Perforating the nuclear boundary – how nuclear pore complexes assemble

Weberruß, Marion; Antonin, Wolfram

doi:10.1242/jcs.194753

Cited by 57 publications

(59 citation statements)

References 84 publications

(122 reference statements)

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“…This phenotype may reflect an early stage of the apoptotic programme, which could be triggered in the HSPA5 knockout cells through ectopic activation of the UPR (Kihlmark et al , ; Pfaffenbach & Lee, ). The clustergram revealed that the perturbation profiles of NUP160 and NUP98 form a distinct cluster compared to the profiles of other components of the NPC (Weberruss & Antonin, ), which is caused by smaller differences across multiple mAb414 staining texture features (Figs C and D, and EV6A). Such a distinction is impossible to detect without multivariate profiling of single cells and is qualitatively confirmed by examining images of phenotypically perturbed cells.…”

Section: Resultsmentioning

confidence: 94%

“…Boxes indicate the 1 st and 3 rd quartile of the data distribution. The whiskers indicate the maximum and minimum datapoints within the 1 st quartile minus 1.5 times the interquartile range (IQR) of the data and the third quartile plus 1.5 times the IQR. Schematic representation of the NPC, adapted from Weberruss and Antonin (Weberruss & Antonin, ). Cells were transfected with three independent plasmids targeting each of the genes NUP62 , HSPA5 , NUP107 or NUP98 . Mean feature profiles were obtained from all transfected cells, or the subset of T(+) cells with a high PV.…”

Section: Resultsmentioning

confidence: 99%

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Large‐scale image‐based profiling of single‐cell phenotypes in arrayed CRISPR‐Cas9 gene perturbation screens

Groot

Lüthi

Lindsay

et al. 2018

Molecular Systems Biology

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High‐content imaging using automated microscopy and computer vision allows multivariate profiling of single‐cell phenotypes. Here, we present methods for the application of the CISPR‐Cas9 system in large‐scale, image‐based, gene perturbation experiments. We show that CRISPR‐Cas9‐mediated gene perturbation can be achieved in human tissue culture cells in a timeframe that is compatible with image‐based phenotyping. We developed a pipeline to construct a large‐scale arrayed library of 2,281 sequence‐verified CRISPR‐Cas9 targeting plasmids and profiled this library for genes affecting cellular morphology and the subcellular localization of components of the nuclear pore complex (NPC). We conceived a machine‐learning method that harnesses genetic heterogeneity to score gene perturbations and identify phenotypically perturbed cells for in‐depth characterization of gene perturbation effects. This approach enables genome‐scale image‐based multivariate gene perturbation profiling using CRISPR‐Cas9.

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Large‐scale image‐based profiling of single‐cell phenotypes in arrayed CRISPR‐Cas9 gene perturbation screens

Groot

Lüthi

Lindsay

et al. 2018

Molecular Systems Biology

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“…Nups are involved in the structural scaffolding of the NPC and in the recruitment of specific carriers and soluble cargos to allow multiple nuclear transport pathways. The NPC structure has been extensively reviewed in recent years and will not be further discussed here [111][112][113].…”

Section: Nuclear Pore and Transportmentioning

confidence: 99%

Caspases rule the intracellular trafficking cartel

2017

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During apoptosis, caspases feast on several hundreds of cellular proteins to orchestrate rapid cellular demise. Indeed, caspases are known to get a taste of every cellular process in one way or another, activating some, but most often shutting them down. Thus, it is not surprising that caspases proteolyze proteins involved in intracellular trafficking with particularly devastating consequences for this important process. This review article focuses on how caspases target the machinery responsible for smuggling goods within and outside the cell.

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“…S1). In the latter 149 case, there is an emerging consensus that NPC assembly begins from the inside of the nucleus on regulators with a potential burst of this assembly mechanism in early G1 (Doucet et al, 2010;154 Otsuka et al, 2016;Weberruss and Antonin, 2016). If the Torsin knockout phenotype does 155 indeed represent a stalling in NPC biogenesis, we would expect to observe the first signs of bleb 156 formation early in G1.…”

Section: Nuclear Envelope Herniations Form In Interphase 144mentioning

confidence: 99%

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

Rampello

Laudermilch

Vishnoi

et al. 2019

Preprint

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32Nuclear envelope herniations (blebs) containing FG-nucleoporins and ubiquitin are the 33 phenotypic hallmark of Torsin ATPase manipulation. Both the dynamics of blebbing and the 34 connection to nuclear pore biogenesis remain poorly understood. We employ a proteomics-based 35 approach to identify MLF2 as a luminal component of the bleb. Using an MLF2-based live cell 36imaging platform, we demonstrate that NE blebbing occurs rapidly and synchronously 37 immediately after nuclear envelope reformation during mitosis. Bleb formation is independent of 38 ubiquitin conjugation within the bleb, but strictly dependent on POM121, a transmembrane 39 nucleoporin essential for interphase nuclear pore biogenesis. Nup358, a late marker for 40 interphase nuclear pore complex (NPC) biogenesis, is underrepresented relative to FG 41 nucleoporins in nuclear envelopes of Torsin-deficient cells. The kinetics of bleb formation, its 42 dependence on POM121, and a reduction of mature NPCs in Torsin deficient cells lead us to 43 conclude that the hallmark phenotype of Torsin manipulation represents the accumulation of 44 stalled NPC assembly intermediates. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 We previously presented a system that resolves both of these limitations by generating a 93 quadruple Torsin deletion HeLa cell line (designated 4TorKO) in which all four Torsin genes 94 (TorsinA, TorsinB, Torsin 2A and Torsin3A) have been deleted using CRISPR/Cas9 genome 95 engineering. This 4TorKO cell line abundantly exhibits the hallmark cellular phenotype of NE 96 blebbing in which the inner nuclear membrane (INM) bulges into the perinuclear space (PNS) to 97 form an omega-shaped herniation. Ubiquitin (Ub) conjugates of the K48 linkage type are 98 enriched in the lumen of the bleb in 4TorKO cells and in mouse models of Torsin dysfunction 99

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Perforating the nuclear boundary – how nuclear pore complexes assemble

Cited by 57 publications

References 84 publications

Large‐scale image‐based profiling of single‐cell phenotypes in arrayed CRISPR‐Cas9 gene perturbation screens

Large‐scale image‐based profiling of single‐cell phenotypes in arrayed CRISPR‐Cas9 gene perturbation screens

Caspases rule the intracellular trafficking cartel

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

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