scholarly journals Nuclear Import of Adeno-Associated Viruses Imaged by High-Speed Single-Molecule Microscopy

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 167
Author(s):  
Samuel L. Junod ◽  
Jason Saredy ◽  
Weidong Yang
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Nuclear Import ◽  
High Speed ◽  
Single Point ◽  
Live Cells ◽  
Nuclear Pore ◽  
Transport Dynamics ◽  
Technical Advances ◽  
Live Cell Microscopy

Understanding the detailed nuclear import kinetics of adeno-associated virus (AAV) through the nuclear pore complex (NPC) is essential for the application of AAV capsids as a nuclear delivery instrument as well as a target for drug development. However, a comprehensive understanding of AAV transport through the sub-micrometer NPCs in live cells calls for new techniques that can conquer the limitations of conventional fluorescence microscopy and electron microscopy. With recent technical advances in single-molecule fluorescence microscopy, we are now able to image the entire nuclear import process of AAV particles and also quantify the transport dynamics of viral particles through the NPCs in live human cells. In this review, we initially evaluate the necessity of single-molecule live-cell microscopy in the study of nuclear import for AAV particles. Then, we detail the application of high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy in tracking the entire process of nuclear import for AAV particles. Finally, we summarize the major findings for AAV nuclear import by using SPEED microscopy.

Distinct roles of nuclear basket proteins in directing the passage of mRNA through the nuclear pore

2021 ◽  
Vol 118 (37) ◽  
pp. e2015621118
Author(s):  
Yichen Li ◽  
Vasilisa Aksenova ◽  
Mark Tingey ◽  
Jingjie Yu ◽  
Ping Ma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Nuclear Export ◽  
Copy Number ◽  
Messenger Rna ◽  
High Speed ◽  
Three Dimensional ◽  
Live Cells ◽  
Nuclear Pore ◽  
Stoichiometric Ratio

The in vivo characterization of the exact copy number and the specific function of each composite protein within the nuclear pore complex (NPC) remains both desirable and challenging. Through the implementation of live-cell high-speed super-resolution single-molecule microscopy, we first quantified the native copies of nuclear basket (BSK) proteins (Nup153, Nup50, and Tpr) prior to knocking them down in a highly specific manner via an auxin-inducible degron strategy. Second, we determined the specific roles that BSK proteins play in the nuclear export kinetics of model messenger RNA (mRNA) substrates. Finally, the three-dimensional (3D) nuclear export routes of these mRNA substrates through native NPCs in the absence of specific BSK proteins were obtained and further validated via postlocalization computational simulations. We found that these BSK proteins possess the stoichiometric ratio of 1:1:1 and play distinct roles in the nuclear export of mRNAs within live cells. The absence of Tpr from the NPC predominantly reduces the probability of nuclear mRNAs entering the NPC for export. Complete depletion of Nup153 and Nup50 results in an mRNA nuclear export efficiency decrease of approximately four folds. mRNAs can gain their maximum successful export efficiency as the copy number of Nup153 increased from zero to only half the full complement natively within the NPC. Lastly, the absence of Tpr or Nup153 seems to alter the 3D export routes of mRNAs as they pass through the NPC. However, the removal of Nup50 alone has almost no impact upon mRNA export route and kinetics.

scholarly journals Nuclear accessibility of β-actin mRNA is measured by 3D single-molecule real-time tracking

2015 ◽  
Vol 209 (4) ◽  
pp. 609-619 ◽  
Author(s):  
Carlas S. Smith ◽  
Stephan Preibisch ◽  
Aviva Joseph ◽  
Sara Abrahamsson ◽  
Bernd Rieger ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Messenger Rna ◽  
Three Dimensional ◽  
Spatial Inhomogeneity ◽  
Live Cells ◽  
Nuclear Pore ◽  
Cell Nuclei ◽  
Entire Volume ◽  
Time Required

Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that β-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 µm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization.

scholarly journals Nuclear import time and transport efficiency depend on importin β concentration

2006 ◽  
Vol 174 (7) ◽  
pp. 951-961 ◽  
Author(s):  
Weidong Yang ◽  
Siegfried M. Musser
Keyword(s):  
Single Molecule ◽  
Nuclear Import ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Transport Efficiency ◽  
Bidirectional Transport ◽  
Pore Complexes ◽  
Transit Speed ◽  
And Control

Although many components and reaction steps necessary for bidirectional transport across the nuclear envelope (NE) have been characterized, the mechanism and control of cargo migration through nuclear pore complexes (NPCs) remain poorly understood. Single-molecule fluorescence microscopy was used to track the movement of cargos before, during, and after their interactions with NPCs. At low importin β concentrations, about half of the signal-dependent cargos that interacted with an NPC were translocated across the NE, indicating a nuclear import efficiency of ∼50%. At high importin β concentrations, the import efficiency increased to ∼80% and the transit speed increased approximately sevenfold. The transit speed and import efficiency of a signal-independent cargo was also increased by high importin β concentrations. These results demonstrate that maximum nucleocytoplasmic transport velocities can be modulated by at least ∼10-fold by the importin β concentration and therefore suggest a potential mechanism for regulating the speed of cargo traffic across the NE.

scholarly journals Tilting and Wobble of Myosin V by High-Speed Single-Molecule Polarized Fluorescence Microscopy

2013 ◽  
Vol 104 (6) ◽  
pp. 1263-1273 ◽  
Author(s):  
John F. Beausang ◽  
Deborah Y. Shroder ◽  
Philip C. Nelson ◽  
Yale E. Goldman
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
High Speed ◽  
Polarized Fluorescence ◽  
Myosin V

scholarly journals Nuclear transport of single molecules

2005 ◽  
Vol 168 (2) ◽  
pp. 233-243 ◽  
Author(s):  
Ulrich Kubitscheck ◽  
David Grünwald ◽  
Andreas Hoekstra ◽  
Daniel Rohleder ◽  
Thorsten Kues ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Nuclear Pore Complex ◽  
Binding Sites ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Far Field ◽  
Permeabilized Cells ◽  
Cytoplasmic Filaments ◽  
Transport Receptor

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 ± 0.2 and 7.1 ± 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs.

Scanning x-ray fluorescence microscopy and principal component analysis

1992 ◽  
Vol 50 (2) ◽  
pp. 1752-1753
Author(s):  
Brian Cross
Keyword(s):  
Principal Component Analysis ◽  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
High Speed ◽  
Image Acquisition ◽  
Principal Component ◽  
Fluorescence Microscope ◽  
Acquisition Rate ◽  
X Ray ◽  
Speed Up

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.

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