label-free-identification-lymphocyte-subtypes-using-three-dimensional

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification ...

Korea Advanced Institute of Science and Technology

Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol(3,4,5)-trisphosphate (PIP3) control cell growth, migration, and other processes by recruiting proteins with pleckstrin homology (PH) domains and possibly other domains to the plasma membrane (PM). However, previous experimental and structural work with PH domains left conflicting evidence about which ones are PIP3 regulated. Here we used live-cell confocal imaging of 130 YFP-conjugated mouse PH domains and found that 20% translocated to the PM in response to receptor-generated PIP3 production. We developed a recursive-learning algorithm to predict PIP3 regulation of 1200 PH domains from different eukaryotes and validated that it accurately predicts PIP3 regulation. Strikingly, this algorithm showed that PIP3 regulation is specified by amino acids across the PH domain, not just the PIP3-binding pocket, and must have evolved several times independently from PIP3-insensitive ancestral PH domains. Finally, our algorithm and live-cell experiments provide a functional survey of PH domains in different species, showing that PI3K regulation increased from approximately two C. elegans and four Drosophila to 40 vertebrate proteins.

Comprehensive identification of PIP3-regulated PH domains from C. elegans to H. sapiens by model prediction and live imaging.

During infection, hepatitis C virus (HCV) NS4B protein remodels host membranes to form HCV replication complexes (RC) which appear as foci under fluorescence microscopy (FM). To understand the role of Rab proteins in forming NS4B foci, cells expressing the HCV replicon were examined biochemically and via FM. First, we show that an isolated NS4B-bound subcellular fraction is competent for HCV RNA synthesis. Further, this fraction is differentially enriched in Rab1, 2, 5, 6 and 7. However, when examined via FM, NS4B foci appear to be selectively associated with Rab5 and Rab7 proteins. Additionally, dominant negative (DN) Rab6 expression impairs Rab5 recruitment into NS4B foci. Further, silencing of Rab5 or Rab7 resulted in a significant decrease in HCV genome replication. Finally, expression of DN Rab5 or Rab7 led to a reticular NS4B subcellular distribution, suggesting that endocytic proteins Rab5 and Rab7, but not Rab11, may facilitate NS4B foci formation.

Endocytic Rab proteins are required for hepatitis C virus replication complex formation.

Phosphoinositide 3-kinases (PI3Ks) and Ras and Rho family small GTPases are key regulators of cell polarization, motility, and chemotaxis. They influence each other's activities by direct and indirect feedback processes that are only partially understood. Here, we show that 21 small GTPase homologs activate PI3K. Using a microscopy-based binding assay, we show that K-Ras, H-Ras, and five homologous Ras family small GTPases function upstream of PI3K by directly binding the PI3K catalytic subunit, p110. In contrast, several Rho family small GTPases activated PI3K by an indirect cooperative positive feedback that required a combination of Rac, CDC42, and RhoG small GTPase activities. Thus, a distributed network of Ras and Rho family small GTPases induces and reinforces PI3K activity, explaining past challenges to elucidate the specific relevance of different small GTPases in regulating PI3K and controlling cell polarization and chemotaxis.

Cooperative activation of PI3K by Ras and Rho family small GTPases.

Protrudin &#x662F; FYVE (Fab 1&#x3001; YOTB&#x3001; Vac 1 &#x548C; EEA1&#xFF09; &#x57DF;&#x5305;&#x542B;&#x86CB;&#x767D;&#x53C2;&#x4E0E;&#x795E;&#x7ECF;&#x5143;&#x8D27;&#x7269;&#x8FD0;&#x8F93;&#x548C;&#x7275;&#x8FDE;&#x7684;&#x9057;&#x4F20;&#x6027;&#x75C9;&#x631B;&#x6027;&#x622A;&#x762B; (HSP) &#x53D1;&#x4F5C;&#x3002;&#x8102;&#x8D28;&#x7ED1;&#x5B9A;&#x57DF; &#xFF08;LBD&#xFF09; &#x5E93;&#x6211;&#x4EEC;&#x57FA;&#x4E8E;&#x56FE;&#x50CF;&#x7684;&#x7B5B;&#x9009;&#x53D1;&#x73B0;&#x65B0;&#x578B;&#x7B49;&#x79BB;&#x5B50;&#x819C; &#xFF08;PM&#xFF09; &#x672C;&#x5730;&#x5316;&#x7684; FYVE &#x57DF;&#x7684; protrudin &#x4E0E;&#x89C4;&#x8303;&#x5DF2;&#x672C;&#x5730;&#x5316;&#x5230;&#x65E9;&#x671F;&#x7684; endosomes &#x7684; FYVE &#x57DF;&#x4E0D;&#x540C;&#x3002;&#x819C;&#x7EA6;&#x675F;&#x529B;&#x7684;&#x8868;&#x9762;&#x7B49;&#x79BB;&#x5B50;&#x4F53;&#x5171;&#x632F; (SPR) &#x5206;&#x6790;&#x7814;&#x7A76;&#x8868;&#x660E;&#x6B64; FYVE &#x57DF;&#x4F18;&#x5148;&#x7ED1;&#x5B9A;&#x78F7;&#x8102;&#x9170;&#x808C;&#x9187;-4&#xFF0C;5-&#x4E8C;&#x78F7;&#x9178; (PtdIns(4,5)P(2))&#xFF0C;&#x4E8C;&#x78F7;&#x8102;&#x9170;&#x808C;&#x9187;-3&#xFF0C;4-&#x78F7;&#x9178; (PtdIns(3,4)P(2)) &#x548C;&#x78F7;&#x8102;&#x9170;&#x808C;&#x9187;-3,4,5-&#x5185; (PtdIns(3,4,5)P(3)) &#x4E0E;&#x4E0D;&#x540C;&#x7684;&#x89C4;&#x8303; FYVE &#x57DF;&#xFF0C;&#x4E13;&#x95E8;&#x5C06;&#x7ED1;&#x5B9A;&#x78F7;&#x8102;&#x9170;&#x808C;&#x9187;-3-&#x78F7;&#x9178; (PtdIns(3)P)&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x8FD9;&#x4E9B;&#x808C;&#x9187; (PtdInsP) &#x5DEE;&#x5F02;&#x89C4;&#x7BA1;&#x7684;&#x901A;&#x8FC7;&#x5176; FYVE &#x57DF; protrudin endosomes &#x548C;&#x4E0B;&#x5348;&#x4E4B;&#x95F4;&#x7A7F;&#x68AD;&#x3002;Protrudin &#x7A81;&#x53D8;&#x4F53;&#x51CF;&#x5C11; PtdInsP &#x7ED1;&#x5B9A;&#x4EB2;&#x548C;&#x529B;&#x672A;&#x80FD;&#x4FC3;&#x8FDB;&#x539F;&#x4EE3;&#x57F9;&#x517B;&#x6D77;&#x9A6C;&#x795E;&#x7ECF;&#x5143;&#x7A81;&#x8D77;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x65B0;&#x578B; PtdInsP &#x9009;&#x62E9;&#x6027;&#x7684; protrudin FYVE &#x57DF;&#x53CA;&#x5176;&#x7EC6;&#x80DE;&#x5B9A;&#x4F4D;&#x548C;&#x5176;&#x5728;&#x7A81;&#x8D77;&#x7684;&#x4F5C;&#x7528;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x3002;

&#x808C;&#x9187;&#x5DEE;&#x5F02;&#x8C03;&#x8282; protrudin FYVE &#x57DF;&#x901A;&#x8FC7;&#x672C;&#x5730;&#x5316;&#x3002;

Protrudin is a FYVE (Fab 1, YOTB, Vac 1 and EEA1) domain-containing protein involved in transport of neuronal cargoes and implicated in the onset of hereditary spastic paraplegia (HSP). Our image-based screening of lipid binding domain (LBD) library revealed novel plasma membrane (PM) localization of the FYVE domain of protrudin unlike canonical FYVE domains that are localized to early endosomes. The membrane binding study by surface plasmon resonance (SPR) analysis showed that this FYVE domain preferentially binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)), phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) unlike canonical FYVE domains that specifically bind phosphatidylinositol-3-phosphate (PtdIns(3)P). Furthermore, we found that these phosphoinositides (PtdInsP) differentially regulate shuttling of protrudin between endosomes and PM via its FYVE domain. Protrudin mutants with reduced PtdInsP-binding affinity failed to promote neurite outgrowth in primary cultured hippocampal neurons. These results suggest that novel PtdInsP selectivity of protrudin-FYVE domain is critical for its cellular localization and its role in neurite outgrowth.

Phosphoinositides differentially regulate protrudin localization through the FYVE domain.

Protrudin est une prot&eacute;ine contenant du domaine FYVE (Fab 1, YOTB, Vac 1 et EEA1) impliqu&eacute; dans le transport de cargaisons neuronales et mis en cause dans l&#39;apparition d&#39;une parapl&eacute;gie spastique h&eacute;r&eacute;ditaire (HSP). Notre criblage imageur de lipidique liaison domaine (LBD) r&eacute;v&eacute;l&eacute; nouvelle membrane plasmique (PM) localisation du domaine FYVE de protrudin &agrave; la diff&eacute;rence des domaines FYVE canoniques qui sont localis&eacute;es &agrave; des endosomes pr&eacute;coces. La membrane liant &eacute;tude en analyse de surface plasmon resonance (SPR) a montr&eacute; que ce domaine FYVE lie pr&eacute;f&eacute;rentiellement le phosphatidylinositol-4, 5-bisphosphate (PtdIns(4,5)P(2)), phosphatidylinositol-3, 4-bisphosphate (PtdIns(3,4)P(2)) et phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) &agrave; la diff&eacute;rence des domaines FYVE canoniques qui lient sp&eacute;cifiquement phosphatidylinositol-3-phosphate (PtdIns(3)P). En outre, nous avons constat&eacute; que ces phosphoinositides (PtdInsP) r&eacute;glementer diff&eacute;remment la navette de protrudin entre les endosomes et PM, par l&#39;interm&eacute;diaire de son domaine FYVE. Protrudin mutants avec PtdInsP-affinit&eacute; r&eacute;duite n&#39;a pas &agrave; promouvoir la croissance des neurites dans des neurones hippocampal cultiv&eacute;s primaires. Ces r&eacute;sultats sugg&egrave;rent que la s&eacute;lectivit&eacute; roman de PtdInsP du domaine de protrudin-FYVE est critique pour sa localisation cellulaire et son r&ocirc;le dans la croissance des neurites.

Phosphoinositides r&eacute;glementer diff&eacute;remment protrudin localisation par le domaine FYVE.

Protrudin ist ein FYVE (Fab 1, YOTB, Vac 1 und EEA1) Dom&auml;ne-haltige Protein Transport von neuronalen Ladungen beteiligt und bei der Entstehung von Heredit&auml;re Spastische Paraplegie (HSP) verwickelt. Unser Image-basierten Screening von Lipid Bindung Dom&auml;ne (LBD) Bibliothek offenbart Roman Plasmamembran (PM)-Lokalisierung der FYVE-Dom&auml;ne des protrudin im Gegensatz zu den kanonischen FYVE-Dom&auml;nen, die auf fr&uuml;hen Endosomen lokalisiert sind. Die Membran binden Studie anhand des Oberfl&auml;chen-Plasmon-Resonanz (SPR)-Analyse zeigte, dass diese FYVE Gebiet bindet bevorzugt Phosphatidylinositol-4, 5-bisphosphat (PtdIns(4,5)P(2)), Phosphatidylinositol-3,4-bisphosphat (PtdIns(3,4)P(2)) und Phosphatidylinositol-3,4,5-Trisphosphate (PtdIns(3,4,5)P(3)) im Gegensatz zu den kanonischen FYVE-Dom&auml;nen, die spezifisch, Phosphatidylinositol-3-Phosphat (PtdIns(3)P) binden. Dar&uuml;ber hinaus fanden wir, dass diese Phosphoinositides (PtdInsP) differentiell regulieren des protrudin zwischen Endosomen und Uhr &uuml;ber seine FYVE Gebiet pendelt. Fehler beim protrudin Mutanten mit reduzierten PtdInsP-Bindungsaffinit&auml;t Neurit Auswuchs in prim&auml;ren kultivierten Neuronen zu f&ouml;rdern. Diese Ergebnisse legen nahe, dass neuartige PtdInsP Selektivit&auml;t des protrudin-FYVE Gebiet entscheidend f&uuml;r deren zellul&auml;re Lokalisierung und seine Rolle im Neurit Auswuchs.

Phosphoinositides Regeln differentiell protrudin Lokalisierung durch die FYVE-Gebiet.

Protrudin &egrave; una proteina di dominio contenenti FYVE (Fab 1, YOTB, Vac 1 ed EEA1) coinvolti nel trasporto di carichi neuronale e implicato nell&#39;insorgenza di paraplegia spastica ereditaria (HSP). Nostro screening basato su immagini di lipidi binding domain (LBD) library ha rivelato romanzo plasma membrana (PM) localizzazione del dominio FYVE di protrudin a differenza dei domini FYVE canoniche che sono localizzate a presto endosomes. L&#39;associazione studio di analisi (SPR) risonanza plasmonica di superficie di membrana hanno mostrato che questo dominio FYVE si lega preferenzialmente fosfatidilinositolo-4,5-bisfosfato (PtdIns(4,5)P(2)), fosfatidilinositolo-3,4-bisfosfato (PtdIns(3,4)P(2)) e fosfatidilinositolo-3, 4,5-trifosfato (PtdIns(3,4,5)P(3)) a differenza dei domini FYVE canoniche che legano specificamente fosfatidilinositolo-3-fosfato (PtdIns(3)P). Inoltre, abbiamo trovato che questi phosphoinositides (PtdInsP) differenzialmente regolano la spola di protrudin tra endosomes e PM tramite il dominio FYVE. Protrudin mutanti con ridotta affinit&agrave; PtdInsP-associazione non &egrave; riuscita a promuovere dei neuriti escrescenza in neuroni ippocampali colti primari. Questi risultati suggeriscono che il romanzo PtdInsP selettivit&agrave; del dominio protrudin-FYVE &egrave; critica per la sua localizzazione cellulare e il suo ruolo nella crescita dei neuriti.

Phosphoinositides differenzialmente regolano la localizzazione protrudin attraverso il dominio FYVE.

Protrudin &#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306E;&#x8CA8;&#x7269;&#x306E;&#x8F38;&#x9001;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3001;&#x907A;&#x4F1D;&#x6027;&#x75D9;&#x6027;&#x5BFE;&#x9EBB;&#x75FA; (HSP) &#x306E;&#x767A;&#x75C7;&#x306B;&#x95A2;&#x4E0E;&#x3059;&#x308B; FYVE (Fab 1&#x3001;YOTB&#x3001;Vac 1 EEA1) &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3092;&#x542B;&#x3080;&#x86CB;&#x767D;&#x8CEA;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x79C1;&#x305F;&#x3061;&#x306E;&#x30A4;&#x30E1;&#x30FC;&#x30B8; &#x30D9;&#x30FC;&#x30B9;&#x306E;&#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x8102;&#x8CEA;&#x7D50;&#x5408;&#x30C9;&#x30E1;&#x30A4;&#x30F3; (LBD) &#x30E9;&#x30A4;&#x30D6;&#x30E9;&#x30EA;&#x306E;&#x5C0F;&#x8AAC;&#x306E;&#x30D7;&#x30E9;&#x30BA;&#x30DE;&#x819C; &#xFF08;PM&#xFF09; &#x306E;&#x30ED;&#x30FC;&#x30AB;&#x30EA;&#x30BC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306E;&#x521D;&#x671F;&#x30A8;&#x30F3;&#x30C9;&#x30BD;&#x30FC;&#x30E0;&#x306B;&#x30ED;&#x30FC;&#x30AB;&#x30E9;&#x30A4;&#x30BA;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x6B63;&#x898F;&#x306E; FYVE &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3068;&#x306F;&#x7570;&#x306A;&#x308A; protrudin FYVE &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E; FYVE &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x304C;&#x512A;&#x5148;&#x7684;&#x30DB;&#x30B9;&#x30D5;&#x30A1;&#x30C1;&#x30B8;&#x30EB;&#x30A4;&#x30CE;&#x30B7;&#x30C8;&#x30FC;&#x30EB; 4, 5-&#x30D3;&#x30B9;&#x30EA;&#x30F3;&#x9178; &#xFF08;PtdIns(4,5)P(2))&#x3001;&#x30D5;&#x30A9;&#x30B9;&#x30D5;&#x30A1;&#x30C1;&#x30B8;&#x30EB;&#x30A4;&#x30CE;&#x30B7;&#x30C8;&#x30FC;&#x30EB;-3, 4-&#x30D3;&#x30B9;&#x30EA;&#x30F3;&#x9178; (PtdIns(3,4)P(2)) &#x304A;&#x3088;&#x3073;&#x30DB;&#x30B9;&#x30D5;&#x30A1;&#x30C1;&#x30B8;&#x30EB;&#x30A4;&#x30CE;&#x30B7;&#x30C8;&#x30FC;&#x30EB;-3,4,5-&#x30C8;&#x30EA;&#x30B9;&#x30EA;&#x30F3;&#x9178; (&#x3068;&#x308A;&#x308F;&#x3051;&#x7D50;&#x5408;&#x30D5;&#x30A9;&#x30B9;&#x30D5;&#x30A1;&#x30C1;&#x30B8;&#x30EB;&#x30A4;&#x30CE;&#x30B7;&#x30C8;&#x30FC;&#x30EB;-3-&#x30EA;&#x30F3;&#x9178; (PtdIns(3)P)&#x3002; &#x6B63;&#x898F;&#x306E; FYVE &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3068;&#x306F;&#x7570;&#x306A;&#x308A; PtdIns(3,4,5)P(3)) &#x3092;&#x7D50;&#x5408;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308B;&#x8868;&#x9762;&#x30D7;&#x30E9;&#x30BA;&#x30E2;&#x30F3;&#x5171;&#x9CF4; (SPR) &#x89E3;&#x6790;&#x7814;&#x7A76;&#x3092;&#x7D50;&#x5408;&#x819C;&#x3092;&#x793A;&#x3057;&#x305F;&#x3055;&#x3089;&#x306B;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30A4;&#x30CE;&#x30B7;&#x30C8;&#x30FC;&#x30EB;&#x30EA;&#x30F3;&#x8102;&#x8CEA; (PtdInsP) &#x7279;&#x7570;&#x7684;&#x30A8;&#x30F3;&#x30C9;&#x30BD;&#x30FC;&#x30E0;&#x3068;&#x5348;&#x5F8C;&#x306E;&#x9593;&#x305D;&#x306E; FYVE &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3092;&#x4ECB;&#x3057;&#x3066; protrudin &#x306E;&#x5F80;&#x5FA9;&#x3092;&#x898F;&#x5236;&#x3092;&#x898B;&#x3064;&#x3051;&#x307E;&#x3059;&#x3002;Protrudin &#x6E1B;&#x5C11; PtdInsP &#x7D50;&#x5408;&#x89AA;&#x548C;&#x6027;&#x3068;&#x5909;&#x7570;&#x4F53;&#x57F9;&#x990A;&#x6D77;&#x99AC;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306E;&#x795E;&#x7D4C;&#x7A81;&#x8D77;&#x4F38;&#x9577;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5931;&#x6557;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x9078;&#x629E;&#x6027; PtdInsP &#x5C0F;&#x8AAC; protrudin FYVE &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x304C;&#x305D;&#x306E;&#x7D30;&#x80DE;&#x306E;&#x5C40;&#x5728;&#x3068;&#x795E;&#x7D4C;&#x7A81;&#x8D77;&#x4F38;&#x9577;&#x306B;&#x304A;&#x3051;&#x308B;&#x305D;&#x306E;&#x5F79;&#x5272;&#x306E;&#x305F;&#x3081;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;

&#x30A4;&#x30CE;&#x30B7;&#x30C8;&#x30FC;&#x30EB;&#x30EA;&#x30F3;&#x8102;&#x8CEA;&#x7279;&#x7570;&#x7684; FYVE &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306B;&#x3088;&#x308B; protrudin &#x306E;&#x5C40;&#x5728;&#x3092;&#x8ABF;&#x7BC0;&#x3057;&#x307E;&#x3059;&#x3002;

Protrudin &#xC2E0;&#xACBD; &#xD654;&#xBB3C;&#xC758; &#xC218;&#xC1A1;&#xC5D0; &#xAD00;&#xC5EC; &#xD558; &#xACE0; &#xC720;&#xC804; &#xACBD;&#xB828; &#xC801;&#xC778; &#xD558;&#xBC18;&#xC2E0; (HSP)&#xC758; &#xBC1C;&#xBCD1;&#xC5D0; &#xC5F0;&#xB8E8; (&#xD339; 1, YOTB, Vac 1&#xACFC; EEA1) FYVE &#xB3C4;&#xBA54;&#xC778;&#xC5D0; &#xD3EC;&#xD568; &#xB41C; &#xB2E8;&#xBC31;&#xC9C8; &#xC774;&#xB2E4;. &#xC9C0;&#xC9C8; &#xBC14;&#xC778;&#xB529; &#xB3C4;&#xBA54;&#xC778; (LBD) &#xB77C;&#xC774;&#xBE0C;&#xB7EC;&#xB9AC;&#xC758; &#xC774;&#xBBF8;&#xC9C0;&#xC5D0; &#xAE30;&#xCD08;&#xB97C; &#xB454; &#xC6B0;&#xB9AC;&#xC758; &#xC2EC;&#xC0AC; &#xC18C;&#xC124; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778; (&#xC624;&#xD6C4;) &#xC9C0;&#xC5ED;&#xD654;&#xB97C; &#xAD00;&#xB828; &#xD30C;&#xC77C;&#xC774; &#xC815;&#xC2DD; FYVE &#xB3C4;&#xBA54;&#xC778;&#xC744; &#xCD08;&#xAE30; endosomes &#xC9C0;&#xC5ED;&#xD654;&#xC640; &#xB2EC;&#xB9AC; protrudin&#xC758; FYVE &#xB3C4;&#xBA54;&#xC778;&#xC744; &#xACF5;&#xAC1C; &#xD588;&#xB2E4;. &#xD45C;&#xBA74; &#xD50C;&#xB77C;&#xC2A4;&#xBAAC; &#xACF5;&#xBA85; (SPR) &#xBD84;&#xC11D; &#xD558; &#xC5EC; &#xC5F0;&#xAD6C;&#xB97C; &#xBC14;&#xC778;&#xB529; &#xB9C9;&#xC774; &#xBCF4;&#xC600;&#xB2E4; FYVE &#xB3C4;&#xBA54;&#xC778;&#xC774; &#xC6B0;&#xC120;&#xC801;&#xC73C;&#xB85C; phosphatidylinositol-4, 5-bisphosphate (PtdIns(4,5)P(2)), phosphatidylinositol-3, 4-bisphosphate (Ptdins(3,4)p(2))&#xC640; phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) &#xD2B9;&#xD788; &#xBC14;&#xC778;&#xB529; phosphatidylinositol-3-&#xC778;&#xC0B0; (PtdIns(3)P) &#xC815;&#xC2DD; FYVE &#xB3C4;&#xBA54;&#xC778; &#xB2EC;&#xB9AC; &#xBC14;&#xC778;&#xB529;&#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xC774;&#xB7EC;&#xD55C; phosphoinositides (PtdInsP) &#xCC28;&#xB3D9; &#xCEA0;&#xCF54;&#xB354;&#xC778; FYVE &#xB3C4;&#xBA54;&#xC778;&#xC744; &#xD1B5;&#xD574; &#xC0AC;&#xC774; endosomes protrudin&#xC758; &#xADDC;&#xC81C;&#xB97C; &#xCC3E;&#xC558;&#xC2B5;&#xB2C8;&#xB2E4;. &#xAC10;&#xC18C; &#xB41C; PtdInsP &#xBC14;&#xC778;&#xB529; &#xC120;&#xD638;&#xB3C4; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; protrudin neurite &#xD30C;&#xC0DD;&#xBB3C; &#xAE30;&#xBCF8; &#xAD50;&#xC591;&#xB41C; hippocampal &#xB274;&#xB7F0;&#xC5D0;&#xC11C; &#xD64D;&#xBCF4; &#xD558;&#xC9C0; &#xBABB;&#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xACB0;&#xACFC; &#xC120;&#xD0DD; PtdInsP &#xC18C;&#xC124;&#xB3C4; protrudin FYVE &#xB3C4;&#xBA54;&#xC778;&#xC740; &#xADF8; &#xC138;&#xD3EC; &#xC9C0;&#xC5ED;&#xD654; &#xBC0F; neurite &#xD30C;&#xC0DD;&#xBB3C;&#xC758; &#xC5ED;&#xD560;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

Phosphoinositides &#xCC28;&#xB3D9; protrudin &#xC9C0;&#xC5ED;&#xD654; FYVE &#xB3C4;&#xBA54;&#xC778;&#xC744; &#xD1B5;&#xD574; &#xC870;&#xC808; &#xD569;&#xB2C8;&#xB2E4;.

Protrudin es una prote&iacute;na que contiene el dominio FYVE (Fab 1, YOTB, Vac 1 y EEA1) implicados en el transporte de cargas neuronales e implicado en la aparici&oacute;n de paraplegia esp&aacute;stica hereditaria (HSP). Nuestra proyecci&oacute;n basada en im&aacute;genes de la biblioteca de l&iacute;pidos binding domain (LBD) revel&oacute; novela membrana del plasma (PM) localizaci&oacute;n del dominio FYVE de protrudin a diferencia de los dominios FYVE can&oacute;nicos que se localizan a endosomas tempranos. La membrana vinculante estudio analizando plasm&oacute;n superficial (SPR) de resonancia demostr&oacute; que este dominio FYVE preferencial une el Fosfatidilinositol-4, 5-bifosfato (PtdIns(4,5)P(2)), fosfatidilinositol-3,4-bifosfato (PtdIns(3,4)P(2)) y fosfatidilinositol-3,4,5-trifosfato (PtdIns(3,4,5)P(3)) a diferencia de los dominios FYVE can&oacute;nicos que se unen espec&iacute;ficamente fosfatidilinositol-3-fosfato (PtdIns(3)P). Adem&aacute;s, encontramos que estos fosfoinos&iacute;tidos (PtdInsP) regulan diferencialmente realizar trayectos de protrudin entre endosomas y PM v&iacute;a su dominio FYVE. Protrudin mutantes con PtdInsP-afinidad reducida no promover neurita consecuencia en las neuronas de hipocampales cultivos primarias. Estos resultados sugieren que la selectividad de PtdInsP novela de dominio protrudin-FYVE es cr&iacute;tica para su localizaci&oacute;n celular y su papel en consecuencia neurita.

Fosfoinos&iacute;tidos regulan diferencialmente protrudin localizaci&oacute;n a trav&eacute;s del dominio FYVE.

Upon phagocytosis, Legionella pneumophila translocates numerous effector proteins into host cells to perturb cellular metabolism and immunity, ultimately establishing intracellular survival and growth. VipD of L. pneumophila belongs to a family of bacterial effectors that contain the N-terminal lipase domain and the C-terminal domain with an unknown function. We report the crystal structure of VipD and show that its C-terminal domain robustly interferes with endosomal trafficking through tight and selective interactions with Rab5 and Rab22. This domain, which is not significantly similar to any known protein structure, potently interacts with the GTP-bound active form of the two Rabs by recognizing a hydrophobic triad conserved in Rabs. These interactions prevent Rab5 and Rab22 from binding to downstream effectors Rabaptin-5, Rabenosyn-5 and EEA1, consequently blocking endosomal trafficking and subsequent lysosomal degradation of endocytic materials in macrophage cells. Together, this work reveals endosomal trafficking as a target of L. pneumophila and delineates the underlying molecular mechanism.

VipD of Legionella pneumophila targets activated Rab5 and Rab22 to interfere with endosomal trafficking in macrophages.

Legionella pneumophila, a human intracellular pathogen, encodes about 290 effector proteins that are translocated into host cells through a secretion machinery. Some of these proteins have been shown to manipulate or subvert cellular processes during infection, but functional roles of a majority of them remain unknown. Lpg0393 is a newly identified Legionella effector classified as a hypothetical protein. Through X-ray crystallographic analysis, we show that Lpg0393 contains a Vps9-like domain, which is structurally most similar to the catalytic core of human Rabex-5 that activates the endosomal Rab proteins Rab5, Rab21 and Rab22. Consistently, Lpg0393 exhibited a guanine-nucleotide exchange factor activity toward the endosomal Rabs. This work identifies the first example of a bacterial guanine-nucleotide exchange factor that is active towards the Rab5 sub-cluster members, implying that the activation of these Rab proteins might be advantageous for the intracellular survival of Legionella. 

Lpg0393 of Legionella pneumophila is a guanine-nucleotide exchange factor for Rab5, Rab21 and Rab22.

The release of infectious hepatitis C virus (HCV) particles from infected cells remains poorly characterized. We previously demonstrated that virus release is dependent on the endosomal sorting complex required for transport (ESCRT). Here, we show a critical role of trans-Golgi network (TGN)-endosome trafficking during the assembly, but principally the secretion, of infectious virus. This was demonstrated by both small interfering RNA (siRNA)-mediated silencing of TGN-associated adaptor proteins and a panel of dominant negative (DN) Rab GTPases involved in TGN-endosome trafficking steps. Importantly, interfering with factors critical for HCV release did not have a concomitant effect on secretion of triglycerides, ApoB, or ApoE, indicating that particles are likely released from Huh7 cells via pathways distinct from that of very-low-density lipoprotein (VLDL). Finally, we show that HCV NS2 perturbs TGN architecture, redistributing TGN membranes to closely associate with HCV core protein residing on lipid droplets. These findings support the notion that HCV hijacks TGN-endosome trafficking to facilitate particle assembly and release. Moreover, although essential for assembly and infectivity, the trafficking of mature virions is seemingly independent of host lipoproteins.

Release of Infectious Hepatitis C Virus from Huh7 Cells Occurs via a trans-Golgi Network-to-Endosome Pathway Independent of Very-Low-Density Lipoprotein Secretion.

Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration.

PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front.

Optical diffraction tomography (ODT) provides label-free three-dimensional (3D) refractive index (RI) measurement of biological samples. However, due to the nature of the RI values of biological specimens, ODT has limited access to molecular specific information. Here, we present an optical setup combining ODT with three-channel 3D fluorescence microscopy, to enhance the molecular specificity of the 3D RI measurement. The 3D RI distribution and 3D deconvoluted fluorescence images of HeLa cells and NIH-3T3 cells are measured, and the cross-correlative analysis between RI and fluorescence of live cells are presented.

Correlative three-dimensional fluorescence and refractive index tomography: bridging the gap between molecular specificity and quantitative bioimaging.

Salmonella uses Type 3 secretion systems (T3SSs) to deliver virulence factors, called effectors, into host cells during infection. The T3SS effectors promote invasion into host cells and the generation of a replicative niche. SopB is a T3SS effector that plays an important role in Salmonella pathogenesis through its lipid phosphatase activity. Here, we show that SopB mediates the recruitment of Rho GTPases (RhoB, RhoD, RhoH, and RhoJ) to bacterial invasion sites. RhoJ contributes to Salmonella invasion, and RhoB and RhoH play an important role in Akt activation. R-Ras1 also contributes to SopB-dependent Akt activation by promoting the localised production of PI(3,4)P /PI(3,4,5)P . Our studies reveal new signalling factors involved in SopB-dependent Salmonella infection.

Article	Year
Comprehensive identification of PIP3-regulated PH domains from C. elegans to H. sapiens by model prediction and live imaging. Molecular cell\| PubMed ID: 18471983	2008
Endocytic Rab proteins are required for hepatitis C virus replication complex formation. Virology\| PubMed ID: 20005553	2010
Cooperative activation of PI3K by Ras and Rho family small GTPases. Molecular cell\| PubMed ID: 22683270	2012
Phosphoinositides differentially regulate protrudin localization through the FYVE domain. The Journal of biological chemistry\| PubMed ID: 23043110	2012
VipD of Legionella pneumophila targets activated Rab5 and Rab22 to interfere with endosomal trafficking in macrophages. PLoS pathogens\| PubMed ID: 23271971	2012
Lpg0393 of Legionella pneumophila is a guanine-nucleotide exchange factor for Rab5, Rab21 and Rab22. PloS one\| PubMed ID: 25821953	2015
Release of Infectious Hepatitis C Virus from Huh7 Cells Occurs via a trans-Golgi Network-to-Endosome Pathway Independent of Very-Low-Density Lipoprotein Secretion. Journal of virology\| PubMed ID: 27226379	2016
PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 27555588	2016
Correlative three-dimensional fluorescence and refractive index tomography: bridging the gap between molecular specificity and quantitative bioimaging. Biomedical optics express\| PubMed ID: 29296497	2017
Salmonella exploits host Rho GTPase signalling pathways through the phosphatase activity of SopB. Cellular microbiology\| PubMed ID: 30010242	2018

Wei Sun Park

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