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Parvoviruses cause nuclear envelope breakdown by activating key enzymes of mitosis.

Porwal M, Cohen S, Snoussi K, Popa-Wagner R, Anderson F, Dugot-Senant N, Wodrich H, Dinsart C, Kleinschmidt JA, Panté N, Kann M - PLoS Pathog. (2013)

Bottom Line: Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases.Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC.PKC activation then triggered activation of cdk-2, which became further activated by caspase-3.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Virology, University of Giessen, Giessen, Germany ; Univ. de Bordeaux, Microbiologie fondamentale et Pathogénicité, UMR 5234, Bordeaux, France ; CNRS, Microbiologie fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.

ABSTRACT
Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.

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Related in: MedlinePlus

Quantification of fluorescence intensity of the markers microinjected with or without H1 into U2OS cells.Quantifications of the fluorescences presented as videos in the supporting information. The microinjected markers are indicated on top of each panel (D10-Tx: Dextran 10-Texas Red labelled, D40-FITC: Dextran 40, FITC-labelled, ab-A647: unrelated IgG, Alexa.647-labelled). The grey lines show the cytoplasmic, the red line the nuclear fluorescence. The y-axis depicts the intensity given in arbitrary units, the x-axis the time after microinjection in seconds. A. Microinjection of buffer with marker proteins. D10-Tx equilibrated between cytoplasm and nucleus directly after microinjection while D40-FITC and ab-A647 stayed excluded from nucleus. B. Microinjection of H1 with marker proteins. D40-Tx and ab-A647 entered the nucleus simultaneously 240 seconds after microinjection and reached the equilibrium. C. Microinjection of Ca++ with marker proteins. D40-Tx and ab-A647 entered the nucleus simultaneously 200 seconds after microinjection also reaching the equilibrium. In summery the panels show that both H1 and Ca++ triggered sudden NEBD approx. 2–3 min after microinjection. Dextran 40 and the antibodies entered the nucleus at the same time indicating a catastrophic-like destruction of the barrier as it was also seen for nuclear escape of 100 kDa cargos and chromatin in permeabilized cells.
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ppat-1003671-g010: Quantification of fluorescence intensity of the markers microinjected with or without H1 into U2OS cells.Quantifications of the fluorescences presented as videos in the supporting information. The microinjected markers are indicated on top of each panel (D10-Tx: Dextran 10-Texas Red labelled, D40-FITC: Dextran 40, FITC-labelled, ab-A647: unrelated IgG, Alexa.647-labelled). The grey lines show the cytoplasmic, the red line the nuclear fluorescence. The y-axis depicts the intensity given in arbitrary units, the x-axis the time after microinjection in seconds. A. Microinjection of buffer with marker proteins. D10-Tx equilibrated between cytoplasm and nucleus directly after microinjection while D40-FITC and ab-A647 stayed excluded from nucleus. B. Microinjection of H1 with marker proteins. D40-Tx and ab-A647 entered the nucleus simultaneously 240 seconds after microinjection and reached the equilibrium. C. Microinjection of Ca++ with marker proteins. D40-Tx and ab-A647 entered the nucleus simultaneously 200 seconds after microinjection also reaching the equilibrium. In summery the panels show that both H1 and Ca++ triggered sudden NEBD approx. 2–3 min after microinjection. Dextran 40 and the antibodies entered the nucleus at the same time indicating a catastrophic-like destruction of the barrier as it was also seen for nuclear escape of 100 kDa cargos and chromatin in permeabilized cells.

Mentions: In order to link our in vivo infection data with the results obtained in permeabilized cells we used microinjection of H1. By time lapse microscopy we visualized the kinetics and extend of NEBD. For this approach we first co-injected differential fluorescently labelled marker molecules of increasing size alone or together with H1 into the cytoplasm of U2OS cells. Following injection in the absence of H1 a 10 kDa marker molecule (5 nm in diameter) equilibrated rapidly between nucleus and cytoplasm because its small size allows diffusion across the NPC barrier (Fig. 10A). A 40 kDa marker (10 nm in diameter) and a 150 kDa marker (IgG antibodies; 15 nm in diameter) stayed cytoplasmic, which is in agreement with the size exclusion limit of the NPC (Figure 10A and supplemental movie S1). Both 40 kDa and 150 kDa marker rapidly equilibrated in the cytoplasm. Given the linear relation between diameter and diffusion this observation indicates that also H1 (26 nm diameter) can reach the NE within less than a minute.


Parvoviruses cause nuclear envelope breakdown by activating key enzymes of mitosis.

Porwal M, Cohen S, Snoussi K, Popa-Wagner R, Anderson F, Dugot-Senant N, Wodrich H, Dinsart C, Kleinschmidt JA, Panté N, Kann M - PLoS Pathog. (2013)

Quantification of fluorescence intensity of the markers microinjected with or without H1 into U2OS cells.Quantifications of the fluorescences presented as videos in the supporting information. The microinjected markers are indicated on top of each panel (D10-Tx: Dextran 10-Texas Red labelled, D40-FITC: Dextran 40, FITC-labelled, ab-A647: unrelated IgG, Alexa.647-labelled). The grey lines show the cytoplasmic, the red line the nuclear fluorescence. The y-axis depicts the intensity given in arbitrary units, the x-axis the time after microinjection in seconds. A. Microinjection of buffer with marker proteins. D10-Tx equilibrated between cytoplasm and nucleus directly after microinjection while D40-FITC and ab-A647 stayed excluded from nucleus. B. Microinjection of H1 with marker proteins. D40-Tx and ab-A647 entered the nucleus simultaneously 240 seconds after microinjection and reached the equilibrium. C. Microinjection of Ca++ with marker proteins. D40-Tx and ab-A647 entered the nucleus simultaneously 200 seconds after microinjection also reaching the equilibrium. In summery the panels show that both H1 and Ca++ triggered sudden NEBD approx. 2–3 min after microinjection. Dextran 40 and the antibodies entered the nucleus at the same time indicating a catastrophic-like destruction of the barrier as it was also seen for nuclear escape of 100 kDa cargos and chromatin in permeabilized cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814971&req=5

ppat-1003671-g010: Quantification of fluorescence intensity of the markers microinjected with or without H1 into U2OS cells.Quantifications of the fluorescences presented as videos in the supporting information. The microinjected markers are indicated on top of each panel (D10-Tx: Dextran 10-Texas Red labelled, D40-FITC: Dextran 40, FITC-labelled, ab-A647: unrelated IgG, Alexa.647-labelled). The grey lines show the cytoplasmic, the red line the nuclear fluorescence. The y-axis depicts the intensity given in arbitrary units, the x-axis the time after microinjection in seconds. A. Microinjection of buffer with marker proteins. D10-Tx equilibrated between cytoplasm and nucleus directly after microinjection while D40-FITC and ab-A647 stayed excluded from nucleus. B. Microinjection of H1 with marker proteins. D40-Tx and ab-A647 entered the nucleus simultaneously 240 seconds after microinjection and reached the equilibrium. C. Microinjection of Ca++ with marker proteins. D40-Tx and ab-A647 entered the nucleus simultaneously 200 seconds after microinjection also reaching the equilibrium. In summery the panels show that both H1 and Ca++ triggered sudden NEBD approx. 2–3 min after microinjection. Dextran 40 and the antibodies entered the nucleus at the same time indicating a catastrophic-like destruction of the barrier as it was also seen for nuclear escape of 100 kDa cargos and chromatin in permeabilized cells.
Mentions: In order to link our in vivo infection data with the results obtained in permeabilized cells we used microinjection of H1. By time lapse microscopy we visualized the kinetics and extend of NEBD. For this approach we first co-injected differential fluorescently labelled marker molecules of increasing size alone or together with H1 into the cytoplasm of U2OS cells. Following injection in the absence of H1 a 10 kDa marker molecule (5 nm in diameter) equilibrated rapidly between nucleus and cytoplasm because its small size allows diffusion across the NPC barrier (Fig. 10A). A 40 kDa marker (10 nm in diameter) and a 150 kDa marker (IgG antibodies; 15 nm in diameter) stayed cytoplasmic, which is in agreement with the size exclusion limit of the NPC (Figure 10A and supplemental movie S1). Both 40 kDa and 150 kDa marker rapidly equilibrated in the cytoplasm. Given the linear relation between diameter and diffusion this observation indicates that also H1 (26 nm diameter) can reach the NE within less than a minute.

Bottom Line: Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases.Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC.PKC activation then triggered activation of cdk-2, which became further activated by caspase-3.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Virology, University of Giessen, Giessen, Germany ; Univ. de Bordeaux, Microbiologie fondamentale et Pathogénicité, UMR 5234, Bordeaux, France ; CNRS, Microbiologie fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.

ABSTRACT
Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.

Show MeSH
Related in: MedlinePlus