniques needed to image the embryos under physiological conditions, including how to hold mouse embryos without agarose embedding, how to transfer embryos without air exposure and how to construct environmental chambers for live imaging by digital scanned light-sheet microscopy (DSLM). Computational tools include manual and semiautomatic tracking programs that are developed for analyzing the large 4D data sets acquired with this system. Note that this protocol does not include details of how to build the light-sheet microscope itself. Time-lapse imaging ends within 12 h, with subsequent tracking analysis requiring 3-6 d. Other than some mouse-handling skills, this protocol requires no advanced skills or knowledge. Light-sheet microscopes are becoming more widely available, and thus the techniques outlined in this paper should be helpful for investigating mouse embryogenesis.

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12/10/14 | Live imaging of endogenous PSD-95 using ENABLED: a conditional strategy to fluorescently label endogenous proteins.
Fortin DA, Tillo SE, Yang G, Rah J, Melander JB, Bai S, Soler-Cedeño O, Qin M, Zemelman BV, Guo C, Mao T, Zhong H
Journal of Neuroscience. 2014 Dec 10;34(50):16698-712. doi: 10.1523/JNEUROSCI.3888-14.2014

Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo.

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09/16/13 | Live imaging of nervous system development and function using light-sheet microscopy.
Lemon WC, Keller PJ
Molecular Reproduction and Development. 2015 Jul;82(7-8):605-18. doi: 10.1002/mrd.22258

In vivo imaging applications typically require carefully balancing conflicting parameters. Often it is necessary to achieve high imaging speed, low photo-bleaching, and photo-toxicity, good three-dimensional resolution, high signal-to-noise ratio, and excellent physical coverage at the same time. Light-sheet microscopy provides good performance in all of these categories, and is thus emerging as a particularly powerful live imaging method for the life sciences. We see an outstanding potential for applying light-sheet microscopy to the study of development and function of the early nervous system in vertebrates and higher invertebrates. Here, we review state-of-the-art approaches to live imaging of early development, and show how the unique capabilities of light-sheet microscopy can further advance our understanding of the development and function of the nervous system. We discuss key considerations in the design of light-sheet microscopy experiments, including sample preparation and fluorescent marker strategies, and provide an outlook for future directions in the field.

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02/06/25 | Live imaging of the extracellular matrix with a glycan-binding fluorophore.
Fiore A, Yu G, Northey JJ, Patel R, Ravenscroft TA, Ikegami R, Kolkman W, Pratik Kumar , Dilan TL, Ruetten VM, Ahrens MB, Shroff H, Wang S, Weaver VM, Pedram K
Nat Methods. 2025 Feb 06:. doi: 10.1038/s41592-024-02590-2

All multicellular systems produce and dynamically regulate extracellular matrices (ECMs) that play essential roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small-molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, non-perturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

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07/08/13 | Live imaging of whole mouse embryos during gastrulation: migration analyses of epiblast and mesodermal cells.
Ichikawa T, Nakazato K, Keller PJ, Kajiura-Kobayashi H, Stelzer EH, Mochizuki A, Nonaka S
PLoS One. 2013 Jul 8;8(7):e64506. doi: 10.1371/journal.pone.0064506

During gastrulation in the mouse embryo, dynamic cell movements including epiblast invagination and mesodermal layer expansion lead to the establishment of the three-layered body plan. The precise details of these movements, however, are sometimes elusive, because of the limitations in live imaging. To overcome this problem, we developed techniques to enable observation of living mouse embryos with digital scanned light sheet microscope (DSLM). The achieved deep and high time-resolution images of GFP-expressing nuclei and following 3D tracking analysis revealed the following findings: (i) Interkinetic nuclear migration (INM) occurs in the epiblast at embryonic day (E)6 and 6.5. (ii) INM-like migration occurs in the E5.5 embryo, when the epiblast is a monolayer and not yet pseudostratified. (iii) Primary driving force for INM at E6.5 is not pressure from neighboring nuclei. (iv) Mesodermal cells migrate not as a sheet but as individual cells without coordination.

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05/26/20 | Live-cell imaging in the era of too many microscopes.
Lemon WC, McDole K
Current Opinion in Cell Biology. 2020 May 26;66:34-42. doi: 10.1016/j.ceb.2020.04.008

At the time of this writing, searching Google Scholar for 'light-sheet microscopy' returns almost 8500 results; over three-quarters of which were published in the last 5 years alone. Searching for other advanced imaging methods in the last 5 years yields similar results: 'super-resolution microscopy' (>16 000), 'single-molecule imaging' (almost 10 000), SPIM (Single Plane Illumination Microscopy, 5000), and 'lattice light-sheet' (1300). The explosion of new imaging methods has also produced a dizzying menagerie of acronyms, with over 100 different species of 'light-sheet' alone, from SPIM to UM (Ultra microscopy) to SiMView (Simultaneous MultiView) to iSPIM (inclined SPIM, not to be confused with iSPIM, inverted SPIM). How then is the average biologist, without an advanced degree in physics, optics, or computer science supposed to make heads or tails of which method is best suited for their needs? Let us also not forget the plight of the optical physicist, who at best might need help with obtaining healthy samples and keeping them that way, or at worst may not realize the impact their newest technique could have for biologists. This review will not attempt to solve all these problems, but instead highlight some of the most recent, successful mergers between biology and advanced imaging technologies, as well as hopefully provide some guidance for anyone interested in journeying into the world of live-cell imaging.

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