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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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Cellular PKC and cdk-2 become activated by H1 and PKCα but not PKCβ is required for NEBD.A, B, C: Activation of PKC, cdk-2 and caspase-3 in permeabilized HeLa cells by H1. Y-axis: activity in arbitrary units. The tested activity is indicated on top of each panel. The columns show the mean values of three independent experiments. The variation bars show the range between the highest and the lowest value. 1. permeabilized cells. 2. permeabilized cells+H1. 3. permeabilized cells+H1+H89. 4. permeabilized cells+H1+Roscovitine. 5. permeabilized cells+H1+zVAD-fmk. 6. H1 without cells. 7. permeabilized cells+H1+thapsigargin. 8. non-permeabilized cells. n.d. not determined. The panels show that permeabilization decrease the activity of all three enzymes and that H1 activates the activities of PKC and cdk-2, while an effect on caspase-3 is doubtful. Inhibition of PKC also reduced activity of cdk2 but not of caspase_3 despite of its inhibitor specificity shown in the supporting information. Thapsigargin pre-treatment, leading to Ca++ depletion inhibits PKC and cdk2 implying that a Ca++-dependent PKC is involved. D. PV H1-mediated NEBD is inhibited by PKCα but not PKCβ. Quantification of PI-stained chromatin of permeabilized HeLa cells to which 300 H1 per permeabilized cell were added. The bars depict 95% CI. Red, dotted line: buffer (n = 28); red line: H1 (n = 14); green dashed line: H1 using PKCα-inhibited cells (n = 19); black dashed line: H1 using PKCβ-inhibited cells. Collectively, the data show that Ca++-dependent PKCα is required for NEBD, which is consistent with the PV-mediated activation of a Ca++-dependent PKC. PKCα subsequently activated cdk-2, which was also shown essential for NEBD. Caspase-3 was not significantly activated by PV but its activity was however essential for NEBD.
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ppat-1003671-g009: Cellular PKC and cdk-2 become activated by H1 and PKCα but not PKCβ is required for NEBD.A, B, C: Activation of PKC, cdk-2 and caspase-3 in permeabilized HeLa cells by H1. Y-axis: activity in arbitrary units. The tested activity is indicated on top of each panel. The columns show the mean values of three independent experiments. The variation bars show the range between the highest and the lowest value. 1. permeabilized cells. 2. permeabilized cells+H1. 3. permeabilized cells+H1+H89. 4. permeabilized cells+H1+Roscovitine. 5. permeabilized cells+H1+zVAD-fmk. 6. H1 without cells. 7. permeabilized cells+H1+thapsigargin. 8. non-permeabilized cells. n.d. not determined. The panels show that permeabilization decrease the activity of all three enzymes and that H1 activates the activities of PKC and cdk-2, while an effect on caspase-3 is doubtful. Inhibition of PKC also reduced activity of cdk2 but not of caspase_3 despite of its inhibitor specificity shown in the supporting information. Thapsigargin pre-treatment, leading to Ca++ depletion inhibits PKC and cdk2 implying that a Ca++-dependent PKC is involved. D. PV H1-mediated NEBD is inhibited by PKCα but not PKCβ. Quantification of PI-stained chromatin of permeabilized HeLa cells to which 300 H1 per permeabilized cell were added. The bars depict 95% CI. Red, dotted line: buffer (n = 28); red line: H1 (n = 14); green dashed line: H1 using PKCα-inhibited cells (n = 19); black dashed line: H1 using PKCβ-inhibited cells. Collectively, the data show that Ca++-dependent PKCα is required for NEBD, which is consistent with the PV-mediated activation of a Ca++-dependent PKC. PKCα subsequently activated cdk-2, which was also shown essential for NEBD. Caspase-3 was not significantly activated by PV but its activity was however essential for NEBD.

Mentions: We next asked if there is a coordinated activation of the enzymes. We permeabilized cells, added H1 in the presence of the different inhibitors and analyzed the activity of i) PKC (α,β,γ,δ,ε,μ,θ,ζ), ii) cdk-2 and iii) caspase-3 in the lysates (Fig. 9A, B, C). We have chosen to analyze cdk-2 as it is needed during earlier steps of mitosis as G1/S phase transition. Its enzymatic activity could have been essential for NEBD as cdk-2 activates cdk-1 [49]. Cdk-1 in turn is required for lamin hyper phosphorylation, needed for lamin depolymerisation [50].


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)

Cellular PKC and cdk-2 become activated by H1 and PKCα but not PKCβ is required for NEBD.A, B, C: Activation of PKC, cdk-2 and caspase-3 in permeabilized HeLa cells by H1. Y-axis: activity in arbitrary units. The tested activity is indicated on top of each panel. The columns show the mean values of three independent experiments. The variation bars show the range between the highest and the lowest value. 1. permeabilized cells. 2. permeabilized cells+H1. 3. permeabilized cells+H1+H89. 4. permeabilized cells+H1+Roscovitine. 5. permeabilized cells+H1+zVAD-fmk. 6. H1 without cells. 7. permeabilized cells+H1+thapsigargin. 8. non-permeabilized cells. n.d. not determined. The panels show that permeabilization decrease the activity of all three enzymes and that H1 activates the activities of PKC and cdk-2, while an effect on caspase-3 is doubtful. Inhibition of PKC also reduced activity of cdk2 but not of caspase_3 despite of its inhibitor specificity shown in the supporting information. Thapsigargin pre-treatment, leading to Ca++ depletion inhibits PKC and cdk2 implying that a Ca++-dependent PKC is involved. D. PV H1-mediated NEBD is inhibited by PKCα but not PKCβ. Quantification of PI-stained chromatin of permeabilized HeLa cells to which 300 H1 per permeabilized cell were added. The bars depict 95% CI. Red, dotted line: buffer (n = 28); red line: H1 (n = 14); green dashed line: H1 using PKCα-inhibited cells (n = 19); black dashed line: H1 using PKCβ-inhibited cells. Collectively, the data show that Ca++-dependent PKCα is required for NEBD, which is consistent with the PV-mediated activation of a Ca++-dependent PKC. PKCα subsequently activated cdk-2, which was also shown essential for NEBD. Caspase-3 was not significantly activated by PV but its activity was however essential for NEBD.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003671-g009: Cellular PKC and cdk-2 become activated by H1 and PKCα but not PKCβ is required for NEBD.A, B, C: Activation of PKC, cdk-2 and caspase-3 in permeabilized HeLa cells by H1. Y-axis: activity in arbitrary units. The tested activity is indicated on top of each panel. The columns show the mean values of three independent experiments. The variation bars show the range between the highest and the lowest value. 1. permeabilized cells. 2. permeabilized cells+H1. 3. permeabilized cells+H1+H89. 4. permeabilized cells+H1+Roscovitine. 5. permeabilized cells+H1+zVAD-fmk. 6. H1 without cells. 7. permeabilized cells+H1+thapsigargin. 8. non-permeabilized cells. n.d. not determined. The panels show that permeabilization decrease the activity of all three enzymes and that H1 activates the activities of PKC and cdk-2, while an effect on caspase-3 is doubtful. Inhibition of PKC also reduced activity of cdk2 but not of caspase_3 despite of its inhibitor specificity shown in the supporting information. Thapsigargin pre-treatment, leading to Ca++ depletion inhibits PKC and cdk2 implying that a Ca++-dependent PKC is involved. D. PV H1-mediated NEBD is inhibited by PKCα but not PKCβ. Quantification of PI-stained chromatin of permeabilized HeLa cells to which 300 H1 per permeabilized cell were added. The bars depict 95% CI. Red, dotted line: buffer (n = 28); red line: H1 (n = 14); green dashed line: H1 using PKCα-inhibited cells (n = 19); black dashed line: H1 using PKCβ-inhibited cells. Collectively, the data show that Ca++-dependent PKCα is required for NEBD, which is consistent with the PV-mediated activation of a Ca++-dependent PKC. PKCα subsequently activated cdk-2, which was also shown essential for NEBD. Caspase-3 was not significantly activated by PV but its activity was however essential for NEBD.
Mentions: We next asked if there is a coordinated activation of the enzymes. We permeabilized cells, added H1 in the presence of the different inhibitors and analyzed the activity of i) PKC (α,β,γ,δ,ε,μ,θ,ζ), ii) cdk-2 and iii) caspase-3 in the lysates (Fig. 9A, B, C). We have chosen to analyze cdk-2 as it is needed during earlier steps of mitosis as G1/S phase transition. Its enzymatic activity could have been essential for NEBD as cdk-2 activates cdk-1 [49]. Cdk-1 in turn is required for lamin hyper phosphorylation, needed for lamin depolymerisation [50].

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