Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

University of Toledo

The evolution of the ancestral eukaryotic flagellum is an example of a cellular organelle that became dispensable in some modern eukaryotes while remaining an essential motile and sensory apparatus in others. To help define the repertoire of specialized proteins needed for the formation and function of cilia, we used comparative genomics to analyze the genomes of organisms with prototypical cilia, modified cilia, or no cilia and identified approximately 200 genes that are absent in the genomes of nonciliated eukaryotes but are conserved in ciliated organisms. Importantly, over 80% of the known ancestral proteins involved in cilia function are included in this small collection. Using Drosophila as a model system, we then characterized a novel family of proteins (OSEGs: outer segment) essential for ciliogenesis. We show that osegs encode components of a specialized transport pathway unique to the cilia compartment and are related to prototypical intracellular transport proteins.

Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis.

We previously reported that acute agonist activation of G(i/o)-coupled receptors inhibits adenylate cyclase (AC) type VIII activity, whereas agonist withdrawal following chronic activation of these receptors induces AC-VIII superactivation. Three splice variants of AC-VIII have been identified, which are called AC-VIII-A, -B and -C (with AC-VIII-B missing the glycosylation domain and AC-VIII-C lacking most of the C1b area). We report here that AC-VIII-A and -B, but not -C, are inhibited by acute mu-opioid and dopaminergic type D2 receptor activation, indicating that the C1b area of AC-VIII has an important role in AC inhibition by G(i/o)-coupled receptor activation. On the other hand the glycosylation sites in AC-VIII did not play a role in AC-VIII regulation. Although AC-VIII-A and -C differed in their capacity to be inhibited by acute agonist exposure, agonist withdrawal after prolonged treatment led to a similar superactivation of all three splice variants, with no significant change in AC-VIII expression. AC-VIII superactivation was not affected by pre-incubation with a cell permeable cAMP analogue, indicating that the superactivation does not depend on the agonist-induced reduction in cAMP levels. The superactivated AC-VIII-A, -B and -C were similarly re-inhibited by re-application of agonist (morphine or quinpirole), returning the activity to control levels. These results demonstrate marked differences in the agonist inhibition of the AC-VIII splice variants before, but not after, superactivation.

Regulation of adenylate cyclase type VIII splice variants by acute and chronic Gi/o-coupled receptor activation.

It was shown previously that chronic exposure to opiate agonists increases adenylyl cyclase (AC) activity, a phenomenon termed AC superactivation (or supersensitization). More recently, we showed that acute Gi/o- coupled receptor activation inhibits the activity of several AC isozymes, including Ca2+/calmodulin-stimulated AC-I and -VIII, whereas chronic receptor activation induces their superactivation. Here, we report that both acute Mu-opioid receptor-induced inhibition and chronic induced superactivation of AC-I and -VIII are pertussis toxin sensitive. In addition, we show that proteins that interfere with the activity of Gbetagamma subunits (Gbetagamma scavengers) strongly attenuate the acute inhibition of AC-I and -VIII and the superactivation of AC-I, and abolish the superactivation of AC-VIII. Based on these results, we suggest that Gbetagamma is involved in the acute inhibition and chronic agonist-induced superactivation of AC types I and VIII.

Inhibition and superactivation of the calcium-stimulated isoforms of adenylyl cyclase: role of Gbetagamma dimers.

Die Zentriol ist die Kernstruktur des Zentrosom und Zilie. Zentriol Vervielf&auml;ltigung auf einmal pro Zellzyklus zu beschr&auml;nken hat ernste Konsequenzen und wird h&auml;ufig bei Krebs beobachtet. Trotz der medizinischen Bedeutung wird der Mechanismus der Zentriol Bildung schlecht verstanden. ASL wurde zuvor berichtet, ein centrosomal Protein, das f&uuml;r Zentrosom Funktion wesentlich zu sein. Hier erkennen wir MecD, einen schweren Verlust of Function-Allel des Gens asl, und zeigen, dass es f&uuml;r Zentriol und Zilien Bildung erforderlich ist. Ebenso Cep152, die Asl-Ortholog bei Wirbeltieren, ist essentiell f&uuml;r Zilien Bildung und seine Funktion kann teilweise von der Drosophila-Asl gerettet werden. Das Studium der Asl Lokalisierung legt nahe, dass es eng verbunden mit der Wand Zentriol ist, aber nicht Teil der Zentriol-Struktur ist. Durch die Analyse der Biogenese Centrosomes in Zellen, die Asl ausgelaugt, fanden wir, dass w&auml;hrend Pericentriolar materiell (PCM) Funktion leicht betroffen ist, ist Asl f&uuml;r Tochter Zentriol Bildung wichtig. Die klare mehrere centriolar Marker in MecD Mutanten nahe, dass Asl fr&uuml;h in Zentriol Duplikation ankommt.

Taufliege asterless und vertebrate Cep152 sind Orthologen notwendig f&uuml;r Zentriol Vervielf&auml;ltigung.

&#x4E2D;&#x5FC3;&#x7C92;&#x662F;&#x4E2D;&#x5FC3;&#x4F53;&#x548C;&#x7EA4;&#x6BDB;&#x7684;&#x6838;&#x5FC3;&#x7ED3;&#x6784;&#x3002;&#x9650;&#x5236;&#x7EC6;&#x80DE;&#x5668;&#x8FDB;&#x884C;&#x590D;&#x5236;&#x5230;&#x6BCF;&#x4E2A;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x7684;&#x4E00;&#x6B21;&#x5931;&#x8D25;&#x5E26;&#x6765;&#x4E25;&#x91CD;&#x7684;&#x540E;&#x679C;&#xFF0C;&#x901A;&#x5E38;&#x5728;&#x764C;&#x75C7;&#x4E2D;&#x89C2;&#x5BDF;&#x5230;&#x3002;&#x5C3D;&#x7BA1;&#x5176;&#x533B;&#x7597;&#x7684;&#x91CD;&#x8981;&#x6027;&#xFF0C;&#x4E2D;&#x5FC3;&#x7C92;&#x7684;&#x5F62;&#x6210;&#x673A;&#x7406;&#x662F;&#x751A;&#x5C11;&#x3002;Asl &#x4EE5;&#x524D;&#x636E;&#x8BF4; centrosomal &#x86CB;&#x767D;&#x8D28;&#x4E2D;&#x5FC3;&#x4F53;&#x529F;&#x80FD;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x627E;&#x51FA; mecD&#xFF0C;&#x4E25;&#x91CD;&#x635F;&#x5931;&#x7684;&#x51FD;&#x6570;&#x7B49;&#x4F4D;&#x57FA;&#x56E0;&#x7684; asl &#x57FA;&#x56E0;&#xFF0C;&#x5E76;&#x8BC1;&#x660E;&#x5B83;&#x662F;&#x5FC5;&#x9700;&#x7684;&#x7EC6;&#x80DE;&#x5668;&#x8FDB;&#x884C;&#x548C;&#x7EA4;&#x6BDB;&#x5F62;&#x6210;&#x3002;&#x540C;&#x6837;&#xFF0C;Cep152&#xFF0C;&#x5728;&#x810A;&#x690E;&#x52A8;&#x7269;&#xFF0C;&#x7FD4;&#x5347; ortholog &#x7EA4;&#x6BDB;&#x5F62;&#x6210;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#xFF0C;&#x5176;&#x529F;&#x80FD;&#x53EF;&#x4EE5;&#x7531;&#x679C;&#x8747;&#x7FD4;&#x5347;&#x90E8;&#x5206;&#x6551;&#x51FA;&#x3002;Asl &#x5B9A;&#x4F4D;&#x7684;&#x7814;&#x7A76;&#x8868;&#x660E;&#x5B83;&#x4E0E;&#x7EC6;&#x80DE;&#x5668;&#x8FDB;&#x884C;&#x9694;&#x79BB;&#x5899;&#xFF0C;&#x5BC6;&#x5207;&#x5173;&#x8054;&#xFF0C;&#x4F46;&#x4E0D;&#x662F;&#x7EC6;&#x80DE;&#x5668;&#x8FDB;&#x884C;&#x7ED3;&#x6784;&#x7684;&#x4E00;&#x90E8;&#x5206;&#x3002;&#x901A;&#x8FC7;&#x5206;&#x6790;&#x751F;&#x7269;&#x7EC6;&#x80DE;&#x8017;&#x5C3D;&#x7684; Asl &#x4E2D;&#x5FC3;&#x4F53;&#x7684;&#x53D1;&#x751F;&#xFF0C;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#xFF0C;&#x867D;&#x7136; pericentriolar &#x6750;&#x6599; (PCM) &#x529F;&#x80FD;&#x8F7B;&#x5EA6;&#x53D7;&#x5F71;&#x54CD;&#xFF0C;Asl &#x662F;&#x5973;&#x513F;&#x7EC6;&#x80DE;&#x5668;&#x8FDB;&#x884C;&#x5F62;&#x6210;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;&#x7F3A;&#x4E4F;&#x660E;&#x786E;&#x7684;&#x51E0;&#x4E2A; mecD &#x7A81;&#x53D8;&#x4F53;&#x7684; centriolar &#x6807;&#x8BB0;&#x8868;&#x660E; Asl &#x662F;&#x65E9;&#x671F;&#x7EC6;&#x80DE;&#x5668;&#x8FDB;&#x884C;&#x91CD;&#x590D;&#x7684;&#x5173;&#x952E;&#x3002;

&#x679C;&#x8747; asterless &#x548C;&#x810A;&#x690E;&#x52A8;&#x7269; Cep152 &#x662F; orthologs &#x7EC6;&#x80DE;&#x5668;&#x8FDB;&#x884C;&#x91CD;&#x590D;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;

The centriole is the core structure of centrosome and cilium. Failure to restrict centriole duplication to once per cell cycle has serious consequences and is commonly observed in cancer. Despite its medical importance, the mechanism of centriole formation is poorly understood. Asl was previously reported to be a centrosomal protein essential for centrosome function. Here we identify mecD, a severe loss-of-function allele of the asl gene, and demonstrate that it is required for centriole and cilia formation. Similarly, Cep152, the Asl ortholog in vertebrates, is essential for cilia formation and its function can be partially rescued by the Drosophila Asl. The study of Asl localization suggests that it is closely associated with the centriole wall, but is not part of the centriole structure. By analyzing the biogenesis of centrosomes in cells depleted of Asl, we found that, while pericentriolar material (PCM) function is mildly affected, Asl is essential for daughter centriole formation. The clear absence of several centriolar markers in mecD mutants suggests that Asl is critical early in centriole duplication.

Drosophila asterless and vertebrate Cep152 Are orthologs essential for centriole duplication.

Le centriole est la structure de base de centrosome et cil. Impossibilit&#xE9; de limiter la duplication de centriole cycle cellulaire une fois par a de graves cons&#xE9;quences et il est couramment observ&#xE9; dans le cancer. En d&#xE9;pit de son importance m&#xE9;dicale, le m&#xE9;canisme de formation centriole est mal comprise. Asl a &#xE9;t&#xE9; pr&#xE9;c&#xE9;demment d&#xE9;clar&#xE9;e &#xE0; une prot&#xE9;ine essentielle pour la fonction centrosomale centrosome. Ici nous identifions MECD, une grave perte de la fonction all&#xE8;le du g&#xE8;ne d&#x26;#39;altitude, et de d&#xE9;montrer qu&#x26;#39;elle est n&#xE9;cessaire pour centriole et la formation de cils. De m&#xEA;me, Cep152, l&#x26;#39;orthologue Asl chez les vert&#xE9;br&#xE9;s, est essentiel pour la formation des cils et sa fonction peut &#xEA;tre partiellement sauv&#xE9; par l&#x26;#39;Asl drosophile. L&#x26;#39;&#xE9;tude de la localisation Asl sugg&#xE8;re qu&#x26;#39;il est &#xE9;troitement associ&#xE9; &#xE0; la paroi centriole, mais ne fait pas partie de la structure centriole. En analysant la biogen&#xE8;se des centrosomes dans les cellules appauvries de l&#x26;#39;ASL, nous avons constat&#xE9; que, tandis que p&#xE9;ricentriolaire mat&#xE9;riau (PCM) est fonction l&#xE9;g&#xE8;rement touch&#xE9;s, Asl est essentiel pour la formation de centriole fille. L&#x26;#39;absence claire de plusieurs marqueurs dans centriolaires mutants MECD sugg&#xE8;re que Asl est essentiel au d&#xE9;but de centriole duplication.

Drosophila Asterless et des vert&#xE9;br&#xE9;s Cep152 Etes Orthologues Indispensable pour Centriole Duplication

Il Centriolo &egrave; la struttura di base del centrosoma e cilio. Fallimento per limitare la duplicazione di una volta per ciclo cellulare Centriolo ha gravi conseguenze ed &egrave; comunemente osservato nel cancro. Nonostante la sua importanza medica, il meccanismo di formazione Centriolo &egrave; scarsamente compreso. ASL in precedenza &egrave; stato segnalato per essere una proteina centrosomal essenziale per la funzione centrosoma. Qui ci identifichiamo mecD, un grave perdita della funzione allele del gene della asl e dimostrare che &egrave; necessaria per la formazione Centriolo e le ciglia. Analogamente, Cep152, l&#39;Asl ortholog nei vertebrati, &egrave; essenziale per la formazione di ciglia e la sua funzione pu&ograve; essere parzialmente salvata dall&#39;Asl della drosofila. Lo studio della localizzazione Asl suggerisce che esso &egrave; strettamente associato con il muro Centriolo, ma non &egrave; parte della struttura del Centriolo. Analizzando la biogenesi di cntrosome nelle cellule impoverite di Asl, abbiamo scoperto che, mentre la funzione (PCM) materiale pericentriolar &egrave; leggermente influenzato, Asl &egrave; essenziale per la formazione di figlia Centriolo. La chiara assenza di diversi marcatori centriolar mecD mutanti suggerisce che la Asl &egrave; critico all&#39;inizio della duplicazione Centriolo.

Drosofila vertebrati e asterless Cep152 sono orthologs essenziali per la duplicazione Centriolo.

&#x4E2D;&#x5FC3;&#x5C0F;&#x4F53;&#x306F;&#x4E2D;&#x5FC3;&#x4F53;&#x3068;&#x7E4A;&#x6BDB;&#x306E;&#x30B3;&#x30A2;&#x69CB;&#x9020;&#x3067;&#x3059;&#x3002; 1&#x56DE;&#x306E;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x306B;&#x4E2D;&#x5FC3;&#x5C0F;&#x4F53;&#x306E;&#x8907;&#x88FD;&#x3092;&#x5236;&#x9650;&#x3059;&#x308B;&#x969C;&#x5BB3;&#x304C;&#x6DF1;&#x523B;&#x306A;&#x5F71;&#x97FF;&#x3092;&#x6301;&#x3063;&#x3066;&#x304A;&#x308A;&#x3001;&#x4E00;&#x822C;&#x7684;&#x306A;&#x764C;&#x3067;&#x89B3;&#x5BDF;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x533B;&#x7642;&#x306E;&#x91CD;&#x8981;&#x6027;&#x306B;&#x3082;&#x304B;&#x304B;&#x308F;&#x3089;&#x305A;&#x3001;&#x4E2D;&#x5FC3;&#x5C0F;&#x4F53;&#x5F62;&#x6210;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306F;&#x3042;&#x307E;&#x308A;&#x7406;&#x89E3;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x3002; ASL&#x306F;&#x3001;&#x4EE5;&#x524D;&#x306B;&#x4E2D;&#x5FC3;&#x4F53;&#x6A5F;&#x80FD;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x306A;&#x4E2D;&#x5FC3;&#x4F53;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x5831;&#x544A;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;mecD&#x3001;ASL&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x6DF1;&#x523B;&#x306A;&#x6A5F;&#x80FD;&#x55AA;&#x5931;&#x578B;&#x5BFE;&#x7ACB;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x8B58;&#x5225;&#x3057;&#x3001;&#x305D;&#x308C;&#x306F;&#x4E2D;&#x5FC3;&#x5C0F;&#x4F53;&#x3068;&#x7E4A;&#x6BDB;&#x5F62;&#x6210;&#x306B;&#x5FC5;&#x8981;&#x3068;&#x3055;&#x308C;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x540C;&#x69D8;&#x306B;&#x3001;Cep152&#x3001;&#x810A;&#x690E;&#x52D5;&#x7269;&#x306B;&#x304A;&#x3051;&#x308B;ASL&#x30AA;&#x30EB;&#x30BD;&#x30ED;&#x30B0;&#x306F;&#x3001;&#x7E4A;&#x6BDB;&#x306E;&#x5F62;&#x6210;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3042;&#x308A;&#x3001;&#x305D;&#x306E;&#x6A5F;&#x80FD;&#x306F;&#x90E8;&#x5206;&#x7684;&#x306B;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;ASL&#x306B;&#x3088;&#x3063;&#x3066;&#x6551;&#x52A9;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002; ASL&#x5C40;&#x5728;&#x306E;&#x7814;&#x7A76;&#x306F;&#x3001;&#x305D;&#x308C;&#x304C;&#x5BC6;&#x63A5;&#x306B;&#x4E2D;&#x5FC3;&#x5C0F;&#x4F53;&#x306E;&#x58C1;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x308B;&#x304C;&#x3001;&#x4E2D;&#x5FC3;&#x5C0F;&#x4F53;&#x69CB;&#x9020;&#x306E;&#x4E00;&#x90E8;&#x3067;&#x306F;&#x3042;&#x308A;&#x307E;&#x305B;&#x3093;&#x3002; ASL&#x306E;&#x67AF;&#x6E07;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;&#x4E2D;&#x5FC3;&#x4F53;&#x306E;&#x751F;&#x5408;&#x6210;&#x3092;&#x5206;&#x6790;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x6211;&#x3005;&#x306F;&#x4E2D;&#x5FC3;&#x4F53;&#x5468;&#x8FBA;&#x7269;&#x8CEA;&#xFF08;PCM&#xFF09;&#x95A2;&#x6570;&#x306F;&#x3001;&#x8EFD;&#x5EA6;&#x306E;&#x5F71;&#x97FF;&#x3092;&#x53D7;&#x3051;&#x3066;&#x3044;&#x308B;&#x9593;&#x306B;&#x3001;ASL&#x304C;&#x5A18;&#x4E2D;&#x5FC3;&#x5C0F;&#x4F53;&#x5F62;&#x6210;&#x306E;&#x305F;&#x3081;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x308F;&#x304B;&#x3063;&#x305F;&#x3002; mecD&#x5909;&#x7570;&#x4F53;&#x306E;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;centriolar&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x306E;&#x660E;&#x78BA;&#x306A;&#x6B20;&#x5982;&#x306F;&#x3001;ASL&#x304C;&#x65E9;&#x671F;&#x306B;&#x4E2D;&#x5FC3;&#x5C0F;&#x4F53;&#x306E;&#x8907;&#x88FD;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;

&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;Asterless&#x3068;&#x810A;&#x690E;&#x52D5;&#x7269;Cep152&#x306F;&#x4E2D;&#x5FC3;&#x5C0F;&#x4F53;&#x306E;&#x8907;&#x88FD;&#x306E;&#x305F;&#x3081;&#x306E;&#x30AA;&#x30EB;&#x30BD;&#x30ED;&#x30B0;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3042;&#x308B;

&#xC911;&#xC2EC;&#xC18C;&#xCCB4; centrosome cilium &#xD575;&#xC2EC; &#xAD6C;&#xC870; &#xC774;&#xB2E4;. &#xC911;&#xC2EC;&#xC18C;&#xCCB4; &#xC911;&#xBCF5; &#xC138;&#xD3EC; &#xC8FC;&#xAE30; &#xB9C8;&#xB2E4; &#xC81C;&#xD55C; &#xC2E4;&#xD328; &#xC2EC;&#xAC01;&#xD55C; &#xACB0;&#xACFC;&#xAC00;&#xC9C0;&#xACE0; &#xD558; &#xACE0; &#xC554;&#xC5D0; &#xC77C;&#xBC18;&#xC801;&#xC73C;&#xB85C; &#xAD00;&#xCC30; &#xD588;&#xB2E4;. &#xC758;&#xB8CC;&#xC758; &#xC911;&#xC694;&#xC131;&#xC5D0;&#xB3C4; &#xBD88;&#xAD6C; &#xD558; &#xACE0; &#xC911;&#xC2EC;&#xC18C;&#xCCB4; &#xD615;&#xC131;&#xC758; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC740; &#xC81C;&#xB300;&#xB85C; &#xC774;&#xD574; &#xB41C;&#xB2E4;. Asl&#xB294; &#xC774;&#xC804;&#xC5D0; centrosomal &#xB2E8;&#xBC31;&#xC9C8; centrosome &#xAE30;&#xB2A5;&#xC5D0; &#xD544;&#xC218;&#xC801;&#xC778; &#xAC83;&#xC73C;&#xB85C; &#xBCF4;&#xACE0; &#xB429;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; mecD, asl &#xC720;&#xC804;&#xC790;&#xC758; &#xC2EC;&#xAC01;&#xD55C; &#xAE30;&#xB2A5; &#xC190;&#xC2E4; &#xB300;&#xB9BD; &#xC720;&#xC804;&#xC790; &#xC2DD;&#xBCC4; &#xACE0; &#xC911;&#xC2EC;&#xC18C;&#xCCB4;&#xC640; &#xC18D;&#xB208;&#xC379; &#xD615;&#xC131;&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xC784;&#xC744; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xB9C8;&#xCC2C;&#xAC00;&#xC9C0;&#xB85C;, Cep152, &#xCC99;&#xCD94; &#xB3D9;&#xBB3C;, Asl ortholog &#xC18D;&#xB208;&#xC379; &#xD615;&#xC131;&#xC5D0; &#xD544;&#xC218;&#xC801; &#xC774;&#xBA70; &#xADF8; &#xAE30;&#xB2A5;&#xC740; &#xBD80;&#xBD84;&#xC801;&#xC73C;&#xB85C; Drosophila Asl&#xC5D0; &#xC758;&#xD574; &#xAD6C;&#xCD9C; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. Asl &#xC9C0;&#xC5ED;&#xD654; &#xC5F0;&#xAD6C; &#xC81C;&#xC548; &#xADF8;&#xAC83; &#xC911;&#xC2EC;&#xC18C;&#xCCB4; &#xBCBD;&#xC5D0; &#xBC00;&#xC811; &#xD558; &#xAC8C; &#xC5F0;&#xAD00; &#xD558;&#xC9C0;&#xB9CC; &#xC911;&#xC2EC;&#xC18C;&#xCCB4; &#xAD6C;&#xC870;&#xC5D0; &#xC18D;&#xD558;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. Asl&#xC758; &#xACE0;&#xAC08; &#xC140;&#xC5D0; centrosomes &#xC18D; &#xBD84;&#xC11D; &#xD558; &#xC5EC; &#xC6B0;&#xB9AC;, pericentriolar &#xC18C;&#xC7AC; (PCM) &#xAE30;&#xB2A5;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xC57D;&#xAC04; &#xB3D9;&#xC548; Asl &#xB538; &#xC911;&#xC2EC;&#xC18C;&#xCCB4; &#xD615;&#xC131;&#xC744; &#xC704;&#xD574; &#xD544;&#xC218;&#xC801;&#xC785;&#xB2C8;&#xB2E4; &#xBC1C;&#xACAC;. &#xC5EC;&#xB7EC; &#xAC1C;&#xC758; centriolar &#xB9C8;&#xCEE4; mecD &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC758; &#xBA85;&#xD655;&#xD55C; &#xBD80;&#xC7AC; Asl &#xC911;&#xC2EC;&#xC18C;&#xCCB4; &#xC911;&#xBCF5; &#xCD08;&#xAE30;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xB098;&#xC654;&#xB2E4;.

Drosophila asterless &#xBC0F; &#xCC99;&#xCD94; cep152&#xB294; orthologs &#xC911;&#xC2EC;&#xC18C;&#xCCB4; &#xC911;&#xBCF5;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;.

El centr&#xED;olo es la estructura b&#xE1;sica de centrosoma y cilio. Omisi&#xF3;n de restringir el centr&#xED;olo la duplicaci&#xF3;n de una vez por ciclo celular tiene consecuencias graves y se observa con frecuencia en el c&#xE1;ncer. A pesar de su importancia m&#xE9;dica, el mecanismo de formaci&#xF3;n centr&#xED;olo es poco conocida. El nivel del mar se hab&#xED;a informado anteriormente que es una prote&#xED;na esencial para la funci&#xF3;n centrosomal centrosoma. Aqu&#xED; identificamos MECD, una grave p&#xE9;rdida de la funci&#xF3;n alelo del gen de nivel del mar, y demostrar que se requiere para centr&#xED;olo y la formaci&#xF3;n de los cilios. Del mismo modo, Cep152, el ortholog Asl en los vertebrados, es esencial para la formaci&#xF3;n de cilios y su funci&#xF3;n puede ser parcialmente rescatado por la ASL de Drosophila. El estudio de la localizaci&#xF3;n Asl sugiere que est&#xE1; estrechamente asociado con la pared centr&#xED;olo, pero no es parte de la estructura centr&#xED;olo. Mediante el an&#xE1;lisis de la biog&#xE9;nesis de los centrosomas en c&#xE9;lulas agotadas de nivel del mar, se encontr&#xF3; que, mientras que el material pericentriolar (PCM), la funci&#xF3;n est&#xE1; levemente afectado, el ASL es esencial para la formaci&#xF3;n de su hija centr&#xED;olo. La clara ausencia de varios marcadores centriolares en mutantes del MECD sugiere que el ASL es cr&#xED;tico al inicio centr&#xED;olo duplicaci&#xF3;n.

Drosophila y vertebrados Asterless Cep152 son Orthologs esencial para la duplicaci&#xF3;n de centr&#xED;olos

We have investigated the expression and function of the Sox15 transcription factor during the development of the external mechanosensory organs of Drosophila. We find that Sox15 is expressed specifically in the socket cell, and have identified the transcriptional cis-regulatory module that controls this activity. We show that Suppressor of Hairless [Su(H)] and the POU-domain factor Ventral veins lacking (Vvl) bind conserved sites in this enhancer and provide critical regulatory input. In particular, we find that Vvl contributes to the activation of the enhancer following relief of Su(H)-mediated default repression by the Notch signaling event that specifies the socket cell fate. Loss of Sox15 gene activity was found to severely impair the electrophysiological function of mechanosensory organs, due to both cell-autonomous and cell-non-autonomous effects on the differentiation of post-mitotic cells in the bristle lineage. Lastly, we find that simultaneous loss of both Sox15 and the autoregulatory activity of Su(H) reveals an important role for these factors in inhibiting transcription of the Pax family gene shaven in the socket cell, which serves to prevent inappropriate expression of the shaft differentiation program. Our results indicate that the later phases of socket cell differentiation are controlled by multiple transcription factors in a collaborative, and not hierarchical, manner.

Complex interplay of three transcription factors in controlling the tormogen differentiation program of Drosophila mechanoreceptors.

Most animals have two centrioles in spermatids (the distal and proximal centrioles), but insect spermatids seem to contain only one centriole (Fuller 1993), which functionally resembles the distal centriole. Using fluorescent centriolar markers, we identified a structure near the fly distal centriole that is reminiscent of a proximal centriole (i.e., proximal centriole-like, or PCL). We show that the PCL exhibits several features of daughter centrioles. First, a single PCL forms near the proximal segment of the older centriole. Second, the centriolar proteins SAS-6, Ana1, and Bld10p/Cep135 are in the PCL. Third, PCL formation depends on SAK/PLK4 and SAS-6. Using a genetic screen for PCL defect, we identified a mutation in the gene encoding the conserved centriolar protein POC1, which is part of the daughter centriole initiation site (Kilburn et al. 2007) in Tetrahymena. We conclude that the PCL resembles an early intermediate structure of a forming centriole, which may explain why no typical centriolar structure is observed under electron microscopy. We propose that, during the evolution of insects, the proximal centriole was simplified by eliminating the later steps in centriole assembly. The PCL may provide a unique model to study early steps of centriole formation.

A proximal centriole-like structure is present in Drosophila spermatids and can serve as a model to study centriole duplication.

Centrioles are conserved microtubule-based organelles with 9-fold symmetry that are essential for cilia and mitotic spindle formation. A conserved structure at the onset of centriole assembly is a "cartwheel" with 9-fold radial symmetry and a central tubule in its core. It remains unclear how the cartwheel is formed. The conserved centriole protein, SAS-6, is a cartwheel component that functions early in centriole formation. Here, combining biochemistry and electron microscopy, we characterize SAS-6 and show that it self-assembles into stable tetramers, which serve as building blocks for the central tubule. These results suggest that SAS-6 self-assembly may be an initial step in the formation of the cartwheel that provides the 9-fold symmetry. Electron microscopy of centrosomes identified 25-nm central tubules with repeating subunits and show that SAS-6 concentrates at the core of the cartwheel. Recombinant and native SAS-6 self-oligomerizes into tetramers with approximately 6-nm subunits, and these tetramers are components of the centrosome, suggesting that tetramers are the building blocks of the central tubule. This is further supported by the observation that elevated levels of SAS-6 in Drosophila cells resulted in higher order structures resembling central tubule morphology. Finally, in the presence of embryonic extract, SAS-6 tetramers assembled into high density complexes, providing a starting point for the eventual in vitro reconstruction of centrioles.

Self-assembling SAS-6 multimer is a core centriole building block.

The cellular and developmental program connecting the centrosome and cilium duplication cycle.

Centrosomes are conserved organelles that are essential for accurate cell division and cilium formation. A centrosome consists of a pair of centrioles surrounded by a protein network of pericentriolar material (PCM) that is essential for the centrosome's function. In this study, we show that Sas-4 provides a scaffold for cytoplasmic complexes (named S-CAP), which include CNN, Asl and D-PLP, proteins that are all found in the centrosomes at the vicinity of the centriole. When Sas-4 is absent, nascent procentrioles are unstable and lack PCM, and functional centrosomes are not generated. When Sas-4 is mutated, so that it cannot form S-CAP complexes, centrosomes are present but with dramatically reduced levels of PCM. Finally, purified S-CAP complexes or recombinant Sas-4 can bind centrosomes stripped of PCM, whereas recombinant CNN or Asl cannot. In summary, PCM assembly begins in the cytosol where Sas-4 provides a scaffold for pre-assembled cytoplasmic complexes before tethering of the complexes in a centrosome.

Sas-4 provides a scaffold for cytoplasmic complexes and tethers them in a centrosome.

Regulated centrosome biogenesis is required for accurate cell division and for maintaining genome integrity. Centrosomes consist of a centriole pair surrounded by a protein network known as pericentriolar material (PCM). PCM assembly is a tightly regulated, critical step that determines the size and capability of centrosomes. Here, we report a role for tubulin in regulating PCM recruitment through the conserved centrosomal protein Sas-4. Tubulin directly binds to Sas-4; together they are components of cytoplasmic complexes of centrosomal proteins. A Sas-4 mutant, which cannot bind tubulin, enhances centrosomal protein complex formation and has abnormally large centrosomes with excessive activity. These results suggest that tubulin negatively regulates PCM recruitment. Whereas tubulin-GTP prevents Sas-4 from forming protein complexes, tubulin-GDP promotes it. Thus, the regulation of PCM recruitment by tubulin depends on its GTP/GDP-bound state. These results identify a role for tubulin in regulating PCM recruitment independent of its well-known role as a building block of microtubules. On the basis of its guanine-bound state, tubulin can act as a molecular switch in PCM recruitment.

Tubulin nucleotide status controls Sas-4-dependent pericentriolar material recruitment.

Centrioles are the key foundation of centrosomes and cilia, yet a molecular understanding of how they form has only recently begun to emerge. Building a fully functional centriole that can form a centrosome and cilium requires two cell cycles. Centriole building starts with procentriole nucleation, a process that is coordinated by the conserved proteins Plk4/Zyg-1, and Asterless/Cep152. Subsequently, Sas-6, a conserved procentriole protein, self-assembles to provide nine-fold symmetry to the centriole scaffold. The procentriole then continues to elongate into a centriole, a process controlled by Sas-4/CPAP and CP110. Then, centrioles recruit Sas-4-mediated pre-assembled centrosomal complexes from the cytoplasm to form the pericentriolar material (PCM). Finally, CP110 and its interacting proteins are involved in controlling the timing of centriole templating of the cilium.

Article	Year
Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis. Cell\| PubMed ID: 15137945	2004
Regulation of adenylate cyclase type VIII splice variants by acute and chronic Gi/o-coupled receptor activation. The Biochemical journal\| PubMed ID: 15537392	2005
Inhibition and superactivation of the calcium-stimulated isoforms of adenylyl cyclase: role of Gbetagamma dimers. Journal of molecular neuroscience : MN\| PubMed ID: 16186630	2005
Drosophila asterless and vertebrate Cep152 Are orthologs essential for centriole duplication. Genetics\| PubMed ID: 18854586	2008
Complex interplay of three transcription factors in controlling the tormogen differentiation program of Drosophila mechanoreceptors. Developmental biology\| PubMed ID: 19232522	2009
A proximal centriole-like structure is present in Drosophila spermatids and can serve as a model to study centriole duplication. Genetics\| PubMed ID: 19293139	2009
Self-assembling SAS-6 multimer is a core centriole building block. The Journal of biological chemistry\| PubMed ID: 20083610	2010
The cellular and developmental program connecting the centrosome and cilium duplication cycle. Seminars in cell & developmental biology\| PubMed ID: 20117227	2010
Sas-4 provides a scaffold for cytoplasmic complexes and tethers them in a centrosome. Nature communications\| PubMed ID: 21694707	2011
Tubulin nucleotide status controls Sas-4-dependent pericentriolar material recruitment. Nature cell biology\| PubMed ID: 22729084	2012
Building a centriole. Current opinion in cell biology\| PubMed ID: 23199753	2013

Tomer Avidor-Reiss

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