correlative-light-electron-microscopy-clem-for-tracking-imaging-viral

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

European Molecular Biology Laboratory

&#x590D;&#x6742;&#x7684;&#x4EBA;&#x7C7B; Nup107 160 nucleoporin &#x590D;&#x6742;&#x7684;&#x6838;&#x5B54;&#x7684;&#x5F62;&#x6210;&#x4E2D;&#x53D1;&#x6325;&#x7740;&#x91CD;&#x8981;&#x4F5C;&#x7528;&#xFF0C;&#x5E76;&#x88AB;&#x672C;&#x5730;&#x5316;&#x4E3A;&#x9002;&#x5EA6;&#x5728;&#x7EC6;&#x80DE;&#x6709;&#x4E1D;&#x5206;&#x88C2;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#xFF0C;Seh1&#xFF0C;Nup107-160 &#x590D;&#x6742;&#x7684;&#x4E00;&#x4E2A;&#x7EC4;&#x6210;&#x90E8;&#x5206;&#xFF0C;&#x67D3;&#x8272;&#x4F53;&#x5BF9;&#x9F50;&#x548C;&#x9694;&#x79BB;&#x529F;&#x80FD;&#x901A;&#x8FC7;&#x8C03;&#x8282; Aurora B &#x53CA;&#x5176;&#x4ED6;&#x67D3;&#x8272;&#x4F53;&#x4E58;&#x5BA2;&#x590D;&#x5408;&#x86CB;&#x767D;&#x4E1D;&#x7684;&#x672C;&#x5730;&#x5316;&#x3002;&#x672C;&#x5730;&#x5316;&#x7684;&#x8868;&#x8FBE;&#x7684;&#x7740;&#x4E1D;-E &#x4E0D;&#x53D7; Seh1 &#x67AF;&#x7AED;&#x548C;&#x7535;&#x955C;&#x5206;&#x6790;&#x8868;&#x660E;&#x5FAE;&#x7BA1;&#x7EBA;&#x9524;&#x9644;&#x4EF6;&#x5B8C;&#x597D;&#x3002;Seh1 &#x8017;&#x5C3D;&#x7684;&#x5355;&#x5143;&#x683C;&#x663E;&#x793A;&#x53D7;&#x635F;&#x7684; Aurora B &#x672C;&#x5730;&#x5316;&#xFF0C;&#x8FD9;&#x4F1A;&#x5BFC;&#x81F4;&#x5728; biorientation &#x548C; midbody &#x4E0E;&#x4E3B;&#x8F74;&#x6210;&#x7EC4;&#x7EC7;&#x7684;&#x4E25;&#x91CD;&#x7F3A;&#x9677;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x590D;&#x6742;&#x7684;&#x6709;&#x4E1D;&#x5206;&#x88C2; Nup107 &#x7684;&#x4E3B;&#x8981;&#x529F;&#x80FD;&#x662F;&#x786E;&#x4FDD;&#x4E1D;&#x5728;&#x4E2D;&#x56FD;&#x5171;&#x4EA7;&#x515A;&#x7684;&#x6B63;&#x786E;&#x5B9A;&#x4F4D;&#x3002;

&#x590D;&#x6742;&#x7684; Nup107 160 nucleoporin &#x4FC3;&#x8FDB;&#x6709;&#x4E1D;&#x5206;&#x88C2;&#x4E8B;&#x4EF6;&#x901A;&#x8FC7;&#x672C;&#x5730;&#x5316;&#x72B6;&#x6001;&#x7684;&#x67D3;&#x8272;&#x4F53;&#x4E58;&#x5BA2;&#x590D;&#x5408;&#x4F53;&#x7684;&#x63A7;&#x5236;&#x3002;

The human Nup107-160 nucleoporin complex plays a major role in formation of the nuclear pore complex and is localized to kinetochores in mitosis. Here we report that Seh1, a component of the Nup107-160 complex, functions in chromosome alignment and segregation by regulating the centromeric localization of Aurora B and other chromosome passenger complex proteins. Localization of CENP-E is not affected by Seh1 depletion and analysis by electron microscopy showed that microtubule kinetochore attachments are intact. Seh1-depleted cells show impaired Aurora B localization, which results in severe defects in biorientation and organization of the spindle midzone and midbody. Our results indicate that a major function of the Nup107 complex in mitosis is to ensure the proper localization of the CPC at the centromere.

The Nup107-160 nucleoporin complex promotes mitotic events via control of the localization state of the chromosome passenger complex.

La nucl&eacute;oporine Nup107-160 humain complexe joue un r&ocirc;le majeur dans la formation du pore nucl&eacute;aire complexe et est localis&eacute;e &agrave; kin&eacute;tochores lors de la mitose. Nous rapportons ici que Seh1, un composant du complexe Nup107-160, fonctions dans l&#39;alignement des chromosomes et la s&eacute;gr&eacute;gation en r&eacute;gulant la localisation centrom&eacute;rique d&#39;Aurora B et d&#39;autres prot&eacute;ines complexes de passager de chromosome. Localisation de CENP-E n&#39;est pas affect&eacute;e par l&#39;appauvrissement Seh1 et analyse au microscope &eacute;lectronique ont montr&eacute; que les attachements de microtubules kin&eacute;tochores sont intacts. Seh1 d&eacute;pl&eacute;tion des cellules montrent avec facult&eacute;s affaiblies localisation Aurora B, qui se traduit par graves anomalies dans la biorientation et l&#39;organisation du milieu de l&#39;axe et des &eacute;cailles. Nos r&eacute;sultats indiquent qu&#39;une fonction majeure de la Nup107 complexe lors de la mitose est d&#39;assurer la bonne localisation du CPC au centrom&egrave;re.

Le complexe de nucl&eacute;oporine Nup107-160 favorise mitotiques &eacute;v&eacute;nements via le contr&ocirc;le de l&#39;&eacute;tat de la localisation du passager chromosomique complex.

Das komplexe menschliche Nup107-160-Nucleoporin spielt eine wichtige Rolle bei Bildung der nuklearen Pore, die komplexe und auf Kinetochores in Mitose lokalisiert ist. Hier berichten wir, dass Seh1, eine Komponente des Komplexes Nup107-160, Funktionen im Chromosom Ausrichtung und Segregation durch die Regulierung der Centromerisches Lokalisierungs von Aurora B und andere Chromosom Passagier komplexe Proteine. Lokalisierung von CENP-E richtet sich nicht nach Seh1 Ersch&ouml;pfung und Analyse durch Elektronenmikroskopie zeigte, dass Microtubule Kinetochor Anlagen intakt sind. Seh1-abgereichertem Zellen zeigen gest&ouml;rter Aurora B-Lokalisierung, was zu schweren defekten in Biorientation und Organisation der Spindel Midzone und Midbody f&uuml;hrt. Unsere Ergebnisse zeigen, dass eine wichtige Funktion von der Nup107 Komplex in Mitose ist f&uuml;r die korrekte Lokalisierung des CPC an die Zentromere.

Die Nup107-160-Nucleoporin-Komplex f&ouml;rdert mitotische Ereignisse &uuml;ber die Kontrolle des Staates Lokalisierung des Chromosoms Reisenden komplexe.

L&#39;umana Nup107-160 nucleoporin complesso svolge un ruolo importante nella formazione del poro nucleare complesso ed &egrave; localizzato ai cinetocori nella mitosi. Riportiamo che Seh1, un componente del complesso Nup107-160, funzioni nel cromosoma allineamento e segregazione disciplinando la localizzazione centromerica di Aurora B e altre proteine complesse di cromosoma passeggeri. Localizzazione di CENP-E non &egrave; influenzata dall&#39;esaurimento Seh1 e analisi di microscopia elettronica ha mostrato che gli allegati del cinetocore microtubulo sono intatti. Le cellule Seh1-impoverito Visualizza alterata localizzazione Aurora B, che si traduce in gravi difetti nell&#39;organizzazione del midzone mandrino e midbody e biorientation. I nostri risultati indicano che una funzione importante della Nup107 complesso nella mitosi &egrave; di garantire la corretta localizzazione della CPC presso il centromero.

Il nucleoporin Nup107-160 complesso promuove eventi mitotici attraverso il controllo dello stato di localizzazione del passeggero cromosomica complesso.

&#x8907;&#x96D1;&#x306A;&#x4EBA;&#x9593;&#x306E; Nup107 160 &#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30DD;&#x30EA;&#x30F3;&#x86CB;&#x767D;&#x8CEA;&#x6838;&#x819C;&#x5B54;&#x8907;&#x5408;&#x4F53;&#x306E;&#x5F62;&#x6210;&#x306B;&#x5927;&#x304D;&#x306A;&#x5F79;&#x5272;&#x3092;&#x62C5;&#x3046;&#x3057;&#x3001;&#x6709;&#x7CF8;&#x5206;&#x88C2;&#x306E;&#x5F15;&#x3063;&#x3071;&#x308A;&#x306B;&#x30ED;&#x30FC;&#x30AB;&#x30E9;&#x30A4;&#x30BA;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x305D;&#x306E; Seh1&#x3001;Nup107 160 &#x30B3;&#x30F3;&#x30D7;&#x30EC;&#x30C3;&#x30AF;&#x30B9;&#x306E;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x3001;&#x6A5F;&#x80FD;&#x67D3;&#x8272;&#x4F53;&#x306E;&#x6574;&#x5217;&#x3068;&#x5206;&#x96E2;&#x306E;&#x30AA;&#x30FC;&#x30ED;&#x30E9; B &#x3068;&#x4ED6;&#x306E;&#x67D3;&#x8272;&#x4F53;&#x65C5;&#x5BA2;&#x306E;&#x8907;&#x96D1;&#x306A;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x30BB;&#x30F3;&#x30C8;&#x30ED;&#x30E1;&#x30A2;&#x306E;&#x5C40;&#x5728;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;CENP E &#x306E;&#x5C40;&#x5728; Seh1 &#x67AF;&#x6E07;&#x306B;&#x3088;&#x308A;&#x5F71;&#x97FF;&#x3055;&#x308C;&#x305A;&#x3001;&#x5206;&#x6790;&#x96FB;&#x5B50;&#x9855;&#x5FAE;&#x93E1;&#x306B;&#x3088;&#x308B;&#x5FAE;&#x5C0F;&#x7BA1;&#x306E;&#x52D5;&#x539F;&#x4F53;&#x306E;&#x6DFB;&#x4ED8;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x304C;&#x6B63;&#x5E38;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;Seh1 &#x67AF;&#x6E07;&#x7D30;&#x80DE;&#x969C;&#x5BB3;&#x306E;&#x30AA;&#x30FC;&#x30ED;&#x30E9; B &#x306E;&#x30ED;&#x30FC;&#x30AB;&#x30E9;&#x30A4;&#x30BA;&#x306F;&#x3001;biorientation &#x304A;&#x3088;&#x3073;&#x30B9;&#x30D4;&#x30F3;&#x30C9;&#x30EB; midzone &#x3068;&#x534A;&#x7403;&#x9593;&#x5206;&#x96E2;&#x306E;&#x7D44;&#x7E54;&#x306B;&#x6DF1;&#x523B;&#x306A;&#x6B20;&#x9665;&#x3067;&#x7D50;&#x679C;&#x3092;&#x8868;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x6709;&#x7CF8;&#x5206;&#x88C2;&#x306E;&#x8907;&#x96D1;&#x306A; Nup107 &#x306E;&#x4E3B;&#x8981;&#x306A;&#x6A5F;&#x80FD;&#x30AF;&#x30EA;&#x30C3;&#x30AF;&#x5358;&#x4FA1;&#x52D5;&#x539F;&#x4F53;&#x3067;&#x306E;&#x9069;&#x5207;&#x306A;&#x30ED;&#x30FC;&#x30AB;&#x30E9;&#x30A4;&#x30BA;&#x3092;&#x78BA;&#x4FDD;&#x3059;&#x308B;&#x305F;&#x3081;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;

&#x8907;&#x96D1;&#x306A; Nup107 160 &#x306E;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30DD;&#x30EA;&#x30F3;&#x86CB;&#x767D;&#x8CEA;&#x7D30;&#x80DE;&#x5206;&#x88C2;&#x306E;&#x30A4;&#x30D9;&#x30F3;&#x30C8;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x8907;&#x96D1;&#x306A;&#x67D3;&#x8272;&#x4F53;&#x65C5;&#x5BA2;&#x306E;&#x5C40;&#x5728;&#x72B6;&#x614B;&#x306E;&#x5236;&#x5FA1;&#x3092;&#x4FC3;&#x9032;&#x3057;&#x307E;&#x3059;&#x3002;

&#xBCF5;&#xC7A1; &#xD55C; &#xC778;&#xAC04;&#xC758; Nup107-160 nucleoporin &#xBCF5;&#xC7A1; &#xD55C; &#xD575; &#xAE30; &#xACF5; &#xD615;&#xC131;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560; &#xD558; &#xACE0; &#xC720;&#xC0AC; &#xBD84;&#xC5F4;&#xC5D0; &#xC788;&#xB294; kinetochores&#xB97C; &#xC9C0;&#xC5ED;&#xD654; &#xB429;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; &#xC624;&#xB85C;&#xB77C; B&#xC640; &#xB2E4;&#xB978; &#xC5FC;&#xC0C9;&#xCCB4; &#xC2B9;&#xAC1D; &#xBCF5;&#xC7A1; &#xD55C; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; centromeric &#xC9C0;&#xC5ED;&#xD654;&#xB97C; &#xADDC;&#xC81C; &#xD568;&#xC73C;&#xB85C;&#xC368; &#xADF8; Seh1, Nup107-160 &#xBCF5;&#xC7A1; &#xD55C; &#xAD6C;&#xC131;, &#xC5FC;&#xC0C9;&#xCCB4; &#xB9DE;&#xCDA4; &#xBC0F; &#xBD84;&#xB9AC; &#xD568;&#xC218;&#xB97C; &#xBCF4;&#xACE0; &#xD569;&#xB2C8;&#xB2E4;. &#xC9C0;&#xC5ED;&#xD654; &#xD30C;&#xC77C;&#xC774; CENP E Seh1 &#xACE0;&#xAC08;&#xC5D0; &#xC758;&#xD574; &#xC601;&#xD5A5;&#xC744; &#xBC1B;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4; &#xBC0F; &#xC804;&#xC790; &#xD604;&#xBBF8;&#xACBD;&#xC73C;&#xB85C; &#xBD84;&#xC11D; microtubule kinetochore &#xCCA8;&#xBD80; &#xD30C;&#xC77C; &#xADF8;&#xB300;&#xB85C; &#xB098;&#xD0C0;&#xB0AC;&#xB2E4;. Seh1 &#xACE0;&#xAC08; &#xC138;&#xD3EC; &#xC7A5;&#xC560; &#xC624;&#xB85C;&#xB77C; B &#xC9C0;&#xC5ED;&#xD654; biorientation &#xBC0F; &#xC2A4;&#xD540; &#xB4E4; midzone&#xC640; midbody&#xC758; &#xC870;&#xC9C1;&#xC5D0; &#xC2EC;&#xAC01;&#xD55C; &#xACB0;&#xD568;&#xC744; &#xD45C;&#xC2DC; &#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; &#xC720;&#xC0AC; &#xBD84;&#xC5F4;&#xC5D0; &#xBCF5;&#xC7A1; &#xD55C; nup107&#xC758; &#xC8FC;&#xC694; &#xAE30;&#xB2A5;&#xC740; centromere&#xC5D0;&#xC11C; &#xD074;&#xB9AD; &#xB2F9; &#xBE44;&#xC6A9;&#xC758; &#xC801;&#xC808; &#xD55C; &#xC9C0;&#xC5ED;&#xD654;&#xB97C; &#xBCF4;&#xC7A5; &#xD558;&#xB294; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;.

&#xBCF5;&#xC7A1; &#xD55C; Nup107-160 nucleoporin &#xC9C0;&#xC5ED;&#xD654; &#xAD6D;&#xAC00;&#xC758; &#xBCF5;&#xC7A1; &#xD55C; &#xC5FC;&#xC0C9;&#xCCB4; &#xC2B9;&#xAC1D;&#xC758; &#xC81C;&#xC5B4;&#xB97C; &#xD1B5;&#xD574; mitotic &#xC774;&#xBCA4;&#xD2B8; &#xCD94;&#xC9C4;.

El nucleoporin de Nup107-160 humano complejo desempe&ntilde;a un papel importante en la formaci&oacute;n del complejo poro nuclear y se localiza a cinetocoros en la mitosis. Aqu&iacute; divulgamos Seh1, un componente del complejo Nup107-160, funciones en la alineaci&oacute;n del cromosoma y segregaci&oacute;n regulando la localizaci&oacute;n centrom&eacute;rica de Aurora B y otras prote&iacute;nas complejas del pasajero del cromosoma. Localizaci&oacute;n de CENP-E no se ve afectado por el agotamiento de la Seh1 y an&aacute;lisis por microscopia electr&oacute;nica demostr&oacute; que los accesorios cinetocoro de microt&uacute;bulos est&aacute;n intactos. Seh1-agotado c&eacute;lulas muestran deterioro localizaci&oacute;n de Aurora B, que se traduce en defectos graves en biorientation y organizaci&oacute;n de la midzone del huso y medio cuerpo. Nuestros resultados indican que una funci&oacute;n importante de la Nup107 complejo en mitosis es asegurar la correcta localizaci&oacute;n de los CPC en el centr&oacute;mero.

El Nup107-160 nucleoporin complejo promueve eventos mit&oacute;ticos mediante control del estado de la localizaci&oacute;n del pasajero cromosoma complejo.

Production of RanGTP around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing factors. Here, we show that the NLS protein ISWI, a known chromatin-remodeling ATPase, is a RanGTP-dependent microtubule (MT)-associated protein. Recombinant ISWI induces MT nucleation, stabilization, and bundling in vitro. In Xenopus culture cells and egg extract, ISWI localizes within the nucleus in interphase and on spindles during mitosis. Depletion of ISWI in egg extracts does not affect spindle assembly, but in anaphase spindle MTs disappear and chromosomes do not segregate. We show directly that ISWI is required for the RanGTP-dependent stabilization of MTs during anaphase independently of its effect on chromosomes. ISWI depletion in Drosophila S2 cells induces defects in spindle MTs and chromosome segregation in anaphase, and the cells eventually stop growing. Our results demonstrate that distinctly from its role in spindle assembly, RanGTP maintains spindle MTs in anaphase through the local activation of ISWI and that this is essential for proper chromosome segregation.

ISWI is a RanGTP-dependent MAP required for chromosome segregation.

The development of the endomembrane system was a major step in eukaryotic evolution. Membrane coats, which exhibit a unique arrangement of beta-propeller and alpha-helical repeat domains, play key roles in shaping eukaryotic membranes. Such proteins are likely to have been present in the ancestral eukaryote but cannot be detected in prokaryotes using sequence-only searches. We have used a structure-based detection protocol to search all proteomes for proteins with this domain architecture. Apart from the eukaryotes, we identified this protein architecture only in the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum, many members of which share a compartmentalized cell plan. We determined that one such protein is partly localized at the membranes of vesicles formed inside the cells in the planctomycete Gemmata obscuriglobus. Our results demonstrate similarities between bacterial and eukaryotic compartmentalization machinery, suggesting that the bacterial PVC superphylum contributed significantly to eukaryogenesis.

The compartmentalized bacteria of the planctomycetes-verrucomicrobia-chlamydiae superphylum have membrane coat-like proteins.

RanGTP mediates nuclear import and mitotic spindle assembly by dissociating import receptors from nuclear localization signal (NLS) bearing proteins. We investigated the interplay between import receptors and the transmembrane nucleoporin Pom121. We found that Pom121 interacts with importin alpha/beta and a group of nucleoporins in an NLS-dependent manner. In vivo, replacement of Pom121 with an NLS mutant version resulted in defective nuclear transport, induction of aberrant cytoplasmic membrane stacks and decreased cell viability. We propose that the NLS sites of Pom121 affect its function in NPC assembly both by influencing nucleoporin interactions and pore membrane structure.

NLS-mediated NPC functions of the nucleoporin Pom121.

Nuclear pore complexes (NPCs) are large macromolecular structures embedded in the nuclear envelope (NE), where they facilitate exchange of molecules between the cytoplasm and the nucleoplasm. In most cell types, NPCs are evenly distributed around the NE. However, the mechanisms dictating NPC distribution are largely unknown. Here, we used the model organism Caenorhabditis elegans to identify genes that affect NPC distribution during early embryonic divisions. We found that down-regulation of the Sm proteins, which are core components of the spliceosome, but not down-regulation of other splicing factors, led to clustering of NPCs. Down-regulation of Sm proteins also led to incomplete disassembly of NPCs during mitosis, but had no effect on lamina disassembly, suggesting that the defect in NPC disassembly was not due to a general defect in nuclear envelope breakdown. We further found that these mitotic NPC remnants persisted on an ER membrane that juxtaposes the mitotic spindle. At the end of mitosis, the remnant NPCs moved toward the chromatin and the reforming NE, where they ultimately clustered by forming membrane stacks perforated by NPCs. Our results suggest a novel, splicing-independent, role for Sm proteins in NPC disassembly, and point to a possible link between NPC disassembly in mitosis and NPC distribution in the subsequent interphase.

Sm protein down-regulation leads to defects in nuclear pore complex disassembly and distribution in C. elegans embryos.

Mitosis in metazoa requires nuclear envelope (NE) disassembly and reassembly. NE disassembly is driven by multiple phosphorylation events. Mitotic phosphorylation of the protein BAF reduces its affinity for chromatin and the LEM family of inner nuclear membrane proteins; loss of this BAF-mediated chromatin-NE link contributes to NE disassembly. BAF must reassociate with chromatin and LEM proteins at mitotic exit to reform the NE; however, how its dephosphorylation is regulated is unknown. Here, we show that the C. elegans protein LEM-4L and its human ortholog Lem4 (also called ANKLE2) are both required for BAF dephosphorylation. They act in part by inhibiting BAF's mitotic kinase, VRK-1, in vivo and in vitro. In addition, Lem4/LEM-4L interacts with PP2A and is required for it to dephosphorylate BAF during mitotic exit. By coordinating VRK-1- and PP2A-mediated signaling on BAF, Lem4/LEM-4L controls postmitotic NE formation in a function conserved from worms to humans.

Coordination of kinase and phosphatase activities by Lem4 enables nuclear envelope reassembly during mitosis.

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.

Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.

The division of cellular space into functionally distinct membrane-defined compartments has been one of the major transitions in the history of life. Such compartmentalization has been claimed to occur in members of the Planctomycetes, Verrucomicrobiae, and Chlamydiae bacterial superphylum. Here we have investigated the three-dimensional organization of the complex endomembrane system in the planctomycete bacteria Gemmata obscuriglobus. We reveal that the G. obscuriglobus cells are neither compartmentalized nor nucleated as none of the spaces created by the membrane invaginations are closed; instead, they are all interconnected. Thus, the membrane organization of G. obscuriglobus, and most likely all PVC members, is not different from, but an extension of, the "classical" Gram-negative bacterial membrane system. Our results have implications for our definition and understanding of bacterial cell organization, the genesis of complex structure, and the origin of the eukaryotic endomembrane system.

Three-dimensional reconstruction of bacteria with a complex endomembrane system.

We report the presence of a membranous tubulovesicular network in the planctomycete bacterium Gemmata obscuriglobus. This endomembrane system interacts with membrane coat proteins and is capable of protein internalization and degradation. Taken together, this suggests that the planctomycetal bacterium could illuminate the emergence of complex endomembrane systems. 

A bacterial tubulovesicular network.

Production of the GTP-bound form of the Ran GTPase (RanGTP) around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing proteins. Several NLS proteins have been identified as spindle assembly factors, but the complexity of the process led us to search for additional proteins with distinct roles in spindle assembly.

CHD4 is a RanGTP-dependent MAP that stabilizes microtubules and regulates bipolar spindle formation.

Infections are associated with extensive platelet consumption, representing a high risk for health. However, the mechanism coordinating the rapid regeneration of the platelet pool during such stress conditions remains unclear. Here, we report that the phenotypic hematopoietic stem cell (HSC) compartment contains stem-like megakaryocyte-committed progenitors (SL-MkPs), a cell population that shares many features with multipotent HSCs and serves as a lineage-restricted emergency pool for inflammatory insults. During homeostasis, SL-MkPs are maintained in a primed but quiescent state, thus contributing little to steady-state megakaryopoiesis. Even though lineage-specific megakaryocyte transcripts are expressed, protein synthesis is suppressed. In response to acute inflammation, SL-MkPs become activated, resulting in megakaryocyte protein production from pre-existing transcripts and a maturation of SL-MkPs and other megakaryocyte progenitors. This results in an efficient replenishment of platelets that are lost during inflammatory insult. Thus, our study reveals an emergency machinery that counteracts life-threatening platelet depletions during acute inflammation.

Inflammation-Induced Emergency Megakaryopoiesis Driven by Hematopoietic Stem Cell-like Megakaryocyte Progenitors.

Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase-activating protein (GAP) GRAF1 (also known as ARHGAP26), facilitates rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid-phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, which were distinct from clathrin-coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane, resulting in a corresponding decrease in the microdomain association of Cdc42. However, Cdc42 captured in its active state was, through a GAP-domain-mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers with defective endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism. 

Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF1.

The mechanism of endoplasmic reticulum (ER) morphogenesis is incompletely understood. ER tubules are shaped by the reticulons (RTNs) and DP1/Yop1p family members, but the mechanism of ER sheet formation is much less clear. Here, we characterize TMEM170A, a human transmembrane protein, which localizes in ER and nuclear envelope membranes. Silencing or overexpressing TMEM170A in HeLa K cells alters ER shape and morphology. Ultrastructural analysis reveals that downregulation of TMEM170A specifically induces tubular ER formation, whereas overexpression of TMEM170A induces ER sheet formation, indicating that TMEM170A is a newly discovered ER-sheet-promoting protein. Additionally, downregulation of TMEM170A alters nuclear shape and size, decreases the density of nuclear pore complexes (NPCs) in the nuclear envelope and causes either a reduction in inner nuclear membrane (INM) proteins or their relocalization to the ER. TMEM170A interacts with RTN4, a member of the reticulon family; simultaneous co-silencing of TMEM170A and RTN4 rescues ER, NPC and nuclear-envelope-related phenotypes, implying that the two proteins have antagonistic effects on ER membrane organization, and nuclear envelope and NPC formation.

Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells.

Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation. 

Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development.

Nanoparticles which surface adsorb proteins in an uncontrolled and non-reproducible manner will have limited uses as nanomedicinal products. A promising approach to avoid nanoparticle non-specific interactions with proteins is to design bio-hybrids by purposely pre-forming a protein corona around the inorganic cores. Here, we investigate, in vitro and in vivo, the newly acquired bio-identity of superparamagnetic iron oxide nanoparticles (SPIONs) upon their functionalization with a pre-formed and well-defined bovine serum albumin (BSA) corona. Cellular uptake, intracellular particle distribution and cytotoxicity were studied in two cell lines: adherent and non-adherent cells. BSA decreases nanoparticle internalization in both cell lines and protects the iron core once they have been internalized. The physiological response to the nanoparticles is then in vivo evaluated by oral administration to Caenorhabditis elegans, which was selected as a model of a functional intestinal barrier. Nanoparticle biodistribution, at single particle resolution, is studied by transmission electron microscopy. The analysis reveals that the acidic intestinal environment partially digests uncoated SPIONs but does not affect BSA-coated ones. It also discloses that some particles could enter the nematode's enterocytes, likely by endocytosis which is a different pathway than the one described for the worm nutrients.

Bio-identity and fate of albumin-coated SPIONs evaluated in cells and by the C. elegans model.

Gold nanoparticles (AuNPs) are present in many man-made products and cosmetics and are also used by the food and medical industries. Tight regulations regarding the use of mammalian animals for product testing can hamper the study of the specific interactions between engineered nanoparticles and biological systems. Invertebrate models, such as the nematode Caenorhabditis elegans (C. elegans), can offer alternative approaches during the early phases of nanoparticle discovery. Here, we thoroughly evaluated the biodistribution of 11-nm and 150-nm citrate-capped AuNPs in the model organism C. elegans at multiple scales, moving from micrometric to nanometric resolution and from the organismal to cellular level. We confirmed that the nanoparticles were not able to cross the intestinal and dermal barriers. We investigated the effect of AuNPs on the survival and reproductive performance of C. elegans, and correlated these effects with the uptake of AuNPs in terms of their number, surface area, and metal mass. In general, exposure to 11-nm AuNPs resulted in a higher toxicity than the larger 150-nm AuNPs. NP aggregation inside C. elegans was determined using absorbance microspectroscopy, which allowed the plasmonic properties of AuNPs to be correlated with their confinement inside the intestinal lumen, where anatomical traits, acidic pH and the presence of biomolecules play an essential role on NP aggregation. Finally, quantitative PCR of selected molecular markers indicated that exposure to AuNPs did not significantly affect endocytosis and intestinal barrier integrity.

In vivo testing of gold nanoparticles using the Caenorhabditis elegans model organism.

Cryo-electron microscopy (cryo-EM) techniques have made a huge advancement recently, providing close to atomic resolution of the structure of protein complexes. Interestingly, this imaging technique can be performed in cells, giving access to the molecular machines in their natural context, therefore bridging structural and cell biology. However, in situ structural electron microscopy faces one major challenge, which is the ability to focus on specific subcellular regions to capture the objects of interest. Correlative light and electron microscopy (CLEM) is one very efficient solution for this. Here we present a sample preparation technique that enables cryo-sections of vitrified cell monolayers in an orientation that places the cryo-section parallel to the fluorescence imaging plane. The main advantage of this approach is that it exploits the potentials of CLEM for cryo-EM investigation, for selecting specific cells of interest in a heterogeneous population, or for finding identified subcellular regions on sections.

Article	Year
The Nup107-160 nucleoporin complex promotes mitotic events via control of the localization state of the chromosome passenger complex. Molecular biology of the cell\| PubMed ID: 19864462	2009
ISWI is a RanGTP-dependent MAP required for chromosome segregation. The Journal of cell biology\| PubMed ID: 20008562	2009
The compartmentalized bacteria of the planctomycetes-verrucomicrobia-chlamydiae superphylum have membrane coat-like proteins. PLoS biology\| PubMed ID: 20087413	2010
NLS-mediated NPC functions of the nucleoporin Pom121. FEBS letters\| PubMed ID: 20624389	2010
Sm protein down-regulation leads to defects in nuclear pore complex disassembly and distribution in C. elegans embryos. Developmental biology\| PubMed ID: 22426005	2012
Coordination of kinase and phosphatase activities by Lem4 enables nuclear envelope reassembly during mitosis. Cell\| PubMed ID: 22770216	2012
Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS pathogens\| PubMed ID: 23236278	2012
Three-dimensional reconstruction of bacteria with a complex endomembrane system. PLoS biology\| PubMed ID: 23700385	2013
A bacterial tubulovesicular network. Journal of cell science\| PubMed ID: 24259664	2014
CHD4 is a RanGTP-dependent MAP that stabilizes microtubules and regulates bipolar spindle formation. Current biology : CB\| PubMed ID: 24268414	2013
Inflammation-Induced Emergency Megakaryopoiesis Driven by Hematopoietic Stem Cell-like Megakaryocyte Progenitors. Cell stem cell\| PubMed ID: 26299573	2015
Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF1. Journal of cell science\| PubMed ID: 26446261	2015
Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells. Journal of cell science\| PubMed ID: 26906412	2016
Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development. Cell\| PubMed ID: 27397507	2016
Bio-identity and fate of albumin-coated SPIONs evaluated in cells and by the C. elegans model. Acta biomaterialia\| PubMed ID: 27427227	2016
In vivo testing of gold nanoparticles using the Caenorhabditis elegans model organism. Acta biomaterialia\| PubMed ID: 28161575	2017
A new method for cryo-sectioning cell monolayers using a correlative workflow. Methods in cell biology\| PubMed ID: 28528643	2017

Rachel Santarella-Mellwig

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