An In Vitro System to Study Nuclear Envelope Breakdown

Marino, Joseph; Champion, Lysie; Wandke, Cornelia; Horváth, Péter; Mayr, Monika I.; Kutay, Ulrike

doi:10.1016/b978-0-12-417160-2.00012-6

Cited by 6 publications

(15 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To test whether PLK1 supports NPC disassembly, we applied a previously developed in vitro system that recapitulates mitotic NPC disintegration on nuclei of semi-permeabilized HeLa cells upon addition of mitotic HeLa cell extracts ( Laurell et al., 2011 , Marino et al., 2014 ). This quantitative visual assay allows studying both the kinetics of NE permeabilization based on nuclear influx of a fluorescently labeled dextran and the release of GFP-labeled nucleoporins from NPCs by time-lapse confocal microscopy ( Figure 1 A).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Mitotic Disassembly of Nuclear Pore Complexes Involves CDK1- and PLK1-Mediated Phosphorylation of Key Interconnecting Nucleoporins

et al. 2017

Self Cite

View full text Add to dashboard Cite

SummaryDuring interphase, the nuclear envelope (NE) serves as a selective barrier between cytosol and nucleoplasm. When vertebrate cells enter mitosis, the NE is dismantled in the process of nuclear envelope breakdown (NEBD). Disassembly of nuclear pore complexes (NPCs) is a key aspect of NEBD, required for NE permeabilization and formation of a cytoplasmic mitotic spindle. Here, we show that both CDK1 and polo-like kinase 1 (PLK1) support mitotic NPC disintegration by hyperphosphorylation of Nup98, the gatekeeper nucleoporin, and Nup53, a central nucleoporin linking the inner NPC scaffold to the pore membrane. Multisite phosphorylation of Nup53 critically contributes to its liberation from its partner nucleoporins, including the pore membrane protein NDC1. Initial steps of NPC disassembly in semi-permeabilized cells can be reconstituted by a cocktail of mitotic kinases including cyclinB-CDK1, NIMA, and PLK1, suggesting that the unzipping of nucleoporin interactions by protein phosphorylation is an important principle underlying mitotic NE permeabilization.

show abstract

Section: Resultsmentioning

confidence: 99%

“…In vitro NPC disassembly on semi-permeabilized HeLa cells in presence of mitotic HeLa cell extract was performed as described ( Marino et al., 2014 ). Briefly, HeLa cells expressing the indicated GFP-tagged nucleoporin were seeded into an Ibidi imaging chamber 12–24 hr before the NEBD assay.…”

Section: Methodsmentioning

confidence: 99%

Mitotic Disassembly of Nuclear Pore Complexes Involves CDK1- and PLK1-Mediated Phosphorylation of Key Interconnecting Nucleoporins

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…Examining the binding interactions of proteins that localize to the cytosolic face of internal membranes may also be appropriate [Hawthorne et al 2016]. It may also be possible to minimize changes to the concentrations of biomolecules of interest through the careful selection of the composition of the extracellular buffer, such as carrying out the experiments in Xenopus egg extract [Marino et al 2014]. …”

Section: Resultsmentioning

confidence: 99%

“…Permeabilization has been used to allow manipulation of cytosolic ion concentrations [Altschuld et al, 1985;Smolen et al, 1986], to allow introduction of large macromolecules [Lorenz et al, 2008], and to allow removal of macromolecules [Lineberger et al, 1989]. In many of these experiments, the cell structures that remained following digitonin permeabilization continued to behave similarly to intact cells, allowing researchers to characterize subcellular processes using approaches not available in intact cells or in in vitro reconstitution systems [Marino et al, 2014;Schulz, 1990;Subedi et al, 2014].…”

Section: Introductionmentioning

confidence: 99%

Permeabilization activated reduction in fluorescence: A novel method to measure kinetics of protein interactions with intracellular structures

et al. 2016

View full text Add to dashboard Cite

Understanding kinetic information is fundamental in understanding biological function. Advanced imaging technologies have fostered the development of kinetic analyses in cells. We have developed Permeabilization Activated Reduction in Fluorescence (PARF) analysis for determination of apparent t1/2 and immobile fraction, describing the dissociation of a protein of interest from intracellular structures. To create conditions where dissociation events are observable, cells expressing a fluorescently-tagged protein are permeabilized with digitonin, diluting the unbound protein into the extracellular media. As the media volume is much larger than the cytosolic volume, the concentration of the unbound pool decreases drastically, shifting the system out of equilibrium--favoring dissociation events. Loss of bound protein is observed as loss of fluorescence from intracellular structures and can be fit to an exponential decay. We compared PARF dissociation kinetics with previously published equilibrium kinetics as determined by FRAP. PARF dissociation rates agreed with the equilibrium-based FRAP analysis predictions of the magnitude of those rates. When used to investigate binding kinetics of a panel of cytoskeletal proteins, PARF analysis revealed that filament stabilization resulted in slower fluorescence loss. Additionally, commonly used “general” F-actin labels display differences in kinetic properties, suggesting that not all fluorescently-tagged actin labels interact with the actin network in the same way. We also observed differential dissociation kinetics for GFP-VASP depending on which cellular structure was being labeled. These results demonstrate that PARF analysis of non-equilibrium systems reveals kinetic information without the infrastructure investment required for other quantitative approaches such as FRAP, photoactivation, or in vitro reconstitution assays.

show abstract

“…The role of Cdk1 in NPC disassembly has been most clearly shown in studies using a powerful in vitro system (Linder et al, 2017; Marino et al, 2014). These studies have implicated the Plk1 and Nek kinases working in coordination with Cdk1 to phosphorylate the core NPC components Nup98 and Nup53.…”

Section: Discussionmentioning

confidence: 99%

Cyclin B1-Cdk1 facilitates MAD1 release from the nuclear pore to ensure a robust spindle checkpoint

Jackman

Marcozzi

Barbiero

et al. 2020

Journal of Cell Biology

View full text Add to dashboard Cite

How the cell rapidly and completely reorganizes its architecture when it divides is a problem that has fascinated researchers for almost 150 yr. We now know that the core regulatory machinery is highly conserved in eukaryotes, but how these multiple protein kinases, protein phosphatases, and ubiquitin ligases are coordinated in space and time to remodel the cell in a matter of minutes remains a major question. Cyclin B1-Cdk is the primary kinase that drives mitotic remodeling; here we show that it is targeted to the nuclear pore complex (NPC) by binding an acidic face of the kinetochore checkpoint protein, MAD1, where it coordinates NPC disassembly with kinetochore assembly. Localized cyclin B1-Cdk1 is needed for the proper release of MAD1 from the embrace of TPR at the nuclear pore so that it can be recruited to kinetochores before nuclear envelope breakdown to maintain genomic stability.

show abstract

An In Vitro System to Study Nuclear Envelope Breakdown

Cited by 6 publications

References 19 publications

Mitotic Disassembly of Nuclear Pore Complexes Involves CDK1- and PLK1-Mediated Phosphorylation of Key Interconnecting Nucleoporins

Mitotic Disassembly of Nuclear Pore Complexes Involves CDK1- and PLK1-Mediated Phosphorylation of Key Interconnecting Nucleoporins

Permeabilization activated reduction in fluorescence: A novel method to measure kinetics of protein interactions with intracellular structures

Cyclin B1-Cdk1 facilitates MAD1 release from the nuclear pore to ensure a robust spindle checkpoint

Contact Info

Product

Resources

About