methods-for-studying-mechanisms-action-antipsychotic-drugs

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in <em>Caenorhabditis elegans</em>

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

Harvard Medical School

McLean Hospital

The Caenorhabditis elegans genome encodes a series of hedgehog-related genes, which are thought to have evolved and diverged from an ancestral Hh gene. They are classified into several families based on their N-terminal domains. Here, we analyze the expression and function of a member of the warthog gene family, wrt-5, that lacks the Hint/Hog domain. wrt-5 is expressed in seam cells, the pharynx, pharyngeal-intestinal valve cells, neurons, neuronal support cells, the excretory cell, and the reproductive system. WRT-5 protein is secreted into the extracellular space during embryogenesis. Furthermore, during larval development, WRT-5 protein is secreted into the pharyngeal lumen and the pharyngeal expression changes in a cyclical manner in phase with the molting cycle. Deletion mutations in wrt-5 cause embryonic lethality, which are temperature sensitive and more severe at 15 degrees C than at 25 degrees C. Animals that hatch exhibit variable abnormal morphology, for example, bagging worms, blistering, molting defects, or Roller phenotypes. We examined hypodermal cell junctions using the AJM-1Colon, two colonsGFP marker in the wrt-5 mutant background and observed cell boundary abnormalities in the arrested embryos. AJM-1Colon, two colonsGFP protein is also misplaced in pharyngeal muscle cells in the absence of WRT-5. In conclusion, we show that wrt-5 is an essential gene that - despite its lack of a Hint domain - has multiple functions in C. elegans and is implicated in cell shape integrity.

The hedgehog-related gene wrt-5 is essential for hypodermal development in Caenorhabditis elegans.

The Caenorhabditis elegans genome encodes ten proteins that share similarity with Hedgehog through the C-terminal Hint/Hog domain. While most genes are members of larger gene families, qua-1 is a single copy gene. Here we show that orthologs of qua-1 exist in many nematodes, including Brugia malayi, which shared a common ancestor with C. elegans about 300 million years ago. The QUA-1 proteins contain an N-terminal domain, the Qua domain, that is highly conserved, but whose molecular function is not known. We have studied the expression pattern of qua-1 in C. elegans using a qua-1::GFP transcriptional fusion. qua-1 is mainly expressed in hyp1 to hyp11 hypodermal cells, but not in seam cells. It is also expressed in intestinal and rectal cells, sensilla support cells, and the P cell lineage in L1. The expression of qua-1::GFP undergoes cyclical changes during development in phase with the molting cycle. It accumulates prior to molting and disappears between molts. Disruption of the qua-1 gene function through an internal deletion that causes a frame shift with premature stop in the middle of the gene results in strong lethality. The animals arrest in the early larval stages due to defects in molting. Electron microscopy reveals double cuticles due to defective ecdysis, but no obvious defects are seen in the hypodermis. Qua domain-only::GFP and full-length QUA-1::GFP fusion constructs are secreted and associated with the overlying cuticle, but only QUA-1::GFP rescues the mutant phenotype. Our results suggest that both the Hint/Hog domain and Qua domain are critically required for the function of QUA-1.

The hedgehog-related gene qua-1 is required for molting in Caenorhabditis elegans.

The Caenorhabditis elegans genome encodes ten proteins that share sequence similarity with the Hedgehog signaling molecule through their C-terminal autoprocessing Hint/Hog domain. These proteins contain novel N-terminal domains, and C. elegans encodes dozens of additional proteins containing only these N-terminal domains. These gene families are called warthog, groundhog, ground-like and quahog, collectively called hedgehog (hh)-related genes. Previously, the expression pattern of seventeen genes was examined, which showed that they are primarily expressed in the ectoderm.

Comprehensive analysis of gene expression patterns of hedgehog-related genes.

Intraflagellar &#x8FD0;&#x8F93;&#xFF0C;&#x4E00;&#x76EE;&#x4E86;&#x7136;&#x3002;

Intraflagellar transport at a glance.

Transports intraflagellaire Regards sur l&#x26;#39;

Intraflagellar Transport auf einen Blick.

Trasporto dell&#39;omologo in un colpo d&#39;occhio.

&#x4E00;&#x76EE;&#x3067;&#x97AD;&#x6BDB;&#x5185;&#x8F38;&#x9001;

&#xD55C; &#xB208;&#xC5D0; intraflagellar &#xC804;&#xC1A1;&#xC785;&#xB2C8;&#xB2E4;.

Intraflagellar transporte de un vistazo

&#x7EA4;&#x6BDB;&#x8FDB;&#x884C;&#x7EC4;&#x88C5;&#x548C;&#x7EF4;&#x62A4;&#x7531; intraflagellar &#x4F20;&#x8F93; (&#x5B66;&#x9662;)&#xFF0C;&#x4F9D;&#x8D56;&#x7535;&#x673A;&#x7684; multiprotein &#x914D;&#x5408;&#x7269;&#xFF0C;&#x79F0;&#x4E3A; IFT &#x7C92;&#x5B50;&#xFF0C;&#x6CBF; axoneme &#x53CC;&#x5411;&#x8FD0;&#x52A8;&#x3002;&#x79C0;&#x4E3D;&#x9690;&#x6746;&#x7EBF;&#x866B;&#x7684;&#x611F;&#x5B98;&#x7EA4;&#x6BDB;&#x8868;&#x793A;&#x5B66;&#x4E60;&#x65C5;&#x6E38;&#x5B66;&#x9662;&#x65C5;&#x6E38;&#x5B66;&#x9662;&#x548C;&#x591A;&#x4E2A;&#x776B;&#x7A81;&#x53D8;&#x4F53;&#x4F53;&#x5185;&#x5EF6;&#x65F6;&#x8367;&#x5149;&#x663E;&#x5FAE;&#x955C;&#x68C0;&#x6D4B;&#x65B9;&#x6CD5;&#x7684;&#x53EF;&#x7528;&#x6027;&#x56E0;&#x975E;&#x5E38;&#x6709;&#x7528;&#x5BF9;&#x8C61;&#x3002;&#x5728;&#x6B64;&#x7CFB;&#x7EDF;&#x4E2D;&#x6709; 60 &#x7684;&#x611F;&#x89C9;&#x795E;&#x7ECF;&#x5143;&#xFF0C;&#x6BCF;&#x4E2A;&#x5BF9;&#x5176;&#x6811;&#x7A81;&#x7684;&#x7ED3;&#x5C40;&#x6709;&#x611F;&#x5B98;&#x7EA4;&#x6BDB;&#x548C;&#x65C5;&#x6E38;&#x5B66;&#x9662;&#x673A;&#x68B0;&#x64CD;&#x4F5C;&#x5728;&#x8FD9;&#x4E9B;&#x7ED3;&#x6784;&#x4E2D;&#x7684;&#x5927;&#x591A;&#x6570;&#x7EC4;&#x4EF6;&#x5DF2;&#x786E;&#x5B9A;&#x4F7F;&#x7528;&#x6B63;&#x5411;&#x548C;&#x53CD;&#x5411;&#x9057;&#x4F20;&#x65B9;&#x6CD5;&#x3002;&#x901A;&#x8FC7;&#x5206;&#x6790;&#x6CBF;&#x751F;&#x6D3B;&#x91CE;&#x751F;&#x578B;&#x53CA;&#x7A81;&#x53D8;&#x578B;&#x52A8;&#x7269;&#x3001; &#x4E24;&#x4E2A;&#x987A;&#x884C;&#x548C;&#x9006;&#x884C; IFT &#x7535;&#x673A;&#x88AB;&#x786E;&#x5B9A;&#x4E00;&#x4E2A;&#x8303;&#x56F4;&#x5185;&#x7684;&#x7EA4;&#x6BDB;&#x7684; IFT &#x7387;&#xFF0C;&#x5EFA;&#x7ACB;&#x4E86;&#x4E24;&#x4E2A;&#x987A;&#x884C;&#x9A71;&#x52A8;&#x86CB;&#x767D;-2 &#x7535;&#x673A;&#x529F;&#x80FD;&#x534F;&#x8C03;&#x548C;&#x8F93;&#x8FD0;&#x7279;&#x6027;&#x7684;&#x6240;&#x6709;&#x5DF2;&#x77E5;&#x7684; IFT &#x7C92;&#x5B50;&#x6210;&#x5206;&#x7684;&#x7279;&#x70B9;&#x662F;&#x3002;&#x8FD9;&#x4E24;&#x79CD;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x548C;&#x7EAF;&#x5316;&#xFF0C;&#x987A;&#x884C;&#x9A71;&#x52A8;&#x86CB;&#x767D;&#x7535;&#x673A;&#x548C;&#x7279;&#x70B9;&#x662F;&#x5176;&#x751F;&#x5316;&#x7279;&#x6027;&#xFF0C;&#x4F7F;&#x7528; MT &#x6ED1;&#x7FD4;&#x548C;&#x5355;&#x5206;&#x5B50;&#x52A8;&#x529B;&#x68C0;&#x5B9A;&#x6CD5;&#x3002;&#x5728;&#x672C;&#x7AE0;&#x4E2D;&#xFF0C;&#x6211;&#x4EEC;&#x53EF;&#x4EE5;&#x603B;&#x7ED3;&#x5982;&#x4F55;&#x7684;&#x9057;&#x4F20;&#x5B66;&#x3001; &#x7EC6;&#x80DE;&#x751F;&#x7269;&#x5B66;&#x3001; &#x7535;&#x5B50;&#x663E;&#x5FAE;&#x955C;&#x548C;&#x751F;&#x7269;&#x5316;&#x5B66;&#x7684;&#x5DE5;&#x5177;&#x7528;&#x4E8E;&#x5256;&#x6790;&#x7EC4;&#x6210;&#x548C;&#x7535;&#x673A;&#x5B66;&#x9662;&#x7684;&#x4F5C;&#x7528;&#x673A;&#x7406;&#x548C;&#x7EBF;&#x866B;&#x4E2D;&#x7684;&#x5B66;&#x9662;&#x7C92;&#x5B50;&#x3002;

Intraflagellar &#x8FD0;&#x8F93;&#x4E2D;&#x7EBF;&#x866B;&#x611F;&#x5B98;&#x7EA4;&#x6BDB;&#x7684;&#x5206;&#x6790;&#x3002;

Cilia are assembled and maintained by intraflagellar transport (IFT), the motor-dependent, bidirectional movement of multiprotein complexes, called IFT particles, along the axoneme. The sensory cilia of Caenorhabditis elegans represent very useful objects for studying IFT because of the availability of in vivo time-lapse fluorescence microscopy assays of IFT and multiple ciliary mutants. In this system there are 60 sensory neurons, each having sensory cilia on the endings of their dendrites, and most components of the IFT machinery operating in these structures have been identified using forward and reverse genetic approaches. By analyzing the rate of IFT along cilia within living wild-type and mutant animals, two anterograde and one retrograde IFT motors were identified, the functional coordination of the two anterograde kinesin-2 motors was established and the transport properties of all the known IFT particle components have been characterized. The anterograde kinesin motors have been heterologously expressed and purified, and their biochemical properties have been characterized using MT gliding and single molecule motility assays. In this chapter, we summarize how the tools of genetics, cell biology, electron microscopy, and biochemistry are being used to dissect the composition and mechanism of action of IFT motors and IFT particles in C. elegans.

Analysis of intraflagellar transport in C. elegans sensory cilia.

Cilia sont assembl&#xE9;s et entretenus par les transports en intraflagellaire (IFT), le moteur-d&#xE9;pendante, le mouvement bidirectionnel de complexes multiprot&#xE9;iques, appel&#xE9;es particules IFT, le long de l&#x26;#39;axon&#xE8;me. Le cils sensoriels de Caenorhabditis elegans repr&#xE9;senter des objets tr&#xE8;s utiles pour l&#x26;#39;&#xE9;tude de l&#x26;#39;IFT en raison de la disponibilit&#xE9; des in vivo time-lapse essais de microscopie par fluorescence de l&#x26;#39;IFT et de multiples mutants ciliaires. Dans ce syst&#xE8;me, il ya 60 neurones sensoriels, chacun ayant des cils sensoriels sur les terminaisons de leurs dendrites, et la plupart des composants de l&#x26;#39;exploitation des machines IFT dans ces structures ont &#xE9;t&#xE9; identifi&#xE9;es &#xE0; l&#x26;#39;aide avant et arri&#xE8;re des approches g&#xE9;n&#xE9;tiques. En analysant le taux de l&#x26;#39;IFT le long des cils dans les animaux vivants de type sauvage et mutant, deux ant&#xE9;rograde et avec une orbite r&#xE9;trograde moteurs IFT ont &#xE9;t&#xE9; identifi&#xE9;s, la coordination fonctionnelle des deux ant&#xE9;rogrades kin&#xE9;sine-2 moteurs a &#xE9;t&#xE9; &#xE9;tabli et les propri&#xE9;t&#xE9;s de transport de tous les particules IFT connu composants ont &#xE9;t&#xE9; caract&#xE9;ris&#xE9;s. Les moteurs ant&#xE9;rograde kin&#xE9;sine ont &#xE9;t&#xE9; exprim&#xE9;e de mani&#xE8;re h&#xE9;t&#xE9;rologue et purifi&#xE9;es, et leurs propri&#xE9;t&#xE9;s biochimiques ont &#xE9;t&#xE9; caract&#xE9;ris&#xE9;s &#xE0; l&#x26;#39;aide MT vol &#xE0; voile et simples tests de motilit&#xE9; mol&#xE9;cule. Dans ce chapitre, nous r&#xE9;sumons la fa&#xE7;on dont les outils de la g&#xE9;n&#xE9;tique, la biologie cellulaire, la microscopie &#xE9;lectronique, et de la biochimie sont utilis&#xE9;s pour diss&#xE9;quer la composition et le m&#xE9;canisme d&#x26;#39;action des moteurs et des particules IFT IFT chez C. elegans.

Analyse du transport intraflagellaire en C. Elegans cils sensoriels

Zilien sind montiert und betreut von intraflagellar Transport (IFT), der Motor-abh&auml;ngigen, bidirektionale Bewegung der multiprotein komplexe, genannt IFT Partikel, entlang dem Axonem. Die sensorischen Zilien Caenorhabditis elegans repr&auml;sentieren sehr n&uuml;tzliche Objekte f&uuml;r IFT wegen der Verf&uuml;gbarkeit von in-vivo Zeitraffer Mikroskopie Fluoreszenzassays IFT und mehrere Zilien Mutanten zu studieren. In diesem System gibt es 60 sensorische Neuronen, jeweils sensorische Zilien auf die Endungen ihrer Dendriten, und die meisten Komponenten der IFT-Maschinerie Betrieb in diesen Strukturen mit vorw&auml;rts- und genetische Ans&auml;tze identifiziert wurden. Durch die Analyse der Rate von IFT entlang Zilien innerhalb lebender Wildtyp und Mutanten Tiere, zwei Anterograde und eine retrograde IFT Motoren identifiziert wurden, wurde die funktionelle Koordination der zwei Anterograde Kinesin-2 Motoren und die Transporteigenschaften aller bekannten IFT Partikel Komponenten gepr&auml;gt haben. Die Anterograde Kinesin Motoren heterologously ausgedr&uuml;ckt und gereinigt wurden und ihre biochemischen Eigenschaften haben mit MT gleiten und Einzelmolek&uuml;l-Motilit&auml;t-Assays gekennzeichnet worden. In diesem Kapitel fassen wir wie die Werkzeuge der Genetik, Zellbiologie, Elektronenmikroskopie und Biochemie werden genutzt, um die Zusammensetzung zu sezieren und Wirkmechanismus von IFT-Motoren und IFT Teilchen in C. Elegans.

Analyse intraflagellar Transport in C. Elegans sensorische Zilien.

Ciglia &egrave; assemblati e mantenuto da trasporto dell&#39;omologo (IFT), il motore-dipendente, movimento bidirezionale di complessi multiproteici, chiamato particelle IFT, lungo l&#39;Assonema. Le ciglia sensoriali dei elegans di Caenorhabditis rappresentano oggetti molto utili per studiare IFT a causa della disponibilit&agrave; di dosaggi microscopia fluorescenza time-lapse in vivo di IFT e pi&ugrave; mutanti ciliare. In questo sistema ci sono 60 neuroni sensoriali, ciascuno con ciglia sensoriali sulle terminazioni dei loro dendriti, e la maggior parte dei componenti delle macchine IFT operano in queste strutture sono stati identificati utilizzando approcci genetici avanti e indietro. Analizzando il tasso di IFT lungo ciglia all&#39;interno di animali viventi wild-type e mutanti, due anterograda e uno retrogradi IFT motori sono stati identificati, &egrave; stato istituito il coordinamento funzionale dei due anterograda chinesina-2 motori e le propriet&agrave; di trasporto di tutti i noti componenti della particella IFT sono state caratterizzate. I motori chinesina anterograda sono stati heterologously espressi e purificati, e loro propriet&agrave; biochimiche sono state caratterizzate mediante scorrimento MT e dosaggi di motilit&agrave; di singola molecola. In questo capitolo, riassumiamo come vengono utilizzati gli strumenti di genetica, biologia cellulare, microscopia elettronica e biochimica per sezionare la composizione e meccanismo d&#39;azione di IFT motori e IFT particelle in c. elegans.

Analisi del trasporto dell&#39;omologo in c. elegans ciglia sensoriali.

&#x7E4A;&#x6BDB;&#x306F;&#x3001;&#x8EF8;&#x7CF8;&#x306B;&#x6CBF;&#x3063;&#x3066;&#x97AD;&#x6BDB;&#x5185;&#x8F38;&#x9001;&#xFF08;IFT&#xFF09;&#x3001;IFT&#x7C92;&#x5B50;&#x3068;&#x547C;&#x3070;&#x308C;&#x308B;&#x591A;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x8907;&#x5408;&#x4F53;&#x306E;&#x904B;&#x52D5;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3001;&#x53CC;&#x65B9;&#x5411;&#x306E;&#x52D5;&#x304D;&#x3001;&#x306B;&#x3088;&#x3063;&#x3066;&#x7D44;&#x307F;&#x7ACB;&#x3066;&#x3001;&#x7DAD;&#x6301;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002; Caenorhabditis elegans&#x306E;&#x611F;&#x899A;&#x7E4A;&#x6BDB;&#x305F;&#x3081;IFT&#x3001;&#x8907;&#x6570;&#x306E;&#x7E4A;&#x6BDB;&#x306E;&#x5909;&#x7570;&#x4F53;&#x306E;in vivo&#x3067;&#x306E;&#x30BF;&#x30A4;&#x30E0;&#x30E9;&#x30D7;&#x30B9;&#x86CD;&#x5149;&#x9855;&#x5FAE;&#x93E1;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x6709;&#x7528;&#x6027;&#x306E;IFT&#x3092;&#x7814;&#x7A76;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x975E;&#x5E38;&#x306B;&#x6709;&#x7528;&#x306A;&#x30AA;&#x30D6;&#x30B8;&#x30A7;&#x30AF;&#x30C8;&#x3092;&#x8868;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3067;&#x4ECA;&#x5F8C;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x907A;&#x4F1D;&#x7684;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3092;&#x9006;&#x306B;&#x7279;&#x5B9A;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3053;&#x308C;&#x3089;&#x306E;&#x69CB;&#x9020;&#x4F53;&#x306E;60&#x611F;&#x899A;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306F;&#x3001;&#x305D;&#x306E;&#x6A39;&#x72B6;&#x7A81;&#x8D77;&#x306E;&#x7D42;&#x672B;&#x306B;&#x305D;&#x308C;&#x305E;&#x308C;&#x304C;&#x611F;&#x899A;&#x7E4A;&#x6BDB;&#x3001;&#x304A;&#x3088;&#x3073;IFT&#x6A5F;&#x68B0;&#x306E;&#x52D5;&#x4F5C;&#x306E;&#x307B;&#x3068;&#x3093;&#x3069;&#x306E;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x91CE;&#x751F;&#x578B;&#x304A;&#x3088;&#x3073;&#x5909;&#x7570;&#x52D5;&#x7269;&#x306E;&#x751F;&#x6D3B;&#x306E;&#x4E2D;&#x3067;&#x7E4A;&#x6BDB;&#x306B;&#x6CBF;&#x3063;&#x3066;IFT&#x306E;&#x5272;&#x5408;&#x3092;&#x5206;&#x6790;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x4E8C;&#x3064;&#x306E;&#x9806;&#x884C;&#x4E00;&#x9006;&#x884C;IFT&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x304C;&#x540C;&#x5B9A;&#x3055;&#x308C;&#x305F;&#x3001;&#x4E8C;&#x3064;&#x306E;&#x9806;&#x884C;&#x6027;&#x30AD;&#x30CD;&#x30B7;&#x30F3;-2&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x306E;&#x6A5F;&#x80FD;&#x306E;&#x9023;&#x643A;&#x3092;&#x78BA;&#x7ACB;&#x3057;&#x3001;&#x3059;&#x3079;&#x3066;&#x306E;&#x65E2;&#x77E5;&#x306E;IFT&#x7C92;&#x5B50;&#x306E;&#x8F38;&#x9001;&#x7279;&#x6027;&#x307E;&#x3057;&#x305F;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x304C;&#x7279;&#x5FB4;&#x3065;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x3002;&#x9806;&#x884C;&#x6027;&#x30AD;&#x30CD;&#x30B7;&#x30F3;&#x30E2;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x7570;&#x7A2E;&#x767A;&#x73FE;&#x3001;&#x7CBE;&#x88FD;&#x3057;&#x3001;&#x305D;&#x306E;&#x751F;&#x5316;&#x5B66;&#x7684;&#x7279;&#x6027;&#x306F;MT&#x30B0;&#x30E9;&#x30A4;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x3068;&#x5358;&#x4E00;&#x5206;&#x5B50;&#x306E;&#x904B;&#x52D5;&#x6027;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x3092;&#x7528;&#x3044;&#x3066;&#x7279;&#x5FB4;&#x3065;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x7AE0;&#x3067;&#x306F;&#x3001;&#x907A;&#x4F1D;&#x5B66;&#x3001;&#x7D30;&#x80DE;&#x751F;&#x7269;&#x5B66;&#x3001;&#x96FB;&#x5B50;&#x9855;&#x5FAE;&#x93E1;&#x3001;&#x751F;&#x5316;&#x5B66;&#x306E;&#x30C4;&#x30FC;&#x30EB;&#x306F;&#x3001;&#x7D44;&#x6210;&#x3068;IFT&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x3068;C. elegans&#x306E;IFT&#x7C92;&#x5B50;&#x306E;&#x4F5C;&#x7528;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x5206;&#x6790;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x65B9;&#x6CD5;&#x3092;&#x307E;&#x3068;&#x3081;&#x3002;

C. elegans&#x306E;&#x611F;&#x899A;&#x7E4A;&#x6BDB;&#x306E;&#x97AD;&#x6BDB;&#x5185;&#x8F38;&#x9001;&#x306E;&#x89E3;&#x6790;

&#xC18D;&#xB208;&#xC379; &#xC870;&#xB9BD; &#xBC0F; intraflagellar &#xC804;&#xC1A1; (IFT) &#xBAA8;&#xD130; &#xC885;&#xC18D;&#xC744; axoneme &#xB530;&#xB77C; IFT &#xC785;&#xC790; &#xB77C;&#xB294; multiprotein &#xB2E8;&#xC9C0;&#xC758; &#xC591;&#xBC29;&#xD5A5; &#xC6C0;&#xC9C1;&#xC784;&#xC5D0; &#xC758;&#xD574; &#xC720;&#xC9C0; &#xB429;&#xB2C8;&#xB2E4;. Caenorhabditis elegans&#xC758; &#xAC10;&#xAC01; &#xC18D;&#xB208;&#xC379; IFT IFT &#xC5EC;&#xB7EC; &#xC18D;&#xB208;&#xC379; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC758; in vivo &#xC2DC;&#xAC04; &#xACBD;&#xACFC; &#xD615;&#xAD11; &#xD604;&#xBBF8;&#xACBD; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xAC00;&#xC6A9;&#xC131; &#xB54C;&#xBB38;&#xC5D0; &#xACF5;&#xBD80;&#xC5D0; &#xB300; &#xD55C; &#xB9E4;&#xC6B0; &#xC720;&#xC6A9;&#xD55C; &#xAC1C;&#xCCB4;&#xB97C; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. &#xC774; &#xC2DC;&#xC2A4;&#xD15C;&#xC5D0;&#xB294; &#xAC01;&#xAC01; &#xADF8;&#xB4E4;&#xC758; dendrites&#xC758; &#xC5D4;&#xB529;&#xC5D0; &#xAC10;&#xAC01; &#xC18D;&#xB208;&#xC379; &#xAC00;&#xC9C0; 60 &#xAC10;&#xAC01; &#xC2E0;&#xACBD; &#xC138;&#xD3EC; &#xBC0F; &#xC815;&#xBC29;&#xD5A5; &#xBC0F; &#xC5ED;&#xBC29;&#xD5A5; &#xC720;&#xC804;&#xC801; &#xBC29;&#xBC95;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC774;&#xB7EC;&#xD55C; &#xAD6C;&#xC870;&#xC5D0;&#xC11C; IFT &#xAE30;&#xACC4;&#xC758; &#xB300;&#xBD80;&#xBD84;&#xC758; &#xAD6C;&#xC131; &#xC694;&#xC18C; &#xD655;&#xC778; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC0B4;&#xC544;&#xC788;&#xB294; &#xC57C;&#xC0DD; &#xD615; &#xBC0F; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xB3D9;&#xBB3C;, &#xB450; &#xCC38;&#xAC00;&#xC790; &#xBC0F; &#xD1F4;&#xD589; &#xC131; IFT &#xBAA8;&#xD130; &#xD655;&#xC778; &#xB418;&#xC5C8;&#xB2E4; &#xD558;&#xB098; &#xB0B4; &#xC18D;&#xB208;&#xC379;&#xC744; &#xB530;&#xB77C; IFT&#xC758; &#xC18D;&#xB3C4; &#xBD84;&#xC11D; &#xD558; &#xC5EC; &#xB450; &#xCC38;&#xAC00;&#xC790; kinesin 2 &#xBAA8;&#xD130;&#xC758; &#xAE30;&#xB2A5; &#xC870;&#xC815; &#xC124;&#xB9BD; &#xD558; &#xACE0; &#xBAA8;&#xB4E0; &#xC54C;&#xB824;&#xC9C4; IFT &#xC785;&#xC790; &#xAD6C;&#xC131; &#xC694;&#xC18C; &#xC804;&#xC1A1; &#xC18D;&#xC131; &#xD2B9;&#xC9D5; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xCC38;&#xAC00;&#xC790; kinesin &#xBAA8;&#xD130; heterologously &#xD45C;&#xD604; &#xB418;&#xC5C8;&#xACE0; &#xC815;&#xD654;, &#xADF8;&#xB9AC;&#xACE0; &#xADF8;&#xB4E4;&#xC758; &#xC0DD; &#xD654; &#xD559;&#xC801; &#xC18D;&#xC131; &#xC0B0; &#xD65C;&#xACF5; &#xD558; &#xACE0; &#xB2E8;&#xC77C; &#xBD84;&#xC790;&#xC758; &#xC6B4;&#xB3D9; &#xC131; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xD2B9;&#xC9D5; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xC7A5;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC;&#xB294; &#xC720;&#xC804;&#xD559;, &#xC138;&#xD3EC; &#xC0DD;&#xBB3C;&#xD559;, &#xC804;&#xC790; &#xD604;&#xBBF8;&#xACBD; &#xAC80;&#xC0AC; &#xBC95;, &#xADF8;&#xB9AC;&#xACE0; &#xC0DD;&#xD654;&#xD559;&#xC758; &#xB3C4;&#xAD6C; &#xAD6C;&#xC131; &#xD574; &#xBD80;&#xB97C; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xBC29;&#xBC95;&#xACFC; IFT&#xC758; &#xD589;&#xB3D9;&#xC758; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xBAA8;&#xD130;&#xC640; C. elegans&#xC758; IFT &#xC785;&#xC790; &#xC694;&#xC57D;.

C. elegans &#xAC10;&#xAC01; &#xC18D;&#xB208;&#xC379; intraflagellar &#xC804;&#xC1A1;&#xC758; &#xBD84;&#xC11D;.

Los cilios son ensamblados y mantenido por intraflagellar transporte (IFT), el motor-dependiente, el movimiento bidireccional de complejos multiproteicos, llamadas part&#xED;culas TIF, a lo largo del axonema. Los cilios sensoriales de Caenorhabditis elegans representan objetos muy &#xFA;tiles para el estudio de IFT, debido a la disponibilidad de in vivo lapso de tiempo, los ensayos de microscop&#xED;a de fluorescencia de IFT y varios mutantes ciliares. En este sistema hay 60 neuronas sensoriales, cada uno de los cilios sensoriales que tiene sobre las terminaciones de sus dendritas, y la mayor&#xED;a de los componentes de la operaci&#xF3;n de maquinaria IFT en estas estructuras se han identificado con marcha adelante y atr&#xE1;s los enfoques gen&#xE9;ticos. Al analizar la tasa de IFT junto a los cilios que viven los animales de tipo salvaje y mutante, dos anter&#xF3;grada y retr&#xF3;grada motores un IFT se identificaron, la coordinaci&#xF3;n funcional de las dos anter&#xF3;grada kinesin-2 motores se estableci&#xF3; y las propiedades de transporte de todas las part&#xED;culas IFT conocido componentes se han caracterizado. El anter&#xF3;grada motores quinesina han heter&#xF3;loga expresada y se purific&#xF3;, y sus propiedades bioqu&#xED;micas han sido caracterizados mediante MT delta y simples ensayos de la motilidad mol&#xE9;cula. En este cap&#xED;tulo, resumimos c&#xF3;mo las herramientas de la gen&#xE9;tica, biolog&#xED;a celular, microscop&#xED;a electr&#xF3;nica, y la bioqu&#xED;mica se est&#xE1;n utilizando para diseccionar la composici&#xF3;n y mecanismo de acci&#xF3;n de los motores y las part&#xED;culas IFT IFT en C. elegans.

An&#xE1;lisis de los Transportes intraflagellar en C. elegans cilios sensoriales

&#x611F;&#x5B98;&#x7EA4;&#x6BDB;&#x8FDB;&#x884C;&#x7EC4;&#x88C5;&#x548C;&#x7EF4;&#x62A4;&#x6240;&#x4F9D;&#x8D56;&#x7684;&#x9A71;&#x52A8;&#x86CB;&#x767D;-2 intraflagellar &#x8FD0;&#x8F93; &#xFF08;&#x65C5;&#x6E38;&#x5B66;&#x9662;&#xFF09;&#x3002;&#x6211;&#x4EEC;&#x8C03;&#x67E5;&#x662F;&#x5426;&#x4E24;&#x4E2A;&#x79C0;&#x4E3D;&#x9690;&#x6746;&#x7EBF;&#x866B;&#x5FAE;&#x7BA1; &alpha;-&#x548C; &beta;-&#x86CB;&#x767D; isotypes&#xFF0C;&#x7F3A;&#x4E4F;&#x5B83;&#x4EEC;&#x7EA4;&#x6BDB;&#x7684;&#x8FDC;&#x7AEF;&#x6BB5;&#x7684;&#x7A81;&#x53D8;&#x4F53;&#x901A;&#x8FC7;&#x786E;&#x5B9A;&#x4F20;&#x9012;&#x7ED9;&#x4ED6;&#x4EEC;&#x7684;&#x5927;&#x4F1A;&#x7F51;&#x7AD9;&#x7531;&#x65C5;&#x6E38;&#x5B66;&#x9662;&#x3002;&#x5B88;&#x6052;&#x6B8B;&#x7559;&#x5728;&#x8FD9;&#x4E24;&#x4E2A; tubulins &#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x7834;&#x574F;&#x7A33;&#x5B9A;&#x5FAE;&#x7BA1;&#x8FDC;&#x7AEF;&#x5355;&#x7EBF;&#x6001;&#x3002;&#x4E00;&#x4E2A;&#x540C;&#x79CD;&#xFF0C;TBB-4&#xFF0C;&#x5C06;&#x6C47;&#x7F16;&#x6210;&#x5FAE;&#x7BA1;&#x7684; axoneme &#x6838;&#x5FC3;&#x548C;&#x8FDC;&#x7AEF;&#x90E8;&#x5206;&#x7684;&#x63D0;&#x793A;&#x5728;&#x90A3;&#x91CC;&#x5FAE;&#x7BA1;&#x63D0;&#x793A;&#x8DDF;&#x8E2A; EB1 &#x627E;&#x5230;&#xFF0C;&#x5219;&#x548C;&#x672C;&#x5730;&#x5316;&#x6240;&#x6709;&#x6CBF;&#x7EA4;&#x6BDB;&#xFF0C;&#x800C;&#x5176;&#x4ED6;&#xFF0C;TBA-5&#xFF0C;&#x96C6;&#x4E2D;&#x5728;&#x8FDC;&#x7AEF; singlets&#x3002;IFT &#x68C0;&#x6D4B;&#x3001; &#x8367;&#x5149;&#x6062;&#x590D;&#x540E;&#x6F02;&#x767D;&#x5206;&#x6790;&#x548C;&#x5EFA;&#x6A21;&#x8868;&#x660E;&#x8FD9;&#x4E9B;&#x5FAE;&#x7BA1;&#x86CB;&#x767D;&#x4E9A;&#x5355;&#x4F4D;&#x7531;&#x5B66;&#x9662;&#x673A;&#x68B0; sub-stoichiometric &#x6570;&#x5B57;&#x8FDE;&#x7EED;&#x8FD0;&#x8F93;&#x53EF;&#x4EE5;&#x4FDD;&#x6301;&#x611F;&#x5B98;&#x7EA4;&#x6BDB;&#x5728;&#x5176;&#x7A33;&#x6001;&#x7684;&#x957F;&#x5EA6;&#x3002;

Intraflagellar &#x8FD0;&#x8F93;&#x63D0;&#x4F9B;&#x611F;&#x5B98;&#x7EA4;&#x6BDB;&#x4E2D;&#x95F4;&#x548C;&#x8FDC;&#x7AEF;&#x90E8;&#x5206;&#x5FAE;&#x7BA1;&#x86CB;&#x767D; isotypes&#x3002;

Sensory cilia are assembled and maintained by kinesin-2-dependent intraflagellar transport (IFT). We investigated whether two Caenorhabditis elegans Î±- and Î²-tubulin isotypes, identified through mutants that lack their cilium distal segments, are delivered to their assembly sites by IFT. Mutations in conserved residues in both tubulins destabilize distal singlet microtubules. One isotype, TBB-4, assembles into microtubules at the tips of the axoneme core and distal segments, where the microtubule tip tracker EB1 is found, and localizes all along the cilium, whereas the other, TBA-5, concentrates in distal singlets. IFT assays, fluorescence recovery after photobleaching analysis and modelling indicate that the continual transport of sub-stoichiometric numbers of these tubulin subunits by the IFT machinery can maintain sensory cilia at their steady-state length.

Intraflagellar transport delivers tubulin isotypes to sensory cilium middle and distal segments.

Les cils sensoriels sont assembl&#xE9;s et entretenus par la kin&#xE9;sine-2-d&#xE9;pendante transport intraflagellaire (IFT). Nous avons &#xE9;tudi&#xE9; si deux Caenorhabditis elegans a-et &#x3B2;-tubuline isotypes, identifi&#xE9;s par des mutants qui n&#x26;#39;ont pas leurs cil segments distaux, sont livr&#xE9;s &#xE0; leurs sites d&#x26;#39;assemblage par IFT. Des mutations dans les r&#xE9;sidus conserv&#xE9;s dans les deux tubulines d&#xE9;stabiliser les microtubules singulet distales. Un isotype, TBB-4, assemble en microtubules dans les conseils de l&#x26;#39;&#xE2;me axon&#xE8;me et segments distaux, o&#xF9; la pointe de microtubules traqueur EB1 est trouv&#xE9;, et localise tout au long du cil, tandis que l&#x26;#39;autre, TBA-5, se concentre dans les maillots distales. Dosages IFT, la r&#xE9;cup&#xE9;ration de fluorescence apr&#xE8;s photoblanchiment analyse et la mod&#xE9;lisation indiquent que le transport continuel de sous-stoechiom&#xE9;triques nombre de ces sous-unit&#xE9;s de tubuline par la machine IFT peut maintenir les cils sensoriels &#xE0; leur &#xE9;tat d&#x26;#39;&#xE9;quilibre longueur.

Transports intraflagellaire Rend isotypes de tubuline au Moyen-cil sensorielle et segments distaux

Sensorische Zilien sind montiert und betreut von Kinesin-2-abh&auml;ngige intraflagellar Transport (IFT). Wir untersuchten, ob zwei Caenorhabditis Elegans &alpha;- und &beta; Isotypen, identifiziert durch Mutanten, die ihre Zilie distalen Segmente, Fehlen von IFT an ihre Baugruppe Websites &uuml;bermittelt werden. Mutationen im konservierten R&uuml;ckst&auml;nden in beiden Tubulins destabilisieren distale Singulett Mikrotubuli. Ein Isotype, TBB-4, assembliert in Mikrotubuli an den Spitzen des Axonem Kern und der distalen Segmente, wo der Microtubule-Tipp-Tracker ES1 gefunden wird, und entlang der Zilie lokalisiert, w&auml;hrend andererseits TBA-5, konzentriert, im distalen Einzellinsen. IFT-assays, Fluoreszenz-Wiederherstellung nach Photobleaching Analyse und Modellierung anzugeben, dass der kontinuierliche Transport von sub-stoichiometric zahlen diese Tubulin-Untereinheiten der IFT-Maschine sensorische Zilien bei ihren station&auml;ren L&auml;nge beibehalten kann.

Intraflagellar Transport liefert Tubulin-Isotypen sensorische Zilie mittleren und distalen Segmente.

Ciglia sensoriali &egrave; assemblati e mantenuto da trasporto dell&#39;omologo chinesina-2-dipendente (IFT). Abbiamo studiato se due Caenorhabditis elegans &alpha; - e &beta;-tubulina isotipi, identificati attraverso mutanti che la mancanza dei segmenti distali cilio, vengono consegnati ai loro siti di assemblaggio da IFT. Mutazioni in residui conservati entrambi tubulins destabilizzano microtubuli singoletto distale. Un isotipo, TBB-4, assembla in microtubuli alle punte del nucleo assonema e segmenti distali, dove il tracker di punta del microtubule EB1 &egrave; trovato e si localizza lungo le ciglia, mentre l&#39;altra, TBA-5, concentra in Camiciole distale. IFT assays, recupero di fluorescenza dopo photobleaching analisi e modellizzazione indicano che il continuo trasporto di sub-stoichiometric numeri di queste subunit&agrave; di tubulina della macchina IFT pu&ograve; mantenere ciglia sensoriali a loro lunghezza steady-state.

Trasporto dell&#39;omologo batte isotipi di tubulina a segmenti medio e distale sensoriali cilio.

&#x611F;&#x899A;&#x7E4A;&#x6BDB;&#x306F;&#x3001;&#x30AD;&#x30CD;&#x30B7;&#x30F3;-2&#x4F9D;&#x5B58;&#x6027;&#x97AD;&#x6BDB;&#x5185;&#x8F38;&#x9001;&#xFF08;IFT&#xFF09;&#x306B;&#x3088;&#x3063;&#x3066;&#x7D44;&#x307F;&#x7ACB;&#x3066;&#x3089;&#x308C;&#x3001;&#x7DAD;&#x6301;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x79C1;&#x305F;&#x3061;&#x306F;&#x5F7C;&#x3089;&#x306E;&#x7E4A;&#x6BDB;&#x9060;&#x4F4D;&#x306E;&#x30BB;&#x30B0;&#x30E1;&#x30F3;&#x30C8;&#x3092;&#x6B20;&#x304F;&#x5909;&#x7570;&#x4F53;&#x306B;&#x3088;&#x3063;&#x3066;&#x8B58;&#x5225;&#x3055;&#x308C;&#x308B;2&#x3064;&#x306E;&#x7DDA;&#x866B;&#xFF08;Caenorhabditis elegans&#xFF09;&#x3B1;-&#x304A;&#x3088;&#x3073;&#x3B2;-&#x30C1;&#x30E5;&#x30FC;&#x30D6;&#x30EA;&#x30F3;&#x30A2;&#x30A4;&#x30BD;&#x30BF;&#x30A4;&#x30D7;&#x306F;&#x3001;IFT&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x306E;&#x30B5;&#x30A4;&#x30C8;&#x306B;&#x914D;&#x4FE1;&#x3055;&#x308C;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x3092;&#x6307;&#x5B9A;&#x3057;&#x307E;&#x3059;&#x3002;&#x8ABF;&#x67FB;&#x4E21;&#x65B9;&#x306E;&#x30C1;&#x30E5;&#x30FC;&#x30D6;&#x30EA;&#x30F3;&#x306B;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x305F;&#x6B8B;&#x57FA;&#x306E;&#x5909;&#x7570;&#x306F;&#x3001;&#x9060;&#x4F4D;&#x30B7;&#x30F3;&#x30B0;&#x30EC;&#x30C3;&#x30C8;&#x5FAE;&#x5C0F;&#x7BA1;&#x3092;&#x4E0D;&#x5B89;&#x5B9A;&#x306B;&#x3059;&#x308B;&#x3002; One&#x30A2;&#x30A4;&#x30BD;&#x30BF;&#x30A4;&#x30D7;&#x3001;TBB-4&#x306F;&#x3001;&#x8EF8;&#x7CF8;&#x30B3;&#x30A2;&#x3068;&#x5FAE;&#x5C0F;&#x7BA1;&#x306E;&#x5148;&#x7AEF;&#x30C8;&#x30E9;&#x30C3;&#x30AB;&#x30FC;EB1&#x304C;&#x767A;&#x898B;&#x3055;&#x308C;&#x305F;&#x9060;&#x4F4D;&#x306E;&#x30BB;&#x30B0;&#x30E1;&#x30F3;&#x30C8;&#x306E;&#x5148;&#x7AEF;&#x306B;&#x5FAE;&#x5C0F;&#x7BA1;&#x306B;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EB;&#x3057;&#x3001;&#x3059;&#x3079;&#x3066;&#x306E;&#x7E4A;&#x6BDB;&#x306B;&#x6CBF;&#x3063;&#x3066;&#x5C40;&#x5728;&#x3057;&#x3001;&#x4ED6;&#x306E;&#x3001;TBA-5&#x306E;&#x306B;&#x5BFE;&#x3057;&#x3001;&#x9060;&#x30B7;&#x30F3;&#x30B0;&#x30EC;&#x30C3;&#x30C8;&#x306B;&#x96C6;&#x4E2D;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002; IFT&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306F;&#x3001;&#x5206;&#x6790;&#x3068;&#x30E2;&#x30C7;&#x30EA;&#x30F3;&#x30B0;&#x3092;&#x9000;&#x8272;&#x5F8C;&#x86CD;&#x5149;&#x56DE;&#x5FA9;&#x306F;IFT&#x6A5F;&#x68B0;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C1;&#x30E5;&#x30FC;&#x30D6;&#x30EA;&#x30F3;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E;&#x30B5;&#x30D6;&#x5316;&#x5B66;&#x91CF;&#x8AD6;&#x6570;&#x306E;&#x7D99;&#x7D9A;&#x7684;&#x306A;&#x8F38;&#x9001;&#x304C;&#x305D;&#x306E;&#x5B9A;&#x5E38;&#x72B6;&#x614B;&#x306E;&#x9577;&#x3055;&#x3067;&#x611F;&#x899A;&#x7E4A;&#x6BDB;&#x3092;&#x7DAD;&#x6301;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x97AD;&#x6BDB;&#x5185;&#x8F38;&#x9001;&#x306F;&#x3001;&#x611F;&#x899A;&#x7E4A;&#x6BDB;&#x306E;&#x4E2D;&#x6771;&#x304A;&#x3088;&#x3073;&#x9060;&#x4F4D;&#x306E;&#x30BB;&#x30B0;&#x30E1;&#x30F3;&#x30C8;&#x306B;&#x30C1;&#x30E5;&#x30FC;&#x30D6;&#x30EA;&#x30F3;&#x30A2;&#x30A4;&#x30BD;&#x30BF;&#x30A4;&#x30D7;&#x3092;&#x63D0;&#x4F9B;

&#xAC10;&#xAC01; &#xC18D;&#xB208;&#xC379; &#xC870;&#xB9BD; &#xD558; &#xACE0; kinesin 2 &#xC885;&#xC18D; intraflagellar &#xC804;&#xC1A1; (IFT)&#xC5D0; &#xC758;&#xD574; &#xC720;&#xC9C0; &#xB429;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; IFT &#xC5B4;&#xC148;&#xBE14;&#xB9AC; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0; &#xC790;&#xC2E0;&#xC758; cilium &#xC6D0;&#xC2EC; &#xC138;&#xADF8;&#xBA3C;&#xD2B8; &#xBD80;&#xC871; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xD1B5;&#xD574; &#xC2DD;&#xBCC4; &#xB41C; &#xB450; Caenorhabditis elegans &alpha; &#xBC0F; &beta; tubulin isotypes&#xB294; &#xBC30;&#xB2EC; &#xC5EC;&#xBD80;&#xB97C; &#xC870;&#xC0AC; &#xD588;&#xB2E4;. &#xB450; tubulins &#xBCF4;&#xC874;&#xB41C; &#xC794;&#xB958;&#xBB3C;&#xC758; &#xBCC0;&#xC774; &#xB9D0; &#xCD08; &#xB0B4;&#xC758; microtubules &#xBD88;&#xC548;&#xC815;. &#xD558;&#xB098;&#xC758; isotype TBB 4 microtubules axoneme &#xCF54;&#xC5B4; &#xBC0F; &#xC6D0;&#xC2EC; &#xC138;&#xADF8;&#xBA3C;&#xD2B8;&#xC758; &#xB05D;&#xC5D0; &#xC870;&#xB9BD; microtubule &#xD301; &#xCD94;&#xC801;&#xAE30; EB1 &#xBC1C;&#xACAC; &#xB418; &#xACE0; &#xBC18;&#xBA74;, cilium &#xB530;&#xB77C; localizes &#xB2E4;&#xB978;, &#xBBF8;&#xC815; 5 &#xC9D1;&#xC911; &#xC6D0;&#xC2EC; singlets&#xC5D0;. IFT &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;, &#xD615;&#xAD11; &#xBCF5;&#xAD6C; &#xD6C4; photobleaching &#xBD84;&#xC11D; &#xBC0F; &#xBAA8;&#xB378;&#xB9C1; IFT &#xAE30;&#xACC4;&#xC5D0; &#xC758;&#xD574; &#xC774;&#xB7EC;&#xD55C; tubulin subunits&#xC758; sub-stoichiometric &#xC22B;&#xC790;&#xC758; &#xC9C0;&#xC18D;&#xC801;&#xC778; &#xAD50;&#xD1B5; &#xC548;&#xC815; &#xC0C1;&#xD0DC;&#xC758; &#xAE38;&#xC774;&#xC5D0; &#xAC10;&#xAC01; &#xC18D;&#xB208;&#xC379;&#xC744; &#xC720;&#xC9C0;&#xD560; &#xC218; &#xC788;&#xC74C;&#xC744; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;.

Intraflagellar &#xC804;&#xC1A1; tubulin isotypes &#xAC10;&#xAC01; cilium &#xC911;&#xAC04; &#xBC0F; &#xC6D0;&#xC2EC; &#xC138;&#xADF8;&#xBA3C;&#xD2B8;&#xC5D0; &#xC81C;&#xACF5;&#xD569;&#xB2C8;&#xB2E4;.

Cilios sensoriales est&#xE1;n montados y mantenidos por kinesin-2-dependiente de intraflagellar transporte (IFT). Hemos investigado si los dos Caenorhabditis elegans alfa-tubulina y &#x3B2;-isotipos, identificados a trav&#xE9;s de los mutantes que carecen de los segmentos distales cilio, se entregan a sus lugares de concentraci&#xF3;n de IFT. Las mutaciones en los residuos conservados en ambas tubulinas desestabilizar a los microt&#xFA;bulos distales singlete. Un isotipo, TBB-4, se constituye en los microt&#xFA;bulos en las puntas del n&#xFA;cleo axonema y segmentos distales, donde la punta de seguimiento de los microt&#xFA;bulos EB1 se encuentra, y se localiza a lo largo del cilio, mientras que el otro, TBA-5, se concentra en singletes distales. Ensayos TIF, la recuperaci&#xF3;n despu&#xE9;s de fluorescencia photobleaching an&#xE1;lisis y la modelizaci&#xF3;n indican que el transporte continuo de la sub-estequiom&#xE9;tricas n&#xFA;mero de estas subunidades de tubulina por la maquinaria IFT puede mantener cilios sensoriales en su estado estacionario longitud.

Transporte intraflagellar Ofrece isotipos de tubulina Medio Cilio sensorial y los segmentos distales

&#x6211;&#x4EEC;&#x5206;&#x6790;&#x4E86;&#x7406;&#x89E3;&#x76F8;&#x5BF9;&#x5F88;&#x5DEE;&#x7684; IFT &#x808C; (DYNC2 &#x7C7B;)-&#x9A71;&#x52A8;&#x4E2D;&#x7EBF;&#x866B;&#x7EA4;&#x6BDB;&#x7684;&#x9006;&#x884C; IFT &#x901A;&#x8DEF;&#xFF0C;&#x6240;&#x4EA7;&#x751F;&#x7684;&#x7ED3;&#x679C;&#x4EE4;&#x4EBA;&#x60CA;&#x8BB6;&#x5728;&#x65C5;&#x6E38;&#x5B66;&#x9662;&#x7684;&#x5F53;&#x524D;&#x6A21;&#x578B;&#x7684;&#x4E0A;&#x4E0B;&#x6587;&#x4E2D;&#x3002;C.&#x7EBF;&#x866B;&#x808C;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x4E0E; intraflagellar &#x8FD0;&#x8F93; &#xFF08;&#x5B66;&#x9662;&#xFF09; &#x7684;&#x68C0;&#x6D4B;&#x65B9;&#x6CD5;&#x5EFA;&#x8BAE;&#x5E38;&#x89C4;&#x65C5;&#x6E38;&#x5B66;&#x9662;&#x808C;&#x52A0;&#x4E09;&#x5149;&#x80BD;&#x94FE;&#x53C2;&#x52A0;&#x5B66;&#x9662;&#xFF0C;&#x4F46;&#x6CA1;&#x6709;&quot;&#x5FC5;&#x8981;&quot;&#x7684;&#x4E2D;&#x95F4;&#x94FE;&#x5305;&#x542B;&#x57FA;&#x672C;&#x91CD; (&#x8F0B;-3&#xFF09;&#xFF0C;&#x5149;&#x4E2D;&#x95F4; &#xFF08;XBX-1)&#x3002;IFT &#x5316;&#x9A8C; XBX 1::YFP &#x7684;&#x5EFA;&#x8BAE;&#x4F5C;&#x4E3A;&#x8FDC;&#x7AEF;&#x7684;&#x7EA4;&#x6BDB;&#x8D27;&#x7269;&#x8FD0;&#x5F80; IFT &#x808C;&#xFF0C;&#x65B9;&#x6CD5;&#x662F;&#xFF1A; &#x7535;&#x52A8;&#x673A;&#x9A71;&#x52A8;&#x86CB;&#x767D;-2&#xFF0C;&#x4F46;&#x72EC;&#x7ACB;&#x5B66;&#x9662;-&#x7C92;&#x5B50;/BBSome &#x914D;&#x5408;&#x7269;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x60CA;&#x8BB6;&#x5730;&#x53D1;&#x73B0;&#xFF0C;&#x672C; OLQ (&#x5916;&#x5507;&#x8C61;&#x9650;&#xFF09; &#x4E0A;&#x7684;&#x7EA4;&#x6BDB;&#x7684;&#x5B50;&#x96C6;&#x795E;&#x7ECF;&#x5143;&#x88C5;&#x914D;&#x4E0E;&#x5E38;&#x89C4;&quot;&#x8F0B;-3&quot;&#x65C5;&#x6E38;&#x5B66;&#x9662;-&#x808C;&#xFF0C;&#xFF0C;&#x53EF;&#x4EE5;&#x6697;&#x793A;&#x6709;&#x8FD9;&#x4E9B;&#x7EA4;&#x6BDB;&#x4E2D;&#x7684;&#x7B2C;&#x4E8C;&#x4E2A; IFT &#x808C;&#x4EE3;&#x7406;&#x65E0;&#x5173;&#x3002;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x57FA;&#x56E0;&#x7F16;&#x7801;&#x808C;&#x91CD;&#x94FE;&#x3001; &#x534E;&#x4FE1;-3 &#x548C;&#x4E24;&#x6761;&#x8F7B;&#x94FE;&#xFF0C;OLQ &#x795E;&#x7ECF;&#x5143;&#xFF0C;&#x53EF;&#x80FD;&#x6784;&#x6210; OLQ &#x795E;&#x7ECF;&#x5143;&#x7EA4;&#x6BDB; IFT &#x808C;&#x60C5;&#x7ED3;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x5F3A;&#x8C03;&#x51E0;&#x4E2A;&#x4EE4;&#x4EBA;&#x60CA;&#x8BB6;&#x7684;&#x9006;&#x884C; IFT &#x9700;&#x8981;&#x7684;&#x529F;&#x80FD;&#xFF0C;&#x6F84;&#x6E05;&#x3002;

C.&#x7EBF;&#x866B;&#x7EA4;&#x6BDB;&#x7684;&#x9006;&#x884C;&#x7684;&#x5B66;&#x9662;&#x673A;&#x68B0;&#xFF1A; &#x4E24;&#x4E2A;&#x5B66;&#x9662;&#x808C;&#x914D;&#x5408;&#x7269;&#x5417;&#xFF1F;

We analyzed the relatively poorly understood IFT-dynein (class DYNC2)-driven retrograde IFT pathway in C. elegans cilia, which yielded results that are surprising in the context of current models of IFT. Assays of C. elegans dynein gene expression and intraflagellar transport (IFT) suggest that conventional IFT-dynein contains essential heavy (CHE-3), light-intermediate (XBX-1), plus three light polypeptide chains that participate in IFT, but no "essential" intermediate chain. IFT assays of XBX-1::YFP suggest that IFT-dynein is transported as cargo to the distal tip of the cilium by kinesin-2 motors, but independent of the IFT-particle/BBSome complexes. Finally, we were surprised to find that the subset of cilia present on the OLQ (outer labial quadrant) neurons assemble independently of conventional "CHE-3" IFT-dynein, implying that there is a second IFT-dynein acting in these cilia. We have found a novel gene encoding a dynein heavy chain, DHC-3, and two light chains, in OLQ neurons, which could constitute an IFT-dynein complex in OLQ neuronal cilia. Our results underscore several surprising features of retrograde IFT that require clarification.

The retrograde IFT machinery of C. elegans cilia: two IFT dynein complexes?

Nous avons analys&#xE9; la relativement mal comprise IFT-dyn&#xE9;ine (classe DYNC2) ax&#xE9; sur la voie r&#xE9;trograde IFT chez C. elegans cils, ce qui a abouti &#xE0; des r&#xE9;sultats qui &#xE9;tonnent dans le contexte des mod&#xE8;les actuels de l&#x26;#39;IFT. Les analyses de C. elegans l&#x26;#39;expression des g&#xE8;nes de dyn&#xE9;ine et le transport intraflagellaire (IFT) sugg&#xE8;rent que conventionnelle IFT-dyn&#xE9;ine contient lourde essentielle (CHE-3), la lumi&#xE8;re interm&#xE9;diaire (XBX-1), major&#xE9; de trois cha&#xEE;nes polypeptidiques l&#xE9;g&#xE8;res qui participent &#xE0; l&#x26;#39;IFT, mais pas &#xAB;essentielle&#xBB; de la cha&#xEE;ne interm&#xE9;diaire. Dosages IFT de XBX-1 :: YFP sugg&#xE8;rent que IFT-dyn&#xE9;ine est transport&#xE9; par cargo vers l&#x26;#39;extr&#xE9;mit&#xE9; distale du cil par la kin&#xE9;sine-2 moteurs, mais ind&#xE9;pendant des complexes IFT-particle/BBSome. Enfin, nous avons &#xE9;t&#xE9; surpris de constater que le sous-ensemble de l&#x26;#39;actuelle cils sur les OLQ (externe quadrant labiale) neurones assembler ind&#xE9;pendamment de classique &#x26;quot;CHE-3&#x26;quot; IFT-dyn&#xE9;ine, ce qui implique qu&#x26;#39;il ya une seconde IFT-dyn&#xE9;ine agissant dans ces cils. Nous avons trouv&#xE9; un nouveau g&#xE8;ne codant pour une cha&#xEE;ne lourde de dyn&#xE9;ine, DHC-3, et deux cha&#xEE;nes l&#xE9;g&#xE8;res, dans les neurones OLQ, ce qui pourrait constituer un complexe IFT-dyn&#xE9;ine dans OLQ cils neuronale. Nos r&#xE9;sultats soulignent plusieurs caract&#xE9;ristiques &#xE9;tonnantes de l&#x26;#39;IFT r&#xE9;trograde qui n&#xE9;cessitent des &#xE9;claircissements.

La machinerie IFT r&#xE9;trograde de C. Elegans Cilia: Deux IFT complexes dyn&#xE9;ine?

Wir analysierten die relativ Verst&auml;ndnism&auml;ngel IFT-Dynein (Klasse DYNC2)-gesteuerte retrograde IFT Weg in C. Elegans Zilien, die Resultate gebracht sind &uuml;berraschend im Kontext der aktuellen Modelle der IFT. Proben von C. Elegans Dynein-Genexpression und intraflagellar Transport (IFT) schlagen vor, da&szlig; konventionelle IFT-Dynein wesentlich schwere (CHE-3), Licht-Mittelstufe (XBX-1), enth&auml;lt sowie drei leichte Polypeptidketten, die Teilnahme an IFT, aber keine &quot;wesentlichen&quot; Fortgeschrittene Kette. IFT Proben von XBX-1::YFP zufolge IFT-Dynein als Fracht zur distalen Spitze der Zilie durch Kinesin-2 Motoren, aber unabh&auml;ngig von der IFT-Partikel/BBSome-komplexe transportiert wird. Schlie&szlig;lich waren wir &uuml;berrascht feststellen, dass die Teilmenge der Cilien auf der OLQ (&auml;u&szlig;ere labial Quadrant) Neuronen montieren unabh&auml;ngig von konventionellen &quot;CHE-3&quot; IFT-Dynein, was bedeutet, dass es eine zweite IFT-Dynein handeln diese Cilien. Wir haben festgestellt, dass eine neuartige gen eine schwere Kette von Dynein, DHC-3 und zwei leichten Ketten, in OLQ Neuronen, die einen IFT-Dynein-Komplex in OLQ neuronale Zilien darstellen k&ouml;nnte. Unsere Ergebnisse unterstreichen mehrere &uuml;berraschende Features der retrograden IFT, f&uuml;r die Kl&auml;rung erforderlich.

Die retrograde IFT-Maschinen von C. Elegans Zilien: zwei IFT Dynein-komplexe?

Abbiamo analizzato l&#39;IFT-dineina relativamente poco capito (classe DYNC2)-driven pathway IFT retrograda in c. elegans cilia, che ha dato risultati che sono sorprendenti nel contesto dei modelli attuali di IFT. Analisi di espressione genica di c. elegans dineina e trasporto dell&#39;omologo (IFT) suggeriscono che convenzionale IFT-dineina contiene essenziali pesanti (-3), luce-intermedio (XBX-1), oltre a tre catene del polipeptide leggero che partecipano IFT, ma nessuna catena intermedia &quot;essenziale&quot;. Dosaggi IFT di XBX-1::YFP suggeriscono che IFT-dineina &egrave; trasportato come carico alla punta distale delle ciglia da chinesina-2 motori, ma indipendente dei complessi IFT-particella/BBSome. Infine, siamo stati sorpresi di trovare che il sottoinsieme delle ciglia presenti su OLQ (quadrante esterno labiale) neuroni assemblare indipendentemente dalle convenzionale &quot;-3&quot; IFT-dineina, che implica che c&#39;&egrave; una seconda recitazione IFT-dineina in queste ciglia. Abbiamo trovato un romanzo gene codifica una catena pesante della dineina, DHC-3 e due catene leggere, nei neuroni OLQ, che potrebbero costituire un complesso IFT-dineina in cilia neuronale OLQ. I nostri risultati sottolineano diversi caratteristiche sorprendenti di IFT retrograda che richiedono chiarimenti.

Il macchinario IFT retrogrado di c. elegans cilia: due complessi di dineina IFT?

&#x6211;&#x3005;&#x306F;&#x6BD4;&#x8F03;&#x7684;&#x8CA7;&#x5F31;IFT-&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#xFF08;&#x30AF;&#x30E9;&#x30B9;DYNC2&#xFF09;&#x4E3B;&#x5C0E;&#x578B;&#x306E;IFT&#x306E;&#x73FE;&#x5728;&#x306E;&#x30E2;&#x30C7;&#x30EB;&#x306E;&#x30B3;&#x30F3;&#x30C6;&#x30AD;&#x30B9;&#x30C8;&#x5185;&#x3067;&#x9A5A;&#x304F;&#x3079;&#x304D;&#x306A;&#x7D50;&#x679C;&#x3092;&#x3082;&#x305F;&#x3089;&#x3057;&#x305F;C. elegans&#x306E;&#x7E4A;&#x6BDB;&#x306B;&#x304A;&#x3051;&#x308B;&#x9006;&#x884C;&#x6027;IFT&#x7D4C;&#x8DEF;&#x3092;&#x7406;&#x89E3;&#x3057;&#x5206;&#x6790;&#x3057;&#x305F;&#x3002; C. elegans&#x306E;&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x73FE;&#x3068;&#x97AD;&#x6BDB;&#x5185;&#x8F38;&#x9001;&#x306E;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306F;&#x3001;&#xFF08;IFT&#xFF09;&#x3001;&#x5F93;&#x6765;&#x306E;IFT-&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#x304C;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3001;&#xFF08;CHE-3&#xFF09;&#x306E;&#x91CD;&#xFF08;XBX-1&#xFF09;&#x5149;&#x306E;&#x4E2D;&#x9593;&#x306B;&#x52A0;&#x3048;&#x3066;&#x3001;IFT&#x306B;&#x53C2;&#x52A0;&#x3059;&#x308B;3&#x3064;&#x306E;&#x5149;&#x306E;&#x30DD;&#x30EA;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x9396;&#x3092;&#x542B;&#x3093;&#x3067;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x306A;&#x3044;&#x304C;&#x3001;&#x305D;&#x308C;&#x26;quot;&#x5FC5;&#x9808;&#x26;quot;&#x4E2D;&#x9593;&#x9396;&#x3002; XBX-1&#x306E;IFT&#x30A2;&#x30C3;&#x30BB;&#x30A4;:: YFP&#x306F;IFT-&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#x306F;&#x30AD;&#x30CD;&#x30B7;&#x30F3;-2&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x306B;&#x3088;&#x3063;&#x3066;&#x7E4A;&#x6BDB;&#x306E;&#x9060;&#x4F4D;&#x5148;&#x7AEF;&#x306B;&#x8CA8;&#x7269;&#x3068;&#x3057;&#x3066;&#x8F38;&#x9001;&#x304C;&#x3001;IFT-particle/BBSome&#x8907;&#x5408;&#x4F53;&#x3068;&#x306F;&#x72EC;&#x7ACB;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x305F;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x6211;&#x3005;&#x306F;OLQ&#xFF08;&#x5916;&#x5074;&#x5507;&#x306E;&#x8C61;&#x9650;&#xFF09;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x4E0A;&#x306B;&#x5B58;&#x5728;&#x3059;&#x308B;&#x7E4A;&#x6BDB;&#x306E;&#x30B5;&#x30D6;&#x30BB;&#x30C3;&#x30C8;&#x306F;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x7E4A;&#x6BDB;&#x306B;&#x304A;&#x3051;&#x308B;&#x7B2C;&#x4E8C;IFT-&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#x4F5C;&#x7528;&#x304C;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x610F;&#x5473;&#x3057;&#x3001;&#x72EC;&#x7ACB;&#x3057;&#x3066;&#x5F93;&#x6765;&#x306E; &#x26;quot;CHE-3&#x26;quot; IFT-&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#x306E;&#x7D44;&#x307F;&#x7ACB;&#x3066;&#x308B;&#x3053;&#x3068;&#x3092;&#x77E5;&#x3063;&#x3066;&#x9A5A;&#x3044;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;OLQ&#x795E;&#x7D4C;&#x7E4A;&#x6BDB;&#x306B;IFT-&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#x8907;&#x5408;&#x4F53;&#x3092;&#x69CB;&#x6210;&#x3067;&#x3057;&#x305F;&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#x91CD;&#x9396;&#x3001;OLQ&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306B;&#x304A;&#x3051;&#x308B;DHC-3&#x3001;&#x304A;&#x3088;&#x3073;2&#x3064;&#x306E;&#x8EFD;&#x9396;&#x3092;&#x30B3;&#x30FC;&#x30C9;&#x3059;&#x308B;&#x65B0;&#x898F;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x767A;&#x898B;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;&#x660E;&#x78BA;&#x5316;&#x3092;&#x5FC5;&#x8981;&#x3068;&#x9006;&#x884C;IFT&#x306E;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x9A5A;&#x304F;&#x3079;&#x304D;&#x7279;&#x5FB4;&#x3092;&#x5F37;&#x8ABF;&#x3059;&#x308B;&#x3002;

C. elegans&#x306E;&#x7E4A;&#x6BDB;&#x306E;&#x9006;&#x884C;&#x6027;IFT&#x6A5F;&#x68B0;&#xFF1A;&#x4E8C;IFT&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#x8907;&#x5408;&#x4F53;&#xFF1F;

&#xC6B0;&#xB9AC;&#xB294; &#xC0C1;&#xB300;&#xC801;&#xC73C;&#xB85C; &#xC800;&#xC870;&#xD55C; &#xC774;&#xD574; IFT &#xD68C; (DYNC2 &#xD074;&#xB798;&#xC2A4;) &#xBD84;&#xC11D;-C. elegans &#xC18D;&#xB208;&#xC379;&#xC5D0; &#xC5ED;&#xD589; IFT &#xD1B5;&#xB85C; &#xAD6C;&#xB3D9;, IFT&#xC758; &#xD604;&#xC7AC; &#xBAA8;&#xB378;&#xC758; &#xB9E5;&#xB77D;&#xC5D0;&#xC11C;&#xC758; &#xC678;&#xB294; &#xC5B4;&#xB5A4; &#xACB0;&#xACFC; &#xAD74;&#xBCF5;. C. elegans &#xD68C; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604; &#xBC0F; intraflagellar &#xC804;&#xC1A1; (IFT)&#xC758; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8; &#xAE30;&#xC874;&#xC758; IFT &#xD68C; IFT&#xC5D0; &#xCC38;&#xC5EC; &#xD558;&#xB294; 3 &#xBE5B; polypeptide &#xC0AC;&#xC2AC; &#xD558;&#xC9C0;&#xB9CC; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4; &quot;&#xD544;&#xC218;&quot; &#xC911;&#xAC04; &#xCCB4;&#xC778; &#xD50C;&#xB7EC;&#xC2A4; &#xD544;&#xC218; &#xBB34;&#xAC70;&#xC6B4; (3 &#xCCB4;), &#xBE5B;-&#xC911;&#xAE09; (XBX-1)&#xC744; &#xD3EC;&#xD568; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;. XBX 1::yfp&#xC758; IFT &#xBD84;&#xC11D; &#xC2E4;&#xD5D8; kinesin 2 &#xBAA8;&#xD130; IFT-&#xC785;&#xC790;/BBSome &#xB2E8;&#xC9C0;&#xC758; &#xB3C5;&#xB9BD;&#xC5D0; &#xC758;&#xD574; IFT &#xD68C;&#xB294; cilium&#xC758; &#xB9D0; &#xCD08; &#xB05D;&#xC5D0; &#xD654;&#xBB3C; &#xCE78;&#xC73C;&#xB85C; &#xC6B4;&#xC1A1; &#xB418;&#xB294; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &#xC6B0;&#xB9AC;&#xB294; OLQ (&#xC678;&#xBD80; &#xC785;&#xC220; &#xC0AC;&#xBD84;&#xBA74;)&#xC5D0; &#xC18D;&#xB208;&#xC379;&#xC758; &#xD558;&#xC704; &#xC9D1;&#xD569;&#xC744; &#xCC3E;&#xC744; &#xC218; &#xB180; &#xB790; &#xB2E4; &#xC2E0;&#xACBD; &#xD68C;-&#xAE30;&#xC874;&#xC758; &quot;CHE&quot; 3 IFT,&#xC774; &#xC18D;&#xB208;&#xC379;&#xC5D0; &#xB450; &#xBC88;&#xC9F8; IFT &#xD68C; &#xC5F0;&#xAE30; &#xC554;&#xC2DC; &#xB3C5;&#xB9BD;&#xC801;&#xC73C;&#xB85C; &#xC870;&#xB9BD;. &#xC6B0;&#xB9AC; &#xD68C; &#xBB34;&#xAC70;&#xC6B4; &#xCCB4;&#xC778;, DHC 3 &#xBC0F; OLQ &#xC2E0;&#xACBD; &#xC18D;&#xB208;&#xC379;&#xC5D0; IFT &#xD68C; &#xBCF5;&#xC7A1; &#xD55C; &#xAD6C;&#xC131; OLQ &#xC2E0;&#xACBD;&#xC5D0; &#xC788;&#xB294; &#xB450; &#xAC1C;&#xC758; &#xACBD; &#xC1C4; &#xC778;&#xCF54;&#xB529; &#xC0C8;&#xB85C;&#xC6B4; &#xC720;&#xC804;&#xC790;&#xB97C; &#xBC1C;&#xACAC; &#xD588;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; &#xBA85;&#xD655;&#xD55C; &#xC124;&#xBA85;&#xC744; &#xC694;&#xAD6C; &#xD558;&#xB294; &#xC5ED;&#xD589; IFT&#xC758; &#xB180;&#xB77C;&#xC6B4; &#xAE30;&#xB2A5; &#xBC11;&#xC904;.

C. elegans &#xC18D;&#xB208;&#xC379;&#xC758; &#xC5ED;&#xD589; IFT &#xAE30;&#xACC4;: &#xB450; IFT &#xD68C; &#xB2E8;&#xC9C0;?

Se analiz&#xF3; la relativamente poco conocida IFT-dine&#xED;na (clase DYNC2) impulsada por v&#xED;a retr&#xF3;grada IFT en C. elegans, los cilios, que arroj&#xF3; resultados que son sorprendentes en el contexto de los modelos actuales de IFT. Los ensayos de C. elegans, la expresi&#xF3;n de genes dine&#xED;na y el transporte intraflagellar (IFT) sugieren que los convencionales IFT-dine&#xED;na contiene pesados &#x200B;&#x200B;esenciales (CHE-3), la luz-intermedio (XBX-1), adem&#xE1;s de tres cadenas de polip&#xE9;ptidos de luz que participan en el IFT, pero no &#x26;quot;esencial&#x26;quot; de la cadena intermedia. Ensayos TIF de XBX-1 :: YFP sugieren que IFT-dine&#xED;na es transportado como carga a la punta distal del cilio por kinesin-2 motores, pero independiente de los complejos IFT-particle/BBSome. Por &#xFA;ltimo, se sorprendieron al encontrar que el subconjunto de los cilios presentes en las OLQ (cuadrante labial externa), las neuronas independientemente de montaje convencional &#x26;quot;CHE-3&#x26;quot; IFT-dine&#xED;na, lo que implica que hay una segunda IFT-dine&#xED;na actuaci&#xF3;n en estos cilios. Hemos encontrado un nuevo gen que codifica una cadena pesada de dine&#xED;na, DHC-3, y dos cadenas ligeras, en las neuronas OLQ, lo que podr&#xED;a constituir un complejo IFT-dine&#xED;na de los cilios OLQ neuronal. Nuestros resultados ponen de relieve varios rasgos sorprendentes de la IFT retr&#xF3;grada que requieren aclaraci&#xF3;n.

La maquinaria IFT retr&#xF3;grada de C. Elegans, Cilia: Dos TIF Complejos dine&#xED;na?

The objective of this study was to investigate the spectral behavior of the relationship between reflectance and chlorophyll content and to develop a technique for non-destructive chlorophyll estimation and distribution in leaves using hyperspectral imaging. The hyperspectral imaging data cube of cucumber (Cucumis sativus) leaves in the range of 450-850 nm was investigated and preprocessed. Sixty optical signatures or indices as a function of the associated reflectance (R(λ)) at the special wavelength (λ) nm which proposed in the literatures were used to predict the total chlorophyll content in cucumber leaves. Finally, R(710)/R(760), (R(780)-R(710))/(R(780)-R(680)), (R(750)-R(705))/(R(750)+R(705)), (R(680)-R(430))/(R(680)+R(430)), R(860)/(R(550)×R(708)), (R(695-705))(-1)-(R(750-800))(-1), and REP-LEM (a index based on red edge position and estimated with a linear extrapolation method) were identified as optimum indices. Red-edge waveband (680-780 nm) appeared in all these optimum indices, indicating the importance of REP (red edge position) in chlorophyll estimation. When (R(695-705))(-1)-(R(750-800))(-1), the best index was applied to an independent validation set, chlorophyll content (r=0.8286) were reasonably well predicted, indicating model robustness. Depending on the sample, this technique enables to identify and characterize the relative content of various chlorophyll that distribution in the cucumber leaves. The map shows a relatively low level of chlorophyll at margins. Higher level can be noticed in the regions along the main veins and in some areas exhibiting dark green tissue. Our results indicate that hyperspectral imaging has considerable promise for predicting pigments in leaves and, the pigments can be detected in situ in living plant samples non-destructively.

In vivo noninvasive detection of chlorophyll distribution in cucumber (Cucumis sativus) leaves by indices based on hyperspectral imaging.

More than 3.2 million litres of vinegar is consumed every day in China. There are many types of vinegar in China. How to control the quality of vinegar is problem. Near infrared spectroscopy (NIR) transmission technique was applied to achieve this purpose. Ninety-five vinegar samples from 14 origins covering 11 provinces in China were collected. They were classified into mature vinegar, aromatic vinegar, rice vinegar, fruit vinegar, and white vinegar. Fruit vinegar and white vinegar were separated from the other traditional categories in the two-dimension principal component space of NIR after principle component analysis (PCA). Least-squares support vector machine (LS-SVM) as the pattern recognition was firstly applied to identify mature vinegar, aromatic vinegar, rice vinegar in this study. The top two principal components (PCs) were extracted as the input of LS-SVM classifiers by principal component analysis (PCA). The best experimental results were obtained using the radial basis function (RBF) LS-SVM classifier with σ=0.8. The accuracies of identification were more than 85% for three traditional vinegar categories. Compared with the back propagation artificial neural network (BP-ANN) approach, LS-SVM algorithm showed its excellent generalisation for identification results. As total acid content (TAC) is highly connecting with the quality of vinegar, NIR was used to prediction the TAC of samples. LS-SVM was applied to building the TAC prediction model based on spectral transmission rate. Compared with partial least-square (PLS) model, LS-SVM model gave better precision and accuracy in predicting TAC. The determination coefficient for prediction (R(p)) of the LS-SVM model was 0.919 and root mean square error for prediction (RMSEP) was 0.3226. This work demonstrated that near infrared spectroscopy technique coupled with LS-SVM could be used as a quality control method for vinegar.

Rapid detecting total acid content and classifying different types of vinegar based on near infrared spectroscopy and least-squares support vector machine.

Glioblastoma multiforme (GBM) is the most common form of malignant glioma, characterized by genetic instability and unpredictable clinical behavior. GBM is marked by an extremely poor prognosis with median overall survival of 12~14 months. In this study, we detected the CD137L-expressing cells and IL-17-expressing cells in tumor tissues resected from patients with GBM. Expression of CD137L and IL-17 were assessed by immunohistochemistry, and the prognostic value of CD137L and IL-17 expression within the tumor tissues were assessed by Cox regression and Kaplan-Meier analysis. Immunohistochemical detection showed that positive cells of CD137L and IL-17 in glioblastoma tissue samples were 46.3% (19/ 41) and 73.2% (30/41) respectively. Expression of CD137L was not correlated with overall survival of GBM patients (P=0.594), while significantly longer survival rate was seen in patients with high expression of IL-17, compared to those with low expression of IL-17 (P=0.007). In addition, we also found that IL-17 expression was significantly correlated with Progression-free survival (PFS) (P=0.016) and death rate (P=0.01). Furthermore, multivariate Cox proportional hazard analyses revealed that IL-17 (P=0.018) and PFS (P=0.028) were independent factors affecting the overall survival probability. Kaplan-Meier analysis showed that PFS of high expression of IL-17 group were significantly longer (P=0.004) than low expression group with GBM. It is concluded that high levels of IL-17 expression in the tumor tissues may be a good prognostic marker for patients with GBM.

Analysis of CD137L and IL-17 expression in tumor tissue as prognostic indicators for gliblastoma.

We report a genome-wide RNA interference (RNAi) screen for Suppressors of Clozapine-induced Larval Arrest (scla genes) in Caenorhabditis elegans, the first genetic suppressor screen for antipsychotic drug (APD) targets in an animal. The screen identifies 40 suppressors, including the Î±-like nicotinic acetylcholine receptor (nAChR) homolog acr-7. We validate the requirement for acr-7 by showing that acr-7 knockout suppresses clozapine-induced larval arrest and that expression of a full-length translational GFP fusion construct rescues this phenotype. nAChR agonists phenocopy the developmental effects of clozapine, while nAChR antagonists partially block these effects. ACR-7 is strongly expressed in the pharynx, and clozapine inhibits pharyngeal pumping. acr-7 knockout and nAChR antagonists suppress clozapine-induced inhibition of pharyngeal pumping. These findings suggest that clozapine activates ACR-7 channels in pharyngeal muscle, leading to tetanus of pharyngeal muscle with consequent larval arrest. No APDs are known to activate nAChRs, but a number of studies indicate that Î±7-nAChR agonists may prove effective for the treatment of psychosis. Î±-like nAChR signaling is a mechanism through which clozapine may produce its therapeutic and/or toxic effects in humans, a hypothesis that could be tested following identification of the mammalian ortholog of C. elegans acr-7.

A Genome-Wide RNAi Screen in Caenorhabditis elegans Identifies the Nicotinic Acetylcholine Receptor Subunit ACR-7 as an Antipsychotic Drug Target.

Nous rapportons un &eacute;cran d&#39;interf&eacute;rence (ARNi) RNA G&eacute;nome-large d&#39;arrestation larvaire suppresseurs de Clozapine-induit (g&egrave;nes scla) chez Caenorhabditis elegans, le premier &eacute;cran de suppresseur g&eacute;n&eacute;tique pour les cibles de m&eacute;dicament antipsychotique (DGPA) chez un animal. L&#39;&eacute;cran indique 40 suppresseurs, y compris le semblable &alpha; nicotinic acetylcholine receptor (CNCDH) homologue acr-7. Nous validons l&#39;exigence pour acr-7 en montrant que l&#39;acr-7 knockout supprime induite par la clozapine larvaire arrestation et que l&#39;expression d&#39;une pleine longueur translationnelle construction de fusion de GFP sauve ce ph&eacute;notype. cause d&#39;agonistes CNCDH les effets sur le d&eacute;veloppement de la clozapine, tandis que les antagonistes de la CNCDH partiellement bloquent ces effets. ACR-7 est fortement exprim&eacute;e dans le pharynx, et inhibe la clozapine pompage pharyng&eacute;e. ACR-7 knockout et CNCDH antagonistes suppriment l&#39;inhibition induite par la clozapine de pompage pharyng&eacute;e. Ces r&eacute;sultats sugg&egrave;rent que la clozapine active ACR-7 canaux en muscle pharyng&eacute;, menant au t&eacute;tanos du muscle pharyng&eacute; avec l&#39;arrestation de larve qui en d&eacute;coule. Aucun APDs ne sont connus pour activer nAChR, mais un certain nombre d&#39;&eacute;tudes indique que les agonistes &alpha;7-CNCDH peuvent s&#39;av&eacute;rer efficaces pour le traitement de la psychose. CNCDH &alpha;-comme la signalisation est un m&eacute;canisme par lequel clozapine peut produire ses effets th&eacute;rapeutiques et/ou toxiques chez les humains, une hypoth&egrave;se qui pourrait &ecirc;tre mis &agrave; l&#39;essai apr&egrave;s identification de l&#39;orthologue mammif&egrave;res de c. elegans acr-7.

Un &eacute;cran de RNAi Genome-Wide chez Caenorhabditis elegans identifie l&#39;ac&eacute;tylcholine nicotinique r&eacute;cepteur sous-unit&eacute; ACR-7 comme une cible de m&eacute;dicament antipsychotique.

Riportiamo una schermata di interferenza (RNAi) RNA genome-wide per arresto larvale soppressori di clozapina-indotta (scla geni) in Caenorhabditis elegans, la prima schermata di genetica soppressore per bersagli di farmaci antipsicotici (APD) in un animale. Schermo identifica 40 soppressori, tra cui il &alpha;-come nicotinici acetilcolina recettore (nAChR) omologo acr-7. Noi di convalidare il requisito per acr-7 mostrando che acr-7 ko sopprime indotta da clozapina arresto larvale e quell&#39;espressione di un full-length costrutto di fusione di GFP traslazionale Salva questo fenotipo. fenocopia agonisti nAChR gli effetti dello sviluppo della clozapina, mentre gli antagonisti nAChR parzialmente bloccano questi effetti. ACR-7 &egrave; fortemente espresso nella faringe e clozapina inibisce il pompaggio pharyngeal. ACR-7 ko e nAChR antagonisti sopprimono l&#39;inibizione indotta da clozapina di pompaggio pharyngeal. Questi risultati suggeriscono che clozapina attiva i canali ACR-7 nel muscolo faringeo, portando a tetano del muscolo faringeo con conseguente arresto allo stadio larvale. No APD sono conosciuti per attivare nAChRs, ma numerosi studi indicano che &alpha;7-nAChR agonisti possono risultare efficace per il trattamento di psicosi. &Alpha;-come nAChR di segnalazione &egrave; un meccanismo attraverso il quale Clozapina pu&ograve; produrre i suoi effetti terapeutici o tossici negli esseri umani, un&#39;ipotesi che potrebbe essere testato a seguito di identificazione dei mammiferi ortholog di c. elegans acr-7.

Una schermata di RNAi Genome-Wide in Caenorhabditis elegans identifica i nicotinici acetilcolina recettore subunit&agrave; ACR-7 come un Target di farmaco antipsicotico.

Morchella esculenta, an edible medicinal mushroom native to China, is recognized as an unparalleled resource of healthy foods and drug discovery. This study firstly investigated the antioxidant activity of Morchella esculenta extracellular polysaccharides (MEEP). An in vitro antioxidant assay showed that MEEP exhibited strong hydroxyl radical scavenging activity and moderate 1,1-diphenyl-2-picryldydrazyl radical scavenging activity and reductive power. For antioxidant testing in vivo, MEEP were orally administered over a period of 60 days in a d-galactose induced aged mice model. Administration of the polysaccharides inhibited significantly the formation of malondialdehyde livers and serums, and raised the activities of antioxidant enzymes and the total antioxidant capacity in a dose-dependent manner. Furthermore, we also observed that MEEPs markedly enhanced the body's immune system by measuring macrophage phagocytosis and splenocyte proliferation in d-galactose induced mice. These findings suggest that EPs from Morchella esculenta are a promising source of natural antioxidants and immunoenhancing drugs.

Evaluation of the antioxidant activity of extracellular polysaccharides from Morchella esculenta.

Design and fabrication of an ammonia sensor operating at room temperature based on pigment-sensitized TiO2 films was described. TiO2 was prepared by sol-gel method and deposited on glass slides containing gold electrodes. Then, the film immersed in a 2.5×10(-4)M ethanol solution of cyanidin to absorb the pigment. The hybrid organic-inorganic formed film here can detect ammonia reversibly at room temperature. The relative change resistance of the films at a potential difference of 1.5V is determined when the films are exposed to atmospheres containing ammonia vapors with concentrations over the range 10-50 ppm. The relative change resistance, S, of the films increased almost linearly with increasing concentrations of ammonia (r=0.92). The response time to increasing concentrations of the ammonia is about 180-220 s, and the corresponding values for decreasing concentrations 240-270 s. At low humidity, ammonia could be ionized by the cyanidin on the TiO2 film and thereby decrease in the proton concentration at the surface. Consequently, more positively charged holes at the surface of the TiO2 have to be extracted to neutralize the adsorbed cyanidin and water film. The resistance response to ammonia of the sensors was nearly independent on temperature from 10 to 50°C. These results are not actually as good as those reported in the literature, but this preliminary work proposes simpler and cheaper processes to realize NH3 sensor for room temperature applications.

A new sensor for ammonia based on cyanidin-sensitized titanium dioxide film operating at room temperature.

A new colorimetric gas-sensor array based on four natural pigments, that were extracted from spinach (Spinacia oleracea), red radish (Raphanus sativus L.), winter jasmine (Jasminum nudiflorum), and black rice (Oryza sativa L. indica), was developed for pork freshness evaluation. A colour change profile for each sample was obtained by differentiating the images of the sensor array before and after exposure to the odour of sample. The total viable count (TVC) per gram of pork was obtained by classical microbiological plating methods, and the biogenic amines were measured by HPLC. Biogenic amine index (BAI) for the determination of meat freshness was developed from the sum of putrescine and cadaverine. The colour change profiles were analysed using principal component analysis and correlated with conventional methods (BAI, TVC). A partial least squares (PLS) prediction model was obtained with r=0.854 and 0.933 for BAI and TVC, respectively.

Limin Hao

Department of Psychiatry

Harvard Medical School

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Limin Hao

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Psychiatry

Harvard Medical School

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Department of Psychiatry