scholarly journals Phagocytosis is mediated by two-dimensional assemblies of the F-BAR protein GAS7

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Kyoko Hanawa-Suetsugu ◽  
Yuzuru Itoh ◽  
Maisarah Ab Fatah ◽  
Tamako Nishimura ◽  
Kazuhiro Takemura ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Critical Role ◽  
Super Resolution ◽  
Two Dimensional ◽  
Coated Pits ◽  
Bar Domain ◽  
Bar Domain Proteins ◽  
Phagocytic Cups ◽  
Super Resolution Microscopy ◽  
Flat Membranes

Abstract Phagocytosis is a cellular process for internalization of micron-sized large particles including pathogens. The Bin-Amphiphysin-Rvs167 (BAR) domain proteins, including the FCH-BAR (F-BAR) domain proteins, impose specific morphologies on lipid membranes. Most BAR domain proteins are thought to form membrane invaginations or protrusions by assembling into helical submicron-diameter filaments, such as on clathrin-coated pits, caveolae, and filopodia. However, the mechanism by which BAR domain proteins assemble into micron-scale phagocytic cups was unclear. Here, we show that the two-dimensional sheet-like assembly of Growth Arrest-Specific 7 (GAS7) plays a critical role in phagocytic cup formation in macrophages. GAS7 has the F-BAR domain that possesses unique hydrophilic loops for two-dimensional sheet formation on flat membranes. Super-resolution microscopy reveals the similar assemblies of GAS7 on phagocytic cups and liposomes. The mutations of the loops abolishes both the membrane localization of GAS7 and phagocytosis. Thus, the sheet-like assembly of GAS7 plays a significant role in phagocytosis.

scholarly journals A putative N-BAR-domain protein is crucially required for the development of hyphae tip appressorium-like structure and its plant infection in Magnaporthe oryzae

2019 ◽  
Vol 1 (1) ◽  
Author(s):  
Lili Lin ◽  
Xiaomin Chen ◽  
Ammarah Shabbir ◽  
Si Chen ◽  
Xuewen Chen ◽  
...  
Keyword(s):  
Magnaporthe Oryzae ◽  
Lipid Membranes ◽  
Cellular Membrane ◽  
Gene Replacement ◽  
Bar Domain ◽  
Plant Infection ◽  
Expression Of Genes ◽  
Peripheral Proteins ◽  
Bar Domain Proteins ◽  
Replacement Technique

Abstract Membrane remodeling modulates many biological processes. The binding of peripheral proteins to lipid membranes results in membrane invaginations and protrusions, which regulate essential intra-cellular membrane and extra-cellular trafficking events. Proteins that bind and re-shape bio-membranes have been identified and extensively investigated. The Bin/Amphiphysin/Rvs (BAR) domain proteins are crescent-shape and play a conserved role in tubulation and sculpturing of cell membranes. We deployed targeted gene replacement technique to functionally characterize two hypothetical proteins (MoBar-A and MoBar-B) containing unitary N-BAR domain in Magnaporthe oryzae. The results obtained from phenotypic examinations showed that MoBAR-A deletion exerted a significant reduction in the growth of the defective ∆Mobar-A strain. Also, MoBAR-A disruption exclusively compromised hyphae-mediated infection. Additionally, the targeted replacement of MoBAR-A suppressed the expression of genes associated with the formation of hyphae tip appressorium-like structure in M. oryzae. Furthermore, single as well as combined deletion of MoBAR-A and MoBAR-B down-regulated the expression of nine different membrane-associated genes. From these results, we inferred that MoBAR-A plays a key and unique role in the pathogenesis of M. oryzae through direct or indirect regulation of the development of appressorium-like structures developed by hyphae tip. Taken together, these results provide unique insights into the direct contribution of the N-BAR domain proteins to morphological, reproduction, and infectious development of M. oryzae.

scholarly journals Adsorption Of Bar-domain Proteins To Charged Lipid Membranes Causes Deformations And Lipid Demixing

2009 ◽  
Vol 96 (3) ◽  
pp. 95a
Author(s):  
George Khelashvili ◽  
Daniel Harries ◽  
Harel Weinstein
Keyword(s):  
Lipid Membranes ◽  
Bar Domain ◽  
Bar Domain Proteins

scholarly journals NanoJ-SQUIRREL: quantitative mapping and minimisation of super-resolution optical imaging artefacts

2017 ◽  
Author(s):  
Siân Culley ◽  
David Albrecht ◽  
Caron Jacobs ◽  
Pedro Matos Pereira ◽  
Christophe Leterrier ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Original Data ◽  
Focal Volume ◽  
Coated Pits ◽  
Superresolution Imaging ◽  
Cell Structures ◽  
Image Artefacts ◽  
Localisation Microscopy ◽  
Super Resolution Microscopy

Most super-resolution microscopy methods depend on steps that contribute to the formation of image artefacts. Here we present NanoJ-SQUIRREL, an ImageJ-based analytical approach providing a quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same focal volume, this approach generates a quantitative map of super-resolution defects, as well as methods for their correction. To illustrate its broad applicability to super-resolution approaches we apply our method to Localization Microscopy, STED and SIM images of a variety of in-cell structures including microtubules, poxviruses, neuronal actin rings and clathrin coated pits. We particularly focus on single-molecule localisation microscopy, where super-resolution reconstructions often feature imperfections not present in the original data. By showing the quantitative evolution of data quality over these varied sample preparation, acquisition and super-resolution methods we display the potential of NanoJ-SQUIRREL to guide optimization of superresolution imaging parameters.

scholarly journals Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Adrien Mau ◽  
Karoline Friedl ◽  
Christophe Leterrier ◽  
Nicolas Bourg ◽  
Sandrine Lévêque-Fort
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Uniform Illumination ◽  
Optical Sectioning ◽  
Coated Pits ◽  
Laser Illumination ◽  
Quantitative Analyses ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractNon-uniform illumination limits quantitative analyses of fluorescence imaging techniques. In particular, single molecule localization microscopy (SMLM) relies on high irradiances, but conventional Gaussian-shaped laser illumination restricts the usable field of view to around 40 µm × 40 µm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile illumination technique that generates uniform and adaptable illumination. ASTER is also highly compatible with optical sectioning techniques such as total internal reflection fluorescence (TIRF). For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power over fields-of-view up to 200 µm × 200 µm. We demonstrate that ASTER improves clustering analysis and nanoscopic size measurements by imaging nanorulers, microtubules and clathrin-coated pits in COS-7 cells, and β2-spectrin in neurons. ASTER’s sharp and quantitative illumination paves the way for high-throughput quantification of biological structures and processes in classical and super-resolution fluorescence microscopies.

Scanning FCS and super-resolution microscopy on 2D lipid membranes (Conference Presentation)

2020 ◽  
Author(s):  
Uwe Ortmann ◽  
Mariano Gonzalez Pisfil ◽  
Marcelle König ◽  
Rhys Dowler ◽  
Benedikt Krämer ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals How curvature-generating proteins build scaffolds on membrane nanotubes

2016 ◽  
Vol 113 (40) ◽  
pp. 11226-11231 ◽  
Author(s):  
Mijo Simunovic ◽  
Emma Evergren ◽  
Ivan Golushko ◽  
Coline Prévost ◽  
Henri-François Renard ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Coarse Grained ◽  
Molecular Scaffolds ◽  
Bar Domain ◽  
Membrane Tube ◽  
Bar Domain Proteins ◽  
Specific Localization ◽  
A Cell ◽  
Dynamics Simulations ◽  
Cooperative Assembly

Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube’s length. Our work implies that the nature of local protein–membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30–40% of a tube’s surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes.

What do nanometer molecular trajectories tell us about heterogeneous cell surface domaines?

1995 ◽  
Vol 53 ◽  
pp. 786-787
Author(s):  
Watt W. Webb
Keyword(s):  
Cell Surface ◽  
Lipid Membranes ◽  
Surface Proteins ◽  
Protein Diffusion ◽  
Coated Pits ◽  
Cell Surface Proteins ◽  
Membrane Heterogeneity ◽  
Fluorescence Photobleaching Recovery ◽  
Photobleaching Recovery ◽  
Plasma Membrane Heterogeneity

Plasma membrane heterogeneity is implicit in the existence of specialized cell surface organelles which are necessary for cellular function; coated pits, post and pre-synaptic terminals, microvillae, caveolae, tight junctions, focal contacts and endothelial polarization are examples. The persistence of these discrete molecular aggregates depends on localized restraint of the constituent molecules within specific domaines in the cell surface by strong intermolecular bonds and/or anchorage to extended cytoskeleton. The observed plasticity of many of organelles and the dynamical modulation of domaines induced by cellular signaling evidence evanescent intermolecular interactions even in conspicuous aggregates. There is also strong evidence that universal restraints on the mobility of cell surface proteins persist virtually everywhere in cell surfaces, not only in the discrete organelles. Diffusion of cell surface proteins is slowed by several orders of magnitude relative to corresponding protein diffusion coefficients in isolated lipid membranes as has been determined by various ensemble average methods of measurement such as fluorescence photobleaching recovery(FPR).

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Super Resolution ◽  
Structure And Function ◽  
And Function ◽  
Super Resolution Microscopy

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

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