Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process ...

Cornell University

Ca(2+) mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca(2+) sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca(2+) waves that originate most frequently from the tips of extended cell protrusions, as well as Ca(2+) oscillations throughout the cell that usually follow the initiating Ca(2+) wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca(2+) puffs at the region of cell contact. Initiating Ca(2+) waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca(2+) release from endoplasmic reticulum stores and to extracellular Ca(2+), but they do not depend on store-operated Ca(2+) entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca(2+) signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca(2+) response.

Ca2+ waves initiate antigen-stimulated Ca2+ responses in mast cells.

&#x7ECF;&#x52A8;&#x5458;&#x662F;&#x5305;&#x62EC;&#x523A;&#x6FC0;&#x7684;&#x80DE;&#x548C;&#x7EC6;&#x80DE;&#x56E0;&#x5B50;&#x751F;&#x4EA7;&#x4E2D;&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#x7684;&#x8BB8;&#x591A;&#x7EC6;&#x80DE;&#x8FC7;&#x7A0B;&#x7684;&#x6838;&#x5FC3;&#x3002;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x5355;&#x4E2A;&#x5355;&#x5143;&#x683C;&#x7684; IgE &#x7279;&#x5B9A; Ag &#x523A;&#x6FC0;&#x548C;&#x9AD8;&#x901F;&#x6210;&#x50CF;&#x7684;&#x5E38;&#x89C4;&#x6216;&#x57FA;&#x56E0;&#x7F16;&#x7801;&#x7ECF;&#x4F20;&#x611F;&#x5668;&#x5728;&#x5927;&#x9F20;&#x55DC;&#x78B1;&#x7C92;&#x7EC6;&#x80DE;&#x767D;&#x8840;&#x75C5;&#x548C;&#x9AA8;&#x9AD3;&#x7684;&#x5927;&#x9F20;&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#xFF0C;&#x89C2;&#x5BDF;&#x7ECF;&#x6CE2;&#x6700;&#x9891;&#x7E41;&#x6E90;&#x81EA;&#x6269;&#x5C55;&#x5355;&#x5143;&#x683C;&#x51F8;&#x51FA;&#xFF0C;&#x4EE5;&#x53CA;&#x6574;&#x4E2A;&#x5355;&#x5143;&#x683C;&#xFF0C;&#x901A;&#x5E38;&#x9075;&#x5FAA;&#x53D1;&#x8D77;&#x7ECF;&#x6CE2;&#x7ECF;&#x632F;&#x8361;&#x7684;&#x63D0;&#x793A;&#x3002;&#x76F8;&#x6BD4;&#x4E4B;&#x4E0B;&#xFF0C;&#x80A1;&#x4EFD;&#x516C;&#x53F8;&#x5411;&#x5FAE;&#x9488;&#x63D0;&#x793A;&#x5171;&#x8F6D;&#x523A;&#x6FC0;&#x672C;&#x5730;&#x3001; &#x91CD;&#x590D;&#x6027;&#x7ECF;&#x6CE1;&#x8299;&#x5728;&#x7EC6;&#x80DE;&#x95F4;&#x63A5;&#x89E6;&#x7684;&#x533A;&#x57DF;&#x3002;&#x542F;&#x52A8;&#x7ECF;&#x6CE2;&#x5F97;&#x5230;&#x591A;&#x6570;&#x5927;&#x9F20;&#x55DC;&#x78B1;&#x767D;&#x8840;&#x75C5;&#x7EC6;&#x80DE;&#x4E0E;&#x53EF;&#x6EB6;&#x6027; Ag &#x523A;&#x6FC0;&#x548C;&#x6291;&#x5236;&#x5242;&#x7ECF;&#x91CA;&#x653E;&#x5BF9;&#x654F;&#x611F;&#xFF0C;&#x4ECE;&#x5185;&#x8D28;&#x7F51;&#x5546;&#x5E97;&#x548C;&#x5411;&#x80DE;&#x5916;&#x7ECF;&#xFF0C;&#x4F46;&#x5B83;&#x4EEC;&#x4E0D;&#x53D6;&#x51B3;&#x4E8E;&#x5E97;&#x9762;&#x5F0F;&#x7ECF;&#x8425;&#x7ECF;&#x6761;&#x76EE;&#x3002;&#x77AC;&#x65F6;&#x53D7;&#x4F53;&#x7535;&#x4F4D;&#x901A;&#x9053; (TRPC) 1 &#x548C; TRPC3 &#x901A;&#x9053;&#x86CB;&#x767D;&#x7684;&#x77ED;&#x53D1;&#x5939; RNA &#x7684;&#x7EC4;&#x5408;&#x5F0F;&#x51CF;&#x5C11;&#x4E86;&#x94F6;&#x5230;&#x8FD9;&#x4E9B;&#x7EC6;&#x80DE;&#x7684;&#x654F;&#x611F;&#x6027;&#xFF0C;&#x5C06;&#x6CE2;&#x8D77;&#x59CB;&#x7AD9;&#x70B9;&#x4ECE;&#x7A81;&#x8D77;&#x8F6C;&#x79FB;&#x5230;&#x7EC6;&#x80DE;&#x4F53;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x63ED;&#x793A;&#x7A7A;&#x95F4;&#x7F16;&#x7801;&#x7ECF;&#x4FE1;&#x53F7; immunoreceptor &#x6FC0;&#x6D3B;&#xFF0C;&#x5B83;&#x5229;&#x7528; TRPC &#x901A;&#x9053;&#x6307;&#x5B9A;&#x8D77;&#x59CB;&#x7AD9;&#x70B9;&#x7ECF;&#x54CD;&#x5E94;&#x7684;&#x54CD;&#x5E94;&#x3002;

Ca2 + &#x6CE2;&#x542F;&#x52A8;&#x4E2D;&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#x6297;&#x539F;&#x523A;&#x6FC0; Ca2 + &#x53CD;&#x5E94;&#x3002;

Ca(2+) mobilisation est au centre de nombreux processus cellulaires, y compris l&#39;exocytose stimul&eacute;e et la production de cytokines dans les cellules de m&acirc;t. Gr&acirc;ce &agrave; la stimulation des cellules simples par IgE sp&eacute;cifiques Ag et imagerie haute vitesse des capteurs de Ca(2+) conventionnels ou g&eacute;n&eacute;tiquement encod&eacute;s en leuc&eacute;mie basophile rat et les mastocytes de rat de moelle osseuse, nous observons des ondes Ca(2+) qui proviennent plus souvent les pointes des saillies de cellule prolong&eacute;e, ainsi que les oscillations de Ca(2+) tout au long de la cellule qui suivent g&eacute;n&eacute;ralement la vague Ca(2+) responsable. En revanche, Ag, conjugu&eacute; &agrave; l&#39;extr&eacute;mit&eacute; d&#39;une micropipette stimule bouff&eacute;es de Ca(2+) locales, r&eacute;p&eacute;titives &agrave; la zone de contact de la cellule. Initiateurs Ca(2+) vagues sont observ&eacute;es dans la plupart rat basophiles cellules leuc&eacute;miques stimulent avec Ag soluble et sont sensibles aux inhibiteurs de la lib&eacute;ration de Ca(2+) dans les magasins de r&eacute;ticulum endoplasmique et Ca(2+) extracellulaire, mais ils ne d&eacute;pendent pas de magasin fonctionnant Ca(2+) entr&eacute;e. Pr&eacute;cipitation de canal potentiel transient receptor (TRPC) 1 et les prot&eacute;ines de canal TRPC3 par l&#39;ARN en &eacute;pingle &agrave; cheveux court r&eacute;duit la sensibilit&eacute; de ces cellules &agrave; l&#39;Ag et d&eacute;place le site d&#39;initiation de vague de saillies vers le corps cellulaire. Nos r&eacute;sultats r&eacute;v&egrave;lent dans l&#39;espace cod&eacute; Ca(2+) signalisation en r&eacute;ponse &agrave; l&#39;activation immunoreceptor qui utilise les canaux TRPC pour sp&eacute;cifier le site d&#39;initiation de la r&eacute;ponse Ca(2+).

Ondes de Ca2 + lancer stimul&eacute;e par l&#39;antig&egrave;ne r&eacute;ponses du Ca2 + dans les cellules de m&acirc;t.

Ca(2+)-Mobilisierung ist zentral f&uuml;r viele zellul&auml;re Prozesse, einschlie&szlig;lich stimulierte Exozytose und Cytokine Produktion in Mastzellen. Mit einzelligen Stimulation durch IgE-spezifischen Ag und Hochgeschwindigkeitsaufnahmen von konventionellen oder genetisch codierte Ca(2+)-Sensoren in Ratte basophile Leuk&auml;mie und Knochenmark abgeleitete Ratte Mastzellen, beobachten wir Ca(2+) Wellen, die am h&auml;ufigsten von den Spitzen der erweiterten Zelle Vorspr&uuml;nge, sowie Ca(2+)-Schwingungen in die Zelle, die in der Regel die initiierende Ca(2+) Welle folgen stammen. Im Gegensatz dazu stimuliert Ag Konjugation mit der Spitze einer Mikropipette lokale, sich wiederholende Ca(2+) Hauche in der Region der Zelle Kontakt. Initiierende Ca(2+) Wellen sind in den meisten Ratte basophile beobachtet Leuk&auml;mie-Zellen stimuliert mit l&ouml;slichen Ag und sind empfindlich gegen Inhibitoren der Ca(2+) Version von Endoplasmatisches Reticulum Shops und extrazellul&auml;re Ca(2+), aber sie h&auml;ngen nicht Speicher zu bet&auml;tigende Ca(2+) Eintrag. Niederschlag des transienten Rezeptor potenzielle Channel (TRPC) 1 und TRPC3 Kanal-Proteine durch kurze Haarnadel RNA reduziert die Empfindlichkeit dieser Zellen AG und verschiebt die Welle-Einleitung-Website von Vorspr&uuml;ngen auf Zellk&ouml;rper. Unsere Ergebnisse zeigen r&auml;umlich codierten Ca(2+) signalisieren in Reaktion auf Aramburuzabala-Aktivierung, die TRPC-Kan&auml;le, um den Standort der Einleitung der Ca(2+) Antwort angeben, nutzt.

Ca2 + Wellen initiieren Antigen stimuliert Ca2 + Antworten in Mastzellen.

CA(2+) mobilitazione &egrave; centrale in molti processi cellulari, tra cui esocitosi stimolata e produzione di citochine in mastociti. Mediante stimolazione della singola cella di IgE specifiche Ag e imaging ad alta velocit&agrave; di sensori Ca(2+) convenzionali o geneticamente codificati nella Leucemia basofila topo e ratto derivate dal midollo osseo di mastociti, osserviamo onde Ca(2+) che pi&ugrave; frequentemente provengono dalle punte delle protrusioni cellulari estese, come pure Ca(2+) oscillazioni in tutta la cella che solitamente seguono l&#39;onda Ca(2+) avvio. Al contrario, Ag coniugata con la punta di una micropipetta stimola locale, ripetitivi sbuffi di Ca(2+) presso la regione di contatto cell. Avvio Ca(2+) onde sono osservate nel ratto pi&ugrave; basofile cellule leucemiche stimolarono con Ag solubili e sono sensibili agli inibitori di Ca(2+) rilascio dai depositi del reticolo endoplasmatico e a Ca(2+) extracellulare, ma essi non dipendono da negozio gestito Ca(2+) voce. Atterramento di transient receptor potenziale canale (TRPC) 1 e proteine canale TRPC3 da short hairpin RNA riduce la sensibilit&agrave; di queste cellule di Ag e sposta il sito di iniziazione onda da sporgenze per il corpo cellulare. I nostri risultati rivelano spazialmente codificati Ca(2+) segnalazione in risposta all&#39;attivazione immunoreceptor che utilizza canali TRPC per specificare il sito di iniziazione della risposta Ca(2+).

Ca2 + onde avviano Ca2 + risposte antigene-stimolata nei mastociti.

&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x52D5;&#x54E1;&#x523A;&#x6FC0;&#x30A8;&#x30AD;&#x30BD;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x304A;&#x3088;&#x3073;&#x30B5;&#x30A4;&#x30C8;&#x30AB;&#x30A4;&#x30F3;&#x7523;&#x751F;&#x306B;&#x304A;&#x3051;&#x308B;&#x80A5;&#x6E80;&#x7D30;&#x80DE;&#x3092;&#x542B;&#x3080;&#x3001;&#x591A;&#x304F;&#x306E;&#x7D30;&#x80DE;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x4E2D;&#x5FC3;&#x3067;&#x3059;&#x3002;&#x5358;&#x4E00;&#x7D30;&#x80DE;&#x523A;&#x6FC0;&#x306B;&#x3088;&#x308B; IgE &#x56FA;&#x6709; Ag &#x3068;&#x5F93;&#x6765;&#x307E;&#x305F;&#x306F;&#x907A;&#x4F1D;&#x7684;&#x30B3;&#x30FC;&#x30C9;&#x5316;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0; &#x30BB;&#x30F3;&#x30B5;&#x30FC; 1i1015 &#x3068;&#x30E9;&#x30C3;&#x30C8;&#x9AA8;&#x9AC4;&#x7531;&#x6765;&#x30DE;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x3067;&#x306E;&#x9AD8;&#x901F;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x6700;&#x3082;&#x983B;&#x7E41;&#x306B;&#x901A;&#x5E38;&#x958B;&#x59CB;&#x3059;&#x308B;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x6CE2;&#x306B;&#x5F93;&#x3063;&#x3066;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x632F;&#x52D5;&#x304C;&#x30BB;&#x30EB;&#x5168;&#x4F53;&#x3068;&#x540C;&#x69D8;&#x306B;&#x62E1;&#x5F35;&#x7D30;&#x80DE;&#x7A81;&#x8D77;&#x306E;&#x5148;&#x7AEF;&#x304B;&#x3089;&#x767A;&#x4FE1;&#x3055;&#x308C;&#x305F;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x6CE2;&#x3092;&#x89B3;&#x5BDF;&#x3057;&#x307E;&#x3059;&#x3002;&#x5BFE;&#x7167;&#x7684;&#x306B;&#x3001;&#x30D4;&#x30DA;&#x30C3;&#x30C8;&#x306E;&#x5148;&#x7AEF;&#x306B;&#x5171;&#x5F79; Ag &#x30ED;&#x30FC;&#x30AB;&#x30EB;&#x3001;&#x53CD;&#x5FA9;&#x7684;&#x306A;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0; &#x30D1;&#x30D5;&#x7D30;&#x80DE;&#x63A5;&#x89E6;&#x306E;&#x9818;&#x57DF;&#x3092;&#x523A;&#x6FC0;&#x3057;&#x307E;&#x3059;&#x3002;&#x767A;&#x4FE1;&#x5074;&#x306E;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x6CE2;&#x307B;&#x3068;&#x3093;&#x3069;&#x306E;&#x30E9;&#x30C3;&#x30C8;&#x306E;&#x30B9;&#x30C8;&#x30A2;&#x904B;&#x55B6;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0; &#x30A8;&#x30F3;&#x30C8;&#x30EA;&#x306B;&#x306F;&#x4F9D;&#x5B58;&#x3057;&#x307E;&#x305B;&#x3093;&#x304C;&#x3001;&#x767D;&#x8840;&#x75C5;&#x7D30;&#x80DE;&#x53EF;&#x6EB6;&#x6027; Ag &#x3068;&#x523A;&#x6FC0;&#x3057;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x653E;&#x51FA;&#x306E;&#x963B;&#x5BB3;&#x5264;&#x306B;&#x5C0F;&#x80DE;&#x4F53;&#x306E;&#x5E97;&#x304B;&#x3089;&#x3001;&#x7D30;&#x80DE;&#x5916;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x306B;&#x654F;&#x611F;&#x597D;&#x5869;&#x57FA;&#x6027;&#x89B3;&#x6E2C;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x904E;&#x6E21;&#x53D7;&#x5BB9;&#x4F53;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x30C1;&#x30E3;&#x30CD;&#x30EB; (TRPC) 1 &#x304A;&#x3088;&#x3073;&#x77ED;&#x9396;&#x30D8;&#x30A2;&#x30D4;&#x30F3;&#x578B; RNA &#x306B;&#x3088;&#x308B; TRPC3 &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x30CE;&#x30C3;&#x30AF;&#x30C0;&#x30A6;&#x30F3;&#x306F; Ag &#x306B;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D30;&#x80DE;&#x306E;&#x611F;&#x53D7;&#x6027;&#x3092;&#x6E1B;&#x3089;&#x3059;&#x3057;&#x3001;&#x6CE2;&#x306E;&#x958B;&#x59CB;&#x30B5;&#x30A4;&#x30C8;&#x7A81;&#x8D77;&#x304B;&#x3089;&#x7D30;&#x80DE;&#x4F53;&#x3078;&#x30B7;&#x30D5;&#x30C8;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306E;&#x7D50;&#x679C;&#x306F;&#x7A7A;&#x9593;&#x7684;&#x306B;&#x30A8;&#x30F3;&#x30B3;&#x30FC;&#x30C9;&#x3055;&#x308C;&#x305F;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x304C;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x5FDC;&#x7B54;&#x306E;&#x958B;&#x59CB;&#x30B5;&#x30A4;&#x30C8;&#x3092;&#x6307;&#x5B9A;&#x3059;&#x308B; TRPC &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x3092;&#x5229;&#x7528;&#x3057;&#x3066;&#x7D30;&#x80DE;&#x8CEA;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306B;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D9;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306B;&#x5FDC;&#x3048;&#x3066;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#x660E;&#x3089;&#x304B;&#x306B;&#x3002;

Ca 2 + &#x6CE2;&#x30DE;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x306E;&#x6297;&#x539F;&#x523A;&#x6FC0; ca 2 + &#x5FDC;&#x7B54;&#x3092;&#x958B;&#x59CB;&#x3057;&#x307E;&#x3059;&#x3002;

Ca(2+) &#xB3D9;&#xC6D0; &#xB9C8;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC;&#xC5D0; &#xC790;&#xADF9;&#xB41C; exocytosis &#xBC0F; cytokine &#xC0DD;&#xC0B0;&#xC744; &#xD3EC;&#xD568; &#xD55C; &#xB9CE;&#xC740; &#xC138;&#xD3EC; &#xD504;&#xB85C;&#xC138;&#xC2A4; &#xC911;&#xC2EC; &#xC774;&#xB2E4;. IgE &#xAD00;&#xB828; Ag&#xC5D0; &#xC758;&#xD574; &#xB2E8;&#xC77C; &#xC138;&#xD3EC; &#xC790;&#xADF9; &#xBC0F; &#xC950; basophilic &#xBC31;&#xD608;&#xBCD1; &#xBC0F; &#xACE8; &#xC218; &#xC720;&#xB798; &#xC950; &#xB9C8;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xAE30;&#xC874;&#xC758; &#xB610;&#xB294; &#xC720;&#xC804;&#xC790; &#xC778;&#xCF54;&#xB529;&#xB41C; Ca(2+) &#xC13C;&#xC11C;&#xC758; &#xACE0;&#xC18D; &#xC774;&#xBBF8;&#xC9C0;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;, &#xC6B0;&#xB9AC; Ca(2+) &#xD30C;&#xB3C4; &#xD655;&#xC7A5;&#xB41C; &#xC140; &#xB3CC;&#xCD9C;, &#xC77C;&#xBC18;&#xC801;&#xC73C;&#xB85C; &#xC2DC;&#xC791; Ca(2+) &#xC6E8;&#xC774;&#xBE0C;&#xC5D0; &#xB530;&#xB77C; &#xC140;&#xC5D0; &#xAC78;&#xCCD0; Ca(2+) &#xC9C4;&#xB3D9;&#xC758; &#xD301;&#xC5D0;&#xC11C; &#xAC00;&#xC7A5; &#xC790;&#xC8FC; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xAD00;&#xCC30;. &#xB300;&#xC870;&#xC801;&#xC73C;&#xB85C;, Ag&#xB294; micropipette &#xD301;&#xC744; &#xD65C;&#xC6A9; &#xC140; &#xC811;&#xCD09; &#xC9C0;&#xC5ED;&#xC5D0;&#xC11C; &#xC9C0;&#xC5ED;, &#xBC18;&#xBCF5;&#xC801;&#xC778; Ca(2+) &#xD37C;&#xD504; &#xC790;&#xADF9;. &#xC2DC;&#xC791; Ca(2+) &#xD30C;&#xB3C4; &#xAD00;&#xCC30; &#xB300;&#xBD80;&#xBD84;&#xC758; &#xC950;&#xC5D0; basophilic &#xBC31;&#xD608;&#xBCD1; &#xC138;&#xD3EC; &#xC131; Ag&#xC640; &#xC790;&#xADF9; endoplasmic &#xADF8;&#xBB3C; &#xC0C1;&#xC810; &#xBC0F; &#xC138;&#xD3EC; &#xC678; Ca(2+) Ca(2+) &#xCD9C;&#xC2DC;&#xC758; &#xC5B5;&#xC81C;&#xC81C;&#xC5D0; &#xBBFC;&#xAC10; &#xD558;&#xC9C0;&#xB9CC; &#xC800;&#xC7A5;&#xC18C; &#xC6B4;&#xC601; Ca(2+) &#xD56D;&#xBAA9;&#xC5D0; &#xC885;&#xC18D; &#xB418;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC77C;&#xC2DC;&#xC801; &#xC218;&#xC6A9; &#xCCB4; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xCC44;&#xB110; (TRPC) 1, &#xC9E7;&#xC740; &#xD5E4;&#xC5B4;&#xD540; RNA&#xC5D0; &#xC758;&#xD574; TRPC3 &#xCC44;&#xB110; &#xB2E8;&#xBC31;&#xC9C8; &#xCD5C;&#xC800;&#xC758; Ag&#xC5D0;&#xC774; &#xC138;&#xD3EC;&#xC758; &#xAC10;&#xB3C4; &#xC904;&#xC5B4;&#xB4E4;&#xACE0; &#xB3CC;&#xCD9C;&#xC5D0;&#xC11C; &#xC140; &#xBC14;&#xB514; &#xC6E8;&#xC774;&#xBE0C; &#xC2DC;&#xC791; &#xC0AC;&#xC774;&#xD2B8;&#xB97C; &#xC774;&#xB3D9;. &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; &#xACF5;&#xAC04;&#xC801;&#xC73C;&#xB85C; &#xC778;&#xCF54;&#xB529;&#xB41C; Ca(2+) Ca(2+) &#xC751;&#xB2F5;&#xC758; &#xC2DC;&#xC791; &#xC704;&#xCE58;&#xB97C; &#xC9C0;&#xC815; &#xD558;&#xB824;&#xBA74; TRPC &#xCC44;&#xB110;&#xC744; &#xD65C;&#xC6A9; &#xD558; &#xC5EC; immunoreceptor &#xD65C;&#xC131;&#xD654;&#xC5D0; &#xB300; &#xD55C; &#xC751;&#xB2F5;&#xC5D0;&#xC11C; &#xC2E0;&#xD638; &#xACF5;&#xAC1C;.

Ca2 + &#xD30C;&#xB3C4; &#xB9C8;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xD56D; &#xC6D0; &#xC790;&#xADF9; Ca2 + &#xC751;&#xB2F5;&#xC744; &#xC2DC;&#xC791;&#xD569;&#xB2C8;&#xB2E4;.

Movilizaci&oacute;n de CA es esencial para muchos procesos celulares, incluyendo exocitosis estimulada y la producci&oacute;n de citoquinas en las c&eacute;lulas del m&aacute;stil. Utilizando estimulaci&oacute;n unicelular por Ag IgE espec&iacute;fica y proyecci&oacute;n de imagen de alta velocidad de sensores de CA convencionales o gen&eacute;ticamente codificados en leucemia basophilic rata y mastocitos de m&eacute;dula &oacute;sea de la rata, observamos CA ondas que se originan con mayor frecuencia en las puntas de protuberancias extendido celular, as&iacute; como CA oscilaciones a lo largo de la c&eacute;lula que generalmente siguen la onda de CA iniciaci&oacute;n. Por el contrario, Ag conjugado con la punta de una micropipeta estimula locales, repetitivos CA soplos en la regi&oacute;n de contacto de la c&eacute;lula. Iniciaci&oacute;n de CA de ondas se observan en rata m&aacute;s bas&oacute;filas c&eacute;lulas leuc&eacute;micas estimularon con Ag soluble y son sensibles a los inhibidores de la liberaci&oacute;n de CA de ret&iacute;culo endopl&aacute;smico tiendas y CA extracelular, pero que no dependen de la entrada de CA de tienda que funciona. Ca&iacute;da de transitoria receptor potencial canal (TRPC) 1 y prote&iacute;nas de canal TRPC3 por short hairpin RNA reduce la sensibilidad de estas c&eacute;lulas a Ag y cambia el sitio de iniciaci&oacute;n de la onda de protuberancias en el cuerpo de la c&eacute;lula. Nuestros resultados revelan espacial codificado CA se&ntilde;alizaci&oacute;n en respuesta a la activaci&oacute;n de la immunoreceptor que utiliza canales TRPC para especificar el sitio de iniciaci&oacute;n de la respuesta de CA.

Ca2 + ondas inician estimulados por el ant&iacute;geno Ca2 + respuestas en las c&eacute;lulas del m&aacute;stil.

&#x30E1;&#x30BD;&#x30DD;&#x30FC;&#x30E9;&#x30B9;&#x30B7;&#x30EA;&#x30AB;&#x7ACB;&#x65B9;&#x5BFE;&#x79F0;&#x6027;&#x3092;&#x6301;&#x3064;&#x7814;&#x7A76;&#x8005;&#x304B;&#x3089;&#x306E;&#x95A2;&#x5FC3;&#x306F;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x6642;&#x9593;&#x3092;&#x96C6;&#x3081;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x306F;&#x7ACB;&#x65B9;&#x5BFE;&#x79F0;&#x6027; Pm3&#x305;n &#x306B;&#x975E;&#x5E38;&#x306B;&#x9AD8;&#x3044;&#x30E2;&#x30EB;&#x6BD4;&#x3092;&#x6709;&#x3059;&#x308B;&#x30B7;&#x30EA;&#x30AB;&#x30CA;&#x30CE;&#x7C92;&#x5B50;&#x306E;&#x30E1;&#x30BD;&#x591A;&#x5B54;&#x4F53;&#x306E;&#x5BA4;&#x6E29;&#x5408;&#x6210;&#x3092;&#x63D0;&#x793A; (&#x76EE; 50%) 3 &#x30A2;&#x30DF;&#x30CE;&#x30D7;&#x30ED;&#x30D4;&#x30EB; triethoxysilane &#x306E;&#x3002;&#x5408;&#x6210;&#x306F;&#x3001;&#x5805;&#x7262;&#x306A;&#x305F;&#x3068;&#x3048;&#x3070;&#x3001;&#x53F3;&#x6709;&#x6A5F;&#x8272;&#x7D20;&#x306E;&#x69CB;&#x9020;&#x306E;&#x640D;&#x5931;&#x306A;&#x3057;&#x306E;&#x8A31;&#x53EF;&#x3067;&#x3059;&#x3002;&#x306B;&#x3088;&#x308B;&#x7D30;&#x5B54;&#x30A8;&#x30AD;&#x30B9;&#x30D1;&#x30F3;&#x30C0;&#x30FC;&#x5206;&#x5B50;&#x3001;&#x7D30;&#x5B54;&#x30B5;&#x30A4;&#x30BA; 2.7 &#x304B;&#x3089; 5 &#x306B;&#x62E1;&#x5927;&#x3067;&#x304D;&#x308B; nm &#x306E;&#x7C92;&#x5B50;&#x30B5;&#x30A4;&#x30BA;&#x304C;&#x6E1B;&#x5C11;&#x3059;&#x308B;&#x4E00;&#x65B9;&#x3002;&#x7D30;&#x5B54;&#x30A8;&#x30AD;&#x30B9;&#x30D1;&#x30F3;&#x30C0;&#x30FC;&#x3068; co-condensing &#x306E;&#x86CD;&#x5149;&#x67D3;&#x6599;&#x3067;&#x540C;&#x3058;&#x5408;&#x6210;&#x8FFD;&#x52A0;&#x524A;&#x6E1B;&#x5E73;&#x5747;&#x7C92;&#x5F84;&#x3092;&#x3055;&#x3089;&#x306B;&#x30C0;&#x30A6;&#x30F3; 100 nm&#x3002;Peg &#x5316;&#x5F8C;&#x3001;&#x3053;&#x306E;&#x3088;&#x3046;&#x306A;&#x86CD;&#x5149;&#x57FA;&#x30E1;&#x30BD;&#x30DD;&#x30FC;&#x30E9;&#x30B9; &#x30B7;&#x30EA;&#x30AB;&#x30CA;&#x30CE;&#x7C92;&#x5B50;&#x81EA;&#x767A;&#x7684;&#x306B;&#x86CD;&#x5149;&#x9855;&#x5FAE;&#x93E1;&#x306B;&#x3088;&#x3063;&#x3066;&#x793A;&#x3055;&#x308C;&#x308B;&#x3088;&#x3046;&#x306B;&#x7D30;&#x80DE;&#x306B;&#x3088;&#x3063;&#x3066;&#x53D6;&#x308A;&#x4E0A;&#x3052;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x9AD8;&#x7A92;&#x7D20;&#x30E1;&#x30BD;&#x30DD;&#x30FC;&#x30E9;&#x30B9; &#x30B7;&#x30EA;&#x30AB;&#x30CA;&#x30CE;&#x7C92;&#x5B50;&#x7ACB;&#x65B9;&#x3068;&#x7D30;&#x5B54;&#x69CB;&#x9020;&#x3002;

&#x4ECB;&#x5B54;&#x6C27;&#x5316;&#x7845;&#x7ACB;&#x65B9;&#x5BF9;&#x79F0;&#x6027;&#x4E0E;&#x4E00;&#x6BB5;&#x65F6;&#x95F4;&#x5438;&#x5F15;&#x4E86;&#x4ECE;&#x7814;&#x7A76;&#x8005;&#x7684;&#x5174;&#x8DA3;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x76EE;&#x524D;&#x5BA4;&#x6E29;&#x5408;&#x6210;&#x4ECB;&#x5B54;&#x6C27;&#x5316;&#x7845;&#x7EB3;&#x7C73;&#x9897;&#x7C92;&#x5177;&#x6709;&#x5F88;&#x9AD8;&#x7684;&#x6469;&#x5C14;&#x6BD4;&#x4E0E;&#x7ACB;&#x65B9; Pm3&#x305;n &#x5BF9;&#x79F0;&#x6027; (&gt; 50%) &#x7684; 3-&#x6C28;&#x4E19;&#x57FA; triethoxysilane&#x3002;&#x9C81;&#x68D2;&#x7684;&#x5141;&#x8BB8;&#xFF0C;&#x4F8B;&#x5982;&#xFF0C;&#x800C;&#x4E0D;&#x4F1A;&#x4E22;&#x5931;&#x7684;&#x7ED3;&#x6784;&#x7684;&#x6709;&#x673A;&#x67D3;&#x6599;&#x5171;&#x805A;&#x5408;&#x6210;&#x3002;&#x5B54;&#x81A8;&#x80C0;&#x5242;&#x5206;&#x5B50;&#xFF0C;&#x901A;&#x8FC7;&#x5B54;&#x5F84;&#x5927;&#x5C0F;&#x53EF;&#x653E;&#x5927;&#x4ECE; 2.7 &#x5230; 5 nm&#xFF0C;&#x867D;&#x7136;&#x9897;&#x7C92;&#x5C3A;&#x5BF8;&#x51CF;&#x5C0F;&#x3002;&#x6DFB;&#x52A0;&#x5B54;&#x81A8;&#x80C0;&#x673A;&#x548C; co-condensing &#x8367;&#x5149;&#x67D3;&#x6599;&#x5728;&#x540C;&#x4E00;&#x5408;&#x6210;&#x51CF;&#x5C11;&#x4E86;&#x5E73;&#x5747;&#x5927;&#x5C0F;&#x7684;&#x9897;&#x7C92;&#x8FDB;&#x4E00;&#x6B65; 100 nm&#x3002;&#x540E;&#x805A;&#x4E59;&#x4E8C;&#x9187;&#x5316;&#xFF0C;&#x8FD9;&#x79CD;&#x8367;&#x5149;&#x80FA;&#x57FA;&#x4ECB;&#x5B54;&#x6C27;&#x5316;&#x7845;&#x7EB3;&#x7C73;&#x9897;&#x7C92;&#x81EA;&#x53D1;&#x6027;&#x5DF2;&#x88AB;&#x8367;&#x5149;&#x663E;&#x5FAE;&#x955C;&#x6240;&#x793A;&#x7684;&#x5355;&#x5143;&#x683C;&#x3002;

&#x9AD8;&#x5EA6;&#x4E0E;&#x7ACB;&#x65B9;&#x80FA;&#x57FA;&#x4ECB;&#x5B54;&#x6C27;&#x5316;&#x7845;&#x7EB3;&#x7C73;&#x9897;&#x7C92;&#x7684;&#x5B54;&#x9699;&#x7ED3;&#x6784;&#x3002;

Mesoporous silica with cubic symmetry has attracted interest from researchers for some time. Here, we present the room temperature synthesis of mesoporous silica nanoparticles possessing cubic Pm3Ì…n symmetry with very high molar ratios (>50%) of 3-aminopropyl triethoxysilane. The synthesis is robust allowing, for example, co-condensation of organic dyes without loss of structure. By means of pore expander molecules, the pore size can be enlarged from 2.7 to 5 nm, while particle size decreases. Adding pore expander and co-condensing fluorescent dyes in the same synthesis reduces average particle size further down to 100 nm. After PEGylation, such fluorescent aminated mesoporous silica nanoparticles are spontaneously taken up by cells as demonstrated by fluorescence microscopy.

Highly Aminated Mesoporous Silica Nanoparticles with Cubic Pore Structure.

Silice m&eacute;soporeuse avec sym&eacute;trie cubique a suscit&eacute; l&#39;int&eacute;r&ecirc;t des chercheurs pendant un certain temps. Nous pr&eacute;sentons ici la synth&egrave;se de la temp&eacute;rature ambiante de m&eacute;soporeux nanoparticules de silice poss&eacute;dant la sym&eacute;trie cubique de Pm3&#x305;n avec des rapports molaires tr&egrave;s &eacute;lev&eacute;s (&gt; 50 %) des 3-aminopropyl tri&eacute;thoxysilane. La synth&egrave;se est robuste permettant, par exemple, co-condensation des colorants organiques sans perte de structure. Par le biais de mol&eacute;cules expanseur de pore, la taille des pores peut &ecirc;tre agrandie de 2,7 &agrave; 5 nm, tandis que la taille des particules diminue. L&#39;ajout de pore expanseur et co-condensing des colorants fluorescents dans la synth&egrave;se m&ecirc;me r&eacute;duit davantage les taille moyenne des particules jusqu&#39;&agrave; 100 nm. Apr&egrave;s PEGylation, ces nanoparticules de silice m&eacute;soporeux amin&eacute; fluorescents sont spontan&eacute;ment repris par cellules tel que d&eacute;montr&eacute; par microscopie &agrave; fluorescence.

Structure des pores tr&egrave;s nanoparticules silice m&eacute;soporeuse amin&eacute; avec Cubic.

Mesopor&ouml;se Kiesels&auml;ure mit kubischer Symmetrie hat Interesse von den Forschern seit einiger Zeit angezogen. Hier pr&auml;sentieren wir die Raumtemperatur-Synthese von Mesopor&ouml;se Silica-Nanopartikel besitzen kubische Pm3&#x305;n Symmetrie mit sehr hohen molaren Verh&auml;ltnis (&gt; 50 %) von 3-Aminopropyl--Triethoxysilane. Die Synthese ist robust, beispielsweise Co-condensation von organischen Farbstoffen ohne Verlust der Struktur. Mittels Pore-Expander-Molek&uuml;le, die Porengr&ouml;&szlig;e vergr&ouml;&szlig;ert werden kann von 2,7 bis 5 nm, w&auml;hrend die Partikelgr&ouml;&szlig;e abnimmt. Hinzuf&uuml;gen von Pore Leuchtstofff&auml;rbungen, Expander und co-condensing in der gleichen Synthese reduziert mittlere Korngr&ouml;&szlig;e weiter auf 100 nm. Nach PEGylierung werden solche fluoreszierende n&Atilde; Mesopor&ouml;se Silica-Nanopartikel von Zellen wie Fluoreszenzmikroskopie demonstriert spontan aufgegriffen.

Hoch Pore n&Atilde; Mesopor&ouml;se Silica-Nanopartikel mit kubischer Struktur.

Silice mesoporosa con simmetria cubica ha attratto interesse da parte di ricercatori per qualche tempo. Qui, vi presentiamo la sintesi di temperatura di mesoporosa nanoparticelle di silice che possiedono simmetria cubica Pm3&#x305;n con rapporti molari molto elevati (&amp;gt; 50%) di 3-amminopropil trietossisilano. La sintesi &egrave; robusto permettendo, ad esempio, co-condensation di coloranti organici senza perdita della struttura. Mediante molecole di espansore del poro, la dimensione dei pori pu&ograve; essere ingrandita da 2.7 a 5 nm, mentre diminuisce la dimensione delle particelle. Aggiungendo poro expander e co-condensing tinture fluorescenti nella stessa sintesi riduce la dimensione media delle particelle ulteriormente fino a 100 nm. Dopo PEGylation, tali nanoparticelle di silice mesoporosa amminate fluorescenti sono presi spontaneamente dalle cellule come dimostrato dalla microscopia di fluorescenza.

Altamente le nanoparticelle di silice mesoporosa amminate Cubic poro struttura.

Mesoporous &#xC2E4;&#xB9AC; &#xCE74; &#xC785;&#xBC29; &#xB300;&#xCE6D;&#xC73C;&#xB85C; &#xC5BC;&#xB9C8; &#xB3D9;&#xC548; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xAD00;&#xC2EC;&#xC744; &#xB04C;&#xACE0;&#xC788;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xB294; mesoporous&#xC758; &#xC0C1; &#xC628; &#xD569;&#xC131; &#xC2E4;&#xB9AC; &#xCE74; &#xB098;&#xB178; &#xC785;&#xC790;&#xB294; &#xB9E4;&#xC6B0; &#xB192;&#xC740; &#xC5B4; &#xAE08; &#xB2C8; &#xBE44;&#xC728;&#xC774; &#xC785;&#xBC29; Pm3&#x305;n &#xB300;&#xCE6D; &#xC18C;&#xC9C0; &#xC81C;&#xC2DC; (&amp;gt; 50%)&#xC758; 3 aminopropyl triethoxysilane. &#xD569;&#xC131;&#xC740; &#xAC15;&#xB825;&#xD55C; &#xD5C8;&#xC6A9;, &#xC608;&#xB97C; &#xB4E4;&#xC5B4;, &#xAD6C;&#xC870;&#xCCB4;&#xC758; &#xC190;&#xC2E4; &#xC5C6;&#xC774; &#xC720;&#xAE30; &#xC5FC;&#xB8CC;&#xC758; co-condensation. &#xAE30; &#xACF5; &#xD655;&#xC7A5;&#xAE30; &#xBD84;&#xC790;&#xC5D0; &#xC758;&#xD574; &#xAE30; &#xACF5; &#xD06C;&#xAE30; &#xC218; &#xD655;&#xB300; 2.7&#xC5D0;&#xC11C; 5 nm&#xC758; &#xC785;&#xC790; &#xD06C;&#xAE30; &#xAC10;&#xC18C; &#xD558;&#xB294; &#xB3D9;&#xC548;. 100&#xC73C;&#xB85C; &#xD3C9;&#xADE0; &#xC785;&#xC790; &#xD06C;&#xAE30;&#xB97C; &#xB354;&#xC6B1; &#xC904;&#xC5EC; &#xAC19;&#xC740; &#xD569;&#xC131;&#xC5D0;&#xC11C; &#xAE30; &#xACF5; &#xC775; &#xC2A4;&#xD32C; &#xB354; &#xBC0F; co-condensing &#xD615;&#xAD11; &#xC5FC;&#xB8CC;&#xB97C; &#xCD94;&#xAC00; nm. PEGylation, &#xD6C4; &#xAC19;&#xC740; &#xD615;&#xAD11; aminated mesoporous &#xC2E4;&#xB9AC; &#xCE74; &#xB098;&#xB178; &#xC785;&#xC790; &#xD615;&#xAD11; &#xD604;&#xBBF8;&#xACBD; &#xAC80;&#xC0AC; &#xBC95;&#xC5D0; &#xC758;&#xD574; &#xD45C;&#xC2DC; &#xB41C; &#xB300;&#xB85C; &#xC140;&#xC5D0; &#xC758;&#xD574; &#xCC44;&#xD0DD; &#xC800;&#xC808;&#xB85C; &#xB429;&#xB2C8;&#xB2E4;.

&#xB192;&#xC740; Aminated Mesoporous &#xD050;&#xBE45;&#xC73C;&#xB85C; &#xC2E4;&#xB9AC; &#xCE74; &#xB098;&#xB178; &#xAD6C;&#xC870; &#xAE30; &#xACF5;.

Mesoporous silica con simetr&iacute;a c&uacute;bica ha despertado el inter&eacute;s de los investigadores durante alg&uacute;n tiempo. Aqu&iacute;, presentamos la s&iacute;ntesis de la temperatura ambiente de mesoporosos nanopart&iacute;culas de s&iacute;lice que poseen simetr&iacute;a c&uacute;bica Pm3&#x305;n con muy alta proporci&oacute;n molar (&gt; 50%) de 3-aminopropil trietoxisilano. La s&iacute;ntesis es robusto permitiendo, por ejemplo, co-condensation de colorantes org&aacute;nicos sin p&eacute;rdida de estructura. Por medio de mol&eacute;culas de expansor de poro, el tama&ntilde;o del poro puede ampliarse de 2.7 a 5 nm, mientras que disminuye el tama&ntilde;o de part&iacute;cula. Agregando poro tintes fluorescentes de expander y co-condensing en la s&iacute;ntesis misma reduce el tama&ntilde;o promedio de part&iacute;cula m&aacute;s hasta 100 nm. Despu&eacute;s de la pegilaci&oacute;n, tales nanopart&iacute;culas de s&iacute;lice mesoporosa aminated fluorescente espont&aacute;neamente se toman por las c&eacute;lulas seg&uacute;n lo demostrado por microscop&iacute;a de fluorescencia.

Altamente Aminated Mesoporous Silica nanopart&iacute;culas con Cubic estructura porosa.

&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#x6FC0;&#x6D3B;&#x53D1;&#x8D77;&#x7684; IgE &#x53D7;&#x4F53;&#x4ECB;&#x5BFC;&#x6297;&#x539F;&#x4EA4;&#x8054;&#x5206;&#x6CCC;&#x6EB6;&#x9176;&#x4F53;&#x4E2D;&#x7684;&#x8FC7;&#x7A0B;&#x79F0;&#x4E3A;&#x8131;&#x9897;&#x7C92;&#x523A;&#x6FC0;&#x80DE;&#x7684;&#x6210;&#x679C;&#x3002;&#x5F88;&#x591A;&#x5DF2;&#x4E86;&#x89E3;&#x5230;&#x8FD9;&#x4E00;&#x8FDB;&#x7A0B;&#xFF0C;&#x5305;&#x62EC;&#x7ECF;&#x52A8;&#x5458;&#x7684;&#x5173;&#x952E;&#x4F5C;&#x7528;&#x7684;&#x5206;&#x5B50;&#x673A;&#x5236;&#x91CD;&#x8981;&#x4F46;&#x523A;&#x6FC0;&#x7684;&#x7ECF;&#x52A8;&#x5458;&#x548C;&#x9897;&#x7C92;&#x80DE;&#x4E4B;&#x95F4;&#x7684;&#x65F6;&#x7A7A;&#x5173;&#x7CFB;&#x90FD;&#x6CA1;&#x6709;&#x5B8C;&#x5168;&#x641E;&#x6E05;&#x695A;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x4E00;&#x4E2A;&#x65B0;&#x578B;&#x7684;&#x57FA;&#x4E8E;&#x6210;&#x50CF;&#x7684;&#x65B9;&#x6CD5;&#xFF0C;&#x4F7F;&#x7528;&#x5F02;&#x786B;&#x6C30;&#x9178;&#x8367;&#x5149;&#x7D20; (FITC)-&#x8461;&#x805A;&#x7CD6;&#x9897;&#x7C92;&#x80DE;&#x5728; RBL &#x80A5;&#x5927;&#x7684;&#x8BB0;&#x8005;&#x4F5C;&#x4E3A;&#x7EC6;&#x80DE;&#x548C;&#x5229;&#x7528;&#x7684;&#x5F02;&#x786B;&#x6C30;&#x9178;&#x7684; pH &#x654F;&#x611F;&#x6027;&#x3002;&#x6211;&#x4EEC;&#x8868;&#x73B0;&#x51FA;&#x9009;&#x62E9;&#x6027;&#x7684;&#x5F02;&#x786B;&#x6C30;&#x9178;-&#x8461;&#x805A;&#x7CD6;&#xFF0C;&#x79EF;&#x7D2F;&#x7684;&#x5438;&#x6536;&#x6DB2;&#x9636;&#x6BB5;&#xFF0C;&#x4F5C;&#x4E3A;&#x4E00;&#x4E2A;&#x6807;&#x8BB0;&#x4E3A;&#x5206;&#x6CCC;&#x6EB6;&#x9176;&#x4F53;&#xFF0C;&#x4E0E;&#x6211;&#x4EEC;&#x63CF;&#x8FF0;&#x5176;&#x5212;&#x5B9A;&#x4E0D;&#x540C;&#x7684;&#x9AD8;&#x5C14;&#x57FA;&#x4E8B;&#x4EF6;&#xFF0C;&#x5305;&#x62EC;&#x5145;&#x5206;&#x878D;&#x5408;&#x3001; &#x543B;&#x8FD0;&#x884C;&#x77AC;&#x6001;&#x878D;&#x5408;&#x548C;&#x590D;&#x5408;&#x80DE;&#x7684;&#x80FD;&#x529B;&#x3002;&#x4F7F;&#x7528;&#x6B64;&#x65B9;&#x6CD5;&#xFF0C;&#x6211;&#x4EEC;&#x627E;&#x5230;&#x8131;&#x9897;&#x7C92;&#x52A8;&#x529B;&#x5B66;&#x5F3A;&#x70C8;&#x4F9D;&#x8D56;&#x7EC6;&#x80DE;&#x5BF9;&#x886C;&#x5E95;&#x9644;&#x4EF6;&#x7684;&#x6301;&#x7EED;&#x65F6;&#x95F4;&#x3002;&#x6211;&#x4EEC;&#x7ED3;&#x5408;&#x8131;&#x9897;&#x7C92;&#x548C;&#x7ECF;&#x8BC1;&#x660E;&#x7ECF;&#x6CE2;&#x5F00;&#x59CB;&#x5728;&#x6269;&#x5C55;&#x5355;&#x5143;&#x683C;&#x51F8;&#x51FA;&#x7684;&#x7AD9;&#x70B9;&#x548C;&#x6709;&#x9650;&#x7684;&#x6297;&#x539F;&#x523A;&#x6FC0;&#x6761;&#x4EF6;&#x4E0B;&#x80DE;&#x4E4B;&#x95F4;&#x7684;&#x7A7A;&#x95F4;&#x5173;&#x7CFB;&#x7684;&#x52A8;&#x6001;&#x5F71;&#x50CF;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x627E;&#x5230;&#x7684;&#x7A7A;&#x95F4;&#x8FD1;&#x7AEF;&#x7ECF;&#x4FE1;&#x53F7;&#x548C;&#x5206;&#x6CCC;&#x4E8B;&#x4EF6;&#x5173;&#x8054;&#x8D77;&#x6765; TRPC1 &#x901A;&#x9053;&#x4E2D;&#x7ECF;&#x52A8;&#x5458;&#x53C2;&#x4E0E;&#x3002;

&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#x9897;&#x7C92;&#x80DE;&#x7684;&#x65F6;&#x7A7A;&#x5206;&#x8FA8;&#x7387;&#x663E;&#x793A; Ca2 + &#x6CE2;&#x542F;&#x52A8;&#x5173;&#x8054;&#x3002;

Mast cell activation initiated by antigen-mediated crosslinking of IgE receptors results in stimulated exocytosis of secretory lysosomes in the process known as degranulation. Much has been learned about the molecular mechanisms important for this process, including the crucial role of Ca(2+) mobilization, but spatio-temporal relationships between stimulated Ca(2+) mobilization and granule exocytosis are incompletely understood. Here we use a novel imaging-based method that uses fluorescein isothiocyanate (FITC)-dextran as a reporter for granule exocytosis in RBL mast cells and takes advantage of the pH sensitivity of FITC. We demonstrate the selectivity of FITC-dextran, accumulated by fluid-phase uptake, as a marker for secretory lysosomes, and we characterize its capacity to delineate different exocytotic events, including full fusion, kiss-and-run transient fusion and compound exocytosis. Using this method, we find strong dependence of degranulation kinetics on the duration of cell to substrate attachment. We combine imaging of degranulation and Ca(2+) dynamics to demonstrate a spatial relationship between the sites of Ca(2+) wave initiation in extended cell protrusions and exocytosis under conditions of limited antigen stimulation. In addition, we find that the spatially proximal Ca(2+) signaling and secretory events correlate with participation of TRPC1 channels in Ca(2+) mobilization.

Spatiotemporal resolution of mast cell granule exocytosis reveals correlation with Ca2+ wave initiation.

Activation des mastocytes initi&eacute;e par r&eacute;ticulation antig&egrave;ne m&eacute;di&eacute;e par des r&eacute;cepteurs d&#39;IgE r&eacute;sultats dans l&#39;exocytose stimul&eacute;e de lysosomes s&eacute;cr&eacute;trices dans le processus appel&eacute; d&eacute;granulation. A beaucoup appris sur les m&eacute;canismes mol&eacute;culaires importantes pour ce processus, y compris le r&ocirc;le crucial de la mobilisation de le Ca(2+), mais des relations spatio-temporelles entre stimul&eacute;e Ca(2+) mobilisation et exocytose des granules sont encore partiellement comprises. Ici, nous utilisons une nouvelle m&eacute;thode bas&eacute;e sur l&#39;imagerie qui utilise l&#39;isothiocyanate de fluoresc&eacute;ine (FITC)-dextran comme reporter pour l&#39;exocytose des granules dans le m&acirc;t RBL cellules et profite de la sensibilit&eacute; au pH du FITC. Nous d&eacute;montrons la s&eacute;lectivit&eacute; du FITC-dextran, accumul&eacute;e par absorption de la phase liquide, comme marqueur des lysosomes s&eacute;cr&eacute;trices, et nous caract&eacute;riser sa capacit&eacute; afin de d&eacute;limiter les diff&eacute;rents &eacute;v&eacute;nements exocytotique, y compris la fusion compl&egrave;te, kiss-and-run fusion transitoire et exocytose compos&eacute;. En utilisant cette m&eacute;thode, nous trouvons forte d&eacute;pendance de la cin&eacute;tique de la d&eacute;granulation sur la dur&eacute;e de la cellule pour la fixation du substrat. Nous combinons l&#39;imagerie de la d&eacute;granulation et Ca(2+) dynamique pour d&eacute;montrer une relation spatiale entre les sites d&#39;initiation de vague Ca(2+) en cellules longues saillies et exocytose dans des conditions de stimulation de l&#39;antig&egrave;ne limit&eacute;. En outre, on trouve que les Ca(2+) de signalisation dans l&#39;espace proximal et les &eacute;v&eacute;nements s&eacute;cr&eacute;toires sont en corr&eacute;lation avec la participation des canaux TRPC1 dans Ca(2+) mobilisation.

R&eacute;solution spatio-temporelle du mastocyte granules cytotoxiques r&eacute;v&egrave;le la corr&eacute;lation avec le Ca2 + initiation de vague.

Mastzell-Aktivierung von Antigen-vermittelten Vernetzung der IgE-Rezeptoren Ergebnisse in stimulierte Exozytose von sekretorische Lysosomen in den Prozess namens Degranulation initiiert. Gelernt viel &uuml;ber die molekularen Mechanismen, die f&uuml;r diesen Prozess, einschlie&szlig;lich die entscheidende Rolle der Ca(2+) Mobilisierung, wichtige raumzeitlichen Beziehungen zwischen stimulierte Ca(2+) Mobilisierung und Untermodul Exozytose werden allerdings nicht vollst&auml;ndig verstanden. Hier verwenden wir eine neuartige Darstellung-gegr&uuml;ndete Methode, Fluoreszein-Isothiocyanat (FITC)-Dextran als Reporter f&uuml;r Granulat Exozytose in RBL Mast Zellen und nutzt die pH-Wert-Empfindlichkeit der FITC. Wir zeigen die Selektivit&auml;t der FITC-Dextran, durch Aufnahme der Fl&uuml;ssigkeit-Phase, als Marker f&uuml;r sekretorische Lysosomen, angesammelt und wir charakterisieren seine Kapazit&auml;t, verschiedene exocytotic Ereignisse, einschlie&szlig;lich der vollst&auml;ndigen Verschmelzung, Kuss-und-Run transient Fusion und zusammengesetzte Exozytose abzugrenzen. Mit dieser Methode finden wir starke Abh&auml;ngigkeit der Degranulation Kinetik der Dauer der Zelle Substrat-Anlage. Wir kombinieren die Bildgebung der Degranulation und Ca(2+) Dynamik eine r&auml;umliche Beziehung zwischen den Standorten der Ca(2+) Welle Initiation in erweiterten Zelle Vorspr&uuml;nge und Exozytose unter Bedingungen der begrenzten Antigen Stimulation nachweisen. Au&szlig;erdem finden wir, dass die r&auml;umlich proximalen Ca(2+) Signalisierung und sekretorischen Ereignisse mit Beteiligung der TRPC1 Kan&auml;le in Ca(2+) Mobilisierung korrelieren.

Raumzeitliche Aufl&ouml;sung der Mastzelle Untermodul Exozytose zeigt Korrelation mit Ca2 + Welle Einleitung.

Attivazione mastociti iniziata da antigene mediata reticolazione dei recettori IgE risultati in stimolata esocitosi dei lisosomi secretive nel processo noto come degranulazione. Molto &egrave; stato appreso circa i meccanismi molecolari importanti per questo processo, compreso il ruolo cruciale della mobilitazione Ca(2+), ma relazioni spazio-temporali tra stimolato Ca(2+) mobilizzazione ed esocitosi dei granuli in modo incompleto sono comprese. Qui usiamo un nuovo metodo di imaging-based che utilizza l&#39;isotiocianato di fluoresceina (FITC)-Destrano come reporter per esocitosi granulo in albero RBL celle e sfrutta la sensibilit&agrave; di pH di FITC. Dimostriamo la selettivit&agrave; del FITC-Destrano, accumulato dall&#39;assorbimento di liquido-fase, come marcatore per secretive lisosomi, e ci caratterizzano la capacit&agrave; di delineare diversi eventi questo, tra cui fusione completa, bacio e conduzione transitori fusion e composti esocitosi. Utilizzando questo metodo, troviamo forte dipendenza della cinetica di degranulazione su tutta la durata della cella all&#39;attacco del substrato. Combiniamo imaging di degranulazione e Ca(2+) dinamica per dimostrare una relazione spaziale tra i siti di iniziazione onda Ca(2+) in cella estese protrusioni ed esocitosi in condizioni di stimolazione antigene limitata. Inoltre, troviamo che gli eventi secretivi e spazialmente prossimale Ca(2+) segnalazione correlano con la partecipazione di TRPC1 canali Ca(2+) mobilitazione.

Risoluzione spatiotemporal di esocitosi di granuli di mastociti rivela correlazione con Ca2 + iniziazione di onda.

&#x30DE;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x6D3B;&#x6027;&#x5316;&#x6297;&#x539F;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x3001;IgE &#x53D7;&#x5BB9;&#x4F53;&#x67B6;&#x6A4B;&#x3092;&#x7528;&#x3044;&#x305F;&#x7D50;&#x679C;&#x523A;&#x6FC0;&#x30A8;&#x30AD;&#x30BD;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306B;&#x5206;&#x6CCC;&#x306E;&#x30EA;&#x30BD;&#x30BD;&#x30FC;&#x30E0;&#x9846;&#x7C92;&#x3068;&#x3057;&#x3066;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3092;&#x958B;&#x59CB;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x591A;&#x304F;&#x306F;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x52D5;&#x54E1;&#x306E;&#x91CD;&#x5927;&#x306A;&#x5F79;&#x5272;&#x306A;&#x3069;&#x3001;&#x3053;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3067;&#x91CD;&#x8981;&#x306A;&#x5206;&#x5B50;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306B;&#x3064;&#x3044;&#x3066;&#x5B66;&#x7FD2;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x304C;&#x3001;&#x523A;&#x6FC0;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x52D5;&#x54E1;&#x3068;&#x9846;&#x7C92;&#x958B;&#x53E3;&#x5206;&#x6CCC;&#x50CF;&#x306E;&#x6642;&#x7A7A;&#x9593;&#x7684;&#x95A2;&#x4FC2;&#x304C;&#x4E0D;&#x5B8C;&#x5168;&#x306B;&#x7406;&#x89E3;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x30D5;&#x30EB;&#x30AA;&#x30EC;&#x30BB;&#x30A4;&#x30F3;&#x82A5;&#x5B50; &#xFF08;FITC) &#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x65B0;&#x898F;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0; &#x30D9;&#x30FC;&#x30B9;&#x306E;&#x65B9;&#x6CD5;&#x3092;&#x4F7F;&#x7528;-&#x30C7;&#x30AD;&#x30B9;&#x30C8;&#x30E9;&#x30F3;&#x306E;&#x958B;&#x53E3;&#x5206;&#x6CCC; RBL &#x30DE;&#x30B9;&#x30C8;&#x3067;&#x30EC;&#x30DD;&#x30FC;&#x30BF;&#x30FC;&#x3068;&#x3057;&#x3066;&#x7D30;&#x80DE;&#x3057;&#x3001;FITC &#x306E; pH &#x306E;&#x611F;&#x5EA6;&#x3092;&#x6D3B;&#x7528;&#x3057;&#x307E;&#x3059;&#x3002;&#x79C1;&#x305F;&#x3061;&#x306E; FITC-&#x30C7;&#x30AD;&#x30B9;&#x30C8;&#x30E9;&#x30F3;&#x6D41;&#x4F53;&#x76F8;&#x306E;&#x5438;&#x53CE;&#x5206;&#x6CCC;&#x30EA;&#x30BD;&#x30BD;&#x30FC;&#x30E0;&#x306E;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3068;&#x3057;&#x3066;&#x84C4;&#x7A4D;&#x306E;&#x9078;&#x629E;&#x6027;&#x3092;&#x793A;&#x3059;&#x3057;&#x3001;&#x5B8C;&#x5168;&#x878D;&#x5408;&#x3001;&#x30AD;&#x30B9;-&#x5B9F;&#x884C;&#x904E;&#x6E21;&#x878D;&#x5408;&#x8907;&#x5408;&#x30A8;&#x30AD;&#x30BD;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306A;&#x3069;&#x3001;&#x3055;&#x307E;&#x3056;&#x307E;&#x306A;&#x958B;&#x53E3;&#x653E;&#x51FA;&#x30A4;&#x30D9;&#x30F3;&#x30C8;&#x3092;&#x8A18;&#x8FF0;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x306F;&#x3001;&#x305D;&#x306E;&#x5BB9;&#x91CF;&#x3092;&#x7279;&#x5FB4;&#x4ED8;&#x3051;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x5F37;&#x3044;&#x30BB;&#x30EB;&#x57FA;&#x677F;&#x306E;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x6DFB;&#x4ED8;&#x3078;&#x306E;&#x671F;&#x9593;&#x306B;&#x4F9D;&#x5B58;&#x306E;&#x8131;&#x9846;&#x7C92;&#x53CD;&#x5FDC;&#x901F;&#x5EA6;&#x3092;&#x898B;&#x3064;&#x3051;&#x307E;&#x3059;&#x3002;&#x8131;&#x9846;&#x7C92;&#x3068;&#x62E1;&#x5F35;&#x7D30;&#x80DE;&#x7A81;&#x8D77;&#x90E8;&#x306B;&#x304A;&#x3051;&#x308B;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x6CE2;&#x767A;&#x751F;&#x30B5;&#x30A4;&#x30C8;&#x3068;&#x30A8;&#x30AD;&#x30BD;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306E;&#x9650;&#x3089;&#x308C;&#x305F;&#x6297;&#x539F;&#x523A;&#x6FC0;&#x6761;&#x4EF6;&#x4E0B;&#x3067;&#x306E;&#x7A7A;&#x9593;&#x7684;&#x95A2;&#x4FC2;&#x3092;&#x793A;&#x3059;&#x305F;&#x3081;&#x306B;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x52D5;&#x614B;&#x306E;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x3092;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x7A7A;&#x9593;&#x7684;&#x8FD1;&#x4F4D;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0; &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x3068;&#x5206;&#x6CCC;&#x30A4;&#x30D9;&#x30F3;&#x30C8; &#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x52D5;&#x54E1;&#x3067; TRPC1 &#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x306E;&#x53C2;&#x52A0;&#x3092;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x308B;&#x3092;&#x898B;&#x3064;&#x3051;&#x307E;&#x3059;&#x3002;

&#x80A5;&#x6E80;&#x7D30;&#x80DE;&#x9846;&#x7C92;&#x958B;&#x53E3;&#x5206;&#x6CCC;&#x50CF;&#x306E;&#x6642;&#x7A7A;&#x9593;&#x5206;&#x89E3;&#x80FD;&#x3067;&#x306F; ca 2 + &#x6CE2;&#x767A;&#x751F;&#x3068;&#x306E;&#x76F8;&#x95A2;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3059;&#x3002;

&#xB3DB;&#xB300; &#xC138;&#xD3EC; &#xD65C;&#xC131;&#xD654; &#xC790;&#xADF9;&#xB41C; exocytosis degranulation &#xC774;&#xB77C;&#xB294; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0;&#xC11C; &#xBD84; &#xB9AC;&#xC18C;&#xC880;&#xC758; &#xACB0;&#xACFC; IgE &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xD56D; &#xC6D0;-&#xC911;&#xC7AC; &#xAC00;&#xAD50;&#xC5D0; &#xC758;&#xD574; &#xC2DC;&#xC791;. Ca(2+) &#xB3D9;&#xC6D0;&#xC758; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xD3EC;&#xD568; &#xD558; &#xC5EC;&#xC774; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xBD84;&#xC790; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC5D0; &#xB300; &#xD55C; &#xB9CE;&#xC740; &#xBC30;&#xC6E0;&#xB2E4; &#xACE0; &#xD558;&#xC9C0;&#xB9CC; &#xC790;&#xADF9;&#xB41C; Ca(2+) &#xB3D9;&#xC6D0; &#xBC0F;&#xACFC; &#xB9BD; exocytosis spatio &#xC2DC;&#xAC04;&#xC801; &#xAD00;&#xACC4;&#xB294; &#xBD88;&#xC644;&#xC804; &#xD558; &#xAC8C; &#xC774;&#xD574;. &#xC6B0;&#xB9AC; &#xC5EC;&#xAE30; fluorescein isothiocyanate (FITC)&#xB97C; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xC18C;&#xC124; &#xC601;&#xC0C1; &#xAE30;&#xBC18; &#xBC29;&#xBC95;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;-dextran RBL &#xB3DB;&#xB300;&#xC5D0;&#xC11C;&#xACFC; &#xB9BD; exocytosis &#xAE30;&#xC790;&#xB85C; &#xC11C; &#xC138;&#xD3EC;&#xC640; FITC&#xC758; pH &#xBBFC;&#xAC10;&#xC131;&#xC744; &#xD65C;&#xC6A9; &#xD569;&#xB2C8;&#xB2E4;. FITC-dextran, &#xBD84; &#xB9AC;&#xC18C;&#xC880;&#xC5D0; &#xB300; &#xD55C; &#xD45C;&#xC2DD;&#xC73C;&#xB85C; &#xC561;&#xCCB4; &#xC0C1; &#xD1B5;&#xD48D; &#xAD00;&#xC5D0; &#xC758;&#xD574; &#xCD95;&#xC801; &#xB41C; &#xC120;&#xD0DD;&#xB3C4; &#xC2DC;&#xC5F0; &#xD558; &#xACE0; &#xC6B0;&#xB9AC;&#xAC00; &#xC804;&#xCCB4; &#xC735;&#xD569;, &#xBCF5;&#xD569; exocytosis, &#xD0A4;&#xC2A4; &#xC2E4;&#xD589; &#xACFC;&#xB3C4; &#xD4E8;&#xC804; &#xB4F1; &#xB2E4;&#xC591; &#xD55C; exocytotic &#xC774;&#xBCA4;&#xD2B8;&#xC758; &#xC724;&#xACFD;&#xC744; &#xADF8;&#xB9AC; &#xB2E4; &#xC6A9;&#xB7C9; &#xD2B9;&#xC131;&#xD654;. &#xC774; &#xBA54;&#xC11C;&#xB4DC;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;, &#xC6B0;&#xB9AC; &#xC140; &#xAE30;&#xD310; &#xBD80;&#xCC29; &#xAE30;&#xAC04;&#xC5D0; degranulation &#xC18D;&#xB3C4;&#xC758; &#xAC15;&#xD55C; &#xC758;&#xC874; &#xCC3E;&#xC744;. &#xC6B0;&#xB9AC;&#xB294; &#xC774;&#xBBF8;&#xC9D5; degranulation&#xC640; &#xD655;&#xC7A5;&#xB41C; &#xC140; &#xB3CC;&#xCD9C;&#xC5D0;&#xC11C; Ca(2+) &#xC6E8;&#xC774;&#xBE0C; &#xC2DC;&#xC791; &#xC0AC;&#xC774;&#xD2B8; &#xBC0F; exocytosis &#xC81C;&#xD55C; &#xB41C; &#xD56D; &#xC6D0; &#xC790;&#xADF9;&#xC758; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; &#xACF5;&#xAC04; &#xAD00;&#xACC4;&#xB97C; &#xBCF4;&#xC5EC; &#xC8FC;&#xAE30; &#xC704;&#xD574; Ca(2+) &#xC5ED;&#xD559;&#xC758; &#xACB0;&#xD569;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xB294; &#xACF5;&#xAC04;&#xC801; &#xC778;&#xC811; Ca(2+) &#xC2E0;&#xD638; &#xBC0F; &#xBD84; &#xC774;&#xBCA4;&#xD2B8; Ca(2+) &#xB3D9;&#xC6D0; TRPC1 &#xCC44;&#xB110;&#xC758; &#xCC38;&#xAC00;&#xC640; &#xC0C1;&#xAD00; &#xCC3E;&#xC744;.

&#xB9C8;&#xC2A4;&#xD2B8; &#xC140;&#xACFC; &#xB9BD; exocytosis spatiotemporal &#xD574;&#xC0C1;&#xB3C4; Ca2 + &#xC6E8;&#xC774;&#xBE0C; &#xAC1C;&#xC2DC;&#xC640;&#xC758; &#xC0C1;&#xAD00; &#xAD00;&#xACC4;&#xB97C; &#xBCF4;&#xC5EC;&#xC900;&#xB2E4;.

Activaci&oacute;n de mastocitos iniciada por reticulaci&oacute;n ant&iacute;geno mediada por receptores de IgE de resultados en la exocitosis estimulado de lisosomas secretores en el proceso conocido como la degranulaci&oacute;n. Se ha aprendido mucho sobre los mecanismos moleculares importantes para este proceso, incluyendo el papel crucial de la movilizaci&oacute;n de CA, pero incompleto se entienden relaciones espacio-temporales entre movilizaci&oacute;n de CA estimulado y exocitosis de gr&aacute;nulos. Aqu&iacute; utilizamos un m&eacute;todo novedoso basado en la proyecci&oacute;n de imagen que utiliza isotiocianato de fluoresce&iacute;na (FITC)-dextrano como reportero para la exocitosis de gr&aacute;nulos en el m&aacute;stil RBL c&eacute;lulas y se aprovecha de la sensibilidad del pH del FITC. Demostramos la selectividad del FITC-dextrano, acumulado por la absorci&oacute;n de l&iacute;quido-fase, como marcador de lisosomas secretores, y se caracteriza su capacidad para delinear diferentes eventos exocit&oacute;ticas, incluyendo completa fusi&oacute;n, fusi&oacute;n transitoria beso-y-run y exocitosis compuesto. Usando este m&eacute;todo, nos encontramos con fuerte dependencia de la cin&eacute;tica de la degranulaci&oacute;n de la duraci&oacute;n de la c&eacute;lula para la fijaci&oacute;n del sustrato. Combinamos la proyecci&oacute;n de imagen de degranulaci&oacute;n y din&aacute;mica de la CA para demostrar una relaci&oacute;n espacial entre los sitios de iniciaci&oacute;n de onda de CA en extendido celular salientes y exocitosis en condiciones de estimulaci&oacute;n de ant&iacute;geno limitada. Adem&aacute;s, encontramos que la se&ntilde;alizaci&oacute;n espacial proximal de CA y eventos secretores se correlacionan con participaci&oacute;n de los canales de TRPC1 en la movilizaci&oacute;n de CA.

Resoluci&oacute;n espaciotemporal de exocitosis de gr&aacute;nulos de mastocitos revela la correlaci&oacute;n con la iniciaci&oacute;n de la onda de Ca2 +.

&#x7ECF; IgE &#x53D7;&#x4F53;&#x4ECB;&#x5BFC;&#x4FE1;&#x53F7;&#x54CD;&#x5E94;&#x52A8;&#x5458;&#x662F;&#x5728;&#x5F88;&#x591A;&#x65B9;&#x9762;&#x7684;&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#x529F;&#x80FD;&#x7684;&#x5173;&#x952E;&#x5DE5;&#x5E8F;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x603B;&#x7ED3;&#x4E00;&#x4E0B;&#x6211;&#x4EEC;&#x76EE;&#x524D;&#x4E86;&#x89E3;&#x7684;&#x5206;&#x5B50;&#x4F9D;&#x636E;&#x8FD9;&#x4E00;&#x8FDB;&#x7A0B;&#x548C;&#x5B83;&#x5728;&#x751F;&#x7406;&#x4E0A;&#x6709;&#x5173;&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#x751F;&#x7269;&#x5B66;&#x4E2D;&#x626E;&#x6F14;&#x7684;&#x89D2;&#x8272;&#x3002;&#x7531;&#x6297;&#x539F;&#x4FE1;&#x53F7;&#x8FD9;&#x4E9B;&#x914D;&#x5408;&#x542F;&#x52A8;&#x5FEB;&#x6CE2;&#x7ECF;&#x5E38;&#x8DDF;&#x6709;&#x4E00;&#x7CFB;&#x5217;&#x7ECF;&#x632F;&#x8361;&#xFF0C;&#x4F9D;&#x8D56;&#x4E8E;&#x5927;&#x91CF;&#x6D8C;&#x5165;&#x4ECB;&#x5BFC;&#x5185;&#x8D28;&#x7F51;&#x9C81;&#x7C73;&#x90A3;&#x7ECF;&#x4F20;&#x611F;&#x5668;&#x8026;&#x5408;&#x7684;&#x7ECF;&#x7ECF;&#x52A8;&#x5458;&#x7684;&#x65B0;&#x8DE8;&#x754C;&#x901A;&#x9053; STIM1 &#x7684;&#x9499;&#x91CA;&#x653E;&#x7684; IgE &#x53D7;&#x4F53;&#x6FC0;&#x6D3B;&#x6FC0;&#x6D3B;&#x9499;&#x901A;&#x9053;&#x86CB;&#x767D; Orai1&#x3002;&#x9897;&#x7C92;&#x80DE;&#x53D6;&#x51B3;&#x4E8E;&#x8FD9;&#x4E00;&#x8FDB;&#x7A0B;&#xFF0C;&#x6FC0;&#x6D3B;&#x86CB;&#x767D;&#x6FC0;&#x9176; C &#x4E9A;&#x578B;&#xFF0C;&#x4EE5;&#x53CA;&#x4E0E;&#x8FD9;&#x4E9B;&#x4FE1;&#x53F7;&#x7684;&#x6B65;&#x9AA4;&#x7684;&#x5177;&#x4F53;&#x89D2;&#x8272;&#x90FD;&#x5F00;&#x59CB;&#x88AB;&#x4E86;&#x89E3;&#x3002;&#x7ECF;&#x52A8;&#x5458;&#x4E5F;&#x626E;&#x6F14;&#x91CD;&#x8981;&#x89D2;&#x8272;&#x5728;&#x523A;&#x6FC0;&#x80DE;&#x7684;&#x56DE;&#x6536; endosomes &#x548C;&#x65B0;&#x5408;&#x6210;&#x7EC6;&#x80DE;&#x56E0;&#x5B50;&#xFF0C;&#x4EE5;&#x53CA;&#x5728;&#x6297;&#x539F;&#x4ECB;&#x5BFC;&#x5927;&#x9F20;&#x7C98;&#x819C;&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#x8D8B;&#x5316;&#x3002;&#x78F7;&#x8102;&#x9170;&#x808C;&#x9187;&#x4EE3;&#x8C22;&#x5728;&#x6240;&#x6709;&#x8FD9;&#x4E9B;&#x8FDB;&#x7A0B;&#x4E2D;&#x626E;&#x6F14;&#x5173;&#x952E;&#x89D2;&#x8272;&#x548C;&#x6211;&#x4EEC;&#x7A81;&#x51FA;&#x663E;&#x793A;&#x8FD9;&#x4E9B;&#x5728;&#x51E0;&#x4E2A;&#x6848;&#x4EF6;&#x4E2D;&#x7684;&#x89D2;&#x8272;&#x3002;

&#x7ECF;&#x52A8;&#x5458;&#x548C;&#x80A5;&#x5927;&#x7EC6;&#x80DE;&#x529F;&#x80FD;&#x53CA;&#x5176;&#x8C03;&#x63A7;&#x7684;&#x4F5C;&#x7528;&#x3002;

Mobilization of Ca(2+) in response to IgE receptor-mediated signaling is a key process in many aspects of mast cell function. Here we summarize our current understanding of the molecular bases for this process and the roles that it plays in physiologically relevant mast cell biology. Activation of IgE receptor signaling by antigen that crosslinks these complexes initiates Ca(2+) mobilization as a fast wave that is frequently followed by a series of Ca(2+) oscillations which are dependent on Ca(2+) influx-mediated by coupling of the endoplasmic reticulum luminal Ca(2+) sensor STIM1 to the calcium release activated calcium channel protein Orai1. Granule exocytosis depends on this process, together with the activation of protein kinase C isoforms, and specific roles for these signaling steps are beginning to be understood. Ca(2+) mobilization also plays important roles in stimulated exocytosis of recycling endosomes and newly synthesized cytokines, as well as in antigen-mediated chemotaxis of rat mucosal mast cells. Phosphoinositide metabolism plays key roles in all of these processes, and we highlight these roles in several cases.

Roles for ca(2+) mobilization and its regulation in mast cell functions.

Mobilisation des Ca(2+) en r&eacute;ponse &agrave; IgE m&eacute;di&eacute;e par les r&eacute;cepteurs de signalisation est un processus cl&eacute; dans de nombreux aspects de la fonction des cellules de m&acirc;t. Ici nous r&eacute;sumons notre compr&eacute;hension des bases mol&eacute;culaires de ce processus et les r&ocirc;les qu&#39;elle joue dans la biologie des cellules de m&acirc;t physiologiquement pertinents. L&#39;activation du r&eacute;cepteur d&#39;IgE de signalisation par antig&egrave;ne que crosslinks ces complexes initie Ca(2+) mobilisation comme une vague rapide qui est souvent suivie d&#39;une s&eacute;rie d&#39;oscillations Ca(2+) qui d&eacute;pendent de l&#39;afflux induite par couplage du r&eacute;ticulum endoplasmique luminal Ca(2+) capteur de Ca(2+) STIM1 pour le calcium release activ&eacute;e prot&eacute;ine de canal de calcium Orai1. Exocytose des granules d&eacute;pend de ce processus, ainsi que l&#39;activation des isoformes de la prot&eacute;ine kinase C, et les r&ocirc;les sp&eacute;cifiques pour ces &eacute;tapes signalisation commencent &agrave; &ecirc;tre compris. Ca(2+) mobilisation joue &eacute;galement un r&ocirc;le important, stimul&eacute;e par exocytose du recyclage des endosomes et des cytokines nouvellement synth&eacute;tis&eacute;es, ainsi que dans le chimiotactisme des mastocytes muqueux de rat induite par l&#39;antig&egrave;ne. Du m&eacute;tabolisme des phosphoinositides joue un r&ocirc;le cl&eacute; dans l&#39;ensemble de ces processus, et nous mettons en &eacute;vidence ces r&ocirc;les dans plusieurs cas.

R&ocirc;les pour la mobilisation de ca(2+) et de son r&egrave;glement en fonctions des cellules de m&acirc;t.

Mobilisierung der Ca(2+) als Reaktion auf die IgE-Rezeptor-vermittelten Signalisierung ist ein Schl&uuml;sselprozess in vielen Aspekten der Mastzell-Funktion. Hier fassen wir unser gegenw&auml;rtige Verst&auml;ndnis der molekularen Grundlagen f&uuml;r diesen Prozess und die Rollen, die physiologisch relevanten Mast Zellbiologie spielt. Aktivierung des IgE-Rezeptors Signalisierung durch Antigen, dass Querverbindungen, die diese komplexe initiiert Ca(2+) Mobilisierung als schnelle Welle, die h&auml;ufig durch eine Reihe von Ca(2+) Schwingungen, die Ca(2+) Zustrom gefolgt wird-vermittelt durch Kopplung des endoplasmatischen Retikulum luminalen Ca(2+) Sensors abh&auml;ngig sind STIM1 zu Calcium release aktiviert Protein Kalzium-Kanal Orai1. Untermodul Exozytose h&auml;ngt dabei zusammen mit der Aktivierung von Protein-Kinase C-Isoformen, und bestimmte Rollen f&uuml;r diese Signalisierung Schritte zu verstehen beginnen. Ca(2+) Mobilisierung spielt auch eine wichtige Rollen in stimulierte Exozytose von recycling Endosomen und neu synthetisierte Zytokine sowie in Antigen-vermittelte Chemotaxis von Ratte Schleimhaut-Mastzellen. Phosphoinositid-Stoffwechsel spielt eine Schl&uuml;sselrolle in all diese Prozesse, und wir markieren diese Rollen in mehreren F&auml;llen.

Rollen f&uuml;r die Mobilisierung der ca(2+) und seine Verordnung in Mastzell-Funktionen.

Mobilizzazione del Ca(2+) in risposta a IgE mediata dal recettore di segnalazione &egrave; un processo chiave in molti aspetti della funzione dei mastociti. Qui riassumiamo la nostra attuale comprensione delle basi molecolari per questo processo e il ruolo che essa svolge fisiologicamente rilevanti mast cell biology. Attivazione del recettore IgE segnalazione di antigene che le reticolazioni questi complessi avvia la mobilitazione Ca(2+) come un&#39;onda veloce che &egrave; spesso seguita da una serie di oscillazioni Ca(2+) che dipendono da Ca(2+) afflusso-mediata dall&#39;accoppiamento del sensore Ca(2+) STIM1 del lume del reticolo endoplasmatico per il rilascio di calcio attivato proteina canale calcio Orai1. Esocitosi dei granuli dipende da questo processo, insieme con l&#39;attivazione della proteina chinasi C isoforme, e ruoli specifici per questi passaggi segnalazione stanno cominciando a essere capito. CA(2+) mobilitazione svolge anche importanti ruoli in stimolata esocitosi di riciclaggio endosomes e cytokines recentemente sintetizzati, cos&igrave; come in-mediata antigene chemiotassi dei mastociti mucosali ratto. Phosphoinositide metabolismo svolge ruoli chiave in tutti questi processi, e si evidenziano questi ruoli in diversi casi.

Ruoli per ca(2+) mobilizzazione e relativo regolamento in funzioni delle cellule dell&#39;albero.

&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x306E;&#x52D5;&#x54E1; IgE &#x53D7;&#x5BB9;&#x4F53;&#x3092;&#x4ECB;&#x3059;&#x308B;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x5FDC;&#x7B54;&#x3067;&#x80A5;&#x6E80;&#x7D30;&#x80DE;&#x306E;&#x6A5F;&#x80FD;&#x306E;&#x591A;&#x304F;&#x306E;&#x5074;&#x9762;&#x3067;&#x91CD;&#x8981;&#x306A;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3067;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x3053;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3068;&#x751F;&#x7406;&#x5B66;&#x7684;&#x95A2;&#x9023;&#x6027;&#x306E;&#x9AD8;&#x3044;&#x30DE;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x751F;&#x7269;&#x5B66;&#x3067;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x308B;&#x5F79;&#x5272;&#x306E;&#x70BA;&#x306E;&#x5206;&#x5B50;&#x57FA;&#x76E4;&#x306E;&#x5F53;&#x793E;&#x306E;&#x73FE;&#x5728;&#x306E;&#x7406;&#x89E3;&#x3092;&#x8981;&#x7D04;&#x3057;&#x307E;&#x3059;&#x3002;&#xFF78;&#xFF9B;&#xFF7D;&#xFF98;&#xFF9D;&#xFF78;&#x3053;&#x308C;&#x3089;&#x306E;&#x932F;&#x4F53;&#x958B;&#x59CB;&#x3055;&#x308C;&#x308B;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x52D5;&#x54E1;&#x3068;&#x3057;&#x3066;&#x983B;&#x7E41;&#x306B;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x306E;&#x6D41;&#x5165;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x5C0F;&#x80DE;&#x4F53;&#x5185;&#x8154;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0; &#x30BB;&#x30F3;&#x30B5;&#x30FC;&#x306E; STIM1 &#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x653E;&#x51FA;&#x3059;&#x308B;&#x7D50;&#x5408;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x308B;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x632F;&#x52D5;&#x306E;&#x30B7;&#x30EA;&#x30FC;&#x30BA;&#x304C;&#x7D9A;&#x304D;&#x307E;&#x3059;&#x901F;&#x6CE2;&#x306B;&#x3088;&#x308B;&#x6297;&#x539F;&#x3092;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0; IgE &#x53D7;&#x5BB9;&#x4F53;&#x6D3B;&#x6027;&#x5316;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0; &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x86CB;&#x767D;&#x8CEA; Orai1 &#x3092;&#x6D3B;&#x6027;&#x5316;&#x3002;&#x958B;&#x53E3;&#x5206;&#x6CCC;&#x86CB;&#x767D;&#x8CEA;&#x30AD;&#x30CA;&#x30FC;&#x30BC; C &#x30A2;&#x30A4;&#x30BD; &#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x3068;&#x4E00;&#x7DD2;&#x306B;&#x3053;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306B;&#x4F9D;&#x5B58;&#x3001;&#x7279;&#x5B9A;&#x306E;&#x30ED;&#x30FC;&#x30EB;&#x3092;&#x3053;&#x308C;&#x3089;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0;&#x624B;&#x9806;&#x7406;&#x89E3;&#x3055;&#x308C;&#x308B;&#x3057;&#x59CB;&#x3081;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x52D5;&#x54E1;&#x306F;&#x3001;&#x307E;&#x305F;&#x30EA;&#x30B5;&#x30A4;&#x30AF;&#x30EB; &#x30A8;&#x30F3;&#x30C9;&#x30BD;&#x30FC;&#x30E0;&#x3068;&#x65B0;&#x305F;&#x306B;&#x5408;&#x6210;&#x3055;&#x308C;&#x305F;&#x30B5;&#x30A4;&#x30C8;&#x30AB;&#x30A4;&#x30F3;&#x306E;&#x523A;&#x6FC0;&#x306E;&#x30A8;&#x30AD;&#x30BD;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x304A;&#x3088;&#x3073;&#x30E9;&#x30C3;&#x30C8;&#x7C98;&#x819C;&#x578B;&#x80A5;&#x6E80;&#x7D30;&#x80DE;&#x306E;&#x8D70;&#x5316;&#x6027;&#x6297;&#x539F;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x30A4;&#x30CE;&#x30B7;&#x30C8;&#x30FC;&#x30EB;&#x30EA;&#x30F3;&#x8102;&#x8CEA;&#x4EE3;&#x8B1D;&#x306F;&#x3053;&#x308C;&#x3089;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x3059;&#x3079;&#x3066;&#x306E;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x308B;&#x3057;&#x3001;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x30B1;&#x30FC;&#x30B9;&#x3067;&#x3053;&#x308C;&#x3089;&#x306E;&#x30ED;&#x30FC;&#x30EB;&#x3092;&#x5F37;&#x8ABF;&#x8868;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x52D5;&#x54E1;&#x3068;&#x305D;&#x306E;&#x30DE;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x6A5F;&#x80FD;&#x306E;&#x8ABF;&#x7BC0;&#x306E;&#x5F79;&#x5272;&#x3002;

IgE &#xC218;&#xC6A9; &#xCCB4;-&#xC911;&#xC7AC; &#xC2E0;&#xD638;&#xC5D0; &#xB300; &#xD55C; &#xC751;&#xB2F5; ca(2+)&#xC758; &#xB3D9;&#xC6D0; &#xB9C8;&#xC2A4;&#xD2B8; &#xC140; &#xD568;&#xC218;&#xC758; &#xC5EC;&#xB7EC; &#xCE21;&#xBA74;&#xC5D0;&#xC11C; &#xD575;&#xC2EC; &#xACFC;&#xC815; &#xC774;&#xB2E4;. &#xC5EC;&#xAE30;&#xC11C; &#xC6B0;&#xB9AC;&#xB294; &#xC21C;&#xC218; &#xAD00;&#xB828; &#xB3DB;&#xB300; &#xC138;&#xD3EC; &#xC0DD;&#xBB3C;&#xD559;&#xC5D0;&#xC11C; &#xC218;&#xD589; &#xD558;&#xB294; &#xC5ED;&#xD560;&#xACFC;&#xC774; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0; &#xB300; &#xD55C; &#xBD84;&#xC790; &#xAE30;&#xC9C0;&#xC758; &#xC6B0;&#xB9AC;&#xC758; &#xD604;&#xC7AC; &#xC774;&#xD574; &#xC694;&#xC57D;. &#xC774; &#xB2E8;&#xC9C0; &#xC2DC;&#xC791; &#xC790;&#xC8FC; &#xB4A4;&#xC5D0; Ca(2+) endoplasmic &#xADF8;&#xBB3C; luminal Ca(2+) &#xC13C;&#xC11C;&#xC758; &#xCEE4;&#xD50C;&#xB9C1;&#xC5D0; &#xC758;&#xD574; &#xC720;&#xC785; &#xC911;&#xC7AC;&#xC5D0; &#xC758;&#xC874; &#xD558;&#xB294; Ca(2+) &#xC9C4;&#xB3D9; &#xC2DC;&#xB9AC;&#xC988; &#xBE60;&#xB978; &#xD30C;&#xB3C4;&#xB85C; &#xB3D9;&#xC6D0; Ca(2+) crosslinks STIM1 &#xCE7C;&#xC298;&#xC744; &#xB9B4;&#xB9AC;&#xC2A4; antigen&#xC5D0; &#xC758;&#xD574; &#xC2E0;&#xD638; IgE &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xD65C;&#xC131;&#xD654; &#xD65C;&#xC131;&#xD654; &#xCE7C;&#xC298; &#xCC44;&#xB110; &#xB2E8;&#xBC31;&#xC9C8; Orai1. &#xACFC; &#xB9BD; exocytosis &#xB2E8;&#xBC31;&#xC9C8; &#xD0A4; &#xB2C8; &#xC544; &#xC81C; C isoforms&#xC758; &#xD65C;&#xC131;&#xD654;&#xC640; &#xD568;&#xAED8;&#xC774; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4; &#xBC0F; &#xD2B9;&#xC815; &#xC5ED;&#xD560;&#xC5D0; &#xB300; &#xD55C; &#xC774;&#xB7EC;&#xD55C; &#xC2E0;&#xD638; &#xB2E8;&#xACC4; &#xC774;&#xD574; &#xD558;&#xAE30; &#xC2DC;&#xC791; &#xD558;&#xB294;. &#xB610;&#xD55C; Ca(2+) &#xB3D9;&#xC6D0; &#xC7AC;&#xD65C;&#xC6A9; endosomes &#xBC0F; &#xC0C8;&#xB85C; &#xD569;&#xC131;&#xB41C; cytokines&#xC758; &#xC790;&#xADF9;&#xB41C; exocytosis &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xC950; &#xC810; &#xB9C9; &#xB9C8;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC;&#xC758; &#xD56D; &#xC6D0;-&#xC911;&#xC7AC; chemotaxis&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4;. Phosphoinositide &#xC2E0;&#xC9C4; &#xB300;&#xC0AC; &#xBAA8;&#xB450;&#xC774; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC758; &#xC8FC;&#xC694; &#xC5ED;&#xD560; &#xC5F0;&#xADF9;&#xACFC; &#xC6B0;&#xB9AC; &#xC5EC;&#xB7EC; &#xC0AC;&#xB840;&#xC5D0;&#xC11C; &#xC774;&#xB7EC;&#xD55C; &#xC5ED;&#xD560;&#xC744; &#xAC15;&#xC870;.

Ca(2+) &#xB3D9;&#xC6D0; &#xBC0F; &#xB9C8;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC; &#xAE30;&#xB2A5;&#xC5D0;&#xB294; &#xADDC;&#xC81C;&#xC5D0; &#xB300; &#xD55C; &#xC5ED;&#xD560;.

Movilizaci&oacute;n de CA en respuesta a la se&ntilde;alizaci&oacute;n mediada por receptores de IgE es un proceso clave en muchos aspectos de la funci&oacute;n de la c&eacute;lula del m&aacute;stil. Aqu&iacute; resumimos nuestra comprensi&oacute;n actual de las bases moleculares de este proceso y el papel que desempe&ntilde;a en la biolog&iacute;a de la c&eacute;lula de m&aacute;stil fisiol&oacute;gicamente relevantes. Activaci&oacute;n de los receptores de IgE de se&ntilde;alizaci&oacute;n por el ant&iacute;geno que reticulaciones estos complejos inicia movilizaci&oacute;n de CA como una ola r&aacute;pida que con frecuencia va seguida de una serie de oscilaciones de CA que dependen de la CA afluencia-mediada por acoplamiento del ret&iacute;culo endopl&aacute;smico luminal CA sensor STIM1 el calcio liberar activado prote&iacute;na del canal de calcio Orai1. Exocitosis de gr&aacute;nulos depende de este proceso, junto con la activaci&oacute;n de isoformas de la prote&iacute;na quinasa C, y roles espec&iacute;ficos para estos pasos de se&ntilde;alizaci&oacute;n est&aacute;n empezando a entenderse. Movilizaci&oacute;n de CA tambi&eacute;n juega un papel importante en exocitosis estimulada de reciclaje endosomas y citoquinas reci&eacute;n sintetizadas, as&iacute; como en ant&iacute;geno mediada por quimiotactismo de mastocitos mucosa de rata. Metabolismo de los fosfoinositoles juega un papel clave en todos estos procesos, y podemos destacar estas funciones en varios casos.

Papeles para la movilizaci&oacute;n de CA y su regulaci&oacute;n en las funciones de la c&eacute;lula del m&aacute;stil.

Ca(2+) oscillations are a hallmark of mammalian fertilization and play a central role in the activation of development. The calcium required for these oscillations is primarily derived from the endoplasmic reticulum (ER), which accumulates in clusters at the microvillar subcortex during oocyte maturation. The migration of the ER to the cortex during maturation is thought to play an important role in rendering the ER competent to generate the calcium transients, and the redistribution of ER is believed to be primarily mediated by microtubules and microfilaments. We have previously shown that the oocyte- and early embryo-restricted maternal effect gene Mater (Nlrp5) localizes to, and is required for, formation of the oocyte cytoplasmic lattices, a tubulin-containing structure that appears to play an important role in organelle positioning and distribution during oocyte maturation. Given these observations, we hypothesized that Mater may also be required for ER redistribution and Ca(2+) homeostasis in oocytes. To test this hypothesis, we first investigated ER localization in metaphase-II Mater(tm/tm) (hypomorph) oocytes and found ER clusters to be less abundant at the microvillar cortex when compared to wild type oocytes. To examine the potential mechanisms by which MATER mediates ER redistribution, we tested whether tubulin expression levels and localization were affected in the mutant oocytes and found that the Triton-insoluble fraction of tubulin was significantly decreased in Mater(tm/tm) oocytes. To identify potential functional defects associated with these ER abnormalities, we next set out to investigate if the pattern of Ca(2+) oscillations was altered in Mater(tm/tm) oocytes after fertilization in vitro. Intriguingly, Ca(2+) oscillations in Mater(tm/tm) oocytes exhibited a significantly lower first peak amplitude and a higher frequency when compared to wild type oocytes. We then found that the Ca(2+) oscillation defect in Mater(tm/tm) oocytes was likely caused by a reduced amount of Ca(2+) in the ER stores. Taken together, these observations support the hypothesis that MATER is required for ER distribution and Ca(2+) homeostasis in oocytes, likely due to defects in lattice-mediated ER positioning and/or redistribution.

The role of MATER in endoplasmic reticulum distribution and calcium homeostasis in mouse oocytes.

Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3's pore-forming α1E subunit showed altered Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1's lipid and sugar components and CaV2.3's α1E and α2δ subunits. Our results provide a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.

Lipid modulation of calcium flux through CaV2.3 regulates acrosome exocytosis and fertilization.

Mast cells play a key role in allergy and inflammation processes as part of the immune response. The activation of mast cells via antigen binding and cross-linking of IgE receptors initiates the onset of dramatic calcium (Ca(2+)) mobilization dynamics that promote the release of mediators of inflammation and allergy. Ca(2+) signaling in mast cells has been studied extensively using a variety of research tools and techniques. In these studies, a large number of proteins have been identified to participate in various stages of these processes. Here we describe single-cell imaging as an important approach for examining Ca(2+) signaling and exocytosis in mast cells. Single-cell imaging tools have advanced significantly over the last 10 years, in part due to improvements in microscope technology and in part due to the development of a new generation of Ca(2+) indicators and genetically encoded Ca(2+) sensors. The single-cell imaging techniques described here provide the spatial and temporal resolution required to decipher the signaling events that are critical for mast cell functions.

Real-time imaging of Ca(2+) mobilization and degranulation in mast cells.

Despite numerous applications, we lack fundamental understanding of how variables such as nanoparticle (NP) size influence the activity of tethered enzymes. Previously, we showed that biomimetic oriented immobilization yielded higher specific activities versus nonoriented adsorption or carboxyl-amine binding. Here, we standardize NP attachment strategy (oriented immobilization via hexahistidine tags) and composition (Ni-NTA coated gold NPs), to test the impact of NP size (⌀5, 10, 20, and 50 nm) on multilayer formation, activity, and kinetic parameters (kcat, KM, kcat/KM) of enzymes representing three different classes: glucose-6-phosphate isomerase (GPI), an isomerase; Glyceraldehyde-3-phosphate dehydrogenase S (GAPDHS), an oxidoreductase; and pyruvate kinase (PK), a transferase. Contrary to other reports, we observed no trend in kinetic parameters for individual enzymes when found in monolayers (<100% enzyme coverage), suggesting an advantage for oriented immobilization versus other attachment strategies. Saturating the NPs to maximize activity per NP resulted in enzyme multilayer formation. Under these conditions, total activity per NP increased with increasing NP size. Conversely, specific activity for all three enzymes was highest when tethered to the smallest NPs, retaining a remarkable 73-94% of the activity of free/untethered enzymes. Multilayer formations caused a clear trend of kcat decreasing with increasing NP size, yet negligible change in KM. Understanding the fundamental relationships between NP size and tethered enzyme activity enables optimized design of various applications, maximizing activity per NP or activity per enzyme molecule.

Effects of Nanoparticle Size on Multilayer Formation and Kinetics of Tethered Enzymes.

Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices.

Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection.

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo(-/-)) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.

Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis.

Lipids are critical regulators of mammalian sperm function, first helping prevent premature acrosome exocytosis, then enabling sperm to become competent to fertilize at the right place/time through the process of capacitation, and ultimately triggering acrosome exocytosis. Yet because they do not fit neatly into the "DNA--RNA-protein" synthetic pathway, they are understudied and poorly understood. Here, we focus on three lipids or lipid classes-cholesterol, phospholipids, and the ganglioside G(M1)--in context of the modern paradigm of acrosome exocytosis. We describe how these various- species are precisely segregated into membrane macrodomains and microdomains, simultaneously preventing premature exocytosis while acting as foci for organizing regulatory and effector molecules that will enable exocytosis. Although the mechanisms responsible for these domains are poorly defined, there is substantial evidence for their composition and functions. We present diverse ways that lipids and lipid modifications regulate capacitation and acrosome exocytosis, describing in more detail how removal of cholesterol plays a master regulatory role in enabling exocytosis through at least two complementary pathways. First, cholesterol efflux leads to proteolytic activation of phospholipase B, which cleaves both phospholipid tails. The resultant changes in membrane curvature provide a mechanism for the point fusions now known to occur far before a sperm physically interacts with the zona pellucida. Cholesterol efflux also enables G(M1) to regulate the voltage-dependent cation channel, Ca(V)2.3, triggering focal calcium transients required for acrosome exocytosis in response to subsequent whole-cell calcium rises. We close with a model integrating functions for lipids in regulating acrosome exocytosis.

Lipid Regulation of Acrosome Exocytosis.

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo.

Biomimicry Promotes the Efficiency of a 10-Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate.

Secretion of the acrosome, a single vesicle located rostrally in the head of a mammalian sperm, through a process known as "acrosome exocytosis" (AE), is essential for fertilization. However, the mechanisms leading to and regulating this complex process are controversial. In particular, poor understanding of Ca2+ dynamics between sperm subcellular compartments and regulation of membrane fusion mechanisms have led to competing models of AE. Here, we developed a transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE) to investigate the spatial and temporal Ca2+ dynamics in AE in live sperm. AcroSensE combines a genetically encoded Ca2+ indicator (GCaMP) fused with an mCherry indicator to spatiotemporally resolve acrosomal Ca2+ rise (ACR) and membrane fusion events, enabling real-time study of AE. We found that ACR is dependent on extracellular Ca2+ and that ACR precedes AE. In addition, we show that there are intermediate steps in ACR and that AE correlates better with the ACR rate rather than absolute Ca2+ amount. Finally, we demonstrate that ACR and membrane fusion progression kinetics and spatial patterns differ with different stimuli and that sites of initiation of ACR and sites of membrane fusion do not always correspond. These findings support a model involving functionally redundant pathways that enable a highly regulated, multistep AE in heterogeneous sperm populations, unlike the previously proposed "acrosome reaction" model.

A genetically targeted sensor reveals spatial and temporal dynamics of acrosomal calcium and sperm acrosome exocytosis.

The murine epididymis has 10 distinct segments which provide opportunity to identify compartmentalized cell physiological mechanisms underlying sperm maturation. However, despite the essential role of the epididymis in reproduction, remarkably little is known about segment-specific functions of this organ. Here, we investigate the dramatic segmental localization of the ganglioside GM1, a glycosphingolipid already known to play key roles in sperm capacitation and acrosome exocytosis (AE). Frozen tissue sections of epididymides from adult mice were treated with the binding subunit of cholera toxin conjugated to AlexaFluor 488 to label GM1. We report that GM1-enriched vesicles were found exclusively in principal and clear cells of segment 2. These vesicles were also restricted to the lumen of segment 2 and did not appear to flow with the sperm into segment 3, within the limits of detection by confocal microscopy. Interestingly, this segment-specific presence was altered in several azoospermic mouse models and in wildtype mice after efferent duct ligation (EDL). These findings indicate that a lumicrine factor, itself dependent on spermatogenesis, controls this segmental differentiation. RNA sequencing results confirmed global de-differentiation of the proximal epididymal segments in response to EDL. Additionally, GM1 localization on the surface of the sperm head increased as sperm transit through segment 2 and have contact with the GM1-enriched vesicles. This is the first report of segment-specific vesicles and their role in enriching sperm with GM1, a glycosphingolipid known to be critical for sperm function, providing key insights into the segment-specific physiology and function of the epididymis.

Segmental differentiation of the murine epididymis: identification of segment-specific, GM1-enriched vesicles and regulation by luminal fluid factors†.

Roy Cohen

Baker Institute for Animal Health,
College of Veterinary Medicine,
Department of Public and Ecosystem Health,
Baker Institute for Animal Health, College of Veterinary Medicine,
Department of Public and Ecosystem Health, College of Veterinary Medicine

Cornell University

Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm