scholarly journals Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution

2012 ◽  
Vol 125 (3) ◽  
pp. 570-575 ◽  
Author(s):  
A. Loschberger ◽  
S. van de Linde ◽  
M.-C. Dabauvalle ◽  
B. Rieger ◽  
M. Heilemann ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Super Resolution ◽  
Nuclear Pore ◽  
Central Channel ◽  
Nanometer Resolution ◽  
Resolution Imaging

scholarly journals Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Tino Pleiner ◽  
Mark Bates ◽  
Sergei Trakhanov ◽  
Chung-Tien Lee ◽  
Jan Erik Schliep ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Epitope Mapping ◽  
Super Resolution ◽  
Building Blocks ◽  
Single Step ◽  
Nuclear Pore ◽  
Site Specific ◽  
Mapping Strategy ◽  
Resolution Imaging ◽  
Single Domain Antibodies

Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.

scholarly journals Structural interaction between the nuclear pore complex and a specific translocating RNP particle.

1995 ◽  
Vol 129 (5) ◽  
pp. 1205-1216 ◽  
Author(s):  
H Mehlin ◽  
B Daneholt ◽  
U Skoglund
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Pore ◽  
Initial Contact ◽  
Chironomus Tentans ◽  
Central Channel ◽  
Balbiani Ring ◽  
Salivary Gland Cells ◽  
Larval Salivary Gland ◽  
Nuclear Ring ◽  
Precise Area

The transport of Balbiani ring (BR) premessenger RNP particles in the larval salivary gland cells of the dipteran Chironomus tentans can be followed using electron microscopy. A BR RNP particle consists of an RNP ribbon bent into a ringlike structure. Upon translocation through the nuclear pore complex (NPC), the ribbon is straightened and enters the central channel of the NPC with the 5' end of the transcript in the lead. The translocating ribbon is likely to interact with the central channel but, in addition, the remaining portion of the ribbon ring makes contact with the periphery of the NPC. To determine the nature of this latter interaction, we have now studied the connections between the RNP particle and the border of the NPC during different stages of translocation using electron microscope tomography. It was observed that the 3' terminal domain of the ribbon always touches the nuclear ring of the NPC, but the precise area of contact is variable. Sometimes also a region on the opposite side of the ribbon ring reaches the nuclear ring. The pattern of contacts could be correlated to the stage of translocation, and it was concluded that the particle-nuclear ring interactions reflect a rotation of the ribbon ring in front of the central channel, the rotation being secondary to the successive translocation of the ribbon through the channel. The particle's mode of interaction with the NPC suggests that the initial contact between the 5' end domain of the ribbon and the entrance to the central channel is probably crucial to accomplish the ordered translocation of the premessenger RNP particle through the NPC.

High-Resolution Imaging of the Nuclear Pore Complex by AC Scanning Force Microscopy

2000 ◽  
Vol 1 (2) ◽  
pp. 109-114 ◽  
Author(s):  
Reinat Nevo ◽  
Peter Markiewicz ◽  
Ruti Kapon ◽  
Michael Elbaum ◽  
Ziv Reich
Keyword(s):  
High Resolution ◽  
Nuclear Pore Complex ◽  
Scanning Force Microscopy ◽  
Nuclear Pore ◽  
High Resolution Imaging ◽  
Force Microscopy ◽  
Scanning Force ◽  
Resolution Imaging

scholarly journals Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex

2017 ◽  
Vol 130 (7) ◽  
pp. 1299-1306 ◽  
Author(s):  
Jiong Ma ◽  
Joseph M. Kelich ◽  
Samuel L. Junod ◽  
Weidong Yang
Keyword(s):  
Nuclear Pore Complex ◽  
Super Resolution ◽  
Nuclear Pore ◽  
Resolution Mapping ◽  
Super Resolution Mapping

scholarly journals Super-resolution 3D tomography of interactions and competition in the nuclear pore complex

2016 ◽  
Vol 23 (3) ◽  
pp. 239-247 ◽  
Author(s):  
Jiong Ma ◽  
Alexander Goryaynov ◽  
Weidong Yang
Keyword(s):  
Nuclear Pore Complex ◽  
Super Resolution ◽  
Nuclear Pore ◽  
3D Tomography

scholarly journals Allosteric Regulation in Gating the Central Channel of the Nuclear Pore Complex

Cell ◽  
2015 ◽  
Vol 161 (6) ◽  
pp. 1361-1373 ◽  
Author(s):  
Junseock Koh ◽  
Günter Blobel
Keyword(s):  
Nuclear Pore Complex ◽  
Allosteric Regulation ◽  
Nuclear Pore ◽  
Central Channel

scholarly journals An agent-based model for mRNA export through the nuclear pore complex

2014 ◽  
Vol 25 (22) ◽  
pp. 3643-3653 ◽  
Author(s):  
Mohammad Azimi ◽  
Evgeny Bulat ◽  
Karsten Weis ◽  
Mohammad R. K. Mofrad
Keyword(s):  
Nuclear Pore Complex ◽  
Mrna Export ◽  
Nuclear Pore ◽  
Central Channel ◽  
Agent Based Model ◽  
Agent Based ◽  
Transport Dynamics ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread into the central channel. Of note, our results also suggest that using a single location-monitoring mRNA label may be insufficient to correctly capture the time regime of mRNA threading through the pore and subsequent transport. This has implications for future experimental design to study mRNA transport dynamics.

scholarly journals Super-Resolution by Feedback Imaging: Mechanisms of Translocation through the Nuclear Pore Complex

2013 ◽  
Vol 104 (2) ◽  
pp. 120a
Author(s):  
Francesco Cardarelli ◽  
Luca Lanzano ◽  
Enrico Gratton
Keyword(s):  
Nuclear Pore Complex ◽  
Super Resolution ◽  
Nuclear Pore ◽  
Imaging Mechanisms

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.

scholarly journals Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution

2014 ◽  
Vol 127 (20) ◽  
pp. 4351-4355 ◽  
Author(s):  
Anna Löschberger ◽  
Christian Franke ◽  
Georg Krohne ◽  
Sebastian van de Linde ◽  
Markus Sauer
Keyword(s):  
Electron Microscopy ◽  
Nuclear Pore Complex ◽  
Super Resolution ◽  
Nuclear Pore ◽  
Fluorescence And Electron Microscopy
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