imaging-cell-shape-change-in-living-drosophila-embryos

Imaging Cell Shape Change in Living <em>Drosophila</em> Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

imaging-intranuclear-actin-rods-live-heat-stressed-drosophila

Imaging Intranuclear Actin Rods in Live Heat Stressed <em>Drosophila</em> Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Baylor College of Medicine (BCM)

Baylor College of Medicine

Plasmalemmal &#x4FEE;&#x590D;&#x662F;&#x635F;&#x574F;&#x7684;&#x771F;&#x6838;&#x7EC6;&#x80DE;&#x751F;&#x5B58;&#x6240;&#x5FC5;&#x9700;&#x7684;&#x3002;&#x901A;&#x8FC7; plasmalemmal &#x4E2D;&#x65AD;&#x7ECF;&#x6D8C;&#x5165;&#x6FC0;&#x6D3B;&#x86CB;&#x767D;&#x9176;&#x3001; &#x56CA;&#x6CE1;&#x805A;&#x96C6;&#x5728;&#x75C5;&#x7076;&#x90E8;&#x4F4D;&#x548C;&#x819C;&#x878D;&#x5408;&#x86CB;&#x767D; &#xFF1B;&#x7ECF;&#x5927;&#x91CF;&#x6D8C;&#x5165;&#x8FD8;&#x542F;&#x52A8;&#x7ADE;&#x4E89;&#x51CB;&#x4EA1;&#x901A;&#x8DEF;&#x3002;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x7684;&#x67D3;&#x6599;&#x5C4F;&#x969C; (&#x76D6;&#x7AE0;) &#x5F62;&#x6210;&#x6765;&#x8BC4;&#x4F30; plasmalemmal &#x4FEE;&#x590D;&#xFF0C;&#x73B0;&#x5728;&#x62A5;&#x544A;&#x4E0E; neurites B104 &#x6D77;&#x9A6C;&#x7EC6;&#x80DE;&#x63A5;&#x8FD1;&#x65AD; &#xFF08;&lt; 50 &mu; m) &#x5411;&#x7D22;&#x739B;&#x5BC6;&#x5C01;&#x5728;&#x8F83;&#x4F4E;&#x7684;&#x9891;&#x7387;&#x548C;&#x51CF;&#x6162;&#x5230;&#x7EC6;&#x80DE;&#x76F8;&#x6BD4; neurites &#x65AD;&#x638C;&#x66F4;&#x8FDC; (&gt; 50 &mu; m) &#x4ECE;&#x7D22;&#x739B;&#x3002;&#x7C7B;&#x4F3C;&#x7269;&#x7684;&#x8425;&#x5730;&#xFF0C;&#x5305;&#x62EC;&#x86CB;&#x767D;&#x6FC0;&#x9176; A &#xFF08;PKA&#xFF09;-&#x5177;&#x4F53;&#x548C;&#x7279;&#x5B9A; Epac &#x7684;&#x8425;&#x5730;&#xFF0C;&#x6BCF;&#x4E2A;&#x589E;&#x52A0;&#x7684;&#x9891;&#x7387;&#x548C;&#x5BC6;&#x5C01;&#x7387;&#x548C;&#x751A;&#x81F3;&#x53EF;&#x4EE5;&#x542F;&#x52A8;&#x5BC6;&#x5C01;&#x5728;&#x6CA1;&#x6709;&#x7ECF;&#x6D8C;&#x5165;&#x4E24;&#x6A2A;&#x65AD;&#x7684;&#x8DDD;&#x79BB;&#x3002;&#x6B64;&#x5916;&#xFF0C;Epac &#x6FC0;&#x6D3B;&#x53C2;&#x4E0E; plasmalemmal &#x5BC6;&#x5C01;&#x8425;&#x4F9D;&#x8D56;&#x3001; &#x72EC;&#x7ACB; PKA &#x7684;&#x901A;&#x8DEF;&#x3002;&#x9891;&#x7387;&#x548C; plasmalemmal &#x5BC6;&#x5C01;&#x7387;&#x662F;&#x4E00;&#x79CD;&#x5C0F;&#x5206;&#x5B50;&#x6291;&#x5236;&#x5242;&#x6709;&#x9488;&#x5BF9;&#x6027;&#x7684;&#x5BF9;&#x5176;&#x50AC;&#x5316;&#x4E9A;&#x5355;&#x4F4D; (KT5720) &#x591A;&#x80BD;&#x6291;&#x5236;&#x5242;&#x6709;&#x9488;&#x5BF9;&#x6027;&#x7684;&#x5BF9;&#x5176;&#x76D1;&#x7BA1;&#x7684;&#x4E9A;&#x57FA; (PKI) &#x7684;&#x65B0;&#x578B; PKC (nPKC&eta; pseudosubstrate &#x7684;&#x7247;&#x6BB5;&#xFF09; &#x548C;&#x4E00;&#x79CD;&#x6297;&#x6C27;&#x5316;&#x5242; (&#x892A;&#x9ED1;&#x7D20;) &#x6291;&#x5236;&#x5242;&#x7684; PKA &#x4E0B;&#x8DCC;&#x3002;&#x7531;&#x4E8E;&#x8FD9;&#x4E9B;&#x548C;&#x5176;&#x4ED6;&#x6570;&#x636E;&#xFF0C;&#x6211;&#x4EEC;&#x5EFA;&#x8BAE;&#x6A21;&#x578B;&#x5197;&#x4F59;&#x5E76;&#x884C;&#x8DEF;&#x5F84;&#x4F9D;&#x8D56;&#x4E8E; Ca(2+) &#x7684; plasmalemmal &#x76D6;&#x7AE0;&#x7684; nPKCs&#x3001; &#x6E38;&#x79BB;&#x6C27;&#x5316;&#x3001; &#x548C;&#x8425;&#x6FC0;&#x6D3B; PKA &#x548C; Epac &#x90E8;&#x5206;&#x4ECB;&#x5BFC;&#x7684;&#x795E;&#x7ECF;&#x7EC6;&#x80DE;&#x635F;&#x4F24;&#x3002;&#x6211;&#x4EEC;&#x4EA6;&#x5EFA;&#x8BAE;&#x8FD9;&#x4E9B;&#x901A;&#x8DEF;&#x548C;&#x7269;&#x8D28;&#x7684;&#x8FDB;&#x5316;&#x8D77;&#x6E90;&#x662F;&#x4FEE;&#x590D; plasmalemmal &#x635F;&#x574F;&#x7684;&#x771F;&#x6838;&#x7EC6;&#x80DE;&#x4E2D;&#x3002;&#x66F4;&#x597D;&#x5730;&#x7406;&#x89E3;&#x56CA;&#x6CE1;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3001; &#x86CB;&#x767D;&#x8D28;&#x548C;&#x901A;&#x8DEF;&#x7684; plasmalemmal &#x5BC6;&#x5C01;&#x5E94;&#x5EFA;&#x8BAE;&#x65B0;&#x578B;&#x795E;&#x7ECF;&#x4FDD;&#x62A4;&#x7684;&#x6CBB;&#x7597;&#x5916;&#x4F24;&#x6027;&#x795E;&#x7ECF;&#x635F;&#x4F24;&#x548C;&#x795E;&#x7ECF;&#x9000;&#x884C;&#x6027;&#x75BE;&#x75C5;&#x3002;

&#x5BC6;&#x5C01; plasmalemmal &#x635F;&#x4F24;&#x795E;&#x7ECF;&#x5143;&#x548C;&#x5176;&#x4ED6;&#x771F;&#x6838;&#x7EC6;&#x80DE;&#x7684;&#x6A21;&#x578B;&#x3002;

Plasmalemmal repair is necessary for survival of damaged eukaryotic cells. Ca(2+) influx through plasmalemmal disruptions activates calpain, vesicle accumulation at lesion sites, and membrane fusion proteins; Ca(2+) influx also initiates competing apoptotic pathways. Using the formation of a dye barrier (seal) to assess plasmalemmal repair, we now report that B104 hippocampal cells with neurites transected nearer (<50 Î¼m) to the soma seal at a lower frequency and slower rate compared to cells with neurites transected farther (>50 Î¼m) from the soma. Analogs of cAMP, including protein kinase A (PKA)-specific and Epac-specific cAMP, each increase the frequency and rate of sealing and can even initiate sealing in the absence of Ca(2+) influx at both transection distances. Furthermore, Epac activates a cAMP-dependent, PKA-independent, pathway involved in plasmalemmal sealing. The frequency and rate of plasmalemmal sealing are decreased by a small molecule inhibitor of PKA targeted to its catalytic subunit (KT5720), a peptide inhibitor targeted to its regulatory subunits (PKI), an inhibitor of a novel PKC (an nPKCÎ· pseudosubstrate fragment), and an antioxidant (melatonin). Given these and other data, we propose a model for redundant parallel pathways of Ca(2+)-dependent plasmalemmal sealing of injured neurons mediated in part by nPKCs, cytosolic oxidation, and cAMP activation of PKA and Epac. We also propose that the evolutionary origin of these pathways and substances was to repair plasmalemmal damage in eukaryotic cells. Greater understanding of vesicle interactions, proteins, and pathways involved in plasmalemmal sealing should suggest novel neuroprotective treatments for traumatic nerve injuries and neurodegenerative disorders.

A model for sealing plasmalemmal damage in neurons and other eukaryotic cells.

R&eacute;paration plasmiques est n&eacute;cessaire pour la survie des cellules eucaryotes endommag&eacute;es. Ca(2+) influx &agrave; travers les perturbations plasmiques active la calpa&iuml;ne, accumulation de v&eacute;sicules aux sites de la l&eacute;sion et prot&eacute;ines de membrane de fusion ; Ca(2+) afflux initie &eacute;galement des voies apoptotiques concurrentes. Avec la formation d&#39;une barri&egrave;re de colorant (sceau) pour &eacute;valuer la r&eacute;paration plasmiques, nous rapportons maintenant que les cellules hippocampiques B104 avec neurites sillonn&eacute;s rapproche (&lt; 50 &mu;m) sur le soma, joint &agrave; une fr&eacute;quence plus basse et la plus lente par rapport aux cellules avec neurites travers&eacute; plus loin (&gt; 50 &mu;m) de soma. Analogues du cAMP, y compris la prot&eacute;ine kinase A (PKA)-sp&eacute;cifiques et cAMP d&#39;Epac sp&eacute;cifiques, chacun augmentent la fr&eacute;quence et le taux d&#39;apposition du sceau et peut m&ecirc;me lancer d&#39;&eacute;tanch&eacute;it&eacute; en l&#39;absence d&#39;afflux de Ca(2+) &agrave; deux distances de transection. En outre, Epac active une voie d&eacute;pendante de l&#39;AMPc, ind&eacute;pendant de la PKA, impliqu&eacute;e dans l&#39;&eacute;tanch&eacute;it&eacute; plasmiques. La fr&eacute;quence et le taux d&#39;&eacute;tanch&eacute;it&eacute; plasmiques sont diminu&eacute;es par un inhibiteur de la petite mol&eacute;cule de PKA cibl&eacute; sa sous-unit&eacute; catalytique (KT5720), un peptide inhibiteur cibl&eacute; ses sous-unit&eacute;s r&eacute;gulatrices (ICP), un inhibiteur d&#39;un roman PKC (un fragment de Pseudo-substrat nPKC&eta;) et un antioxydant (m&eacute;latonine). Compte tenu de ces et autres donn&eacute;es, nous proposons un mod&egrave;le pour les voies parall&egrave;les redondants d&#39;&eacute;tanch&eacute;it&eacute; plasmiques de Ca(2+) d&eacute;pendant des neurones l&eacute;s&eacute;s m&eacute;di&eacute;e en partie par la nPKCs, l&#39;oxydation cytosolique et cAMP d&#39;activation de la PKA et Epac. Nous proposons &eacute;galement que l&#39;origine &eacute;volutive de ces voies et substances devait r&eacute;parer plasmiques dans les cellules eucaryotes. Une meilleure compr&eacute;hension des interactions de v&eacute;sicule, prot&eacute;ines et voies impliqu&eacute;es dans l&#39;&eacute;tanch&eacute;it&eacute; plasmiques devrait sugg&eacute;rer des traitements neuroprotecteurs roman pour les l&eacute;sions nerveuses traumatiques et les troubles neurod&eacute;g&eacute;n&eacute;ratifs.

Un mod&egrave;le pour l&#39;&eacute;tanch&eacute;it&eacute; des dommages plasmiques dans les neurones et autres cellules eucaryotes.

Plasmalemmal-Reparatur ist notwendig f&uuml;r das &Uuml;berleben der besch&auml;digten eukaryotic Zellen. Ca(2+) Zustrom durch plasmalemmal St&ouml;rungen aktiviert Calpain, Vesikel Ansammlung bei L&auml;sion Sites und Membrane Schmelzverfahrensproteine; Ca(2+) Zustrom initiiert auch konkurrierende Apoptotic Bahnen. Anhand der Bildung von Farbstoff Barriere (Siegel) zur Bewertung der plasmalemmal reparieren wir jetzt Berichten, dass B104 hippocampale Zellen mit Neurites n&auml;her transected (&lt; 50 &mu;m) zu den Soma bei einer niedrigeren frequenz und langsamer im vergleich zu zellen mit Neurites transected weiter zu versiegeln (&gt; 50 &mu;m) aus der Soma. Analoga des Lagers, einschlie&szlig;lich Proteinkinase A (PKA)-spezifische und Epac-spezifische cAMP, jede erh&ouml;hen die H&auml;ufigkeit und die Rate der Abdichtung und kann sogar initiieren, Abdichtung in Ermangelung Ca(2+) Zustrom bei beiden Transection-Entfernungen. Dar&uuml;ber hinaus aktiviert Epac Lager-abh&auml;ngige, PKA-unabh&auml;ngig vom Weg plasmalemmal Abdichtung beteiligt. Die H&auml;ufigkeit und die Rate der plasmalemmal Abdichtung sind durch ein niedermolekularer Inhibitor der PKA gezielt an seine katalytische Untereinheit (KT5720), ein Peptid-Hemmer gezielt an die regulatorischen Untereinheiten (PKI), ein Inhibitor der eine neuartige PKC (ein nPKC&eta;-Pseudosubstrate-Fragment) und ein Antioxidans (Melatonin) verringert. Da diese und andere Daten, schlagen wir ein Modell f&uuml;r redundante parallele Pfade der Ca(2+) abh&auml;ngigen plasmalemmal Versiegelung von verletzten Neuronen, die Teil von nPKCs, Zytosolische Oxidation und cAMP Aktivierung des PKA und Epac vermittelt. Wir schlagen auch vor, dass der evolution&auml;re Ursprung dieser Wege und Stoffe wurde plasmalemmal Schaden in eukaryotischen Zellen zu reparieren. Besseres Verst&auml;ndnis der Vesikel Interaktionen, Proteine und Bahnen beteiligt plasmalemmal Abdichtung sollte Roman neuroprotektive Therapien f&uuml;r traumatischen Nervenverletzungen und Neurodegenerative Erkrankungen stellen.

Ein Modell zur Abdichtung von plasmalemmal Schaden in Neuronen und andere eukaryotic Zellen.

Plasmalemmal riparazione &egrave; necessario per la sopravvivenza delle cellule eucariotiche danneggiate. CA(2+) afflusso attraverso perturbazioni plasmalemmal attiva calpaina, accumulo di vescicola presso siti di lesione e proteine di fusione di membrana; Afflusso CA(2+) avvia anche concorrenti vie apoptotic. Mediante la formazione di una barriera di colorante (seal) per valutare la riparazione plasmalemmal, segnalano ora che le cellule dell&#39;ippocampo B104 con neuriti attraversata pi&ugrave; vicino (&lt; 50 &mu;m) a soma sigillo a una frequenza pi&ugrave; bassa e il ritmo pi&ugrave; lento rispetto alle cellule con neuriti attraversata pi&ugrave; lontano (&amp;gt; 50 &mu;m) da soma. Analoghi del cAMP, tra cui la proteina chinasi A (PKA)-specifiche e specifici dell&#39;Epac cAMP, ciascuno aumentare la frequenza e la velocit&agrave; di saldatura e pu&ograve; anche avviare tenuta in assenza di Ca(2+) afflusso presso entrambe le distanze transezione. Inoltre, Epac attiva un pathway cAMP-dipendente, indipendente dalla PKA, coinvolti nella tenuta di plasmalemmal. La frequenza e il tasso di plasmalemmal di tenuta sono diminuiti di un inibitore di piccola molecola di PKA mirato alla sua subunit&agrave; catalitica (KT5720), un inibitore del peptide mirato alla sua regolamentazione subunit&agrave; (PKI), un inibitore di un romanzo PKC (un frammento di pseudosubstrate nPKC&eta;) e un antiossidante (melatonina). Dati questi e altri dati, proponiamo un modello per percorsi paralleli ridondanti di tenuta plasmalemmal Ca(2+)-dipendente dei neuroni infortunati mediata in parte da nPKCs, ossidazione citosolico e cAMP attivazione della PKA ed Epac. Proponiamo inoltre che l&#39;origine evolutiva di queste sostanze e percorsi era a riparare i danni plasmalemmal in cellule eucariote. Una maggiore comprensione delle interazioni della vescicola, proteine e pathways coinvolti nella tenuta plasmalemmal dovrebbe suggerire trattamenti neuroprotettivi romanzo per lesioni nervose traumatiche e malattie neurodegenerative.

Un modello per la sigillatura di danno plasmalemmal in neuroni e altre cellule eucariote.

&#x539F;&#x5F62;&#x8CEA;&#x306E;&#x4FEE;&#x5FA9;&#x306F;&#x3001;&#x7834;&#x640D;&#x3057;&#x305F;&#x771F;&#x6838;&#x7D30;&#x80DE;&#x306E;&#x751F;&#x5B58;&#x306E;&#x305F;&#x3081;&#x306B;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x6D41;&#x5165;&#x539F;&#x5F62;&#x8CEA;&#x306E;&#x6DF7;&#x4E71;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x30AB;&#x30EB;&#x30D1;&#x30A4;&#x30F3;&#x3001;&#x5C0F;&#x80DE;&#x306E;&#x84C4;&#x7A4D;&#x306F;&#x75C5;&#x5909;&#x306E;&#x30B5;&#x30A4;&#x30C8;&#x3068;&#x819C;&#x878D;&#x5408;&#x86CB;&#x767D;&#x8CEA;&#x304C;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x6D41;&#x5165;&#x3082;&#x7AF6;&#x5408;&#x3059;&#x308B;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x7D4C;&#x8DEF;&#x3092;&#x958B;&#x59CB;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x4ECA; B104 &#x6D77;&#x99AC;&#x7D30;&#x80DE;&#x7A81;&#x8D77;&#x3068;&#x8FD1;&#x3044;&#x5207;&#x96E2;&#x3053;&#x3068;&#x5831;&#x544A;&#x539F;&#x5F62;&#x8CEA;&#x306E;&#x4FEE;&#x7406;&#x3092;&#x8A55;&#x4FA1;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x67D3;&#x6599;&#x30D0;&#x30EA;&#x30A2; &#xFF08;&#x30B7;&#x30FC;&#x30EB;&#xFF09; &#x306E;&#x5F62;&#x6210;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;(&lt; 50 &mu; m&#xFF09; &#x3092;&#x76F8;&#x99AC;&#x30B7;&#x30FC;&#x30EB;&#x4F4E;&#x3044;&#x5468;&#x6CE2;&#x6570;&#x3068;&#x9060;&#x304F;&#x5207;&#x96E2;&#x795E;&#x7D4C;&#x7A81;&#x8D77;&#x306E;&#x7D30;&#x80DE;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x9045;&#x3044;&#x901F;&#x5EA6;&#x3067; &#xFF08;&#x76EE; 50 &mu; m)&#x3001;&#x76F8;&#x99AC;&#x304B;&#x3089;&#x3002;&#x30D7;&#x30ED;&#x30C6;&#x30A4;&#x30F3;&#x30AD;&#x30CA;&#x30FC;&#x30BC; A(PKA) &#x3092;&#x542B;&#x3080;&#x30AD;&#x30E3;&#x30F3;&#x30D7;&#x306E;&#x985E;&#x7E01;&#x4F53;-&#x7279;&#x5B9A;&#x3068; Epac &#x56FA;&#x6709;&#x306E;&#x30AD;&#x30E3;&#x30F3;&#x30D7;&#x3001;&#x5404;&#x5468;&#x6CE2;&#x6570;&#x3068;&#x30B7;&#x30FC;&#x30EB;&#x306E;&#x7387;&#x5897;&#x52A0;&#x3057;&#x3001;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x6D41;&#x5165;&#x306F;&#x4E21;&#x65B9;&#x96E2;&#x65AD;&#x8DDD;&#x96E2;&#x306E;&#x4E0D;&#x5728;&#x3067;&#x30B7;&#x30FC;&#x30EB;&#x3082;&#x958B;&#x59CB;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;Epac &#x539F;&#x5F62;&#x8CEA;&#x306E;&#x30B7;&#x30FC;&#x30EA;&#x30F3;&#x30B0;&#x306B;&#x95A2;&#x4E0E;&#x3059;&#x308B; cAMP &#x4F9D;&#x5B58;&#x3001;PKA &#x4F9D;&#x5B58;&#x7D4C;&#x8DEF;&#x3092;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x3057;&#x307E;&#x3059;&#x3002;&#x5468;&#x6CE2;&#x6570;&#x3068;&#x539F;&#x5F62;&#x8CEA;&#x306E;&#x30B7;&#x30FC;&#x30EA;&#x30F3;&#x30B0;&#x306E;&#x7387;&#x305D;&#x306E;&#x89E6;&#x5A92;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306B; (KT5720), &#x305D;&#x306E;&#x8ABF;&#x7BC0;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8; (PKI) &#x3092;&#x6A19;&#x7684;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x963B;&#x5BB3;&#x5264;&#x5C0F;&#x8AAC; PKC (&#x3001;nPKC&eta; &#x3092; pseudosubstrate &#x30D5;&#x30E9;&#x30B0;&#x30E1;&#x30F3;&#x30C8;) &#x304A;&#x3088;&#x3073;&#x9178;&#x5316;&#x9632;&#x6B62;&#x5264; (&#x30E1;&#x30E9;&#x30C8;&#x30CB;&#x30F3;) &#x306E;&#x963B;&#x5BB3;&#x5264;&#x306E;&#x6A19;&#x7684; PKA &#x306F;&#x5C0F;&#x5206;&#x5B50;&#x963B;&#x5BB3;&#x85AC;&#x306B;&#x3088;&#x3063;&#x3066;&#x6E1B;&#x5C11;&#x3057;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x4ED6;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x3092;&#x4E0E;&#x3048;&#x3001;&#x30E2;&#x30C7;&#x30EB;&#x5197;&#x9577;&#x4E26;&#x5217;&#x7D4C;&#x8DEF; Ca(2+) &#x4F9D;&#x5B58;&#x539F;&#x5F62;&#x8CEA;&#x306E;&#x30B7;&#x30FC;&#x30EA;&#x30F3;&#x30B0;&#x306E;&#x4E00;&#x90E8;&#x304C;&#x5A92;&#x4ECB;&#x3059;&#x308B; nPKCs&#x3001;&#x30BE;&#x30EB;&#x6027;&#x7D30;&#x80DE;&#x8CEA;&#x306E;&#x9178;&#x5316;&#x3068; PKA &#x3068; Epac &#x306E;&#x30AD;&#x30E3;&#x30F3;&#x30D7;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x306B;&#x3088;&#x3063;&#x3066;&#x8CA0;&#x50B7;&#x3057;&#x305F;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x63D0;&#x6848;.&#x6211;&#x3005; &#x3082;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D4C;&#x8DEF;&#x306E;&#x7269;&#x8CEA;&#x306E;&#x9032;&#x5316;&#x306E;&#x8D77;&#x6E90;&#x306F;&#x771F;&#x6838;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;&#x539F;&#x5F62;&#x8CEA;&#x306E;&#x640D;&#x50B7;&#x3092;&#x4FEE;&#x5FA9;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3060;&#x3063;&#x305F;&#x3053;&#x3068;&#x3092;&#x63D0;&#x6848;&#x3059;&#x308B;.&#x5C0F;&#x80DE;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3001;&#x86CB;&#x767D;&#x8CEA;&#x304A;&#x3088;&#x3073;&#x539F;&#x5F62;&#x8CEA;&#x306E;&#x30B7;&#x30FC;&#x30EA;&#x30F3;&#x30B0;&#x306B;&#x95A2;&#x4E0E;&#x3059;&#x308B;&#x7D4C;&#x8DEF;&#x306E;&#x4E00;&#x5C64;&#x306E;&#x7406;&#x89E3;&#x306F;&#x5916;&#x50B7;&#x6027;&#x795E;&#x7D4C;&#x640D;&#x50B7;&#x3084;&#x795E;&#x7D4C;&#x5909;&#x6027;&#x75BE;&#x60A3;&#x306E;&#x305F;&#x3081;&#x306E;&#x65B0;&#x898F;&#x795E;&#x7D4C;&#x4FDD;&#x8B77;&#x6CBB;&#x7642;&#x3092;&#x793A;&#x5506;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x3068;&#x4ED6;&#x306E;&#x771F;&#x6838;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;&#x539F;&#x5F62;&#x8CEA;&#x640D;&#x50B7;&#x3092;&#x30B7;&#x30FC;&#x30EB;&#x3059;&#x308B;&#x305F;&#x3081;&#x30E2;&#x30C7;&#x30EB;&#x3002;

Plasmalemmal &#xBCF5;&#xAD6C; &#xC190;&#xC0C1; &#xB41C; &#xC9C4; &#xD575; &#xC138;&#xD3EC;&#xC758; &#xC0DD;&#xC874;&#xC744; &#xC704;&#xD574; &#xD544;&#xC694; &#xD558;&#xB2E4;. Plasmalemmal &#xC911;&#xB2E8;&#xC744; &#xD1B5;&#xD574; Ca(2+) &#xC720;&#xC785; &#xD65C;&#xC131;&#xD654; calpain, &#xAE30; &#xCD95;&#xC801; &#xBCD1; &#xBCC0; &#xC0AC;&#xC774;&#xD2B8; &#xBC0F; &#xB9C9; &#xC735;&#xD569; &#xB2E8;&#xBC31;&#xC9C8;; Ca(2+) &#xC720;&#xC785;&#xC740; &#xB610;&#xD55C; &#xACBD;&#xC7C1; apoptotic &#xACBD;&#xB85C; &#xC2DC;&#xC791;&#xD569;&#xB2C8;&#xB2E4;. &#xC5FC;&#xB8CC; &#xC7A5;&#xBCBD; (&#xB3C4;&#xC7A5;)&#xC758; &#xD615;&#xC131;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; plasmalemmal &#xC218;&#xB9AC;&#xB97C; &#xD3C9;&#xAC00; &#xD558;&#xAE30; &#xC704;&#xD574;, &#xC6B0;&#xB9AC;&#xAC00; &#xC9C0;&#xAE08; &#xBCF4;&#xACE0; B104 hippocampal &#xC140; neurites transected &#xAC00;&#xAE4C;&#xC774; (&lt; 50 &mu; m)&#xB294; &#xC18C;&#xB9C8;&#xB97C; &#xB0AE;&#xC740; &#xC8FC;&#xD30C;&#xC218;&#xC640; &#xBA40;&#xB9AC; transected neurites &#xC140;&#xC5D0; &#xBE44;&#xD574; &#xB290;&#xB9B0; &#xC18D;&#xB3C4;&#xC5D0; &#xC778;&#xAC10; (&amp;gt; 50 &mu; m)&#xB294; &#xC18C;&#xB9C8;&#xC5D0;&#xC11C;. &#xCEA0;&#xD504;, &#xB2E8;&#xBC31;&#xC9C8; &#xD0A4; &#xB2C8; &#xC544; &#xC81C; A (PKA)&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xB9E4;&#xC6B0; &#xC720;&#xC0AC;-Epac &#xAD00;&#xB828; &#xCEA0;&#xD504; &#xBC0F; &#xAC01; &#xC8FC;&#xD30C;&#xC218; &#xBC0F; &#xC530;&#xB9C1;&#xC758; &#xC18D;&#xB3C4; &#xC99D;&#xAC00; &#xD558; &#xACE0; &#xC2EC;&#xC9C0;&#xC5B4; &#xB450; transection &#xAC70;&#xB9AC;&#xC5D0;&#xC11C; Ca(2+) &#xC720;&#xC785;&#xC758; &#xBD80;&#xC7AC;&#xC5D0; &#xC530;&#xB9C1;&#xC744; &#xC2DC;&#xC791;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, Epac &#xCEA0;&#xD504; &#xC885;&#xC18D;, PKA &#xB3C5;&#xB9BD;&#xC801;&#xC778; &#xACBD;&#xB85C; plasmalemmal &#xC530;&#xB9C1;&#xC5D0; &#xCC38;&#xC5EC;&#xB97C; &#xD65C;&#xC131;&#xD654;&#xD569;&#xB2C8;&#xB2E4;. &#xC18C;&#xB294; &#xCD09;&#xB9E4; &#xB2E8;&#xC704; (KT5720), &#xD3A9; &#xD2F0; &#xB4DC; &#xC5B5;&#xC81C;&#xC81C;&#xB294; &#xADDC;&#xC815; &#xC18C; &#xB2E8;&#xC704; (PKI)&#xB97C; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xC18C;&#xC124; PKC (&#xB294; nPKC&eta; pseudosubstrate &#xC870;&#xAC01;), &#xADF8;&#xB9AC;&#xACE0; &#xC0B0;&#xD654; &#xBC29;&#xC9C0; &#xC81C; (&#xBA5C; &#xB77C; &#xD1A0; &#xB2CC;)&#xC758; &#xC5B5;&#xC81C;&#xC81C;&#xB97C; &#xD0C0;&#xAC9F;&#xC73C;&#xB85C; PKA&#xC758; &#xC791;&#xC740; &#xBD84;&#xC790; &#xC5B5;&#xC81C; &#xBB3C; &#xC8FC;&#xAE30;&#xC640; plasmalemmal &#xC530;&#xB9C1;&#xC758; &#xC18D;&#xB3C4; &#xAC10;&#xC18C;. &#xC774;&#xB4E4;&#xACFC; &#xB2E4;&#xB978; &#xB370;&#xC774;&#xD130;&#xB97C; &#xAC10;&#xC548;&#xD560; &#xB54C;, &#xC6B0;&#xB9AC; &#xC911;&#xC7AC; &#xBD80;&#xBD84;&#xC5D0; nPKCs, cytosolic &#xC0B0;&#xD654;&#xC640; PKA&#xC640; Epac &#xCEA0;&#xD504; &#xD65C;&#xC131;&#xD654; &#xBD80;&#xC0C1;&#xB41C; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; Ca(2+) &#xC885;&#xC18D; plasmalemmal &#xC530;&#xB9C1;&#xC758; &#xC911;&#xBCF5; &#xBCD1;&#xB82C; &#xACBD;&#xB85C; &#xB300; &#xD55C; &#xBAA8;&#xB378;&#xC744; &#xC81C;&#xC548; &#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xB610;&#xD55C; &#xC774;&#xB7EC;&#xD55C; &#xACBD;&#xB85C; &#xBB3C;&#xC9C8;&#xC758; &#xC9C4;&#xD654; &#xADFC;&#xC6D0; &#xC9C4; &#xD575; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; plasmalemmal &#xD53C;&#xD574;&#xB97C; &#xBCF5;&#xAD6C; &#xD558;&#xB294; &#xC81C;&#xC548; &#xD569;&#xB2C8;&#xB2E4;. &#xC774;&#xD574; &#xAE30; &#xC0C1;&#xD638; &#xC791;&#xC6A9;, &#xB2E8;&#xBC31;&#xC9C8;, &#xADF8;&#xB9AC;&#xACE0; &#xACBD;&#xB85C; plasmalemmal &#xC530;&#xB9C1;&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xC678;&#xC0C1; &#xC131; &#xC2E0;&#xACBD; &#xC0C1;&#xD574; &#xBC0F; &#xC2E0;&#xACBD; &#xD1F4;&#xD589; &#xC131; &#xC9C8;&#xD658;&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xC2E0;&#xACBD; &#xBCF4;&#xD638; &#xCE58;&#xB8CC; &#xC81C;&#xC548; &#xD55C;&#xB2E4;.

Plasmalemmal &#xC190;&#xC0C1; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC640; &#xB2E4;&#xB978; &#xC9C4; &#xD575; &#xC138;&#xD3EC;&#xC5D0; &#xC530;&#xB9C1;&#xC744; &#xC704;&#xD55C; &#xBAA8;&#xB378;.

Reparaci&oacute;n plasmalemmal es necesario para la supervivencia de las c&eacute;lulas eucariotas da&ntilde;adas. Afluencia de CA a trav&eacute;s de interrupciones plasmalemmal activa calpain, acumulaci&oacute;n de ves&iacute;cula en sitios de la lesi&oacute;n y prote&iacute;nas de fusi&oacute;n de membrana; Afluencia de CA tambi&eacute;n inicia competencia v&iacute;as apopt&oacute;ticas. Mediante la formaci&oacute;n de una barrera de tinte (sello) para evaluar la reparaci&oacute;n plasmalemmal, ahora nos informan que c&eacute;lulas hipocampales B104 con neuritas transversalmente el m&aacute;s cercano (&lt; 50 &mu;m) para el soma sello en una frecuencia m&aacute;s baja y m&aacute;s lenta en comparaci&oacute;n con las c&eacute;lulas con neuritas transversalmente m&aacute;s lejos (&gt; 50 &mu;m) de soma. An&aacute;logos de campo, incluyendo la prote&iacute;na quinasa A (PKA)-espec&iacute;ficas y campamento de Epac-espec&iacute;ficas, cada uno aumentar la frecuencia y la velocidad de sellado y puede incluso iniciar sellado en la ausencia de afluencia de CA en ambas distancias de corte transversal. Adem&aacute;s, Epac activa una v&iacute;a dependiente de campo, independiente del PKA, involucrada en el lacre plasmalemmal. La frecuencia y la velocidad de sellado plasmalemmal se disminuyen por un inhibidor de la mol&eacute;cula peque&ntilde;a de PKA dirigido a su subunidad catal&iacute;tica (KT5720), un inhibidor del p&eacute;ptido dirigido a sus subunidades reguladoras (PKI), un inhibidor de una novela PKC (un fragmento de pseudosubstrate de nPKC&eta;) y un antioxidante (melatonina). Teniendo en cuenta estos y otros datos, se propone un modelo para v&iacute;as paralelas redundantes de sellado plasmalemmal Ca(2+) dependiente de neuronas lesionadas mediado en parte por isoformas, oxidaci&oacute;n citos&oacute;lica y campo activaci&oacute;n de PKA y Epac. Tambi&eacute;n proponemos que el origen evolutivo de estos caminos y sustancias deb&iacute;a reparar plasmalemmal da&ntilde;o en las c&eacute;lulas eucariotas. Una mayor comprensi&oacute;n de las interacciones de la ves&iacute;cula, prote&iacute;nas y v&iacute;as implicadas en el lacre plasmalemmal debe sugerir tratamientos neuroprotectores novela para lesiones traum&aacute;ticas del nervio y trastornos neurodegenerativos.

Un modelo para el sellado de plasmalemmal da&ntilde;o en las neuronas y otras c&eacute;lulas eucariotas.

Cell-shape change demands cell-surface growth, but how growth is fueled and choreographed is still debated. Here we use cellularization, the first complete cytokinetic event in Drosophila embryos, to show that cleavage furrow ingression is kinetically coupled to the loss of surface microvilli. We modulate furrow kinetics with RNAi against the Rho1-GTPase regulator slam and show that furrow ingression controls the rate of microvillar depletion. Finally, we directly track the microvillar membrane and see it move along the cell surface and into ingressing furrows, independent of endocytosis. Together, our results demonstrate that the kinetics of the ingressing furrow regulate the utilization of a microvillar membrane reservoir. Because membranes of the furrow and microvilli are contiguous, we suggest that ingression drives unfolding of the microvilli and incorporation of microvillar membrane into the furrow. We conclude that plasma membrane folding/unfolding can contribute to the cell-shape changes that promote embryonic morphogenesis. 

The plasma membrane flattens out to fuel cell-surface growth during Drosophila cellularization.

Conformation-based disorders are manifested at the level of protein structure, necessitating an accurate understanding of how misfolded proteins are processed by the cellular proteostasis network. Asparagine-linked glycosylation plays important roles for protein quality control within the secretory pathway. The suspected role for the MAN1B1 gene product MAN1B1, also known as ER mannosidase I, is to function within the ER similar to the yeast ortholog Mns1p, which removes a terminal mannose unit to initiate a glycan-based ER-associated degradation (ERAD) signal. However, we recently discovered that MAN1B1 localizes to the Golgi complex in human cells and uncovered its participation in ERAD substrate retention, retrieval to the ER, and subsequent degradation from this organelle. The objective of the current study was to further characterize the contribution of MAN1B1 as part of a Golgi-based quality control network. Multiple lines of experimental evidence support a model in which neither the mannosidase activity nor catalytic domain is essential for the retention or degradation of the misfolded ERAD substrate Null Hong Kong. Instead, a highly conserved, vertebrate-specific non-enzymatic decapeptide sequence in the luminal stem domain plays a significant role in controlling the fate of overexpressed Null Hong Kong. Together, these findings define a new functional paradigm in which Golgi-localized MAN1B1 can play a mannosidase-independent gatekeeper role in the proteostasis network of higher eukaryotes. 

A Golgi-localized mannosidase (MAN1B1) plays a non-enzymatic gatekeeper role in protein biosynthetic quality control.

Cell surface expansion is a necessary part of cell shape change. One long-standing hypothesis proposes that membrane for this expansion comes from the flattening out of cell surface projections such as microvilli and membrane folds. Correlative EM data of cells undergoing phagocytosis, cytokinesis, and morphogenesis has hinted at the existence of such an unfolding mechanism for decades; but unfolding has only recently been confirmed using live-cell imaging and biophysical approaches. Considering the wide range of cells in which plasma membrane unfolding has now been reported, it likely represents a fundamental mechanism of cell shape change. 

A membrane reservoir at the cell surface: unfolding the plasma membrane to fuel cell shape change.

We combine immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), followed by automated image analysis, to quantify the concentration of nuclear transcription factors, number of transcription factors bound, and number of nascent mRNAs synthesized at individual gene loci. A theoretical model is used to decipher how transcription factor binding modulates the stochastic kinetics of mRNA production. We demonstrate this approach by examining the regulation of hunchback in the early Drosophila embryo. 

Combining protein and mRNA quantification to decipher transcriptional regulation.

Cells store membrane in surface reservoirs of pits and protrusions. These membrane reservoirs facilitate cell shape change and buffer mechanical stress, but we do not know how reservoir dynamics are regulated. During cellularization, the first cytokinesis in Drosophila embryos, a reservoir of microvilli unfolds to fuel cleavage furrow ingression. We find that regulated exocytosis adds membrane to the reservoir before and during unfolding. Dynamic F-actin deforms exocytosed membrane into microvilli. Single microvilli extend and retract in ∼20 s, while the overall reservoir is depleted in sync with furrow ingression over 60-70 min. Using pharmacological and genetic perturbations, we show that exocytosis promotes microvillar F-actin assembly, while furrow ingression controls microvillar F-actin disassembly. Thus, reservoir F-actin and, consequently, reservoir dynamics are regulated by membrane supply from exocytosis and membrane demand from furrow ingression.

Membrane Supply and Demand Regulates F-Actin in a Cell Surface Reservoir.

Environmental stress threatens the fidelity of embryonic morphogenesis. Heat, for example, is a teratogen. Yet how heat affects morphogenesis is poorly understood. Here, we identify a heat-inducible actin stress response (ASR) in Drosophila embryos that is mediated by the activation of the actin regulator Cofilin. Similar to ASR in adult mammalian cells, heat stress in fly embryos triggers the assembly of intra-nuclear actin rods. Rods measure up to a few microns in length, and their assembly depends on elevated free nuclear actin concentration and Cofilin. Outside the nucleus, heat stress causes Cofilin-dependent destabilization of filamentous actin (F-actin) in actomyosin networks required for morphogenesis. F-actin destabilization increases the chance of morphogenesis mistakes. Blocking the ASR by reducing Cofilin dosage improves the viability of heat-stressed embryos. However, improved viability correlates with restoring F-actin stability, not rescuing morphogenesis. Thus, ASR endangers embryos, perhaps by shifting actin from cytoplasmic filaments to an elevated nuclear pool.

ArticleTotal : 2	Year
Publication title Cited by 22	2011
Publication title Cited by 3	2020

Article	Year
A model for sealing plasmalemmal damage in neurons and other eukaryotic cells. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 21106818	2010
The plasma membrane flattens out to fuel cell-surface growth during Drosophila cellularization. Developmental cell\| PubMed ID: 24316147	2013
A Golgi-localized mannosidase (MAN1B1) plays a non-enzymatic gatekeeper role in protein biosynthetic quality control. The Journal of biological chemistry\| PubMed ID: 24627495	2014
A membrane reservoir at the cell surface: unfolding the plasma membrane to fuel cell shape change. Bioarchitecture\| PubMed ID: 24844289	2014
Combining protein and mRNA quantification to decipher transcriptional regulation. Nature methods\| PubMed ID: 26098021	2015
Membrane Supply and Demand Regulates F-Actin in a Cell Surface Reservoir. Developmental cell\| PubMed ID: 27165556	2016
Cofilin-Mediated Actin Stress Response Is Maladaptive in Heat-Stressed Embryos. Cell reports\| PubMed ID: 30917306	2019

Lauren Figard

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