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Lamin A/C Deficiency Enables Increased Myosin2 Bipolar Filament Ensembles Which Promote Divergent Actomyosin Network Anomalies Through Self Organization.
Molecular Biology of the Cell ( IF 3.1 ) Pub Date : 2020-08-20 , DOI: 10.1091/mbc.e20-01-0017-t
O'Neil Wiggan 1 , Jennifer G DeLuca 1 , Timothy J Stasevich 1, 2 , James R Bamburg 1
Affiliation  

Nuclear envelope proteins influence cell cytoarchitecure by poorly understood mechanisms. Here we show that siRNA-mediated silencing of lamin A/C (LMNA) promotes contrasting stress fiber assembly and disassembly in individual cells and within cell populations. We show that LMNA deficient cells have elevated myosin-II bipolar filament accumulations, irregular formation of actin comet tails and podosome-like adhesions, increased steady state nuclear localization of the mechanosensitive transcription factors MKL1 and YAP, and induced expression of some MKL1/Serum Response Factor (SRF) regulated genes such as that encoding myosin-IIA (MYH9). Our studies utilizing live cell imaging and pharmacological inhibition of myosin-II, support a mechanism of deregulated myosin-II self-organizing activity at the nexus of divergent actin cytoskeletal aberrations resultant from LMNA loss. In light of our results, we propose a model of how the nucleus, via linkage to the cytoplasmic actomyosin network, may act to control myosin-II contractile behavior through both mechanical and transcriptional feedback mechanisms.

Movie S1: Time-lapse confocal microscopy of basal z-projection planes for HeLa cells expressing myosin-2A-mCherry and transiently transfected GFP-Lifeact (F-actin labeling). Images were captured at 1 min intervals, 48h post siRNA treatment and the video rate is 20 frames/s. Related to Figure 3.Download Original Video (.5 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-01-0017-t/20200820/media/mc-e20-01-0017-t-s01.,1200,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518b16abb28bb4fbcf0e4cbb2f7ba4f642791cd7eb70e15c3e2e6093f65b0ad28620d438616ad715667142cb7d326c454756c1f2130f2db55f2210d0b893d326c733e59d36698d6251e8c93521a7fafed7838152f925dd0d4cfd5b715b0804d8c0fc20dcf0d885889ddf1c28a065ed198d62c8b802bcc04ab9c58cbd5293e15c3d86c5708f652026da228b8fa79b42630b029d4b37ab9e52e28949357653b71abcbf01fd9ea97bad1dae7956Movie S2: Time-lapse confocal microscopy of control and LMNA siRNA depleted RPE-1 cells depicting F-actin (GFP-Lifeact) comet formations (red circles). Images were captured at 5 sec intervals and the video rate is 10 frames/s. Related to Figure 4.Download Original Video (1.4 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-01-0017-t/20200820/media/mc-e20-01-0017-t-s02.,1200,900,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87cef5b08edfd566280b042467395a7343b853c870340305df500f2eefa469e3f31ea4ead3ea179c33819fa8b77ef35a20a8e6a8f53c01136964e0cff31c393df22e4d63caa0309305f68e3f1731ee1dd1e865b9bb6abc41c64e5580c715d85a8e9Movie S3: Time-lapse confocal microscopy of basal z-projections for LMNA siRNA treated HeLa cells expressing myosin-2A-mCherry and transiently transfected GFP-Lifeact. Red arrow illustrates comet tail with myosin-2A labeling. Images were captured at 90 sec intervals and the video rate is 3 frames/s. Related to Figure 4.Download Original Video (4.3 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-01-0017-t/20200820/media/mc-e20-01-0017-t-s03.,1200,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518b16abb28bb4fbcf0e4cbb2f7ba4f642791cd7eb70e15c3e2e6093f65b0ad28620d438616ad715667142cb7d326c454756c1f2130f2db55f2210d0b893d326c733e59d36698d6251e8c93521a7fafed7838152f925dd0d4cfd5b715b0804d8c0fc20dcf0d885889ddf1c28a065ed198d62cab802bc05be49fad9b2dcd15c8e857378840394f4bf3a7fe7d7656b5fbe302046e6f4f5ad85b30451be73352fad3b89607b8b6e98b0ad2c4983Movie S4: Time-lapse confocal microscopy of basal z-projections for control or LMNA siRNA treated HeLa cells expressing myosin-2A-mCherry. Cells were treated with 100 M blebbistatin and washed as indicated. Images were captured at 7 min intervals and the video rate is 20 frames/s. Related to Figure 8.Download Original Video (1.0 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-01-0017-t/20200820/media/mc-e20-01-0017-t-s04.,1200,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518b16abb28bb4fbcf0e4cbb2f7ba4f642791cd7eb70e15c3e2e6093f65b0ad28620d438616ad715667142cb7d326c454756c1f2130f2db55f2210d0b893d326c733e59d36698d6251e8c93521a7fafed7838152f925dd0d4cfd5b715b0804d8c0fc20dcf0d885889ddf1c28a065ed198d62cdb802bc13cdcba701e44e59086d2a4a0bc2dbc903b1f4d9b5ddd017df21c03b83c2c2872355a2ff62c8b52e74cb357e009b34114343ff1e18d5


中文翻译:

Lamin A / C缺乏症使Myosin2双极丝簇增多,从而通过自我组织促进了不同的肌动球蛋白网络异常。

核被膜蛋白通过了解甚少的机制影响细胞的细胞结构。在这里,我们表明,siRNA介导的层粘连蛋白A / C(LMNA)沉默促进了单个细胞内和细胞群体内相反的应力纤维组装和拆卸。我们显示LMNA缺陷细胞具有升高的肌球蛋白II双极丝积累,肌动蛋白彗星尾巴和足小体样粘连的不规则形成,机械敏感转录因子MKL1和YAP的稳态核定位增加以及某些MKL1 /血清反应的诱导表达因子(SRF)调控的基因,例如编码肌球蛋白IIA(MYH9)的基因。我们利用活细胞成像和肌球蛋白II的药理抑制作用进行研究,支持一种因LMNA丢失导致肌动蛋白II的自组织活性失控的机制。根据我们的结果,我们提出了一个模型,该模型说明了细胞核如何通过与胞质放线菌素网络的连接,通过机械和转录反馈机制来控制肌球蛋白II的收缩行为。

电影S1:表达Myosin-2A-mCherry和瞬时转染的GFP-Lifeact(F-肌动蛋白标记)的HeLa细胞的基础z投影平面的延时共聚焦显微镜。siRNA处理后48小时,以1分钟的间隔捕获图像,视频速率为20帧/秒。相关图3.下载原始视频(.5 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-01-0017-t/20200820/media/mc-e20-01-0017-t-s01.,1200,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518b16abb28bb4fbcf0e4cbb2f7ba4f642791cd7eb70e15c3e2e6093f65b0ad28620d438616ad715667142cb7d326c454756c1f2130f2db55f2210d0b893d326c733e59d36698d6251e8c93521a7fafed7838152f925dd0d4cfd5b715b0804d8c0fc20dcf0d885889ddf1c28a065ed198d62c8b802bcc04ab9c58cbd5293e15c3d86c5708f652026da228b8fa79b42630b029d4b37ab9e52e28949357653b71abcbf01fd9ea97bad1dae7956电影S2:对照和LMNA siRNA耗尽的RPE-1细胞的延时共聚焦显微镜,描绘了F-肌动蛋白(GFP-Lifeact)彗星的形成(红色圆圈)。以5秒的间隔捕获图像,视频速率为10帧/秒。相关图4.下载原始视频(1.4 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-01-0017-t/20200820/media/mc-e20-01-0017-t-s02.,1200,900,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87cef5b08edfd566280b042467395a7343b853c870340305df500f2eefa469e3f31ea4ead3ea179c33819fa8b77ef35a20a8e6a8f53c01136964e0cff31c393df22e4d63caa0309305f68e3f1731ee1dd1e865b9bb6abc41c64e5580c715d85a8e9电影S3: LMNA siRNA处理的HeLa细胞表达肌球蛋白2A-mCherry和瞬时转染的GFP-Lifeact的基础z投影的延时共聚焦显微镜。红色箭头表示带有myosin-2A标签的彗尾。以90秒的间隔捕获图像,视频速率为3帧/秒。相关图4.下载原始视频(4.3 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-01-0017-t/20200820/media/mc-e20-01-0017-t-s03.,1200,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518b16abb28bb4fbcf0e4cbb2f7ba4f642791cd7eb70e15c3e2e6093f65b0ad28620d438616ad715667142cb7d326c454756c1f2130f2db55f2210d0b893d326c733e59d36698d6251e8c93521a7fafed7838152f925dd0d4cfd5b715b0804d8c0fc20dcf0d885889ddf1c28a065ed198d62cab802bc05be49fad9b2dcd15c8e857378840394f4bf3a7fe7d7656b5fbe302046e6f4f5ad85b30451be73352fad3b89607b8b6e98b0ad2c4983电影S4:用于表达myosin-2A-mCherry的对照或LMNA siRNA处理的HeLa细胞的基础z投射的延时共聚焦显微镜检查。细胞用100 M blebbistatin处理并按指示洗涤。以7分钟的间隔捕获图像,视频速率为20帧/秒。相关图8.下载原始视频(1.0 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-01-0017-t/20200820/media/mc-e20-01-0017-t-s04.,1200,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518b16abb28bb4fbcf0e4cbb2f7ba4f642791cd7eb70e15c3e2e6093f65b0ad28620d438616ad715667142cb7d326c454756c1f2130f2db55f2210d0b893d326c733e59d36698d6251e8c93521a7fafed7838152f925dd0d4cfd5b715b0804d8c0fc20dcf0d885889ddf1c28a065ed198d62cdb802bc13cdcba701e44e59086d2a4a0bc2dbc903b1f4d9b5ddd017df21c03b83c2c2872355a2ff62c8b52e74cb357e009b34114343ff1e18d5
更新日期:2020-08-21
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