single-molecule-fret-imaging-for-observing-conformational-dynamics

Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver ...

Nankai University

&#x7BA1;&#x72B6;&#x7F51;&#x7EDC;&#x7684;&#x5185;&#x8D28;&#x7F51; &#xFF08;ER&#xFF09; &#x751F;&#x6210;&#x8981;&#x6C42;&#x4ECB;&#x5BFC; dynamin &#x6837;&#x3001; &#x819C;&#x86CB;&#x767D; atlastin (ATL) &#x7684;&#x540C;&#x578B;&#x819C;&#x878D;&#x5408;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x5DF2;&#x786E;&#x5B9A;&#x6676;&#x4F53;&#x7ED3;&#x6784;&#x6E38;&#x79BB;&#x90E8;&#x5206;&#x7684;&#x4EBA;&#x7C7B; ATL1&#xFF0C;&#x6DF1;&#x5165;&#x8003;&#x8651;&#x819C;&#x878D;&#x5408;&#x7684;&#x673A;&#x5236;&#x3002;&#x7ED3;&#x6784;&#x63ED;&#x793A;&#x4E86;&#x86CB;&#x767D;&#x57DF;&#x548C;&#x5FC3;&#x7535;&#x56FE;&#x87BA;&#x65CB;&#x675F;&#xFF0C;&#x7531;&#x94FE;&#x63A5;&#x5668;&#x533A;&#x57DF;&#x8FDE;&#x63A5;&#x8D77;&#x6765;&#x3002;&#x4E00;&#x4E2A;&#x7ED3;&#x6784;&#x5BF9;&#x5E94;&#x4E8E; prefusion &#x7684;&#x72B6;&#x6001;&#xFF0C;&#x901A;&#x8FC7;&#x5B83;&#x4EEC;&#x7684;&#x86CB;&#x767D;&#x57DF;&#x4E0E;&#x7ED1;&#x5B9A;&#x5728;&#x754C;&#x9762;&#x4E0A;&#x7684;&#x6838;&#x82F7;&#x9178;&#x5F62;&#x6210;&#x4E8C;&#x805A;&#x4F53;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x7684; ATL apposing &#x819C;&#x4E2D;&#x7684;&#x5206;&#x5B50;&#x3002;&#x5176;&#x4ED6;&#x7ED3;&#x6784;&#x5BF9;&#x5E94;&#x5230; GTP &#x6C34;&#x89E3;&#x548C;&#x78F7;&#x9178;&#x76D0;&#x91CA;&#x653E;&#x540E;&#xFF0C;&#x751F;&#x6210;&#x7684; postfusion &#x72B6;&#x6001;&#x3002;&#x4E24;&#x4E2A; ATL &#x5206;&#x5B50;&#x4E09;&#x87BA;&#x65CB;&#x675F;&#x4E0E; prefusion &#x7684;&#x7ED3;&#x6784;&#x76F8;&#x6BD4;&#xFF0C;&#x63A5;&#x53D7;&#x4E0E;&#x86CB;&#x767D;&#x57DF;&#x4E2D;&#xFF0C;&#x53EF;&#x4EE5;&#x5728;&#x4E00;&#x8D77;&#x62C9;&#x819C;&#x4E3B;&#x8981;&#x6784;&#x8C61;&#x53D8;&#x5316;&#x3002;&#x62DF;&#x8BAE;&#x7684;&#x878D;&#x5408;&#x673A;&#x5236;&#x652F;&#x6301;&#x7684;&#x751F;&#x5316;&#x5B9E;&#x9A8C;&#x548C;&#x878D;&#x5408;&#x68C0;&#x5B9A;&#x6CD5;&#x4E0E;&#x91CE;&#x751F;&#x578B;&#x548C;&#x7A81;&#x53D8;&#x4F53;&#x5168;&#x957F;&#x679C;&#x8747; atl&#x3002;&#x8FD9;&#x4E9B;&#x5B9E;&#x9A8C;&#x8FD8;&#x663E;&#x793A;&#x819C;&#x878D;&#x5408;&#x901A;&#x8FC7;&#x4EE5;&#x4E0B;&#x4E24;&#x4E2A;&#x8DE8;&#x819C;&#x6BB5; C &#x7EC8;&#x7AEF;&#x6E38;&#x79BB;&#x5C3E;&#x5DF4;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x663E;&#x793A; ATL1 &#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x5BFC;&#x81F4;&#x9057;&#x4F20;&#x6027;&#x75C9;&#x631B;&#x6027;&#x622A;&#x762B;&#x59A5;&#x534F;&#x540C;&#x578B; ER &#x878D;&#x5408;&#x3002;

Atlastin &#x86CB;&#x767D;&#x7ED3;&#x6784;&#x63D0;&#x4F9B;&#x540C;&#x578B;&#x878D;&#x5408;&#x5185;&#x8D28;&#x7F51;&#x819C;&#x7684;&#x6D1E;&#x5BDF;&#x3002;

The generation of the tubular network of the endoplasmic reticulum (ER) requires homotypic membrane fusion that is mediated by the dynamin-like, membrane-bound GTPase atlastin (ATL). Here, we have determined crystal structures of the cytosolic segment of human ATL1, which give insight into the mechanism of membrane fusion. The structures reveal a GTPase domain and athree-helix bundle, connected by a linker region. One structure corresponds to a prefusion state, in which ATL molecules in apposing membranes interact through their GTPase domains to form a dimer with the nucleotides bound at the interface. The other structure corresponds to a postfusion state generated after GTP hydrolysis and phosphate release. Compared with the prefusion structure, the three-helix bundles of the two ATL molecules undergo a major conformational change relative to the GTPase domains, which could pull the membranes together. The proposed fusion mechanism is supported by biochemical experiments and fusion assays with wild-type and mutant full-length Drosophila ATL. These experiments also show that membrane fusion is facilitated by the C-terminal cytosolic tails following the two transmembrane segments. Finally, our results show that mutations in ATL1 causing hereditary spastic paraplegia compromise homotypic ER fusion.

Structures of the atlastin GTPase provide insight into homotypic fusion of endoplasmic reticulum membranes.

La g&eacute;n&eacute;ration du r&eacute;seau tubulaire du r&eacute;ticulum endoplasmique (ER) n&eacute;cessite la fusion membranaire homotypiques qui est m&eacute;di&eacute;e par l&#39;atlastin de GTPase dynamine-like, li&eacute;e &agrave; la membrane (ATL). Ici, nous avons d&eacute;termin&eacute; les structures cristallines du tron&ccedil;on cytosolique de ATL1 humaine, qui donnent un aper&ccedil;u du m&eacute;canisme de fusion membranaire. Les structures r&eacute;v&egrave;lent une GTPase domaine et atrois-h&eacute;lice bundle, reli&eacute; par une r&eacute;gion de l&#39;&eacute;diteur de liens. Une structure correspond &agrave; un &Eacute;tat prefusion, dans lequel les mol&eacute;cules ATL &agrave; relater les membranes interagissent par le biais de leur domaine GTPase pour former un dim&egrave;re avec les nucl&eacute;otides li&eacute;s &agrave; l&#39;interface. L&#39;autre structure correspond &agrave; un &Eacute;tat post&eacute;rieurement g&eacute;n&eacute;r&eacute; apr&egrave;s que hydrolyse du GTP et phosphate de sortie. Par rapport &agrave; la structure prefusion, les faisceaux de trois h&eacute;lices des deux mol&eacute;cules d&#39;ATL subissent un changement de conformation majeur par rapport &agrave; des domaines de la GTPase, qui pourrait rassembler les membranes. Le m&eacute;canisme de la fusion propos&eacute;e est pris en charge par les exp&eacute;riences biochimiques et des essais de fusion avec le type sauvage et mutante pleine longueur Drosophila ATL. Ces exp&eacute;riences d&eacute;montrent &eacute;galement que la fusion membranaire est facilit&eacute;e par la queue cytosolique C-terminale apr&egrave;s les deux segments transmembranaires. Enfin, nos r&eacute;sultats montrent que les mutations ATL1 entra&icirc;nant une parapl&eacute;gie spastique h&eacute;r&eacute;ditaire compromettent fusion ER homotypiques.

Structures de l&#39;atlastin GTPase donnent un aper&ccedil;u de la fusion homotypique de membranes du r&eacute;ticulum endoplasmique.

Die Generation der tubul&auml;ren Netzes von dem Endoplasmatischen Retikulum (ER) erfordert homotypischen Membrane Fusion, die von der Dynamin-&auml;hnlichen, Membrane-springen GTPase Atlastin (ATL) vermittelt wird. Hier haben wir Kristallstrukturen des Zytosolische Segments des menschlichen ATL1 bestimmt, die Einblick in den Mechanismus der Membrane Fusion. Die Strukturen zeigen ein GTPase Dom&auml;ne und adrei-Helix B&uuml;ndel, verbunden durch eine Linker-Region. Eine Struktur entspricht einem prefusion Zustand, in dem ATL-Molek&uuml;le im apposing Membranen durch ihre GTPase-Domains ein Dimer mit der Nukleotide interagieren, gebunden an der Schnittstelle zu bilden. Die andere Struktur entspricht einem postfusion Zustand nach GTP Hydrolyse und Phosphat release generiert. Verglichen mit der prefusion-Struktur, durchlaufen die drei-Helix-B&uuml;ndel der zwei ATL-Molek&uuml;le eine gr&ouml;&szlig;ere Konformations&auml;nderungen Ver&auml;nderung im Verh&auml;ltnis der GTPase-Dom&auml;nen, die die Membranen an einem Strang ziehen konnte. Der vorgeschlagene Fusion-Mechanismus wird unterst&uuml;tzt durch biochemische Experimente und Fusion-Assays mit Wildtyp und Mutanten in Drosophila-ATL. Diese Experimente zeigen auch, dass die Membrane Fusion durch die C-terminale Zytosolische Schw&auml;nze nach die zwei Transmembran-Segmenten erleichtert wird. Schlie&szlig;lich zeigen unsere Ergebnisse, dass Mutationen im ATL1 verursacht Heredit&auml;re Spastische Paraplegie homotypischen ER Verschmelzung gef&auml;hrden.

Strukturen der Atlastin GTPase geben Einblick in homotypischen Verschmelzung von Endoplasmatisches Reticulum Membranen.

La generazione della rete tubolare del reticolo endoplasmatico (ER) richiede homotypic fusione di membrana che &egrave; mediata dalla dynamin-like, membrana-limiti GTPase atlastin (ATL). Qui, abbiamo determinato strutture cristalline del segmento citosolico di ATL1 umana, che danno la comprensione del meccanismo di fusione della membrana. Le strutture di rivelano un GTPase dominio e atre-helix bundle, collegato da una regione di linker. Una struttura corrisponde ad uno stato di prefusion, in cui molecole ATL in apposite membrane interagiscono attraverso i loro domini GTPase per formare un dimero con i nucleotidi legati all&#39;interfaccia. L&#39;altra struttura corrisponde a un postfusion stato generato dopo la pubblicazione di idrolisi di GTP e fosfato. Confrontato con la struttura prefusion, i tre-helix bundle di due molecole di ATL subiscono un importante cambiamento conformazionale rispetto i domini di GTPase, che potrebbe tirare le membrane insieme. Il meccanismo di fusione proposto &egrave; supportato da esperimenti biochimici e dosaggi di fusione con wild-type e mutanti full-length della drosofila ATL. Questi esperimenti dimostrano anche che la fusione di membrana &egrave; facilitata da code citosoliche C-terminale seguendo i due segmenti transmembrana. Infine, i nostri risultati mostrano che mutazioni ATL1 causando la paraplegia spastica ereditaria compromettano fusion ER homotypic.

Strutture di atlastin GTPase forniscono insight in homotypic fusione delle membrane del reticolo endoplasmatico.

&#x5C0F;&#x80DE;&#x4F53; &#xFF08;ER&#xFF09; &#x306E;&#x7BA1;&#x72B6;&#x306E;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x306E;&#x4E16;&#x4EE3;&#x306F;&#x30C0;&#x30A4;&#x30CA;&#x30DF;&#x30F3;&#x306E;&#x3088;&#x3046;&#x306B;&#x3001;&#x819C;&#x7D50;&#x5408; gtp &#x30A2;&#x30FC;&#x30BC;&#x304F;&#x3067; (ATL) &#x3092;&#x4ECB;&#x3057;&#x305F;&#x30A8;&#x30CD;&#x30EB;&#x30AE;&#x30FC;&#x819C;&#x878D;&#x5408;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x819C;&#x878D;&#x5408;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306B;&#x6D1E;&#x5BDF;&#x529B;&#x3092;&#x4E0E;&#x3048;&#x308B;&#x4EBA;&#x9593; ATL1 &#x306E;&#x30BE;&#x30EB;&#x6027;&#x7D30;&#x80DE;&#x8CEA;&#x306E;&#x30BB;&#x30B0;&#x30E1;&#x30F3;&#x30C8;&#x306E;&#x7D50;&#x6676;&#x69CB;&#x9020;&#x3092;&#x6C7A;&#x5B9A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x69CB;&#x9020;&#x3092; gtp &#x30A2;&#x30FC;&#x30BC; &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3068; 3 &#x30AB;&#x56FD;&#x30D8;&#x30EA;&#x30C3;&#x30AF;&#x30B9; &#x30D0;&#x30F3;&#x30C9;&#x30EB; &#x30EA;&#x30F3;&#x30AB;&#x30FC;&#x9818;&#x57DF;&#x3067;&#x63A5;&#x7D9A;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3001;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3059;&#x3002;1 &#x3064;&#x306E;&#x69CB;&#x9020;&#x306F;&#x819C;&#x3092; apposing &#x3067; ATL &#x5206;&#x5B50;&#x304C;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30A4;&#x30B9;&#x306B;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x3055;&#x308C;&#x305F;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x3068;&#x4E8C;&#x91CF;&#x4F53;&#x3092;&#x5F62;&#x6210;&#x3059;&#x308B;&#x3001;gtp &#x30A2;&#x30FC;&#x30BC; &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x5BFE;&#x8A71; prefusion &#x5DDE;&#x306B;&#x5BFE;&#x5FDC;&#x3057;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x4ED6;&#x306E;&#x69CB;&#x9020;&#x306B;&#x306F; GTP &#x52A0;&#x6C34;&#x5206;&#x89E3;&#x3068;&#x30EA;&#x30F3;&#x9178;&#x306E;&#x30EA;&#x30EA;&#x30FC;&#x30B9;&#x5F8C;&#x306B;&#x751F;&#x6210;&#x3055;&#x308C;&#x305F; postfusion &#x306E;&#x72B6;&#x614B;&#x306B;&#x5BFE;&#x5FDC;&#x3057;&#x307E;&#x3059;&#x3002;Prefusion &#x306E;&#x69CB;&#x9020;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x3001;2 &#x3064;&#x306E; ATL &#x5206;&#x5B50;&#x306E; 3 &#x3064;&#x306E;&#x3089;&#x305B;&#x3093;&#x675F;&#x819C;&#x304C;&#x4E00;&#x7DD2;&#x306B;&#x5F15;&#x3063;&#x5F35;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x306E; gtp &#x30A2;&#x30FC;&#x30BC; &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306B;&#x76F8;&#x5BFE;&#x7684;&#x306A;&#x4E3B;&#x8981;&#x306A;&#x69CB;&#x9020;&#x5909;&#x66F4;&#x3092;&#x53D7;&#x3051;&#x307E;&#x3059;&#x3002;&#x63D0;&#x6848;&#x306E;&#x878D;&#x5408;&#x6A5F;&#x69CB;&#x751F;&#x5316;&#x5B66;&#x7684;&#x5B9F;&#x9A13;&#x3068;&#x91CE;&#x751F;&#x578B;&#x3068;&#x5909;&#x7570;&#x578B;&#x306E;&#x30D5;&#x30EB;&#x30EC;&#x30F3;&#x30B0;&#x30B9; &#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8; ATL. &#x878D;&#x5408;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x3053;&#x308C;&#x3089;&#x306E;&#x5B9F;&#x9A13;&#x306F;&#x307E;&#x305F;&#x3001;&#x819C;&#x878D;&#x5408;&#x6B21;&#x306E; 2 &#x3064;&#x306E;&#x819C;&#x8CAB;&#x901A;&#x30BB;&#x30B0;&#x30E1;&#x30F3;&#x30C8; C &#x30BF;&#x30FC;&#x30DF;&#x30CA;&#x30EB; &#x30BE;&#x30EB;&#x6027;&#x7D30;&#x80DE;&#x8CEA;&#x30C6;&#x30FC;&#x30EB;&#x306B;&#x3088;&#x3063;&#x3066;&#x4FC3;&#x9032;&#x3055;&#x308C;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x6211;&#x3005; &#x306E;&#x7D50;&#x679C;&#x306F; ATL1 &#x7A81;&#x7136;&#x5909;&#x7570;&#x306E;&#x907A;&#x4F1D;&#x6027;&#x75D9;&#x6027;&#x5BFE;&#x9EBB;&#x75FA;&#x306E;&#x539F;&#x56E0;&#x3084;&#x5C0F;&#x80DE;&#x4F53;&#x306E;&#x878D;&#x5408;&#x3092;&#x5371;&#x967A;&#x306B;&#x3055;&#x3089;&#x3059;&#x3053;&#x3068;. &#x3057;&#x307E;&#x3059;&#x3002;

&#x304F; gtp &#x30A2;&#x30FC;&#x30BC;&#x306E;&#x69CB;&#x9020;&#x3084;&#x5C0F;&#x80DE;&#x4F53;&#x819C;&#x878D;&#x5408;&#x306B;&#x6D1E;&#x5BDF;&#x529B;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

Endoplasmic &#xADF8;&#xBB3C; (ER)&#xC758; &#xAD00; &#xB124;&#xD2B8;&#xC6CC;&#xD06C;&#xC758; &#xC0DD;&#xC131;&#xC740; dynamin &#xCC98;&#xB7FC;, &#xB9C9; &#xBC14;&#xC778;&#xB529; GTPase atlastin (ATL)&#xC5D0; &#xC758;&#xD574; &#xC911;&#xC7AC; homotypic &#xB9C9; &#xD4E8;&#xC804;&#xC774; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xB9C9; &#xD4E8;&#xC804;&#xC758; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC5D0; &#xB300; &#xD55C; &#xD1B5;&#xCC30;&#xB825;&#xC744; &#xC8FC;&#xB294; &#xC778;&#xAC04;&#xC758; ATL1 cytosolic &#xC138;&#xADF8;&#xBA3C;&#xD2B8;&#xC758; &#xD06C;&#xB9AC;&#xC2A4;&#xD0C8; &#xAD6C;&#xC870;&#xB97C; &#xACB0;&#xC815; &#xD588;&#xB2E4;. &#xAD6C;&#xC870; &#xACF5;&#xAC1C; GTPase &#xB3C4;&#xBA54;&#xC778;&#xACFC; &#xBC31;&#xC0AC; &#xB098;&#xC120; &#xBC88;&#xB4E4;, &#xB9C1;&#xCEE4; &#xC601;&#xC5ED;&#xC73C;&#xB85C; &#xC5F0;&#xACB0; &#xD569;&#xB2C8;&#xB2E4;. &#xD55C; &#xAD6C;&#xC870;&#xB97C; &#xC778;&#xD130;&#xD398;&#xC774;&#xC2A4;&#xC5D0; &#xBC14;&#xC778;&#xB529;&#xB41C; &#xB274;&#xD074;&#xB808;&#xC624;&#xD2F0;&#xB4DC;&#xC640; &#xC774;&#xD569;&#xCCB4;&#xB97C; &#xD615;&#xC131; GTPase &#xB3C4;&#xBA54;&#xC778; &#xB4E4;&#xC744; &#xD1B5;&#xD574; ATL &#xBD84;&#xC790; apposing &#xC138;&#xD3EC; &#xB9C9;&#xC5D0; &#xC791;&#xC6A9; &#xD558;&#xB294; prefusion &#xC0C1;&#xD0DC;&#xC5D0; &#xD574;&#xB2F9; &#xD569;&#xB2C8;&#xB2E4;. &#xB2E4;&#xB978; &#xAD6C;&#xC870;&#xB294; GTP &#xAC00;&#xC218;&#xBD84;&#xD574; &#xBC0F; &#xC778;&#xC0B0; &#xC5FC; &#xD574;&#xC81C; &#xD6C4;&#xC5D0; &#xC0DD;&#xC131; &#xB418;&#xB294; postfusion &#xC0C1;&#xD0DC;&#xC5D0; &#xD574;&#xB2F9; &#xD569;&#xB2C8;&#xB2E4;. Prefusion &#xAD6C;&#xC870;&#xC640; &#xBE44;&#xAD50; &#xD558;&#xBA74;, &#xB450; &#xAC1C;&#xC758; ATL &#xBD84;&#xC790;&#xC758; 3 &#xB098;&#xC120; &#xBC88;&#xB4E4; &#xD568;&#xAED8; &#xC138;&#xD3EC; &#xB9C9;&#xC744; &#xB04C;&#xC5B4; &#xB2F9;&#xAE38; &#xC218;&#xB3C4; &#xC788;&#xACE0; GTPase &#xB3C4;&#xBA54;&#xC778;&#xC744; &#xAE30;&#xC900;&#xC73C;&#xB85C; &#xC8FC;&#xC694; &#xAD6C;&#xC870;&#xC801; &#xBCC0;&#xD654;&#xB97C; &#xBC1B; &#xB2E4;. &#xC0DD;&#xD654;&#xD559; &#xC2E4;&#xD5D8;&#xC640; &#xC57C;&#xC0DD; &#xD615; &#xBC0F; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xCD08;&#xD30C;&#xB9AC; ATL. &#xC804;&#xCCB4; &#xAE38;&#xC774;&#xC640; &#xC735;&#xD569; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC5D0;&#xC11C; &#xC81C;&#xC548; &#xB41C; &#xD4E8;&#xC804; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xC9C0;&#xC6D0; &#xC774;&#xB7EC;&#xD55C; &#xC2E4;&#xD5D8;&#xB3C4; &#xB9C9; &#xD4E8;&#xC804; C &#xD130;&#xBBF8;&#xB110; cytosolic &#xAF2C;&#xB9AC; &#xB450; &#xB9C9; &#xD6A1;&#xB2E8; &#xC138;&#xADF8;&#xBA3C;&#xD2B8;&#xB97C; &#xB2E4;&#xC74C;&#xC5D0; &#xC758;&#xD574; &#xC6A9;&#xC774; &#xD558; &#xAC8C; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; &#xC720;&#xC804; &#xACBD;&#xB828; &#xC801;&#xC778; &#xD558;&#xBC18;&#xC2E0;&#xC744; &#xC77C;&#xC73C;&#xD0A4;&#xB294; atl1&#xC5D0; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; homotypic ER &#xD4E8;&#xC804; &#xD0C0;&#xD611;&#xC744; &#xBCF4;&#xC5EC;&#xC900;&#xB2E4;.

Atlastin Gtpase&#xC758; &#xAD6C;&#xC870; endoplasmic &#xADF8;&#xBB3C; &#xB9C9;&#xC758; homotypic &#xC735;&#xD569;&#xC5D0; &#xB300; &#xD55C; &#xD1B5;&#xCC30;&#xB825;&#xC744; &#xC81C;&#xACF5;&#xD569;&#xB2C8;&#xB2E4;.

La generaci&oacute;n de la red tubular del ret&iacute;culo endopl&aacute;smico (ER) requiere homot&iacute;picos fusi&oacute;n de membrana que es mediada por la GTPasa Dinamina-como, unida a la membrana atlastin (ATL). Aqu&iacute;, hemos determinado las estructuras cristalinas del segmento citos&oacute;lica de ATL1 humano, que dan la penetraci&oacute;n en el mecanismo de fusi&oacute;n de la membrana. Las estructuras revelan un paquete de dominio y ATRES-h&eacute;lice de GTPasa, conectado por una regi&oacute;n de vinculador. Una estructura corresponde a un estado de prefusi&oacute;n, en el que las mol&eacute;culas de ATL en apposing membranas interact&uacute;an a trav&eacute;s de sus dominios de GTPasa para formar un d&iacute;mero con los nucle&oacute;tidos limitados en la interfaz. La otra estructura corresponde a un estado postfusion que genera despu&eacute;s de hidr&oacute;lisis de GTP y fosfato de liberaci&oacute;n. En comparaci&oacute;n con la estructura de prefusi&oacute;n, los paquetes de tres-h&eacute;lice de las dos mol&eacute;culas ATL sufren un cambio conformacional importante en relaci&oacute;n con los dominios de la GTPasa, que podr&iacute;a reunir las membranas. El mecanismo de la fusi&oacute;n propuesta es apoyado por experimentos bioqu&iacute;micos y an&aacute;lisis de la fusi&oacute;n con el tipo salvaje y mutante integral ATL. del Drosophila Estos experimentos tambi&eacute;n demuestran que la fusi&oacute;n de la membrana es facilitada por las colas citos&oacute;licas c-terminal siguiendo los dos segmentos transmembrana. Finalmente, nuestros resultados demuestran que las mutaciones en ATL1 causando paraplegia esp&aacute;stica hereditaria comprometen homot&iacute;picos fusi&oacute;n de ER.

Estructuras de la GTPasa de atlastin proporcionan la penetraci&oacute;n en homot&iacute;picos fusi&oacute;n de las membranas del ret&iacute;culo endoplasm&aacute;tico.

The homotypic fusion of endoplasmic reticulum (ER) membranes is mediated by atlastin (ATL), which consists of an N-terminal cytosolic domain containing a GTPase module and a three-helix bundle followed by two transmembrane (TM) segments and a C-terminal tail (CT). Fusion depends on a GTP hydrolysis-induced conformational change in the cytosolic domain. Here, we show that the CT and TM segments also are required for efficient fusion and provide insight into their mechanistic roles. The essential feature of the CT is a conserved amphipathic helix. A synthetic peptide corresponding to the helix, but not to unrelated amphipathic helices, can act in trans to restore the fusion activity of tailless ATL. The CT promotes vesicle fusion by interacting directly with and perturbing the lipid bilayer without causing significant lysis. The TM segments do not serve as mere membrane anchors for the cytosolic domain but rather mediate the formation of ATL oligomers. Point mutations in either the C-terminal helix or the TMs impair ATL's ability to generate and maintain ER morphology in vivo. Our results suggest that protein-lipid and protein-protein interactions within the membrane cooperate with the conformational change of the cytosolic domain to achieve homotypic ER membrane fusion.

Lipid interaction of the C terminus and association of the transmembrane segments facilitate atlastin-mediated homotypic endoplasmic reticulum fusion.

Formation of the endoplasmic reticulum (ER) network requires homotypic membrane fusion, which involves a class of atlastin (ATL) GTPases. Purified Drosophila ATL is capable of mediating vesicle fusion in vitro, but such activity has not been reported for any other ATLs. Here, we determined the preliminary crystal structure of the cytosolic segment of Drosophila ATL in a GDP-bound state. The structure reveals a GTPase domain dimer with the subsequent three-helix bundles associating with their own GTPase domains and pointing in opposite directions. This conformation is similar to that of human ATL1, to which GDP and high concentrations of inorganic phosphate, but not GDP only, were included. Drosophila ATL restored ER morphology defects in mammalian cells lacking ATLs, and measurements of nucleotide-dependent dimerization and GTPase activity were comparable for Drosophila ATL and human ATL1. However, purified and reconstituted human ATL1 exhibited no in vitro fusion activity. When the cytosolic segment of human ATL1 was connected to the transmembrane (TM) region and C-terminal tail (CT) of Drosophila ATL, the chimera still exhibited no fusion activity, though its GTPase activity was normal. These results suggest that GDP-bound ATLs may adopt multiple conformations and the in vitro fusion activity of ATL cannot be achieved by a simple collection of functional domains.

Comparison of human and Drosophila atlastin GTPases.

Atlastin (ATL), a membrane-anchored GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes, is required for formation of the tubular network of the peripheral ER. How exactly ATL mediates membrane fusion is only poorly understood. Here we show that fusion is preceded by the transient tethering of ATL-containing vesicles caused by the dimerization of ATL molecules in opposing membranes. Tethering requires GTP hydrolysis, not just GTP binding, because the two ATL molecules are pulled together most strongly in the transition state of GTP hydrolysis. Most tethering events are futile, so that multiple rounds of GTP hydrolysis are required for successful fusion. Supported lipid bilayer experiments show that ATL molecules sitting on the same (cis) membrane can also undergo nucleotide-dependent dimerization. These results suggest that GTP hydrolysis is required to dissociate cis dimers, generating a pool of ATL monomers that can dimerize with molecules on a different (trans) membrane. In addition, tethering and fusion require the cooperation of multiple ATL molecules in each membrane. We propose a comprehensive model for ATL-mediated fusion that takes into account futile tethering and competition between cis and trans interactions.

Cis and trans interactions between atlastin molecules during membrane fusion.

Extended synaptotagmins (E-Syts) mediate lipid exchange between the endoplasmic reticulum (ER) and the plasma membrane (PM). Anchored on the ER, E-Syts bind the PM via an array of C2 domains in a Ca- and lipid-dependent manner, drawing the two membranes close to facilitate lipid exchange. How these C2 domains bind the PM and regulate the ER-PM distance is not well understood. Here, we applied optical tweezers to dissect PM binding by E-Syt1 and E-Syt2. We detected Ca- and lipid-dependent membrane-binding kinetics of both E-Syts and determined the binding energies and rates of individual C2 domains or pairs. We incorporated these parameters in a theoretical model to recapitulate salient features of E-Syt-mediated membrane contacts observed in vivo, including their equilibrium distances and probabilities. Our methods can be applied to study other proteins containing multiple membrane-binding domains linked by disordered polypeptides.

Stepwise membrane binding of extended synaptotagmins revealed by optical tweezers.

Numerous proteins directly or indirectly bind membranes to exert their roles in a wide variety of biological processes. Such membrane binding often occurs in the presence of an external mechanical force. It remains challenging to quantify these interactions using traditional experimental approaches based on a large number of molecules, due to ensemble averaging or the lack of mechanical force. Here we described a new single-molecule approach based on high-resolution optical tweezers to characterize protein-membrane interactions. A single membrane binding protein is attached to the lipid bilayer coated on a silica bead via a flexible polypeptide linker, tethered to another bead via a long DNA handle, and pulled away from the bilayer using optical tweezers. Dynamic protein binding and unbinding is detected by the corresponding changes in the extension of the protein-DNA tether with high spatiotemporal resolution, which reveals the membrane binding affinity, kinetics, and intermediates. We demonstrated the method using C2 domains of extended synaptotagmin 2 (E-Syt2) with a detailed protocol. The method can be widely applied to investigate complex protein-membrane interactions under well-controlled experimental conditions.

Single-Molecule Optical Tweezers Study of Protein-Membrane Interactions.

Lipid transfer proteins (LTPs) transfer lipids between different organelles, and thus play key roles in lipid homeostasis and organelle dynamics. The lipid transfer often occurs at the membrane contact sites (MCS) where two membranes are held within 10-30 nm. While most LTPs act as a shuttle to transfer lipids, recent experiments reveal a new category of eukaryotic LTPs that may serve as a bridge to transport lipids in bulk at MCSs. However, the molecular mechanisms underlying lipid transfer and MCS formation are not well understood. Here, we first review two recent studies of extended synaptotagmin (E-Syt)-mediated membrane binding and lipid transfer using novel approaches. Then we describe mathematical models to quantify the kinetics of lipid transfer by shuttle LTPs based on a lipid exchange mechanism. We find that simple lipid mixing among membranes of similar composition and/or lipid partitioning among membranes of distinct composition can explain lipid transfer against a concentration gradient widely observed for LTPs. We predict that selective transport of lipids, but not membrane proteins, by bridge LTPs leads to osmotic membrane tension by analogy to the osmotic pressure across a semipermeable membrane. A gradient of such tension and the conventional membrane tension may drive bulk lipid flow through bridge LTPs at a speed consistent with the fast membrane expansion observed . Finally, we discuss the implications of membrane tension and lipid transfer in organelle biogenesis. Overall, the quantitative models may help clarify the mechanisms of LTP-mediated MCS formation and lipid transfer.

Quantitative Models of Lipid Transfer and Membrane Contact Formation.

The Synaptotagmin-like Mitochondrial-lipid-binding Protein (SMP) domain is a newly identified lipid transfer module present in proteins that regulate lipid homeostasis at membrane contact sites (MCSs). However, how the SMP domain associates with the membrane to extract and unload lipids is unclear. Here, we performed in vitro DNA brick-assisted lipid transfer assays and in silico molecular dynamics simulations to investigate the molecular basis of the membrane association by the SMP domain of extended synaptotagmin (E-Syt), which tethers the tubular endoplasmic reticulum (ER) to the plasma membrane (PM). We demonstrate that the SMP domain uses its tip region to recognize the extremely curved subdomain of tubular ER and the acidic-lipid-enriched PM for highly efficient lipid transfer. Supporting these findings, disruption of these mechanisms results in a defect in autophagosome biogenesis contributed by E-Syt. Our results suggest a model that provides a coherent picture of the action of the SMP domain at MCSs.

Insights into membrane association of the SMP domain of extended synaptotagmin.

The endoplasmic reticulum (ER), which is composed of a continuous network of tubules and sheets, forms the most widely distributed membrane system in eukaryotic cells. As a result, it engages a variety of organelles by establishing membrane contact sites (MCSs). These contacts regulate organelle positioning and remodeling, including fusion and fission, facilitate precise lipid exchange, and couple vital signaling events. Here, we systematically review recent advances and converging themes on ER-involved organellar contact. The molecular basis, cellular influence, and potential physiological functions for ER/nuclear envelope contacts with mitochondria, Golgi, endosomes, lysosomes, lipid droplets, autophagosomes, and plasma membrane are summarized.

Stay in touch with the endoplasmic reticulum.

Synchronized ferroptosis contributes to nephron loss in acute kidney injury (AKI). However, the propagation signals and the underlying mechanisms of the synchronized ferroptosis for renal tubular injury remain unresolved. Here we report that platelet-activating factor (PAF) and PAF-like phospholipids (PAF-LPLs) mediated synchronized ferroptosis and contributed to AKI. The emergence of PAF and PAF-LPLs in ferroptosis caused the instability of biomembranes and signaled the cell death of neighboring cells. This cascade could be suppressed by PAF-acetylhydrolase (II) (PAFAH2) or by addition of antibodies against PAF. Genetic knockout or pharmacological inhibition of PAFAH2 increased PAF production, augmented synchronized ferroptosis and exacerbated ischemia/reperfusion (I/R)-induced AKI. Notably, intravenous administration of wild-type PAFAH2 protein, but not its enzymatically inactive mutants, prevented synchronized tubular cell death, nephron loss and AKI. Our findings offer an insight into the mechanisms of synchronized ferroptosis and suggest a possibility for the preventive intervention of AKI.

PAFAH2 suppresses synchronized ferroptosis to ameliorate acute kidney injury.

Author Correction: PAFAH2 suppresses synchronized ferroptosis to ameliorate acute kidney injury.

Homotypic membrane fusion of the endoplasmic reticulum (ER) is mediated by dynamin-like GTPase atlastin (ATL). This fundamental process relies on GTP-dependent domain rearrangements in the N-terminal region of ATL (ATL), including the GTPase domain and three-helix bundle (3HB). However, its conformational dynamics during the GTPase cycle remain elusive. Here, we combine single-molecule FRET imaging and molecular dynamics simulations to address this conundrum. Different from the prevailing model, ATL can form a loose crossover dimer upon GTP binding, which is tightened by GTP hydrolysis for membrane fusion. Furthermore, the α-helical motif between the 3HB and transmembrane domain, which is embedded in the surface of the lipid bilayer and self-associates in the crossover dimer, is required for ATL function. To recycle the proteins, Pi release, which disassembles the dimer, activates frequent relative movements between the GTPase domain and 3HB, and subsequent GDP dissociation alters the conformational preference of the ATL monomer for entering the next reaction cycle. Finally, we found that two disease-causing mutations affect human ATL1 activity by destabilizing GTP binding-induced loose crossover dimer formation and the membrane-embedded helix, respectively. These results provide insights into ATL-mediated homotypic membrane fusion and the pathological mechanisms of related disease.

Dissecting the mechanism of atlastin-mediated homotypic membrane fusion at the single-molecule level.

Extended-synaptotagmins (E-Syts) are proteins located on the endoplasmic reticulum (ER) that tether the ER to the plasma membrane (PM) and regulate their lipid homeostasis via its lipid transfer module, the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain. Here, we describe in vitro DNA nanostructure-assisted lipid transfer assays investigating how the SMP domain transports lipids between membranes and associates with the membranes to extract and release lipids. The lipid transfer signal was detected through fluorescence resonance energy transfer (FRET). This method overcomes the limitations of commonly used lipid transfer assays in accurately controlling inter-liposome distance and liposome size, enabling us to further understand the details involved in the process of SMP domain-mediated lipid transfer. Similar platforms can be extended to studying the lipid transfer distance and membrane curvature sensitivity of other lipid transfer proteins.

Xin Bian

State Key Laboratory of Medicinal Chemical Biology,
College of Life Sciences,
Frontiers Science Center for Cell Responses,
State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Frontiers Science Center for Cell Responses

Nankai University

Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin