scholarly journals Torsin ATPases are required to complete nuclear pore complex biogenesis in interphase

2019 ◽  
Author(s):  
Anthony J. Rampello ◽  
Ethan Laudermilch ◽  
Nidhi Vishnoi ◽  
Sarah M. Prohet ◽  
Lin Shao ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Nuclear Pore ◽  
Ubiquitin Conjugation ◽  
Bleb Formation ◽  
Kinetics Of

AbstractNuclear envelope herniations (blebs) containing FG-nucleoporins and ubiquitin are the phenotypic hallmark of Torsin ATPase manipulation. Both the dynamics of blebbing and the connection to nuclear pore biogenesis remain poorly understood. We employ a proteomics-based approach to identify MLF2 as a luminal component of the bleb. Using an MLF2-based live cell imaging platform, we demonstrate that NE blebbing occurs rapidly and synchronously immediately after nuclear envelope reformation during mitosis. Bleb formation is independent of ubiquitin conjugation within the bleb, but strictly dependent on POM121, a transmembrane nucleoporin essential for interphase nuclear pore biogenesis. Nup358, a late marker for interphase nuclear pore complex (NPC) biogenesis, is underrepresented relative to FG nucleoporins in nuclear envelopes of Torsin-deficient cells. The kinetics of bleb formation, its dependence on POM121, and a reduction of mature NPCs in Torsin deficient cells lead us to conclude that the hallmark phenotype of Torsin manipulation represents the accumulation of stalled NPC assembly intermediates.

scholarly journals Torsin ATPase deficiency leads to defects in nuclear pore biogenesis and sequestration of MLF2

2020 ◽  
Vol 219 (6) ◽  
Author(s):  
Anthony J. Rampello ◽  
Ethan Laudermilch ◽  
Nidhi Vishnoi ◽  
Sarah M. Prophet ◽  
Lin Shao ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Myeloid Leukemia ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Nuclear Pore ◽  
Ubiquitin Conjugation ◽  
Bleb Formation ◽  
Kinetics Of

Nuclear envelope herniations (blebs) containing FG-nucleoporins and ubiquitin are the phenotypic hallmark of Torsin ATPase manipulation. Both the dynamics of blebbing and the connection to nuclear pore biogenesis remain poorly understood. We employ a proteomics-based approach to identify myeloid leukemia factor 2 (MLF2) as a luminal component of the bleb. Using an MLF2-based live-cell imaging platform, we demonstrate that nuclear envelope blebbing occurs rapidly and synchronously immediately after nuclear envelope reformation during mitosis. Bleb formation is independent of ubiquitin conjugation within the bleb, but strictly dependent on POM121, a transmembrane nucleoporin essential for interphase nuclear pore biogenesis. Nup358, a late marker for interphase nuclear pore complex (NPC) biogenesis, is underrepresented relative to FG-nucleoporins in nuclear envelopes of Torsin-deficient cells. The kinetics of bleb formation, its dependence on POM121, and a reduction of mature NPCs in Torsin-deficient cells lead us to conclude that the hallmark phenotype of Torsin manipulation represents aberrant NPC intermediates.

scholarly journals Live Cell Imaging Of The Kinetics Of Ligand Binding At The Human Adenosine A3 Receptor

2009 ◽  
Vol 96 (3) ◽  
pp. 677a
Author(s):  
Lauren T. May ◽  
Stephen J. Briddon ◽  
Stephen J. Hill
Keyword(s):  
Ligand Binding ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Adenosine A3 Receptor ◽  
Kinetics Of

scholarly journals Imaging of nanoparticle uptake and kinetics of intracellular trafficking in individual cells

Nanoscale ◽  
2021 ◽  
Author(s):  
Natalia Vtyurina ◽  
Christoffer Åberg ◽  
Anna Salvati
Keyword(s):  
Intracellular Trafficking ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Drug Carriers ◽  
Live Cell ◽  
Nanoparticle Uptake ◽  
Kinetics Of

Live cell imaging is a powerful tool to understand how nano-sized objects, such as drug carriers used for nanomedicine applications, are taken up and trafficked by cells. Here we visualized...

scholarly journals Systematic Deletion and Mitotic Localization of the Nuclear Pore Complex Proteins of Aspergillus nidulans

2006 ◽  
Vol 17 (12) ◽  
pp. 4946-4961 ◽  
Author(s):  
Aysha H. Osmani ◽  
Jonathan Davies ◽  
Hui-Lin Liu ◽  
Aaron Nile ◽  
Stephen A. Osmani
Keyword(s):  
Aspergillus Nidulans ◽  
Nuclear Pore Complex ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Nuclear Transport ◽  
Essential Genes ◽  
Nuclear Pore ◽  
Systematic Analysis ◽  
Insight Into ◽  
Mitotic Chromatin

To define the extent of the modification of the nuclear pore complex (NPC) during Aspergillus nidulans closed mitosis, a systematic analysis of nuclear transport genes has been completed. Thirty genes have been deleted defining 12 nonessential and 18 essential genes. Several of the nonessential deletions caused conditional phenotypes and self-sterility, whereas deletion of some essential genes caused defects in nuclear structure. Live cell imaging of endogenously tagged NPC proteins (Nups) revealed that during mitosis 14 predicted peripheral Nups, including all FG repeat Nups, disperse throughout the cell. A core mitotic NPC structure consisting of membrane Nups, all components of the An-Nup84 subcomplex, An-Nup170, and surprisingly, An-Gle1 remained throughout mitosis. We propose this minimal mitotic NPC core provides a conduit across the nuclear envelope and acts as a scaffold to which dispersed Nups return during mitotic exit. Further, unlike other dispersed Nups, An-Nup2 locates exclusively to mitotic chromatin, suggesting it may have a novel mitotic role in addition to its nuclear transport functions. Importantly, its deletion causes lethality and defects in DNA segregation. This work defines the dramatic changes in NPC composition during A. nidulans mitosis and provides insight into how NPC disassembly may be integrated with mitosis.

scholarly journals Dissecting Torsin/cofactor function at the nuclear envelope: a genetic study

2016 ◽  
Vol 27 (25) ◽  
pp. 3964-3971 ◽  
Author(s):  
Ethan Laudermilch ◽  
Pei-Ling Tsai ◽  
Morven Graham ◽  
Elizabeth Turner ◽  
Chenguang Zhao ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Structural Analysis ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Compositional Analysis ◽  
Immunogold Labeling ◽  
Nuclear Pore ◽  
Functional Link ◽  
Experimental Platform ◽  
Bleb Formation

The human genome encodes four Torsin ATPases, the functions of which are poorly understood. In this study, we use CRISPR/Cas9 engineering to delete all four Torsin ATPases individually and in combination. Using nuclear envelope (NE) blebbing as a phenotypic measure, we establish a direct correlation between the number of inactivated Torsin alleles and the occurrence of omega-shaped herniations within the lumen of the NE. A similar, although not identical, redundancy is observed for LAP1 and LULL1, which serve as regulatory cofactors for a subset of Torsin ATPases. Unexpectedly, deletion of Tor2A in a TorA/B/3A-deficient background results in a stark increase of bleb formation, even though Tor2A does not respond to LAP1/LULL1 stimulation. The robustness of the observed phenotype in Torsin-deficient cells enables a structural analysis via electron microscopy tomography and a compositional analysis via immunogold labeling. Ubiquitin and nucleoporins were identified as distinctively localizing components of the omega-shaped bleb structure. These findings suggest a functional link between the Torsin/cofactor system and NE/nuclear pore complex biogenesis or homeostasis and establish a Torsin-deficient cell line as a valuable experimental platform with which to decipher Torsin function.

scholarly journals Live cell imaging and electron microscopy reveal dynamic processes of BAF-directed nuclear envelope assembly

2008 ◽  
Vol 121 (15) ◽  
pp. 2540-2554 ◽  
Author(s):  
T. Haraguchi ◽  
T. Kojidani ◽  
T. Koujin ◽  
T. Shimi ◽  
H. Osakada ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Dynamic Processes ◽  
Nuclear Envelope Assembly

scholarly journals Formation of the postmitotic nuclear envelope from extended ER cisternae precedes nuclear pore assembly

2011 ◽  
Vol 194 (3) ◽  
pp. 425-440 ◽  
Author(s):  
Lei Lu ◽  
Mark S. Ladinsky ◽  
Tomas Kirchhausen
Keyword(s):  
Nuclear Envelope ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Current Model ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Envelope Formation ◽  
Pore Complexes ◽  
Nuclear Pore Assembly ◽  
A Current

During mitosis, the nuclear envelope merges with the endoplasmic reticulum (ER), and nuclear pore complexes are disassembled. In a current model for reassembly after mitosis, the nuclear envelope forms by a reshaping of ER tubules. For the assembly of pores, two major models have been proposed. In the insertion model, nuclear pore complexes are embedded in the nuclear envelope after their formation. In the prepore model, nucleoporins assemble on the chromatin as an intermediate nuclear pore complex before nuclear envelope formation. Using live-cell imaging and electron microscope tomography, we find that the mitotic assembly of the nuclear envelope primarily originates from ER cisternae. Moreover, the nuclear pore complexes assemble only on the already formed nuclear envelope. Indeed, all the chromatin-associated Nup107–160 complexes are in single units instead of assembled prepores. We therefore propose that the postmitotic nuclear envelope assembles directly from ER cisternae followed by membrane-dependent insertion of nuclear pore complexes.

scholarly journals High Temporal Resolution 3D Live-Cell Imaging of Budding Yeast Meiosis Defines Discontinuous Actin/Telomere-Mediated Chromosome Motion, Correlated Nuclear Envelope Deformation and Actin Filament Dynamics

2021 ◽  
Vol 9 ◽  
Author(s):  
Tadasu Nozaki ◽  
Frederick Chang ◽  
Beth Weiner ◽  
Nancy Kleckner
Keyword(s):  
Nuclear Envelope ◽  
Temporal Resolution ◽  
Meiotic Prophase ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Budding Yeast ◽  
Three Dimensional ◽  
Live Cell ◽  
High Temporal Resolution ◽  
Homolog Pairing

Chromosome movement is prominent at mid-meiotic prophase and is proposed to enhance the efficiency and/or stringency of homolog pairing and/or to help prevent or resolve topological entanglements. Here, we combine fluorescent repressor operator system (FROS) labeling with three-dimensional (3D) live-cell imaging at high spatio-temporal resolution to define the detailed kinetics of mid-meiotic prophase motion for a single telomere-proximal locus in budding yeast. Telomere motions can be grouped into three general categories: (i) pauses, in which the telomere “jiggles in place”; (ii) rapid, straight/curvilinear motion which reflects Myo2/actin-mediated transport of the monitored telomere; and (iii) slower directional motions, most of which likely reflect indirectly promoted motion of the monitored telomere in coordination with actin-mediated motion of an unmarked telomere. These and other findings highlight the importance of dynamic assembly/disassembly of telomere/LINC/actin ensembles and also suggest important roles for nuclear envelope deformations promoted by actin-mediated telomere/LINC movement. The presented low-SNR (signal-to-noise ratio) imaging methodology provides opportunities for future exploration of homolog pairing and related phenomena.

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