Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Helmholtz Centre for Infection Research

Sortilin&#xFF0C;&#x4E5F;&#x79F0;&#x4E3A;&#x795E;&#x7ECF;&#x964D;&#x538B;&#x7D20;&#x53D7;&#x4F53; 3 &#xFF08;NTR3&#xFF09;&#xFF0C;&#x662F;&#x5177;&#x6709;&#x53CC;&#x91CD;&#x529F;&#x80FD;&#x7684;&#x8DE8;&#x819C;&#x86CB;&#x767D;&#x3002;&#x5B83;&#x662F;&#x4F5C;&#x4E3A;&#x4E00;&#x79CD;&#x53D7;&#x4F53; neuromediators &#x548C;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x5728;&#x7EC6;&#x80DE;&#x819C;&#xFF0C;&#x4F46;&#x4E5F;&#x7275;&#x8FDE;&#x5728;&#x7ED1;&#x5B9A;&#x548C;&#x4E00;&#x4E9B;&#x6EB6;&#x9176;&#x4F53;&#x86CB;&#x767D;&#x8D28;&#x8FD0;&#x8F93;&#x3002;&#x4F46;&#x662F;&#xFF0C;sortilin phagosome &#x6210;&#x719F;&#x8FC7;&#x7A0B;&#x7684;&#x4F5C;&#x7528;&#x5DF2;&#x4E0D;&#x524D;&#x8C03;&#x67E5;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x663E;&#x793A;&#x5DE8;&#x566C;&#x7EC6;&#x80DE;&#xFF0C;sortilin &#x4E3B;&#x8981;&#x662F;&#x9AD8;&#x5C14;&#x57FA;&#x5728;&#x672C;&#x5730;&#x5316;&#x548C;&#x8FD0;&#x5F80;&#x4E73;&#x80F6;&#x73E0; phagosomes (&#x67B8;&#x675E;&#x591A;&#x7CD6;)&#x3002;&#x6211;&#x4EEC;&#x5229;&#x7528;&#x6D3B;&#x7EC6;&#x80DE;&#x6210;&#x50CF;&#x548C;&#x7535;&#x5B50;&#x663E;&#x5FAE;&#x955C;&#xFF0C;&#x53D1;&#x73B0;&#x8FD9; sortilin &#x53D6;&#x51B3;&#x4E8E;&#x5B83;&#x7684;&#x7EC6;&#x80DE;&#x8D28;&#x5C3E;&#x5DF4;&#x7684;&#x65B9;&#x5F0F;&#x4F20;&#x9012;&#x5230;&#x67B8;&#x675E;&#x591A;&#x7CD6;&#x3002;&#x6211;&#x4EEC;&#x8FD8;&#x663E;&#x793A;&#xFF0C;sortilin &#x53C2;&#x52A0;&#x9178; sphingomyelinase (ASM) &#x548C; prosaposin (PS) &#x76F4;&#x63A5;&#x9001;&#x4EA4; phagosome&#xFF0C;&#x7ED5;&#x8FC7;&#x6EB6;&#x9176;&#x4F53;&#x8F66;&#x53A2;&#x4E0E;&#x878D;&#x5408;&#x3002;&#x8FDB;&#x4E00;&#x6B65;&#x7684;&#x5206;&#x6790;&#x8BC1;&#x5B9E; ASM &#x548C; PS &#x4E3A;&#x76EE;&#x6807;&#x5411; phagosome sortilin Brefeldin A &#x654F;&#x611F;&#x901A;&#x8DEF;&#x4E2D;&#x3002;&#x4E3B;&#x8981;&#x5206;&#x79BB; Sort1(-/-) &#x5C0F;&#x9F20;&#x7684;&#x5DE8;&#x566C;&#x7EC6;&#x80DE;&#x7684;&#x5206;&#x6790;&#x6307;&#x51FA;&#x5BF9;&#x67B8;&#x675E;&#x591A;&#x7CD6;&#x7684; ASM &#x548C; PS&#xFF0C;&#x4F46;&#x4E0D;&#x662F; pro-cathepsin D&#xFF0C;&#x4EA4;&#x8D27;&#x4E25;&#x91CD;&#x53D7;&#x635F;&#x3002;&#x6211;&#x4EEC;&#x5EFA;&#x8BAE;&#x901A;&#x8DEF;&#x4ECB;&#x5BFC;&#x7684; sortilin &#x6240;&#x9009;&#x5B9A;&#x7684;&#x6EB6;&#x9176;&#x4F53;&#x86CB;&#x767D;&#x8D28;&#x8FD0;&#x5230;&#x9AD8;&#x5C14;&#x57FA;&#x4F9D;&#x8D56;&#x6CBF;&#x7EBF; phagosome &#x5728; phagosomes &#x7684;&#x6210;&#x719F;&#x8FC7;&#x7A0B;&#x3002;

&#x9AD8;&#x5C14;&#x57FA;&#x5411; phagosome &#x8FD0;&#x8F93;&#x9178; sphingomyelinase &#x548C; prosaposin &#x88AB;&#x4ECB;&#x5BFC; sortilin&#x3002;

Sortilin, also known as neurotensin receptor 3 (NTR3), is a transmembrane protein with a dual function. It acts as a receptor for neuromediators and growth factors at the plasma membrane, but it has also been implicated in binding and transport of some lysosomal proteins. However, the role of sortilin during phagosome maturation has not been investigated before. Here, we show that in macrophages, sortilin is mainly localized in the Golgi and transported to latex-bead phagosomes (LBPs). Using live-cell imaging and electron microscopy, we found that sortilin is delivered to LBPs in a manner that depends on its cytoplasmic tail. We also show that sortilin participates in the direct delivery of acid sphingomyelinase (ASM) and prosaposin (PS) to the phagosome, bypassing fusion with lysosomal compartments. Further analysis confirmed that ASM and PS are targeted to the phagosome by sortilin in a Brefeldin-A-sensitive pathway. Analysis of primary macrophages isolated from Sort1(-/-) mice indicated that the delivery of ASM and PS, but not pro-cathepsin D, to LBPs was severely impaired. We propose a pathway mediated by sortilin by which selected lysosomal proteins are transported to the phagosome along a Golgi-dependent route during the maturation of phagosomes.

Golgi-to-phagosome transport of acid sphingomyelinase and prosaposin is mediated by sortilin.

SORTILINE, &eacute;galement connu sous le nom de neurotensine r&eacute;cepteur 3 (NTR3), est une prot&eacute;ine transmembranaire avec une double fonction. Il agit comme un r&eacute;cepteur de neurom&eacute;diateurs et facteurs de croissance &agrave; la membrane plasmique, mais il a aussi &eacute;t&eacute; impliqu&eacute; dans la liaison et le transport de certaines prot&eacute;ines lysosomales. Cependant, le r&ocirc;le de SORTILINE au cours de la maturation du phagosome n&#39;a pas &eacute;t&eacute; &eacute;tudi&eacute; avant. Ici, nous montrons que, dans les macrophages, SORTILINE est principalement localis&eacute;e dans l&#39;appareil de Golgi et transport&eacute;e dans les phagosomes latex-bead (LBPs). &Agrave; l&#39;aide d&#39;imagerie de cellules vivantes et la microscopie &eacute;lectronique, nous avons constat&eacute; que SORTILINE est remis &agrave; LBPs d&#39;une mani&egrave;re qui d&eacute;pend de sa queue cytoplasmique. Nous montrons &eacute;galement que SORTILINE participe &agrave; l&#39;injection directe de la sphingomy&eacute;linase acide (ASM) et prosaposin (PS) dans le phagosome, sans passer par la fusion avec compartiments lysosomes. Une analyse plus approfondie a confirm&eacute; que ASM et PS ciblent le phagosome par SORTILINE dans une voie br&eacute;feldine A-sensible. Analyse des macrophages primaires isol&eacute;s de souris Sort1(-/-) a indiqu&eacute; que la livraison de l&#39;ASM et le PS, mais pas pro-cathepsin D, &agrave; LBPs a &eacute;t&eacute; gravement r&eacute;duite. Nous proposons une voie m&eacute;di&eacute;e par SORTILINE par lequel certaines prot&eacute;ines lysosomiales sont transport&eacute;s vers le phagosome sur un itin&eacute;raire de Golgi-d&eacute;pendante au cours de la maturation des phagosomes.

Transport de Golgi-&agrave;-phagosome sphingomy&eacute;linase acide et prosaposin est m&eacute;di&eacute;e par la SORTILINE.

Sortilin, auch bekannt als Neurotensin Rezeptor 3 (NTR3), ist ein Transmembranprotein mit eine Doppelfunktion. Es fungiert als ein Rezeptor f&uuml;r Neuromediators und Wachstumsfaktoren an die Plasmamembran, aber es hat auch in die Bindung und den Transport von einigen lysosomalen Proteinen verwickelt worden. Allerdings ist die Rolle des Sortilin w&auml;hrend Phagosom Reifung vor nicht untersucht worden. Hier zeigen wir, dass in Makrophagen, Sortilin haupts&auml;chlich in den Golgi lokalisiert und in Latex-Bead Phagosomen (LBPs) transportiert. Mit Leben-Zelle Darstellung und Elektronenmikroskopie, fanden wir, dass Sortilin LBPs in einer Weise zugestellt wird, die auf den zytoplasmatischen Schwanz abh&auml;ngt. Wir zeigen auch, dass Sortilin in der direkten Lieferung der sauren Sphingomyelinase (ASM) und Prosaposin (PS) an die Phagosom beteiligt sich Fusion mit lysosomalen F&auml;cher zu umgehen. Weitere Analyse best&auml;tigt, dass ASM und PS von Sortilin in einen Brefeldin-A-Sensitive-Weg zu den Phagosom ausgerichtet sind. Analyse der prim&auml;ren Makrophagen isoliert von den Sort1(-/-) M&auml;usen ergaben, dass die Lieferung von ASM und PS, aber nicht pro-cathepsin D, an LBPs stark eingeschr&auml;nkt wurde. Wir schlagen einen Weg von Sortilin, die durch die ausgew&auml;hlten lysosomale Proteine in die Phagosom auf einer Golgi-abh&auml;ngige-Route, w&auml;hrend der Reifung der Phagosomen transportiert werden vermittelt.

Golgi-Phagosom-Transport von saure Sphingomyelinase und Prosaposin ist durch Sortilin vermittelt.

Sortilin, noto anche come neurotensina recettore 3 (NTR3), &egrave; una proteina transmembrana con una duplice funzione. Esso agisce come un recettore per neuromediators e fattori di crescita alla membrana del plasma, ma &egrave; stato anche implicato nel trasporto di alcune proteine lisosomiali e vincolante. Tuttavia, il ruolo di sortilin durante la maturazione del Fagosoma non &egrave; stato studiato prima. Qui, ci mostrano che nei macrofagi, sortilin &egrave; principalmente localizzata nel Golgi e trasportati in lattice-bead phagosomes (LBPs). Usando live cell imaging e microscopia elettronica, abbiamo trovato che sortilin &egrave; consegnato a LBPs in un modo che dipende dalla sua coda citoplasmatica. Vi mostriamo anche quello sortilin partecipa la consegna diretta della sfingomielinasi acida (ASM) e prosaposina (PS) per il Fagosoma, bypassando la fusione con compartimenti lisosomiali. Un&#39;ulteriore analisi ha confermato che ASM e PS sono mirati per il Fagosoma da sortilin in un percorso di Gbf1-A-sensibile. Analisi dei macrofagi primari isolati da topi Sort1(-/-) indicano che la consegna di ASM e PS, ma non pro-cathepsin D, LBPs era gravemente compromessa. Proponiamo un percorso mediato da sortilin da cui selezionate proteine lisosomiali vengono trasportate in Fagosoma lungo un itinerario di Golgi-dipendente durante la maturazione del phagosomes.

Trasporto di Golgi-Fagosoma della sfingomielinasi acida e prosaposina &egrave; mediata da sortilin.

&#x30BD;&#x30FC;&#x30C6;&#x30A3;&#x30EA;&#x30F3;&#x3068;&#x3057;&#x3066;&#x3082;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30C6;&#x30F3;&#x30B7;&#x30F3;&#x53D7;&#x5BB9;&#x4F53; 3 (NTR3)&#x3001;&#x30C7;&#x30E5;&#x30A2;&#x30EB;&#x6A5F;&#x80FD;&#x4ED8;&#x304D;&#x819C;&#x8CAB;&#x901A;&#x86CB;&#x767D;&#x8CEA;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;Neuromediators &#x3068;&#x539F;&#x5F62;&#x8CEA;&#x819C;&#x306E;&#x6210;&#x9577;&#x56E0;&#x5B50;&#x306E;&#x53D7;&#x5BB9;&#x4F53;&#x3068;&#x3057;&#x3066;&#x6A5F;&#x80FD;&#x304C;&#x3001;&#x305D;&#x308C;&#x306F;&#x307E;&#x305F;&#x30D0;&#x30A4;&#x30F3;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x3068;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x30E9;&#x30A4;&#x30BD;&#x30BE;&#x30FC;&#x30E0; &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x8F38;&#x9001;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x30BD;&#x30FC;&#x30C6;&#x30A3;&#x30EA;&#x30F3; &#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x6210;&#x719F;&#x904E;&#x7A0B;&#x306E;&#x5F79;&#x5272;&#x524D;&#x306B;&#x8ABF;&#x67FB;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005; &#x30DE;&#x30AF;&#x30ED;&#x30D5;&#x30A1;&#x30FC;&#x30B8;&#x306E;&#x30BD;&#x30FC;&#x30C6;&#x30A3;&#x30EA;&#x30F3;&#x3067;&#x3059;&#x4E3B;&#x306B;&#x30B4;&#x30EB;&#x30B8;&#x4F53;&#x306E;&#x5C40;&#x5728;&#x3057;&#x3001;&#x30E9;&#x30C6;&#x30C3;&#x30AF;&#x30B9; &#x30D3;&#x30FC;&#x30BA; phagosome (&#x307E;&#x3048;&#x304C;&#x304D;) &#x8F38;&#x9001;. &#x3057;&#x307E;&#x3059;&#x3002;&#x4F4F;&#x30BB;&#x30EB;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x6295;&#x5C04;&#x304A;&#x3088;&#x3073;&#x96FB;&#x5B50;&#x9855;&#x5FAE;&#x93E1;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x305D;&#x306E;&#x30BD;&#x30FC;&#x30C6;&#x30A3;&#x30EA;&#x30F3;&#x307E;&#x3048;&#x304C;&#x304D;&#x306B;&#x305D;&#x306E;&#x7D30;&#x80DE;&#x8CEA;&#x30C6;&#x30FC;&#x30EB;&#x306B;&#x4F9D;&#x5B58;&#x65B9;&#x6CD5;&#x3067;&#x914D;&#x4FE1;&#x3055;&#x308C;&#x307E;&#x3059;&#x304C;&#x767A;&#x898B;&#x3057;&#x307E;&#x3059;&#x3002;&#x307E;&#x305F;&#x3001;&#x30BD;&#x30FC;&#x30C6;&#x30A3;&#x30EA;&#x30F3;&#x9178;&#x30B9;&#x30D5;&#x30A3;&#x30F3;&#x30B4;&#x30DF;&#x30A8;&#x30EA;&#x30CA;&#x30FC;&#x30BC; (ASM) &#x3068; prosaposin (PS) &#x306E;&#x76F4;&#x63A5;&#x914D;&#x4FE1;&#x306F;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x306B;&#x53C2;&#x52A0;&#x30E9;&#x30A4;&#x30BD;&#x30BE;&#x30FC;&#x30E0; &#x30B3;&#x30F3;&#x30D1;&#x30FC;&#x30C8;&#x30E1;&#x30F3;&#x30C8;&#x3068;&#x306E;&#x878D;&#x5408;&#x3092;&#x30D0;&#x30A4;&#x30D1;&#x30B9;. &#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306A;&#x308B;&#x5206;&#x6790;&#x306F;&#x3001;ASM &#x3068; PS &#x30BD;&#x30FC;&#x30C6;&#x30A3;&#x30EA;&#x30F3; &#x30D6;&#x30EC;&#x30D5;&#x30A7;&#x30EB;&#x30B8;&#x30F3; A &#x611F;&#x53D7;&#x6027;&#x7D4C;&#x8DEF;&#x3067;&#x306E;&#x3059;&#x308B;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x304C;&#x5BFE;&#x8C61;&#x3068;&#x78BA;&#x8A8D;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;Sort1(-/-) &#x30DE;&#x30A6;&#x30B9;&#x304B;&#x3089;&#x5206;&#x96E2;&#x3057;&#x305F;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x306E;&#x30DE;&#x30AF;&#x30ED;&#x30D5;&#x30A1;&#x30FC;&#x30B8;&#x306E;&#x89E3;&#x6790;&#x3078;&#x307E;&#x3048;&#x304C;&#x304D; ASM &#x3068; PS &#x304C;&#x306A;&#x3044; pro-cathepsin D &#x306E;&#x914D;&#x4FE1;&#x91CD;&#x5EA6;&#x969C;&#x5BB3;&#x8005;&#x304C;&#x793A;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x306B;&#x3088;&#x3063;&#x3066; phagosome &#x306E;&#x6210;&#x719F;&#x306B;&#x4F34;&#x3046;&#x30B4;&#x30EB;&#x30B8;&#x4F9D;&#x5B58;&#x578B;&#x30EB;&#x30FC;&#x30C8;&#x306B;&#x6CBF;&#x3063;&#x3066;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x306B;&#x9078;&#x629E;&#x3057;&#x305F;&#x30EA;&#x30BD;&#x30BD;&#x30FC;&#x30E0; &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x304C;&#x8F38;&#x9001;&#x3055;&#x308C;&#x308B;&#x30BD;&#x30FC;&#x30C6;&#x30A3;&#x30EA;&#x30F3;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x7D4C;&#x8DEF;&#x3092;&#x63D0;&#x6848;&#x3059;&#x308B;.

&#x30B4;&#x30EB;&#x30B8; &#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0; &#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30FC;&#x30C8;&#x9178;&#x30B9;&#x30D5;&#x30A3;&#x30F3;&#x30B4;&#x30DF;&#x30A8;&#x30EA;&#x30CA;&#x30FC;&#x30BC;&#x3068; prosaposin &#x306E;&#x30BD;&#x30FC;&#x30C6;&#x30A3;&#x30EA;&#x30F3;&#x306B;&#x3088;&#x3063;&#x3066;&#x5A92;&#x4ECB;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;

Sortilin, &#xC77C;&#xCEEC;&#xC5B4; neurotensin &#xC218;&#xC6A9; &#xCCB4; 3 (NTR3), &#xB4C0;&#xC5BC; &#xAE30;&#xB2A5;&#xC744; &#xAC00;&#xC9C4; &#xB9C9; &#xD6A1;&#xB2E8; &#xB2E8;&#xBC31;&#xC9C8; &#xC774;&#xB2E4;. &#xADF8;&#xAC83;&#xC740; neuromediators &#xBC0F; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778;&#xC5D0;&#xC11C; &#xC131;&#xC7A5; &#xC778;&#xC790; &#xC218;&#xC6A9; &#xCCB4; &#xC5ED;&#xD560;&#xC744; &#xD558;&#xC9C0;&#xB9CC; &#xBC14;&#xC778;&#xB529; &#xBC0F; &#xC77C;&#xBD80; lysosomal &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC218;&#xC1A1;&#xC5D0;&#xB3C4; &#xC5F0;&#xB8E8; &#xB418;&#xC5B4; &#xC788;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, phagosome &#xC131;&#xC219; &#xD558;&#xB294; &#xB3D9;&#xC548; sortilin&#xC758; &#xC5ED;&#xD560; &#xD558;&#xAE30; &#xC804;&#xC5D0; &#xC870;&#xC0AC; &#xD558;&#xC9C0;&#xB294;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xB294;&#xC5D0;&#xC11C; &#xB300; &#xC2DD; &#xC138;&#xD3EC;, sortilin&#xB294; &#xC8FC;&#xB85C; Golgi &#xC9C0;&#xC5ED;&#xD654; &#xC774;&#xBA70; &#xB77C;&#xD14D;&#xC2A4; &#xBE44;&#xB4DC; phagosomes (LBPs)&#xB85C; &#xC774;&#xC1A1; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xB77C;&#xC774;&#xBE0C; &#xC140; &#xC774;&#xBBF8;&#xC9D5; &#xBC0F; &#xC804;&#xC790; &#xD604;&#xBBF8;&#xACBD;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;, &#xC6B0;&#xB9AC;&#xB294; &#xADF8; sortilin LBPs &#xC138;&#xD3EC;&#xC9C8; &#xAF2C;&#xB9AC;&#xC5D0; &#xC758;&#xC874; &#xD558;&#xB294; &#xBC29;&#xC2DD;&#xC73C;&#xB85C; &#xC804;&#xB2EC; &#xBC1C;&#xACAC;. &#xC6B0;&#xB9AC; &#xB610;&#xD55C; &#xADF8; sortilin phagosome, &#xD558; &#xC0B0; sphingomyelinase (ASM) &#xBC0F; prosaposin (PS)&#xC758; &#xC9C1;&#xC811; &#xBC30;&#xB2EC;&#xC5D0; &#xCC38;&#xC5EC; &#xD558; &#xACE0; &#xC735;&#xD569; lysosomal &#xAD6C;&#xD68D;&#xC744; &#xC6B0;&#xD68C; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xCD94;&#xAC00; &#xBD84;&#xC11D; &#xD655;&#xC778; ASM &#xBC0F; PS&#xC5D0;&#xC11C; phagosome&#xB97C; sortilin Brefeldin A &#xBBFC;&#xAC10;&#xD55C; &#xD1B5;&#xB85C;&#xC5D0; &#xC758;&#xD574; &#xB300;&#xC0C1; &#xB429;&#xB2C8;&#xB2E4;. Sort1(-/-) &#xC950;&#xC5D0;&#xC11C; &#xBD84;&#xB9AC; &#xB41C; &#xC8FC; &#xC138;&#xD3EC; &#xBD84;&#xC11D; Lbps&#xC5D0; ASM &#xBC0F; PS, &#xD558;&#xC9C0;&#xB9CC; &#xD558;&#xC9C0; pro-cathepsin D&#xC758; &#xBC30;&#xB2EC; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4; &#xC2EC;&#xAC01; &#xC7A5;&#xC560;&#xC778; &#xD45C;&#xC2DC;. &#xACBD;&#xB85C;&#xB294; &#xC120;&#xD0DD;&#xD55C; lysosomal &#xB2E8;&#xBC31;&#xC9C8;&#xC740; &#xC774;&#xC1A1; Golgi &#xC885;&#xC18D; &#xACBD;&#xB85C; &#xB530;&#xB77C; phagosome phagosomes &#xC131;&#xC219; &#xD558;&#xB294; &#xB3D9;&#xC548; sortilin&#xC5D0; &#xC758;&#xD574; &#xC911;&#xC7AC; &#xC81C;&#xC548; &#xD55C;&#xB2E4;.

Golgi phagosome &#xC804;&#xC1A1; &#xC0B0; sphingomyelinase&#xC640; prosaposin&#xC758; sortilin&#xC5D0; &#xC758;&#xD574; &#xC911;&#xC7AC;&#xC785;&#xB2C8;&#xB2E4;.

Sortilina, tambi&eacute;n conocido como receptor de neurotensina 3 (NTR3), es una prote&iacute;na transmembrana con una doble funci&oacute;n. Act&uacute;a como un receptor para neuromediadores y factores de crecimiento en la membrana plasm&aacute;tica, sino tambi&eacute;n se ha implicado en enlace y transporte de algunas prote&iacute;nas lisosomales. Sin embargo, no ha investigado el papel de sortilina durante la maduraci&oacute;n del fagosoma antes. Aqu&iacute;, demostramos que en macr&oacute;fagos, sortilina principalmente est&aacute; localizada en el Golgi y transportado al l&aacute;tex-grano fagosomas (LBPs). Usando microscopia electr&oacute;nica y la proyecci&oacute;n de imagen de c&eacute;lulas vivas, encontramos que sortilina se entrega a LBPs de una manera que depende de su Cola citoplasm&aacute;tica. Tambi&eacute;n mostramos que sortilina participa en la prestaci&oacute;n directa de esfingomielinasa &aacute;cida (ASM) y prosaposin (PS) para el fagosoma, evitando la fusi&oacute;n con compartimentos lisosomales. An&aacute;lisis posterior confirm&oacute; que ASM y PS son dirigidos al fagosoma por sortilina en un camino de Brefeldino-A-sensible. An&aacute;lisis de macr&oacute;fagos primarios aislados de ratones Sort1(-/-) indic&oacute; que la entrega de ASM y PS, pero no pro-cathepsin D, LBPs era seriamente deteriorada. Proponemos una v&iacute;a mediada por sortilina por el cual las prote&iacute;nas lisosomales son transportadas al fagosoma a lo largo de una ruta de Golgi-dependiente durante la maduraci&oacute;n de los fagosomas.

Transporte de Golgi-a-fagosoma de esfingomielinasa &aacute;cida y prosaposin est&aacute; mediada por sortilina.

Bacterial and parasitic intracellular pathogens or their secreted products have been shown to induce host cell transcriptional responses, which may benefit the host, favour the microorganism or be unrelated to the infection. In most instances, however, it is not known if the host cell nucleus is proximately required for the development of an intracellular infection. This information can be obtained by the infection of artificially enucleated host cells (cytoplasts). This model, although rather extensively used in studies of viral infection, has only been applied to few bacterial pathogens, which do not include Mycobacterium spp. Here, we investigate the internalization, phagosome biogenesis and survival of M. smegmatis in enucleated type II alveolar epithelial cells. Cytoplasts were infected with M. smegmatis, but the percentage of infection was significantly lower than that of nucleated cells. Scanning electron microscopy indicated that in both cells and cytoplasts, bacteria were internalized by a phagocytosis-like mechanism. Interestingly, phagosome fusion with lysosomes and mycobacterial killing were both more efficient in enucleated than in nucleated cells, a finding that may be correlated with the increased number of autophagic vesicles developed in cytoplasts. We provide evidence that although quantitative changes were observed, the full development of the infection, as well as mycobacterial killing did not require the presence of the host cell nucleus.

Internalization, phagolysosomal biogenesis and killing of mycobacteria in enucleated epithelial cells.

Phagosome maturation is an essential part of the innate and adaptive immune response. Although it is well established that several Ras-related proteins in brain (Rab) proteins become associated to phagosomes, little is known about how these phagosomal Rab proteins influence phagosome maturation. Here, we show a specific role for Rab34 and mammalian uncoordinated 13-2 (Munc13-2) in phagolysosome biogenesis and cargo delivery. Rab34 knockdown impaired the fusion of phagosomes with late endosomes/lysosomes and high levels of active Rab34 promoted this process. We demonstrate that Rab34 enhances phagosome maturation independently of Rab7 and coordinates phagolysosome biogenesis through size-selective transfer of late endosomal/lysosomal cargo into phagosomes. More importantly, we show that Rab34 mediates phagosome maturation through the recruitment of the protein Munc13-2. Finally, we report that the alternative maturation pathway controlled by Rab34 is critical for mycobacterial killing because Rab34 silencing resulted in mycobacterial survival, and Rab34 expression led to mycobacterial killing. Altogether, our studies uncover Rab34/Munc13-2 as a critical part of an alternative Rab7-independent phagosome maturation machinery and lysosome-mediated killing of mycobacteria.

Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34.

Within a cell, the regulation of organelle positioning is considered to be critical in spatio-temporal responses. The position of late endocytic organelles (named here lysosomes for simplicity) is tightly controlled and has a functional impact on processes like endocytosis, phagocytosis and autophagocytosis. The cytoplasmic distribution profile of lysosomes can be easily determined in cells where the cytoplasm/nuclear ratio in a cross-section area is high. However, determining lysosomal position in cells with lower cytoplasm/nuclear ratio, such as macrophages is more challenging. Here, we describe a method that can be efficiently and accurately used to determine the position of organelles in macrophages using confocal microscopy in two-dimensional (2D) images. Using this approach in macrophages, we confirmed previous observations in epithelial cells that both changes in cytoplasmic pH and the levels of active Rab34 induced a re-distribution of lysosomes to the cell centre or periphery. Noteworthy is that this Rab34-dependent re-distribution of lysosomes did not significantly affect the spatial distribution profile of phagolysosomes in the cytoplasm. We conclude that although Rab34 regulates both lysosomal positioning and lysosome to phagosome fusion, the latter effect is not due to the regulation of the cytoplasmic accessibility of lysosomes to phagosomes by Rab34.

Bahram Kasmapour

Research Group Phagosome Biology

Helmholtz Centre for Infection Research

Study of Phagolysosome Biogenesis in Live Macrophages

Bahram Kasmapour

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Research Group Phagosome Biology

Helmholtz Centre for Infection Research

Study of Phagolysosome Biogenesis in Live Macrophages

Research Group Phagosome Biology