FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3035432909> ?p ?o ?g. }

• W3035432909 abstract "Article15 June 2020free access Source DataTransparent process Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B disassembly Yubin Li Corporal Michael J. Crescenz VAMC, Philadelphia, PA, USA Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Minghui Li Corporal Michael J. Crescenz VAMC, Philadelphia, PA, USA Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Department of Rheumatology and Immunology, Tianjin Medical University General Hospital, Tianjin, China Search for more papers by this author Bettina Weigel Cell Fate Engineering and Disease Modeling Group, German Cancer Research Center (DKFZ) and DKFZ-ZMBH Alliance, Heidelberg, Germany HITBR Hector Institute for Translational Brain Research GmbH, Heidelberg, Germany Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Moritz Mall Cell Fate Engineering and Disease Modeling Group, German Cancer Research Center (DKFZ) and DKFZ-ZMBH Alliance, Heidelberg, Germany HITBR Hector Institute for Translational Brain Research GmbH, Heidelberg, Germany Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Victoria P Werth Corporal Michael J. Crescenz VAMC, Philadelphia, PA, USA Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Ming-Lin Liu Corresponding Author [email protected] orcid.org/0000-0001-8827-2024 Corporal Michael J. Crescenz VAMC, Philadelphia, PA, USA Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Yubin Li Corporal Michael J. Crescenz VAMC, Philadelphia, PA, USA Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Minghui Li Corporal Michael J. Crescenz VAMC, Philadelphia, PA, USA Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Department of Rheumatology and Immunology, Tianjin Medical University General Hospital, Tianjin, China Search for more papers by this author Bettina Weigel Cell Fate Engineering and Disease Modeling Group, German Cancer Research Center (DKFZ) and DKFZ-ZMBH Alliance, Heidelberg, Germany HITBR Hector Institute for Translational Brain Research GmbH, Heidelberg, Germany Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Moritz Mall Cell Fate Engineering and Disease Modeling Group, German Cancer Research Center (DKFZ) and DKFZ-ZMBH Alliance, Heidelberg, Germany HITBR Hector Institute for Translational Brain Research GmbH, Heidelberg, Germany Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Victoria P Werth Corporal Michael J. Crescenz VAMC, Philadelphia, PA, USA Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Ming-Lin Liu Corresponding Author [email protected] orcid.org/0000-0001-8827-2024 Corporal Michael J. Crescenz VAMC, Philadelphia, PA, USA Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Author Information Yubin Li1,2, Minghui Li1,2,3, Bettina Weigel4,5,6, Moritz Mall4,5,6, Victoria P Werth1,2 and Ming-Lin Liu *,1,2 1Corporal Michael J. Crescenz VAMC, Philadelphia, PA, USA 2Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 3Department of Rheumatology and Immunology, Tianjin Medical University General Hospital, Tianjin, China 4Cell Fate Engineering and Disease Modeling Group, German Cancer Research Center (DKFZ) and DKFZ-ZMBH Alliance, Heidelberg, Germany 5HITBR Hector Institute for Translational Brain Research GmbH, Heidelberg, Germany 6Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany *Corresponding author. Tel: +1 215 614 1621; E-mail: [email protected] EMBO Rep (2020)21:e48779https://doi.org/10.15252/embr.201948779 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The nuclear lamina is essential for the structural integration of the nuclear envelope. Nuclear envelope rupture and chromatin externalization is a hallmark of the formation of neutrophil extracellular traps (NETs). NET release was described as a cellular lysis process; however, this notion has been questioned recently. Here, we report that during NET formation, nuclear lamin B is not fragmented by destructive proteolysis, but rather disassembled into intact full-length molecules. Furthermore, we demonstrate that nuclear translocation of PKCα, which serves as the kinase to induce lamin B phosphorylation and disassembly, results in nuclear envelope rupture. Decreasing lamin B phosphorylation by PKCα inhibition, genetic deletion, or by mutating the PKCα consensus sites on lamin B attenuates extracellular trap formation. In addition, strengthening the nuclear envelope by lamin B overexpression attenuates NET release in vivo and reduces levels of NET-associated inflammatory cytokines in UVB-irradiated skin of lamin B transgenic mice. Our findings advance the mechanistic understanding of NET formation by showing that PKCα-mediated lamin B phosphorylation drives nuclear envelope rupture for chromatin release in neutrophils. Synopsis Nuclear envelope rupture is crucial for chromatin externalization during NET formation. Nuclear lamin B disassembly, but not cleavage, regulated by PKCα-mediated phosphorylation, is responsible for nuclear envelope rupture and NET release. Lamin B disassembly is responsible for nuclear envelope rupture and chromatin release during NET formation. Nuclear lamin B disassembly is regulated by PKCα-mediated lamin B phosphorylation. Strengthening the nuclear envelope by lamin B overexpression attenuates NET formation. Introduction Neutrophils are the most common leukocytes. Increasing evidence from many studies (Brinkmann et al, 2004; Lood et al, 2016; Soehnlein et al, 2017; Boeltz et al, 2019), including our own (Folkesson et al, 2015), indicates the importance of neutrophils in acute or chronic inflammation in variety of human diseases (Brinkmann et al, 2004; Lood et al, 2016; Soehnlein et al, 2017; Boeltz et al, 2019). NET formation is characterized by nuclear chromatin decondensation (Brinkmann et al, 2004) through peptidyl arginine deiminase type IV (PAD4)-catalyzed histone citrullination (Neeli et al, 2008; Wang et al, 2009) or neutrophil elastase-mediated histone cleavage (Pieterse et al, 2018), followed by chromatin extrusion via the ruptured nuclear envelope. The externalized nuclear chromatin serves as the backbone of NETs that mix with cytosolic contents, i.e., granule proteins and oxidized mitochondrial DNA, and form the extracellular trap structure (Boeltz et al, 2019). Several signaling pathways may be important in the development of NET formation, i.e., production of reactive oxygen species (ROS) through NADPH oxidase (NOX2)-dependent or NADPH oxidase (NOX2)-independent pathways (Douda et al, 2015; Pieterse et al, 2018; Boeltz et al, 2019), activation of protein kinase C (PKC), extracellular-signal-regulated kinase (ERK/MAPK), and phosphatidylinositide 3-kinase (PI3K)/Akt (Boeltz et al, 2019). ROS is thought to be required for the release of myeloperoxidase and neutrophil elastase from granules in activated neutrophils. Then, neutrophil elastase is translocated to the nucleus for chromatin decondensation by histone cleavage in the early stage of NET formation (Fuchs et al, 2007; Papayannopoulos et al, 2010). However, a very recent consensus statement paper by experts in the field suggests that the direct connection between lysosomal membrane instability and NET formation is still under discussion (Boeltz et al, 2019). Pieterse et al (2018) reported that neutrophil elastase may be associated with cleavage of the N-terminal tail, the proteolytic sensitive region, of histones in the late stage of NET formation. Another paper reported that neutrophils from neutrophil elastase-deficient mice could still undergo NET formation (Martinod et al, 2016). In addition to ROS-dependent NET formation, a number of recent studies also reported ROS-independent NET formation induced by certain stimuli (Rochael et al, 2015; Tatsiy & McDonald, 2018). Nuclear envelope rupture is the hallmark and a prerequisite step in the multi-step process of NET formation (Boeltz et al, 2019). However, the relevant cellular and molecular mechanism of nuclear envelope rupture during NET formation is still unclear. NET formation was described as a lytic cell death mechanism (Fuchs et al, 2007; Papayannopoulos et al, 2010; Yipp & Kubes, 2013). However, this notion has been questioned by several recent observations (Amulic et al, 2017; Neubert et al, 2018). Rupture of the nuclear envelope appears to be a distinct process from previously described lysis or dissolution of the nuclear envelope (Amulic et al, 2017; Neubert et al, 2018). In addition, Pilsczek et al (2010) described vital, but not lytic, NET formation in which NETs are released via nuclear budding or vesicle release by an unknown mechanism. The nuclear envelope consists of outer and inner lipid nuclear membranes (ONM and INM) and nuclear lamina, a protein filament meshwork that provides structural scaffold to reinforce nuclear envelope integrity (Goldberg et al, 2008). Nuclear lamins are categorized as either A-type (A, C) or B-type (B1, B2) lamins (Goldberg et al, 2008). A-type lamins form thick filament bundles that contribute to the mechanical stiffness of nuclei (Lammerding et al, 2006; Goldberg et al, 2008; Rowat et al, 2013), whereas B-type lamins form thin but highly organized meshworks that are crucial to the integrity and elasticity of the nuclear envelope (Vergnes et al, 2004; Goldberg et al, 2008), but not stiffness (Lammerding et al, 2006; Swift et al, 2013). Shin et al reported that nuclear stiffness increases with the lamin A:B ratio (Shin et al, 2013), and increasing the lamin A:B ratio by downregulation of lamin B promotes nuclear stiffness in hematopoietic cells (Shin et al, 2013). Lamin B is anchored underneath the INM through the lamin B receptor (LBR) (Olins et al, 2008; Singh et al, 2016). Nuclear envelope breakdown (Hatch & Hetzer, 2014) is a common cellular event in nuclear fragmentation during cell apoptosis (Shimizu et al, 1998), in nuclear division during cell mitosis (Collas et al, 1997; Mall et al, 2012), and in viral nuclear access during viral infection (Park & Baines, 2006). In the cellular processes described above, nuclear lamin B is either proteolytically degraded by caspases (Slee et al, 2001) or disassembled through phosphorylation by protein kinase C (PKC) (Collas et al, 1997; Muranyi et al, 2002). Mature neutrophils are terminally differentiated cells thought to have an unusual nucleus with a paucity of lamin A/C (Olins et al, 2008) and a reduced amount of lamin B (Olins et al, 2008). Recent studies, however, reported that neutrophils do have A-type (Amulic et al, 2017) and B-type (Moisan & Girard, 2006; Rowat et al, 2013) lamins. The role of lamin B in neutrophil biology is unclear and was ignored, and the involvement of lamin B in NET formation has not been investigated. In the current study, we investigated the importance of lamin B for the integrity of the nucleus in neutrophils and cellular mechanisms that regulate nuclear envelope rupture during NET formation in neutrophils from human and mice. We also investigated the effect of strengthening the nuclear envelope by lamin B overexpression on NET formation in vivo and on the presence of NET-associated inflammatory cytokines in UVB-irradiated skin of lamin B transgenic mice. Results Nuclear lamin B is a substantial component of the nuclear envelope that is involved in NET formation Neutrophils are terminally differentiated cells, and the role of nuclear lamina in neutrophil biology is not well understood (Olins et al, 2008). However, nuclear envelope breakdown is required for nuclear chromatin externalization during NET formation. To explore the role of nuclear lamin B, we detected the expression of lamin B in primary human polymorphonuclear neutrophils (pPMNs) and the involvement of lamin B in NET formation (Fig 1A and B). NET formation can be triggered by PMA, the most commonly used NET inducer (Fig 1A; Brinkmann et al, 2004), or by platelet activating factor (PAF), a UVB-induced lipid mediator and naturally existing stimulus of NETs (Damiani & Ullrich, 2016). Confocal microscopy analyses indicated that nuclear lamin B is a substantial component of the nuclear envelope (Fig 1B and C), which is ruptured during NET release (Fig 1B), indicating the involvement of lamin B in the process of NET formation. Figure 1. Nuclear lamin B is a substantial component of nuclear envelope that is involved in NET formation A. Summary and representative analysis of NET formation of pPMNs stimulated by 50 nM PMA for 3 h, detected by fluorescent microplate reader and confocal microscopy, respectively. Scale bar 20 μm. B. Representative confocal microscopy images of pPMNs that were treated without (control) or with 50 nM PMA for 3 h and stained for lamin B and DNA as described in panel (B). The light blue arrows indicate the release of decondensed DNA associated with lamin B molecules from ruptured nuclear envelope. Scale bar 10 μm. C. Schematic cross-section of a cell and portion of the nucleus and the nuclear envelope, as well as the ones with corresponding overexpression of lamin B. The two lipid bilayers of the nuclear envelope are the inner and outer nuclear membranes (INM and ONM, respectively). The meshwork, nuclear lamin B is anchored to INM through lamin B receptor (LBR). D. Representative and summary of immunoblots of lamin B of bone marrow neutrophils from WT and Lmnb1TG mice. Vinculin served as loading control. E. Summary analysis of PAF-induced NET formation in mPMNs from WT vs. Lmnb1TG mice that were stimulated without or with 10 μM PAF for 3 h and then fixed by 2% PFA and stained by SYTOX Green, followed by detection with fluorescent microplate reader. F. The endpoint analysis of NET-DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and Lmnb1TG mice without (control) or with 10 μM PAF in medium containing 1 μM SYTOX Green dye with recording by a microplate reader at the 3-h time point. The NET-DNA release index was reported in comparison with an assigned value of 100% for the total DNA released by neutrophils lysed by 0.5% (v/v) Triton X-100. G, H. Representative images (G) and summary analysis (H) of PAF-induced NET formation in mPMNs from WT vs. Lmnb1TG mice that were stimulated without or with 10 μM PAF for 3 h and stained with both cell-permeable SYTO Red and cell-impermeable SYTOX Green, without fixation. Images were taken by Olympus confocal microscopy, followed by automated quantification of NETs on 5–6 non-overlapping area per well using ImageJ for calculation of % cells with NET formation. (G) Scale bars, 40 μm. Data information: Panels (A, D, E, F, H) were summary analysis of NET formation (A, E), or summary analyses of immunoblots (D), DNA% release index (F) that were calculated based on the arbitrary fluorescent readout unit (A, E), immunoblot arbitrary unit (D). NET-DNA release index calculated based on fluorescent readout (F), or % cells with NET formation by image analysis (H). Data in (A, D, E, F, H) represent mean ± SD (n = 3–5 biological replicates). *P < 0.05, **P < 0.01, ***P < 0.001 between groups as indicated. Comparisons among three or more groups were performed using ANOVA, followed by Student–Newman–Keuls test. Comparison between two groups was analyzed by the Student t test. Source data are available online for this figure. Source Data for Figure 1 [embr201948779-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Lamin B is assembled as a filament meshwork that lies beneath the INM, and the two are associated by an integral protein, lamin B receptor (Fig 1C). Given the importance of lamin B in nuclear envelope integration (Vergnes et al, 2004; Goldberg et al, 2008), one may propose that lamin B may be important in regulating nuclear envelope rupture during NET formation. To test this hypothesis, we first explored the effects of lamin B overexpression on NET formation by using neutrophils from lamin B transgenic Lmnb1TG mice (Fig 1D). We found attenuated NET formation in peritoneal neutrophils from Lmnb1TG mice as compared to those from their WT littermates (Fig 1E–H), analyzed by fluorometric NET quantification (Fig 1E; Sollberger et al, 2016), endpoint analysis of NET-DNA release index (Fig 1F; Douda et al, 2015; Khan et al, 2018), and immunofluorescent imaging analysis of NET formation (Fig 1G and H; Sollberger et al, 2016). On the other hand, lamin B maturation is regulated by farnesylation (Adam et al, 2013), and long-term inhibition of protein farnesylation with farnesyltransferase inhibitor (FTI) can reduce the amount of mature lamin B (Adam et al, 2013). To explore the effects of decreased lamin B, we found that coincubation with FTI can reduce the amount of mature lamin B both in differentiated HL-60 neutrophils (dPMNs) (Fig EV1A) and in RAW 264.7 macrophages (Fig EV1C). Most importantly, we found that decreased lamin B in FTI-pretreated cells could enhance PAF-induced extracellular trap formation both in dPMNs and in RAW264.7 cells (Fig EV1B, D and E). Taken together, the levels of mature lamin B expression in the nucleus affected extracellular trap formation in a negative dose-dependent manner both in neutrophils and macrophages. Click here to expand this figure. Figure EV1. Decreased lamin B expression enhances extracellular traps formation in vitro and time course of PKCα phosphorylation during NET formation (Related to Figs 1 and 3) A, C. Summary and representative immunoblot analysis for the mature lamin B expression in HL-60 dPMNs (A) or RAW264.7 cells (C) that were treated without (0) or with 2 or 10 μM farnesyltransferase inhibitor (FTI) L-744,832 for 48 h. B, D, E. Summary analysis (B, D) and representative images (E) of extracellular trap release in HL-60 dPMNs (B) RAW264.7 cells (E) that were pretreated without or with 2 μM for 48 h, followed by treatment or not with 10 μM PAF for 3 h and then stained with cell-impermeable SYTOX Green, without fixation. Fluorescent and phase-contrast images were taken by Olympus confocal microscopy. The yellow arrows indicate neutrophils with NET formation. Scale bars, 20 μm (E). F, G. Representative immunoblots and the summary analyses of total and phosphorylated PKCα (p-PKCα), in human dPMNs that were treated either by PMA (F) or PAF (G) for 0, 0.5, 1, 2, 3 h. Data information: The summary analyses were calculated based on the arbitrary density of immunoblot images (A, C, F, G), or % DNA release index was analyzed by comparison of the fluorescent intensity of the indicated conditions to an assigned value of 100% for the total DNA released by neutrophils lysed by 0.5% (v/v) Triton X-100 (B, D) as compared to their untreated controls. Data are given as mean ± SD from at least three independent biological replicates. *P < 0.05, **P < 0.01, ***P < 0.01 between different groups as indicated. Comparisons among three or more groups were performed using ANOVA, followed by Student–Newman–Keuls test. Source data are available online for this figure. Download figure Download PowerPoint Therefore, our results demonstrated that nuclear lamin B is a substantial component of the nuclear envelope in neutrophils, similar to other cell types (Vergnes et al, 2004; Goldberg et al, 2008). Nuclear lamin B is important in regulating nuclear envelop breakdown and release of extracellular traps. Nuclear lamin B disassembly, but not proteolytic cleavage, is responsible for nuclear envelope rupture during NET formation Next, we sought to elucidate how lamin B is involved in nuclear envelope rupture during NET formation. Unexpectedly, immunoblot analysis of the time-course studies demonstrated that nuclear lamin B remained as an intact full-length molecule during NET formation in human dPMNs 0–3 h after stimulation by PMA (Fig 2A and B) or PAF (Fig 2C and D), in contrast to the destructively cleaved and fragmented lamin B in apoptotic neutrophils (Fig 2A). Importantly, immunoblot analysis of isolated extracellular NETs (Fig 2E) showed that nuclear lamin B is also released with NETs to the extracellular space and NET-associated lamin B was not degraded, but remained as an intact full-length molecule (Fig 2E), confirming the findings from the whole-cell lysates. In contrast to uncleaved nuclear lamin B, we observed cleaved histone H3 in the neutrophils with NET formation (Fig 2E). Neutrophil elastase may contribute to the cleavage of histone H3 and decondensation of chromatin during NET formation (Konig & Andrade, 2016; Pieterse et al, 2018). Therefore, our results suggested potential disassembly, but not the destructive cleavage, of lamin B is responsible for nuclear envelope rupture and extracellular trap formation during NET formation in neutrophils. Figure 2. Nuclear lamin B disassembly, but not proteolytic cleavage, is responsible for nuclear envelope rupture in neutrophils with NET formation A, B. Representative and summary of immunoblots of full-length lanes of lamin B in human dPMNs that were treated with PMA for 0, 0.5, 1, 2, 3 h during NET formation, or apoptotic dPMNs that were induced by PMA for longer term (12 h) treatment (A). C, D. Representative and summary immunoblots of lamin B in human dPMNs that were treated with PAF for 0, 0.5, 1, 2, 3 h during NET formation. E. Representative confocal microscopy image of a group of pPMNs with NET formation (enclosed by a light blue trapezoid) and their extracellular NETs (indicated by a purple rectangle square) released by these cells, as well as the immunoblot analysis of uncleaved lamin B and cleaved histone H3 with the whole-cell lysates, as well as immunoblot analysis of uncleaved lamin B with lysate of the NETs isolated from conditioned medium of pPMNs with NET formation that were induced by 3 h PMA treatment. F. Representative immunoblots with full-length lanes for lamin B analysis, and pro-caspase-3 and its activated form caspase-3 in human dPMNs with NET formation that were treated with PMA for 0, 0.5, 1, 2, 3 h. G. Representative immunoblots display full-length lamin B (69 kDa) and their cleaved fragments (45 and 25 kDa), and pro-caspase-3 and its activated form caspase-3 in the apoptotic human dPMNs that were treated with PMA for 12, 24 h. Data information: The anti-human lamin B was used in all immunoblots (A–G) and the confocal image (E), and the latter was further detected by FITC-labeled 2nd Ab, scale bar, 20 μm. Anti-human caspase-3 was used (F, G), and β-actin served as loading control (A–D, F, G). Data represent mean ± SD (n = 3 biological replicates) for (B, D). Comparisons among three or more groups were performed using ANOVA, followed by Student–Newman–Keuls test. Source data are available online for this figure. Source Data for Figure 2 [embr201948779-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Caspase-3 mediates apoptosis and cleaves lamin B (Slee et al, 2001) during apoptotic nuclear fragmentation. In the current study, we found that caspase-3 remained inactive as pro-caspase-3 over a 3-h period of time during NET formation (Fig 2F), while activated caspase-3 was detected in apoptotic dPMNs induced by extended PMA treatment (Arroyo et al, 2002) for 12–24 h (Fig 2G). Furthermore, we saw that lamin B was cleaved into 25 and 45 kDa fragments, corresponding to caspase-3 activation during apoptosis (Fig 2G). These experiments further confirmed that destructive proteolytic cleavage is not responsible for lamin B disintegration during nuclear envelope rupture in neutrophils with NET formation. Nuclear translocation and phosphorylation of PKCα during NET formation To address how nuclear lamin B was disassembled during nuclear envelope rupture, we found that cytosolic PKCα was translocated to the nuclear membrane where it may mediate lamin B phosphorylation and nuclear envelope disintegration, similar to the role of PKCα during mitosis (Mall et al, 2012), and viral infection (Park & Baines, 2006). The immunofluorescent image analysis (Fig 3A) demonstrated that total PKCα was gradually translocated from the cytosol into the nucleus over the time during the early period of PMA stimulation from 5 to 30 min (analysis started after 5 min PMA stimulation to allow the neutrophils to adherer for microscopic analysis). PKCα accumulated in the nucleus and resulted in nuclear envelope discontinuity/rupture at 60-min time point (Fig 3A and B), followed by the consequent extrusion of decondensed chromatin from the ruptured/collapsed nuclear envelope at 180 min (Fig 3A), and the subsequent NET formation. Correspondingly, PKCα phosphorylation was gradually increased over the 3-h stimulation period in human primary pPMNs (Fig 3C and D) and dPMNs (Fig EV1F and G) treated with PMA or PAF. Figure 3. Nuclear translocation and phosphorylation of PKCα is accompanied by nuclear envelope rupture during NET formation A. Representative images for the time course of PKCα nuclear translocation, subsequent nuclear envelope rupture, and DNA release in human dPMNs exposed to 50 nM PMA for 5, 15, 30, 60, and 180 min and then stained concomitantly for DNA (DAPI), nuclear lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and PKCα (primary anti-human PKCα, and PE-labeled secondary antibody), followed by confocal fluorescent microscopy analysis. White empty arrows indicate cytoplasmic distribution of PKCα at 5, 15, and 30 min, yellow arrows indicate site of discontinuity/rupture of nuclear envelope at 60 min, light blue arrows display the sites of nuclear envelope rupture and chromatin release at 180 min (A). Scale bars, 20 μm. The time course started 5 min after PMA stimulation in order to allow the adherence of neutrophils on the bottom of dish for immunocytostaining. B. Summary analysis of the nuclear envelope continuity was analyzed based on staining of the nuclear envelope with primary anti-lamin B, and FITC-labeled secondary antibody. C, D. Representative immunoblots of total PKCα and p-PKCα in primary human pPMNs that were treated by PMA (C) or PAF (D) for 0, 0.5, 1, 2, 3 h. Data information: The summary analyses of panel (B) were calculated based on the circumferences/perimeters of the cell nuclei of 7–10 cells from different time points from 3 to 6 independent experiments. Data in (B) represent mean ± SD (n = 3–6 biological replicates). *P < 0.05 between groups as indicated. Comparisons among three or more groups were performed using ANOVA, followed by Student–Newman–Keuls test. Source data are available online for this figure. Source Data for Figure 3 [embr201948779-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint Accumulation of phosphorylated PKCα in the nucleus results in lamin B phosphorylation, disassembly, and nuclear envelope rupture during NET formation To explore the potential role of phosphorylated PKCα (p-PKCα) in nuclear envelope rupture, we first isolated the nucleus of neutrophils that were stimulated by PMA at 0 and 2 h (Fig 4A). We found accumulation of p-PKCα and full-length lamin B in the nuclear fraction of neutrophils with 2-h PMA stimulation (Fig 4A), while there was no detectable total PKCα in the nuclear fraction of neutrophils at time 0 (Fig 4A) in immunoblot analysis. These data are in line with the results obtained by fluorescent microscopy (Fig 3A). Confocal microscopy analysis of a neutrophil in the early stage of NET formation further confirmed the accumulation of p-PKCα in the nucleus, particularly at the rupture site of the nuclear envelope, which had decondensed/swollen chromatin but not yet released to form extracellular traps (b1 cell, Fig 4B). In contrast, another neutrophil in the same image showed a ruptured nuclear envelope with release of decondensed chromatin as the backbone of NETs that contained the mixture of p-PKCα and disassembled nuclear lamin B (b2 cell, Fig 4B). Furthermore, p-PKCα and full-length lamin B were also detected in the immunoblot of the extracellular NETs isolated from cultured human primary pPMNs (Fig 4C). The results of concomitant detection of these two molecules both by immunofluorescent microscopy (Fig 4B) and immunoblots (Fig 4C) indicate a potential active interaction between p-PKCα and lamin B during nuclear envelope rupture. Figure 4. Accumulation of phosphorylated PKCα in nucleus results in nuclear lamin B phosphorylation, disassembly, and the consequent nuclear enve" @default.
• W3035432909 created "2020-06-19" @default.
• W3035432909 creator A5022244610 @default.
• W3035432909 creator A5029793095 @default.
• W3035432909 creator A5030342071 @default.
• W3035432909 creator A5051405817 @default.
• W3035432909 creator A5055279293 @default.
• W3035432909 creator A5064584608 @default.
• W3035432909 date "2020-06-15" @default.
• W3035432909 modified "2023-10-14" @default.
• W3035432909 title "Nuclear envelope rupture and NET formation is driven by PKCα‐mediated lamin B disassembly" @default.
• W3035432909 cites W1564279536 @default.
• W3035432909 cites W1572925458 @default.
• W3035432909 cites W1589413565 @default.
• W3035432909 cites W1602299201 @default.
• W3035432909 cites W161395021 @default.
• W3035432909 cites W1759129926 @default.
• W3035432909 cites W1859576715 @default.
• W3035432909 cites W1965659644 @default.
• W3035432909 cites W1966369019 @default.
• W3035432909 cites W1976541550 @default.
• W3035432909 cites W1982243149 @default.
• W3035432909 cites W1988969017 @default.
• W3035432909 cites W1994227421 @default.
• W3035432909 cites W2008568038 @default.
• W3035432909 cites W2014391550 @default.
• W3035432909 cites W2018860370 @default.
• W3035432909 cites W2023953437 @default.
• W3035432909 cites W2024882180 @default.
• W3035432909 cites W2025884506 @default.
• W3035432909 cites W2026987638 @default.
• W3035432909 cites W2030637965 @default.
• W3035432909 cites W2032936612 @default.
• W3035432909 cites W2039970582 @default.
• W3035432909 cites W2040062051 @default.
• W3035432909 cites W2042168939 @default.
• W3035432909 cites W2052373675 @default.
• W3035432909 cites W2062056826 @default.
• W3035432909 cites W2070299769 @default.
• W3035432909 cites W2074331191 @default.
• W3035432909 cites W2081021921 @default.
• W3035432909 cites W2087851913 @default.
• W3035432909 cites W2090787210 @default.
• W3035432909 cites W2091512988 @default.
• W3035432909 cites W2091862270 @default.
• W3035432909 cites W2096620879 @default.
• W3035432909 cites W2104934425 @default.
• W3035432909 cites W2112543741 @default.
• W3035432909 cites W2113221891 @default.
• W3035432909 cites W2115959782 @default.
• W3035432909 cites W2118217258 @default.
• W3035432909 cites W2118273159 @default.
• W3035432909 cites W2124633704 @default.
• W3035432909 cites W2127910803 @default.
• W3035432909 cites W2136682979 @default.
• W3035432909 cites W2138020499 @default.
• W3035432909 cites W2163340435 @default.
• W3035432909 cites W2168176302 @default.
• W3035432909 cites W2169617094 @default.
• W3035432909 cites W2200350105 @default.
• W3035432909 cites W2215388791 @default.
• W3035432909 cites W2234611476 @default.
• W3035432909 cites W2261479737 @default.
• W3035432909 cites W2301131921 @default.
• W3035432909 cites W2314107066 @default.
• W3035432909 cites W2411235909 @default.
• W3035432909 cites W2437854394 @default.
• W3035432909 cites W2546931104 @default.
• W3035432909 cites W2550084246 @default.
• W3035432909 cites W2570320605 @default.
• W3035432909 cites W2596984373 @default.
• W3035432909 cites W2766872204 @default.
• W3035432909 cites W2791392455 @default.
• W3035432909 cites W2802399006 @default.
• W3035432909 cites W2885558416 @default.
• W3035432909 cites W2888168693 @default.
• W3035432909 cites W2888725678 @default.
• W3035432909 cites W2890771157 @default.
• W3035432909 cites W2891621105 @default.
• W3035432909 cites W2910296364 @default.
• W3035432909 cites W2911027500 @default.
• W3035432909 cites W2945137965 @default.
• W3035432909 cites W2978060160 @default.
• W3035432909 cites W3035432909 @default.
• W3035432909 doi "https://doi.org/10.15252/embr.201948779" @default.
• W3035432909 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7403722" @default.
• W3035432909 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/32537912" @default.
• W3035432909 hasPublicationYear "2020" @default.
• W3035432909 type Work @default.
• W3035432909 sameAs 3035432909 @default.
• W3035432909 citedByCount "32" @default.
• W3035432909 countsByYear W30354329092020 @default.
• W3035432909 countsByYear W30354329092021 @default.
• W3035432909 countsByYear W30354329092022 @default.
• W3035432909 countsByYear W30354329092023 @default.
• W3035432909 crossrefType "journal-article" @default.
• W3035432909 hasAuthorship W3035432909A5022244610 @default.
• W3035432909 hasAuthorship W3035432909A5029793095 @default.
• W3035432909 hasAuthorship W3035432909A5030342071 @default.
• W3035432909 hasAuthorship W3035432909A5051405817 @default.

• next