tracking-cells-gfp-transgenic-zebrafish-using-photoconvertible

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Heidelberg University

&#x5728;&#x7EC6;&#x80DE;&#x6838;&#x4E2D;&#x7684;&#x7C92;&#x5B50;&#x8FD0;&#x52A8;&#x53EF;&#x4EE5;&#x53D7;&#x7C98;&#x5EA6;&#x3001; &#x5B9A;&#x5411;&#x7684;&#x6D41;&#x52A8;&#x3001; &#x4E3B;&#x52A8;&#x8FD0;&#x8F93;&#x6216;&#x5982;&#x67D3;&#x8272;&#x8D28;&#x7F51;&#x7EDC;&#x969C;&#x788D;&#x7684;&#x5B58;&#x5728;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x8C03;&#x67E5;&#x7684;&#x6D41;&#x52A8;&#x6027;&#x5C0F;&#x7684;&#x8367;&#x5149;&#x86CB;&#x767D;&#x662F;&#x5426;&#x53D7;&#x67D3;&#x8272;&#x8D28;&#x5BC6;&#x5EA6;&#x3002;&#x6269;&#x6563;&#x7684;&#x60F0;&#x6027;&#x7684;&#x8367;&#x5149;&#x86CB;&#x767D;&#x7814;&#x7A76;&#x751F;&#x6D3B;&#x7EC6;&#x80DE;&#x7EC6;&#x80DE;&#x6838;&#x8367;&#x5149;&#x76F8;&#x5173;&#x5149;&#x8C31; (FCS) &#x4F7F;&#x7528;&#x53CC;&#x8272;&#x5171;&#x7126;&#x626B;&#x63CF;&#x68C0;&#x6D4B;&#x7CFB;&#x7EDF;&#x3002;&#x6211;&#x4EEC;&#x9996;&#x5148;&#x4ECB;&#x7ECD;&#x5B9E;&#x9A8C;&#x63ED;&#x9732; FCS &#x7279;&#x5B9A;&#x5DE5;&#x4EF6;&#x6240;&#x9047;&#x5230;&#x7684;&#x6D3B;&#x7EC6;&#x80DE;&#x7814;&#x7A76;&#xFF0C;&#x4EE5;&#x53CA;&#x6218;&#x7565;&#xFF0C;&#x4EE5;&#x9632;&#x6B62;&#x5B83;&#x4EEC;&#xFF0C;&#x7279;&#x522B;&#x662F;&#x90A3;&#x4E9B;&#x56E0;&#x8367;&#x5149;&#x7528;&#x4E8E;&#x6821;&#x51C6;&#x7684;&#x7126;&#x5377;&#xFF0C;&#x4EE5;&#x53CA;&#x6E29;&#x5EA6;&#x548C;&#x6536;&#x8D2D;&#x6761;&#x4EF6;&#x7528;&#x4E8E;&#x8367;&#x5149;&#x6CE2;&#x52A8;&#x6D4B;&#x91CF;&#x65B9;&#x6CD5;&#x7684;&#x9009;&#x62E9;&#x3002;&#x5B9A;&#x4E49;&#x4E4B;&#x540E;&#x6700;&#x597D;&#x7684;&#x6536;&#x8D2D;&#x6761;&#x4EF6;&#xFF0C;&#x6211;&#x4EEC;&#x7684;&#x5404;&#x79CD;&#x4EBA;&#x7C7B;&#x7EC6;&#x80DE; GFP &#x7684;&#x6D41;&#x52A8;&#x6027;&#x5185;&#x7EC6;&#x80DE;&#x7684;&#x7EC6;&#x80DE;&#x6838;&#xFF0C;&#x5DEE;&#x522B;&#x5F88;&#x5927;&#xFF0C;&#x4F46;&#x4E0D;&#x4F1A;&#x4E0D;&#x4E0E;&#x67D3;&#x8272;&#x8D28;&#x5BC6;&#x5EA6;&#x5173;&#x8054;&#x663E;&#x793A;&#x3002;&#x6838;&#x5185;&#x5305;&#x6DB5;&#x6269;&#x6563;&#x578B;&#x6D41;&#x52A8;&#x6027;&#x5F3A;&#x70C8;&#x53D6;&#x51B3;&#x4E8E;&#x86CB;&#x767D;&#x5927;&#x5C0F;&#xFF1A; &#x5728;&#x4E00;&#x7CFB;&#x5217;&#x7684;&#x7EFF;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D;-&#x4F4E;&#x805A;&#x7269;&#xFF0C;&#x7528;&#x4F5C;&#x81EA;&#x7531;&#x60F0;&#x6027;&#x7684;&#x8367;&#x5149;&#x793A;&#x8E2A;&#x5242;&#xFF0C;&#x6269;&#x6563;&#x7CFB;&#x6570;&#x4E0B;&#x964D;&#x4ECE;&#x5355;&#x4F53;&#x5230;&#x8FDC;&#x8FDC;&#x8D85;&#x8FC7;&#x9884;&#x671F;&#x5206;&#x5B50;&#x53EF;&#x4EE5;&#x514D;&#x8D39;&#x5728;&#x6C34;&#x6EB6;&#x6DB2;&#x4E2D;&#x7684;&#x56DB;&#x805A;&#x4F53;&#x3002;&#x4ECD;&#x7136;&#xFF0C;&#x6574;&#x4E2A;&#x6838;&#x5185;&#x5305;&#x6DB5;&#x67D3;&#x8272;&#x8D28;&#x7F51;&#x7EDC;&#x662F;&#x81EA;&#x7531;&#x5F00;&#x653E;&#x5C0F;&#x86CB;&#x767D;&#x8D28;&#x7531;&#x56DB;&#x805A;&#x7269;&#x7EFF;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D;-&#x72B6;&#x6001;&#xFF0C;&#x65E0;&#x8BBA;&#x67D3;&#x8272;&#x8D28;&#x5BC6;&#x5EA6;&#x6216;&#x5355;&#x5143;&#x683C;&#x7684;&#x884C;&#x7684;&#x5927;&#x5C0F;&#x3002;&#x751A;&#x81F3;&#x6700;&#x5BC6;&#x96C6;&#x7684;&#x67D3;&#x8272;&#x8D28;&#x533A;&#x57DF;&#x4E0D;&#x6392;&#x9664;&#x514D;&#x8D39;&#x7EFF;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D;&#x5355;&#x4F53;&#x6216;&#x591A;&#x805A;&#x4F53;&#x3002;

&#x5728;&#x751F;&#x6D3B;&#x4E2D;&#x7684;&#x4F4E;&#x805A;&#x7269;&#x8C03;&#x52A8;&#x6620;&#x5C04;&#x7EFF;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D;&#x7EC6;&#x80DE;&#x539F;&#x5B50;&#x6838;&#x3002;

Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers.

Mapping eGFP oligomer mobility in living cell nuclei.

Mouvement des particules dans les noyaux des cellules peut &ecirc;tre affect&eacute;e par la viscosit&eacute;, orient&eacute;es flux, transport actif ou la pr&eacute;sence d&#39;obstacles tels que le r&eacute;seau de la chromatine. Ici, nous examinons si la mobilit&eacute; des petites prot&eacute;ines fluorescentes est affect&eacute;e par la densit&eacute; de la chromatine. Diffusion des prot&eacute;ines fluorescentes inertes a &eacute;t&eacute; &eacute;tudi&eacute;e chez les vivants &agrave; l&#39;aide de la spectroscopie de corr&eacute;lation de fluorescence (FCS) avec un syst&egrave;me de d&eacute;tection de balayage confocal bicolore de noyaux cellulaires. Nous pr&eacute;sentons d&#39;abord exposer les artefacts de FCS sp&eacute;cifiques rencontr&eacute;s dans les &eacute;tudes de cellules vivantes ainsi que des strat&eacute;gies pour les pr&eacute;venir, en particulier ceux qui d&eacute;coulent du choix du fluorophore utilis&eacute;s pour l&#39;&eacute;talonnage de la focal volume, ainsi que la temp&eacute;rature et des conditions d&#39;acquisition utilis&eacute;es pour mesures de fluorescence fluctuation des exp&eacute;riences. Apr&egrave;s avoir d&eacute;fini les meilleures conditions d&#39;acquisition, nous montrons pour diverses lign&eacute;es cellulaires humaines que la mobilit&eacute; des GFP varie consid&eacute;rablement au sein du noyau cellulaire, mais n&#39;est pas corr&eacute;l&eacute;e avec la densit&eacute; de la chromatine. La mobilit&eacute; intranucl&eacute;aire de diffusion d&eacute;pend fortement de la taille de la prot&eacute;ine : dans une s&eacute;rie de GFP-oligom&egrave;res, utilis&eacute;s comme des traceurs fluorescents inertes gratuits, le coefficient de diffusion pass&eacute;e de monom&egrave;re &agrave; t&eacute;tram&egrave;re de beaucoup plus que pr&eacute;vu pour les mol&eacute;cules libres en solution aqueuse. Pourtant, le r&eacute;seau entier de chromatine intranucl&eacute;aire est librement accessible aux petites prot&eacute;ines jusqu&#39;&agrave; la taille d&#39;eGFP-t&eacute;tram&egrave;res, quelle que soit la densit&eacute; de la chromatine ou lign&eacute;e cellulaire. M&ecirc;me les plus denses r&eacute;gions de chromatine n&#39;excluent pas eGFP-monom&egrave;res libres ou multim&egrave;res.

Cartographie eGFP mobilit&eacute; oligom&egrave;re dans la vie de noyaux cellulaires.

Bewegung von Teilchen in Zellkerne kann von Viskosit&auml;t, gerichtete Str&ouml;me, aktiven Transport oder das Vorhandensein von Hindernissen wie das Chromatin-Netzwerk beeinflusst werden. Hier untersuchen wir, ob die Mobilit&auml;t der kleinen fluoreszierende Proteine von der Chromatin-Dichte betroffen ist. Verbreitung der inerten fluoreszierende Proteine wurde untersucht, in lebenden Zellen-Kerne mit Fluoreszenz-Korrelations-Spektroskopie (FCS) mit einem zweifarbigen konfokale Scan Detection-System. Zuerst stellen wir Experimente Verf&uuml;gbarmachen von FCS-spezifische Artefakte gefunden in Leben Zelle Studien sowie Strategien zur Verh&uuml;tung, insbesondere aus der Wahl der Fluorophor verwendet f&uuml;r die Kalibrierung der fokalen Volumen sowie Temperatur und &Uuml;bernahme-Bedingungen verwendet f&uuml;r Fluoreszenzmessungen Fluktuation resultieren. Nach der Definition der besten &Uuml;bernahme-Bedingungen, zeigen wir f&uuml;r verschiedene menschliche Zelllinien, die die Mobilit&auml;t der GFP im Zellkern stark schwankt, aber nicht mit Chromatin Dichte korrelieren. Die intranukle&auml;ren Diffusionsprozess Mobilit&auml;t h&auml;ngt stark von Protein-Gr&ouml;&szlig;e: in einer Reihe von GFP-Oligomere, als freie inert Fluoreszenz Tracer, verringerte sich der Diffusionskoeffizient von dem Monomer auf das Tetramer, viel mehr als erwartet f&uuml;r Molek&uuml;le in w&auml;ssriger L&ouml;sung kostenlos. Dennoch ist das gesamte intranukle&auml;ren Chromatin-Netzwerk f&uuml;r kleine Proteine bis zur Gr&ouml;&szlig;e des eGFP-tetrameren, unabh&auml;ngig von der Chromatin-Dichte oder Zelllinie frei zug&auml;nglich. Selbst die dichtesten Chromatin Regionen ausschlie&szlig;en kostenlose eGFP-Monomere oder Multimere nicht.

Zuordnung eGFP Oligomer Mobilit&auml;t in der lebenden Zelle Kerne.

Movimento delle particelle nei nuclei delle cellule pu&ograve; essere influenzata dalla viscosit&agrave;, flussi diretti, trasporto attivo o la presenza di ostacoli quali la rete della cromatina. Qui Indaghiamo se la mobilit&agrave; delle piccole proteine fluorescenti &egrave; influenzata dalla densit&agrave; della cromatina. Diffusione delle proteine fluorescenti inerte &egrave; stato studiato in vivo delle cellule nuclei mediante spettroscopia di correlazione di fluorescenza (FCS) con un sistema di rilevazione di scansione confocale a due colori. Vi presentiamo primi esperimenti esponendo manufatti FCS-specifiche incontrate nella cella live studi, nonch&eacute; strategie per prevenirli, in particolare quelli derivanti dalla scelta del fluoroforo utilizzato per la taratura del volume focale, cos&igrave; come la temperatura e le condizioni di acquisizione utilizzate per le misurazioni di fluttuazione di fluorescenza. Dopo aver definito le migliori condizioni di acquisto, vi mostriamo per diverse linee di cellule umane che la mobilit&agrave; di GFP varia in modo significativo all&#39;interno del nucleo delle cellule, ma non correlare con densit&agrave; della cromatina. La mobilit&agrave; intranucleari diffusione dipende fortemente dalla dimensione della proteina: in una serie di GFP-oligomeri, usati come traccianti fluorescenti inerte liberi, il coefficiente di diffusione &egrave; diminuito da monomero a tetramero molto pi&ugrave; del previsto per libero molecole in soluzione acquosa. Ancora, la rete intera intranucleari cromatina &egrave; liberamente accessibile per piccole proteine fino alla dimensione di eGFP-tetrameri, indipendentemente dalla densit&agrave; della cromatina o linea cellulare. Anche le regioni di cromatina pi&ugrave; densi non escludono eGFP-monomeri liberi o multimeri.

Mappatura eGFP mobilit&agrave; oligomero nella vita delle cellule di nuclei.

&#x7D30;&#x80DE;&#x6838;&#x306B;&#x304A;&#x3051;&#x308B;&#x7C92;&#x5B50;&#x306E;&#x904B;&#x52D5;&#x306E;&#x5F71;&#x97FF;&#x7C98;&#x5EA6;, &#x6709;&#x5411;&#x30D5;&#x30ED;&#x30FC;&#x3001;&#x80FD;&#x52D5;&#x8F38;&#x9001;&#x307E;&#x305F;&#x306F;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3; &#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x306A;&#x3069;&#x306E;&#x969C;&#x5BB3;&#x7269;&#x306E;&#x5B58;&#x5728;&#x3092;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x5C0F;&#x3055;&#x306A;&#x86CD;&#x5149;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x30E2;&#x30D3;&#x30EA;&#x30C6;&#x30A3; &#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x5BC6;&#x5EA6;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F71;&#x97FF;&#x3092;&#x53D7;&#x3051;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x3092;&#x8ABF;&#x67FB;&#x3057;&#x307E;&#x3059;&#x3002;&#x4E0D;&#x6D3B;&#x6027;&#x86CD;&#x5149;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x62E1;&#x6563;&#x306F;&#x3001;&#x30EA;&#x30D3;&#x30F3;&#x30B0;&#x3067;&#x691C;&#x8A0E;&#x3057;&#x305F;&#x7D30;&#x80DE;&#x306E;&#x86CD;&#x5149;&#x76F8;&#x95A2;&#x5206;&#x5149;&#x6CD5; (FCS) 2 &#x8272;&#x5171;&#x7126;&#x70B9;&#x8D70;&#x67FB;&#x578B;&#x691C;&#x51FA;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x7528;&#x3044;&#x305F;&#x6838;&#x3002;&#x307E;&#x305A;&#x3001;&#x305D;&#x308C;&#x3089;&#x3001;&#x7279;&#x306B;&#x3053;&#x308C;&#x3089;&#x306E;&#x7126;&#x70B9;&#x306E;&#x30DC;&#x30EA;&#x30E5;&#x30FC;&#x30E0;&#x3068;&#x3057;&#x3066;&#x6E29;&#x5EA6;&#x6821;&#x6B63;&#x7528; fluorophore &#x3068;&#x86CD;&#x5149;&#x63FA;&#x3089;&#x304E;&#x6E2C;&#x5B9A;&#x306B;&#x4F7F;&#x7528;&#x8CB7;&#x53CE;&#x6761;&#x4EF6;&#x306E;&#x9078;&#x629E;&#x304B;&#x3089;&#x767A;&#x751F;&#x3092;&#x9632;&#x3050;&#x305F;&#x3081;&#x306E;&#x6226;&#x7565;&#x3068;&#x540C;&#x69D8;&#x306B;&#x30E9;&#x30A4;&#x30D6;&#x7D30;&#x80DE;&#x7814;&#x7A76;&#x3067;&#x906D;&#x9047;&#x3057;&#x305F; FCS &#x56FA;&#x6709;&#x306E;&#x30A2;&#x30FC;&#x30C6;&#x30A3;&#x30D5;&#x30A1;&#x30AF;&#x30C8;&#x306E;&#x516C;&#x958B;&#x5B9F;&#x9A13;&#x3092;&#x63D0;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x6700;&#x9AD8;&#x306E;&#x8CB7;&#x53CE;&#x6761;&#x4EF6;&#x3092;&#x5B9A;&#x7FA9;&#x3057;&#x305F;&#x3089;&#x3001;GFP &#x306E;&#x30E2;&#x30D3;&#x30EA;&#x30C6;&#x30A3;&#x306F;&#x3001;&#x7D30;&#x80DE;&#x6838;&#x5185;&#x3067;&#x5927;&#x5E45;&#x306B;&#x7570;&#x306A;&#x308A;&#x307E;&#x3059;&#x304C;&#x3001;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x5BC6;&#x5EA6;&#x3068;&#x306E;&#x76F8;&#x95A2;&#x95A2;&#x4FC2;&#x306F;&#x306A;&#x3044;&#x5404;&#x7A2E;&#x306E;&#x3072;&#x3068;&#x7D30;&#x80DE;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x6838;&#x5185;&#x5C01;&#x5165;&#x4F53;&#x306E;&#x62E1;&#x6563;&#x79FB;&#x52D5;&#x6027;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x30B5;&#x30A4;&#x30BA;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F37;&#x304F;&#x6C7A;&#x307E;&#x308B;: &#x30B7;&#x30EA;&#x30FC;&#x30BA;&#x306E; GFP-&#x30AA;&#x30EA;&#x30B4;&#x30DE;&#x30FC;&#x7121;&#x6599;&#x306E;&#x4E0D;&#x6D3B;&#x6027;&#x86CD;&#x5149;&#x30C8;&#x30EC;&#x30FC;&#x30B5;&#x30FC;&#x3068;&#x3057;&#x3066;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x308B;&#x62E1;&#x6563;&#x4FC2;&#x6570;&#x6E1B;&#x5C11;&#x30E2;&#x30CE;&#x30DE;&#x30FC;&#x304B;&#x3089;&#x6C34;&#x6EB6;&#x6DB2;&#x4E2D;&#x306E;&#x5206;&#x5B50;&#x306E;&#x7121;&#x6599;&#x306E;&#x3088;&#x308A;&#x3082;&#x306F;&#x308B;&#x304B;&#x306B;&#x4E88;&#x60F3;&#x30C6;&#x30C8;&#x30E9;&#x30DE;&#x30FC;&#x3057;&#x307E;&#x3059;&#x3002;&#x305D;&#x308C;&#x3067;&#x3082;&#x3001;&#x5168;&#x4F53;&#x306E;&#x6838;&#x5185;&#x5C01;&#x5165;&#x4F53;&#x306E;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3; &#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF; eGFP-&#x30C6;&#x30C8;&#x30E9;&#x30DE;&#x30FC; &#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x5BC6;&#x5EA6;&#x306B;&#x304B;&#x304B;&#x308F;&#x3089;&#x305A;&#x30BB;&#x30EB;&#x30E9;&#x30A4;&#x30F3;&#x306E;&#x30B5;&#x30A4;&#x30BA;&#x307E;&#x3067;&#x5C0F;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x305F;&#x3081;&#x306E;&#x81EA;&#x7531;&#x306B;&#x30A2;&#x30AF;&#x30BB;&#x30B9;&#x53EF;&#x80FD;&#x3067;&#x3059;&#x3002;&#x6700;&#x3082;&#x9AD8;&#x5BC6;&#x5EA6;&#x306E;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x5730;&#x57DF;&#x3067;&#x3082;&#x7121;&#x6599; eGFP &#x30E2;&#x30CE;&#x30DE;&#x30FC;&#x307E;&#x305F;&#x306F;&#x591A;&#x91CF;&#x4F53;&#x3092;&#x9664;&#x5916;&#x3057;&#x306A;&#x3044;&#x3067;&#x304F;&#x3060;&#x3055;&#x3044;&#x3002;

EGFP &#x30DE;&#x30C3;&#x30D4;&#x30F3;&#x30B0; &#x30AA;&#x30EA;&#x30B4;&#x30DE;&#x30FC; &#x30E2;&#x30D3;&#x30EA;&#x30C6;&#x30A3; &#x30EA;&#x30D3;&#x30F3;&#x30B0;&#x3067;&#x30BB;&#x30EB;&#x306E;&#x6838;&#x3002;

&#xC138;&#xD3EC; &#xD575;&#xC5D0; &#xC788;&#xB294; &#xC785;&#xC790;&#xC758; &#xC6B4;&#xB3D9; &#xC810;&#xB3C4;, &#xAC10;&#xB3C5;&#xB41C; &#xD750;&#xB984;, &#xD65C;&#xC131; &#xC804;&#xC1A1; &#xB610;&#xB294; chromatin &#xB124;&#xD2B8;&#xC6CC;&#xD06C; &#xB4F1;&#xC758; &#xC7A5;&#xC560;&#xBB3C;&#xC758; &#xC874;&#xC7AC;&#xC5D0; &#xC758;&#xD574; &#xC601;&#xD5A5;&#xC744; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC; &#xC791;&#xC740; &#xD615;&#xAD11; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC774;&#xB3D9;&#xC131; chromatin &#xBC00;&#xB3C4; &#xC601;&#xD5A5;&#xC744; &#xBC1B;&#xB294; &#xC5EC;&#xBD80;&#xB97C; &#xC870;&#xC0AC; &#xD569;&#xB2C8;&#xB2E4;. &#xBD88;&#xD65C;&#xC131; &#xD615;&#xAD11; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xBCF4;&#xAE09;&#xC740; &#xC0DD;&#xD65C;&#xC5D0;&#xC11C; &#xACF5;&#xBD80; &#xD558;&#xB294; &#xC138;&#xD3EC; &#xD575; &#xB450; &#xC0C9; &#xACF5;&#xCD08;&#xC810; &#xB808;&#xC774;&#xC800; &#xC2A4;&#xCE94; &#xD0D0;&#xC9C0; &#xC2DC;&#xC2A4;&#xD15C;&#xC73C;&#xB85C; &#xD615;&#xAD11; &#xC0C1;&#xAD00; &#xBD84;&#xAD11;&#xD559; (FCS)&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;. &#xCC98;&#xC74C; &#xC120;&#xBB3C;&#xC774; &#xD2B9;&#xD788; &#xC628;&#xB3C4; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xCD08;&#xC810; &#xBCFC;&#xB968; &#xAD50;&#xC815;&#xC5D0; &#xC0AC;&#xC6A9; &#xB418;&#xB294; fluorophore &#xBC0F; &#xD615;&#xAD11; &#xBCC0;&#xB3D9; &#xCE21;&#xC815;&#xC5D0; &#xC0AC;&#xC6A9; &#xB418;&#xB294; &#xC778;&#xC218; &#xC870;&#xAC74; &#xC120;&#xD0DD;&#xC5D0;&#xC11C; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xADF8;, &#xADF8;&#xB4E4;&#xC744; &#xBC29;&#xC9C0; &#xD558;&#xB294; &#xC804;&#xB7B5;&#xC73C;&#xB85C; &#xC0B4;&#xC544;&#xC788;&#xB294; &#xC138;&#xD3EC; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; FCS &#xD2B9;&#xC815; &#xC544;&#xD2F0;&#xD329;&#xD2B8;&#xB97C; &#xB178;&#xCD9C; &#xD558;&#xB294; &#xC2E4;&#xD5D8;. &#xCD5C;&#xACE0;&#xC758; &#xC778;&#xC218; &#xC870;&#xAC74;&#xC744; &#xC815;&#xC758; &#xD558; &#xACE0; &#xB098;&#xBA74; &#xC6B0;&#xB9AC;&#xB294; GFP&#xC758; &#xC774;&#xB3D9;&#xC131; &#xC138;&#xD3EC; &#xD575; &#xB0B4;&#xC5D0;&#xC11C; &#xD06C;&#xAC8C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4; &#xC788;&#xC9C0;&#xB9CC; chromatin &#xBC00;&#xB3C4;&#xC640; &#xC5F0;&#xAD00; &#xB418;&#xC9C0; &#xC54A;&#xC740; &#xB2E4;&#xC591; &#xD55C; &#xC778;&#xAC04;&#xC758; &#xC138;&#xD3EC; &#xB77C;&#xC778;&#xC5D0; &#xB300; &#xD55C; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. Intranuclear diffusional &#xC774;&#xB3D9;&#xC131; &#xAC15;&#xB825; &#xD558; &#xAC8C; &#xB2E8;&#xBC31;&#xC9C8; &#xD06C;&#xAE30;&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4;: GFP-&#xC62C;&#xB9AC;&#xACE0;, &#xBB34;&#xB8CC; &#xBD88;&#xD65C;&#xC131; &#xD615;&#xAD11; &#xCD94;&#xC801;&#xAE30;&#xB85C; &#xC0AC;&#xC6A9; &#xB418;&#xB294; &#xC77C;&#xB828;&#xC758; &#xD655;&#xC0B0; &#xACC4;&#xC218;&#xB294; &#xB2E8;&#xC704;&#xCCB4;&#xC5D0;&#xC11C; &#xAC10;&#xC18C; tetramer &#xBD84;&#xC790; &#xC6A9;&#xC561;&#xC5D0; &#xBB34;&#xB8CC;&#xB97C; &#xC608;&#xC0C1; &#xBCF4;&#xB2E4; &#xD6E8;&#xC52C; &#xB354;. &#xADF8;&#xB798;&#xB3C4;, &#xC804;&#xCCB4; intranuclear chromatin &#xB124;&#xD2B8;&#xC6CC;&#xD06C; chromatin &#xBC00;&#xB3C4; &#xB610;&#xB294; &#xC140; &#xB77C;&#xC778;&#xC5D0; &#xAD00;&#xACC4; &#xC5C6;&#xC774; eGFP tetramers &#xD06C;&#xAE30; &#xC791;&#xC740; &#xB2E8;&#xBC31;&#xC9C8;&#xC744; &#xC704;&#xD574; &#xC790;&#xC720;&#xB86D;&#xAC8C; &#xAC00;&#xB2A5;&#xD569;&#xB2C8;&#xB2E4;. &#xBB34;&#xB8CC; eGFP &#xB2E8;&#xC704;&#xCCB4; &#xB610;&#xB294; multimers densest chromatin &#xC601;&#xC5ED;&#xB3C4; &#xC81C;&#xC678; &#xD558;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;.

EGFP &#xB9E4;&#xD551; &#xC0DD;&#xD65C;&#xC5D0; &#xC62C;&#xB9AC;&#xACE0; &#xC774;&#xB3D9;&#xC131; &#xD575; &#xC138;&#xD3EC;.

Movimiento de part&iacute;culas en los n&uacute;cleos de c&eacute;lula puede verse afectada por la viscosidad, flujos dirigidos, transporte activo o la presencia de obst&aacute;culos como la red de cromatina. Aqu&iacute; Investigamos si la movilidad de peque&ntilde;as prote&iacute;nas fluorescentes se ve afectada por la densidad de la cromatina. Difusi&oacute;n de prote&iacute;nas fluorescentes inertes fue estudiado en la vida de n&uacute;cleos usando Espectroscopia de correlaci&oacute;n de fluorescencia (FCS) con un sistema de detecci&oacute;n exploraci&oacute;n confocal bicolor de la c&eacute;lula. Primero presentamos experimentos exponiendo artefactos FCS espec&iacute;ficos encontrados en estudios de c&eacute;lulas vivas, as&iacute; como estrategias para evitar que, en particularlas derivadas de la elecci&oacute;n del fluor&oacute;foro utilizado para la calibraci&oacute;n de la volumen focal, as&iacute; como la temperatura y las condiciones de adquisici&oacute;n utilizadas para la medici&oacute;n de fluctuaci&oacute;n de fluorescencia. Despu&eacute;s de definir las mejores condiciones de adquisici&oacute;n, mostramos para varias l&iacute;neas de c&eacute;lulas humanas que la movilidad de GFP var&iacute;a considerablemente dentro del n&uacute;cleo de la c&eacute;lula, pero no se correlaciona con la densidad de la cromatina. La movilidad difusional intranuclear depende en el tama&ntilde;o de la prote&iacute;na: en una serie de GFP-olig&oacute;meros, utilizados como trazadores fluorescentes inertes libres, el coeficiente de difusi&oacute;n disminuy&oacute; de mon&oacute;mero a la tetr&aacute;mero mucho m&aacute;s de lo esperado para mol&eacute;culas gratis en soluci&oacute;n acuosa. Todav&iacute;a, la red de cromatina intranucleares todo es libremente accesible para peque&ntilde;as prote&iacute;nas hasta el tama&ntilde;o de eGFP-tetr&aacute;meros, independientemente de la densidad de la cromatina o l&iacute;nea celular. Incluso las regiones m&aacute;s densas de la cromatina no excluyen gratis eGFP-mon&oacute;meros o Mult&iacute;meros.

Mapeo eGFP movilidad olig&oacute;mero en vida celular n&uacute;cleos.

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analysis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications.

Parvovirus induced alterations in nuclear architecture and dynamics.

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.

Protein diffusion in mammalian cell cytoplasm.

The habenular neural circuit is attracting increasing attention from researchers in fields as diverse as neuroscience, medicine, behavior, development, and evolution. Recent studies have revealed that this part of the limbic system in the dorsal diencephalon is involved in reward, addiction, and other behaviors and its impairment is associated with various neurological conditions and diseases. Since the initial description of the dorsal diencephalic conduction system (DDC) with the habenulae in its center at the end of the nineteenth century, increasingly sophisticated techniques have resolved much of its anatomy and have shown that these pathways relay information from different parts of the forebrain to the tegmentum, midbrain, and hindbrain. The first part of this review gives a brief historical overview on how the improving experimental approaches have allowed the stepwise uncovering much of the architecture of the habenula circuit as we know it today. Our brain distributes tasks differentially between left and right and it has become a paradigm that this functional lateralization is a universal feature of vertebrates. Moreover, task dependent differential brain activities have been linked to anatomical differences across the left-right axis in humans. A good way to further explore this fundamental issue will be to study the functional consequences of subtle changes in neural network formation, which requires that we fully understand DDC system development. As the habenular circuit is evolutionarily highly conserved, researchers have the option to perform such difficult experiments in more experimentally amenable vertebrate systems. Indeed, research in the last decade has shown that the zebrafish is well suited for the study of DDC system development and the phenomenon of functional lateralization. We will critically discuss the advantages of the zebrafish model, available techniques, and others that are needed to fully understand habenular circuit development.

Habenula circuit development: past, present, and future.

The conserved habenular neural circuit relays cognitive information from the forebrain into the ventral mid- and hindbrain. In zebrafish, the bilaterally formed habenulae in the dorsal diencephalon are made up of the asymmetric dorsal and symmetric ventral habenular nuclei, which are homologous to the medial and lateral nuclei respectively, in mammals. These structures have been implicated in various behaviors related to the serotonergic/dopaminergic neurotransmitter system. The dorsal habenulae develop adjacent to the medially positioned pineal complex. Their precursors differentiate into two main neuronal subpopulations which differ in size across brain hemispheres as signals from left-sided parapineal cells influence their differentiation program. Unlike the dorsal habenulae and despite their importance, the ventral habenulae have been poorly studied. It is not known which genetic programs underlie their development and why they are formed symmetrically, unlike the dorsal habenulae. A main reason for this lack of knowledge is that the vHb origin has remained elusive to date.

Article	Year
Mapping eGFP oligomer mobility in living cell nuclei. PloS one\| PubMed ID: 19347038	2009
Parvovirus induced alterations in nuclear architecture and dynamics. PloS one\| PubMed ID: 19536327	2009
Protein diffusion in mammalian cell cytoplasm. PloS one\| PubMed ID: 21886771	2011
Habenula circuit development: past, present, and future. Frontiers in neuroscience\| PubMed ID: 22536170	2012
The ventral habenulae of zebrafish develop in prosomere 2 dependent on Tcf7l2 function. Neural development\| PubMed ID: 24067090	2013

Nicolas Dross

Biography

JoVE Journal Publications

Other Publications