scholarly journals G4C2 repeat RNA mediates the disassembly of the nuclear pore complex in C9orf72 ALS/FTD

2020 ◽  
Author(s):  
Alyssa N Coyne ◽  
Benjamin L Zaepfel ◽  
Lindsey Hayes ◽  
Boris Fitchman ◽  
Yuval Salzberg ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Complex Function ◽  
Super Resolution ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Neuronal Nuclei ◽  
Molecular Events ◽  
Human Neurons

AbstractNucleocytoplasmic transport, controlled by the nuclear pore complex, has recently emerged as a pathomechanism underlying neurodegenerative diseases including C9orf72 ALS/FTD. However, little is known about the underlying molecular events and the underlying biology in human neurons. Using super resolution structured illumination microscopy of twenty three nucleoporins in nuclei from C9orf72 iPSC derived neurons and postmortem human tissue we identify a unique subset of eight nucleoporins lost from human neuronal nuclei. POM121, an integral transmembrane nucleoporin, appears to coordinate the composition of the nucleoporins within human neuronal nuclei ultimately impacting nucleocytoplasmic transport, and subsequent cellular toxicity in C9orf72 iPSNs. These data suggest that POM121 is a critical nucleoporin in the maintenance of the nuclear localization of specific nucleoporins in human neurons. Moreover, loss of nuclear POM121, as a result of expanded C9orf72 ALS/FTD repeat RNA, initiates a pathological cascade affecting nucleoporin composition within neuronal nuclei, nuclear pore complex function, and overall downstream neuronal survival.

scholarly journals The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Alyssa N. Coyne ◽  
Jeffrey D. Rothstein
Keyword(s):  
Nuclear Pore Complex ◽  
Mammalian Cells ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Structured Illumination Microscopy ◽  
Neuronal Nuclei ◽  
Complex Injury ◽  
Sporadic Als ◽  
Human Neurons

AbstractNuclear pore complex injury has recently emerged as an early and significant contributor to familial and sporadic ALS disease pathogenesis. However, the molecular events leading to this pathological phenomenon characterized by the reduction of specific nucleoporins from neuronal nuclear pore complexes remain largely unknown. This is due in part to a lack of knowledge regarding the biological pathways and proteins underlying nuclear pore complex homeostasis specifically in human neurons. We have recently uncovered that aberrant nuclear accumulation of the ESCRT-III protein CHMP7 initiates nuclear pore complex in familial and sporadic ALS neurons. In yeast and non-neuronal mammalian cells, nuclear relocalization of CHMP7 has been shown to recruit the ESCRT-III proteins CHMP4B, CHMP2B, and VPS4 to facilitate nuclear pore complex and nuclear envelope repair and homeostasis. Here, using super resolution structured illumination microscopy, we find that neither CHMP4B nor CHMP2B are increased in ALS neuronal nuclei. In contrast, VPS4 expression is significantly increased in ALS neuronal nuclei prior to the emergence of nuclear pore injury in a CHMP7 dependent manner. However, unlike our prior CHMP7 knockdown studies, impaired VPS4 function does not mitigate alterations to the NPC and the integral transmembrane nucleoporin POM121. Collectively our data suggest that while alterations in VPS4 subcellular localization appear to be coincident with nuclear pore complex injury, therapeutic efforts to mitigate this pathogenic cascade should be targeted towards upstream events such as the nuclear accumulation of CHMP7 as we have previously described.

scholarly journals Applications of Super Resolution Expansion Microscopy in Yeast

2021 ◽  
Vol 9 ◽  
Author(s):  
Liwen Chen ◽  
Longfang Yao ◽  
Li Zhang ◽  
Yiyan Fei ◽  
Lan Mi ◽  
...  
Keyword(s):  
Super Resolution ◽  
Imaging Study ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Preparation Technique ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Imaging ◽  
Conventional Microscopy ◽  
Related Proteins

Super-resolution microscopy includes multiple techniques in optical microscopy that enable sub-diffraction resolution fluorescence imaging of cellular structures. Expansion microscopy (EXM) is a method of physical expansion to obtain super-resolution images of a biological sample on conventional microscopy. We present images of yeast organelles, applying the combination of super-resolution and ExM techniques. When preparing pre-expanded samples, conventional methods lead to breakage of dividing yeast cells and difficulties in studying division-related proteins. Here, we describe an improved sample preparation technique that avoids such damage. ExM in combination with Airyscan and structured illumination microscopy (SIM) collected sub-cellular structural images of nuclear pore complex, septin, and a-tubulin in yeast. Our method of expansion in yeast is well-suited for super-resolution imaging study of yeast.

Structure, assembly and interactions of the nuclear lamina and the nuclear pore complex

1992 ◽  
Vol 50 (1) ◽  
pp. 490-491
Author(s):  
N. Panté ◽  
M. Jarnik ◽  
E. Heitlinger ◽  
U. Aebi
Keyword(s):  
Nuclear Pore Complex ◽  
Divalent Cations ◽  
Nuclear Lamina ◽  
Dynamic Structure ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Cytoplasmic Filaments ◽  
Radial Spokes ◽  
Nuclear Ring ◽  
Central Plug

The nuclear pore complex (NPC) is a ∼120 MD supramolecular machine implicated in nucleocytoplasmic transport, that is embedded in the double-membraned nuclear envelope (NE). The basic framework of the ∼120 nm diameter NPC consists of a 32 MD cytoplasmic ring, a 66 MD ‘plug-spoke’ assembly, and a 21 MD nuclear ring. The ‘central plug’ seen in en face views of the NPC reveals a rather variable appearance indicating that it is a dynamic structure. Projecting from the cytoplasmic ring are 8 short, twisted filaments (Fig. 1a), whereas the nuclear ring is topped with a ‘fishtrap’ made of 8 thin filaments that join distally to form a fragile, 30-50 nm distal diameter ring centered above the NPC proper (Fig. 1b). While the cytoplasmic filaments are sensitive to proteases, they as well as the nuclear fishtraps are resistant to RNase treatment. Removal of divalent cations destabilizes the distal rings and thereby opens the fishtraps, addition causes them to reform. Protruding from the tips of the radial spokes into perinuclear space are ‘knobs’ that might represent the large lumenal domain of gp210, a membrane-spanning glycoprotein (Fig. 1c) which, in turn, may play a topogenic role in membrane folding and/or act as a membrane-anchoring site for the NPC. The lectin wheat germ agglutinin (WGA) which is known to recognize the ‘nucleoporins’, a family of glycoproteins having O-linked N-acetyl-glucosamine, is found in two locations on the NPC (Fig. 1. d-f): (i) whereas the cytoplasmic filaments appear unlabelled (Fig. 1d&e), WGA-gold labels sites between the central plug and the cytoplasmic ring (Fig. le; i.e., at a radius of 25-35 nm), and (ii) it decorates the distal ring of the nuclear fishtraps (Fig. 1, d&f; arrowheads).

scholarly journals Two-dimensional TIRF-SIM–traction force microscopy (2D TIRF-SIM-TFM)

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Liliana Barbieri ◽  
Huw Colin-York ◽  
Kseniya Korobchevskaya ◽  
Di Li ◽  
Deanna L. Wolfson ◽  
...  
Keyword(s):  
Resolution Enhancement ◽  
Traction Force ◽  
Super Resolution ◽  
Traction Force Microscopy ◽  
Two Dimensional ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Force Microscopy ◽  
Health And Disease ◽  
Spatio Temporal

AbstractQuantifying small, rapidly evolving forces generated by cells is a major challenge for the understanding of biomechanics and mechanobiology in health and disease. Traction force microscopy remains one of the most broadly applied force probing technologies but typically restricts itself to slow events over seconds and micron-scale displacements. Here, we improve >2-fold spatially and >10-fold temporally the resolution of planar cellular force probing compared to its related conventional modalities by combining fast two-dimensional total internal reflection fluorescence super-resolution structured illumination microscopy and traction force microscopy. This live-cell 2D TIRF-SIM-TFM methodology offers a combination of spatio-temporal resolution enhancement relevant to forces on the nano- and sub-second scales, opening up new aspects of mechanobiology to analysis.

scholarly journals Double moiré localized plasmon structured illumination microscopy

Nanophotonics ◽  
2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Ruslan Röhrich ◽  
A. Femius Koenderink
Keyword(s):  
Near Field ◽  
Super Resolution ◽  
Reconstruction Algorithm ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Experimental Realization ◽  
Spatial Frequencies ◽  
Wide Range ◽  
Localized Plasmon ◽  
Plasmonic Grating

AbstractStructured illumination microscopy (SIM) is a well-established fluorescence imaging technique, which can increase spatial resolution by up to a factor of two. This article reports on a new way to extend the capabilities of structured illumination microscopy, by combining ideas from the fields of illumination engineering and nanophotonics. In this technique, plasmonic arrays of hexagonal symmetry are illuminated by two obliquely incident beams originating from a single laser. The resulting interference between the light grating and plasmonic grating creates a wide range of spatial frequencies above the microscope passband, while still preserving the spatial frequencies of regular SIM. To systematically investigate this technique and to contrast it with regular SIM and localized plasmon SIM, we implement a rigorous simulation procedure, which simulates the near-field illumination of the plasmonic grating and uses it in the subsequent forward imaging model. The inverse problem, of obtaining a super-resolution (SR) image from multiple low-resolution images, is solved using a numerical reconstruction algorithm while the obtained resolution is quantitatively assessed. The results point at the possibility of resolution enhancements beyond regular SIM, which rapidly vanishes with the height above the grating. In an initial experimental realization, the existence of the expected spatial frequencies is shown and the performance of compatible reconstruction approaches is compared. Finally, we discuss the obstacles of experimental implementations that would need to be overcome for artifact-free SR imaging.

scholarly journals Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance

2009 ◽  
Vol 185 (3) ◽  
pp. 475-491 ◽  
Author(s):  
Evgeny Onischenko ◽  
Leslie H. Stanton ◽  
Alexis S. Madrid ◽  
Thomas Kieselbach ◽  
Karsten Weis
Keyword(s):  
Nuclear Pore Complex ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Interaction Network ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Binding Partners ◽  
Interaction Partners ◽  
Redundant Functions

The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.

scholarly journals High-fidelity structured illumination microscopy by point-spread-function engineering

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Gang Wen ◽  
Simin Li ◽  
Linbo Wang ◽  
Xiaohu Chen ◽  
Zhenglong Sun ◽  
...  
Keyword(s):  
Point Spread Function ◽  
Super Resolution ◽  
Reconstruction Algorithm ◽  
High Fidelity ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Imaging Tool ◽  
Point Spread ◽  
Spread Function ◽  
Point Spread Function Engineering

AbstractStructured illumination microscopy (SIM) has become a widely used tool for insight into biomedical challenges due to its rapid, long-term, and super-resolution (SR) imaging. However, artifacts that often appear in SIM images have long brought into question its fidelity, and might cause misinterpretation of biological structures. We present HiFi-SIM, a high-fidelity SIM reconstruction algorithm, by engineering the effective point spread function (PSF) into an ideal form. HiFi-SIM can effectively reduce commonly seen artifacts without loss of fine structures and improve the axial sectioning for samples with strong background. In particular, HiFi-SIM is not sensitive to the commonly used PSF and reconstruction parameters; hence, it lowers the requirements for dedicated PSF calibration and complicated parameter adjustment, thus promoting SIM as a daily imaging tool.

The FDTD Analysis of Near-Field Response for Microgroove Structure With Standing Wave Illumination for the Realization of Coherent Structured Illumination Microscopy

2021 ◽  
Author(s):  
Yizhao Guan ◽  
Hiromasa Kume ◽  
Shotaro Kadoya ◽  
Masaki Michihata ◽  
Satoru Takahashi
Keyword(s):  
Standing Wave ◽  
Incident Angle ◽  
Near Field ◽  
Super Resolution ◽  
Structured Illumination ◽  
Field Phase ◽  
Structured Illumination Microscopy ◽  
Depth Measurement ◽  
Field Response ◽  
Depth Dependency

Abstract Microstructures are widely used in the manufacture of functional surfaces. An optical-based super-resolution, non-invasive method is preferred for the inspection of surfaces with massive microstructures. The Structured Illumination Microscopy (SIM) uses standing-wave illumination to reach optical super-resolution. Recently, coherent SIM is being studied. It can obtain not only the super-resolved intensity distribution but also the phase and amplitude distribution of the sample surface beyond the diffraction limit. By analysis of the phase-depth dependency, the depth measurement for microgroove structures with coherent SIM is expected. FDTD analysis is applied for observing the near-field response of microgroove under the standing-wave illumination. The near-field phase shows depth dependency in this analysis. Moreover, the effects from microgroove width, the incident angle, and the relative position between the standing-wave peak and center of the microgroove are investigated. It is found the near-field phase change can measure depth until 200 nm (aspect ratio 1) with an error of up to 20.4 nm in the case that the microgroove width is smaller than half of the wavelength.

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