Visualizing Cytoplasmic Flow During Single-cell Wound Healing in <em>Stentor coeruleus</em>

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

MARINE BIOLOGICAL LABORATORY

Vrije Universiteit Amsterdam

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.

&#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;

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

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which is the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches are addressed, we first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.

A brief introduction to single-molecule fluorescence methods.

Förster Resonance Energy Transfer (FRET) is the phenomenon of non-radiative transfer of electronic excitations from a donor fluorophore to an acceptor, mediated by electronic dipole-dipole coupling. The transfer rate and, as a consequence, efficiency depend non-linearly on the distance between the donor and the acceptor. FRET efficiency can thus be used as an effective and accurate reporter of distance between two fluorophores and changes thereof. Over the last 50 years or so, FRET has been used as a spectroscopic ruler to measure conformations and conformational changes of biomolecules. More recently, FRET has been combined with microscopy, ultimately allowing measurement of FRET between a single donor and a single acceptor pair. In this review, we will explain the physical foundations of FRET and how FRET can be applied to biomolecules. We will highlight the power of the different FRET approaches by focusing on its application to the motor protein kinesin, which undergoes several conformational changes driven by enzymatic action, that ultimately result in unidirectional motion along microtubule filaments, driving active transport in the cell. Single-molecule and ensemble FRET studies of different aspects of kinesin have provided numerous insights into the complex chemomechanical mechanism of this fascinating protein.

Bram Prevo

Physiology Course

MARINE BIOLOGICAL LABORATORY

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Bram Prevo

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

MARINE BIOLOGICAL LABORATORY

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Physiology Course