Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

University of Tennessee

Force-driven conformational changes provide a broad basis for protein extensibility, and multidomain proteins broaden the possibilities further by allowing for a multiplicity of forcibly extended states. Red cell spectrin is prototypical in being an extensible, multidomain protein widely recognized for its contribution to erythrocyte flexibility. Atomic force microscopy has already shown that single repeats of various spectrin family proteins can be forced to unfold reversibly under extension. Recent structural data indicates, however, that the linker between triple-helical spectrin repeats is often a contiguous helix, thus raising questions as to what the linker contributes and what defines a domain mechanically. We have examined the extensible unfolding of red cell spectrins as monomeric constructs of just two, three, or four repeats from the actin-binding ends of both alpha- and beta-chains, i.e., alpha(18-21) and beta(1-4) or their subfragments. In addition to single repeat unfolding evident in sawtooth patterns peaked at relatively low forces (<50 pN at 1 nm/ms extension rates), tandem repeat unfolding is also demonstrated in ensemble-scale analyses of thousands of atomic force microscopy contacts. Evidence for extending two chains and loops is provided by force versus length scatterplots which also indicate that tandem repeat unfolding occurs at a significant frequency relative to single repeat unfolding. Cooperativity in forced unfolding of spectrin is also clearly demonstrated by a common force scale for the unfolding of both single and tandem repeats.

Cooperativity in forced unfolding of tandem spectrin repeats.

&#x5DE8;&#x4EBA;&#x548C;&#x7A33;&#x5B9A;&#x8815;&#x866B;&#x80F6;&#x675F;&#x5F62;&#x6210;&#x4E86;&#x4ECE; poly(ethylene glycol) (PEG)-&#x57FA;&#x4E8E;&#x7684;&#x5D4C;&#x6BB5;&#x5171;&#x805A;&#x7269;&#x4EB2;&#x6709;&#x76F8;&#x6BD4;&#xFF0C;&#x8F83;&#x5C0F;&#x7684;&#x7A0B;&#x5E8F;&#x96C6;&#x7684;&#x91CF;&#x5927;&#x7684;&#x758F;&#x6C34;&#x6027;&#x836F;&#x7269;&#x6216;&#x67D3;&#x6599;&#x6BCF;&#x627F;&#x8FD0;&#x4EBA;&#x4EA4;&#x8D27;&#x7684;&#x6F5C;&#x5728;&#x4F18;&#x52BF;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x5C55;&#x793A;&#x8815;&#x866B;&#x80F6;&#x675F;&#x53EF;&#x4EE5;&#x6307;&#x5411;&#x5355;&#x5143;&#x683C;&#x5185;&#x5316;&#x548C;&#x4EA4;&#x4ED8;&#x7684;&#x65E0;&#x6BD2;&#x7684;&#x67D3;&#x6599;&#xFF0C;&#x4EE5;&#x53CA;&#x7EC6;&#x80DE;&#x6BD2;&#x836F;&#x7269;&#x3002;&#x7EC4;&#x6210;&#x5171;&#x805A;&#x7269;&#x662F;&#x7ED3;&#x675F;&#x7D20;&#x4EE5;&#x8C03;&#x89E3;&#x7684;&#x8815;&#x866B;&#x80F6;&#x675F;&#x4EB2;&#x8F74;&#x627F;&#x8868;&#x9762;&#x548C;&#x7279;&#x5B9A;&#x751F;&#x7269;&#x7D20;&#x7684;&#x53D7;&#x4F53;&#x5BF9;&#x8840;&#x7BA1;&#x5E73;&#x6ED1;&#x808C;&#x7EC6;&#x80DE;&#x9AD8;&#x4EB2;&#x548C;&#x6027;&#x7ED3;&#x5408;&#x3002;&#x539F;&#x59CB;&#x8815;&#x866B;&#x80F6;&#x675F;&#xFF0C;&#x90A3;&#x4E9B;&#x7F3A;&#x4E4F;&#x751F;&#x7269;&#x7D20;&#xFF0C;&#x663E;&#x793A;&#x592A;&#x9891;&#x7E41;&#x548C;&#x975E;&#x7279;&#x5F02;&#x6027;&#x70B9;&#x9644;&#x4EF6;&#x5230;&#x76F8;&#x540C;&#x7684;&#x66F2;&#x9762;&#x3002;&#x7D20;&#x8815;&#x866B;&#x80F6;&#x675F;&#x8BC1;&#x660E;&#x81F3;&#x5C11;&#x4E00;&#x4E2A;&#x6708;&#x7A33;&#x5B9A;&#x5728;&#x6C34;&#x6EB6;&#x6DB2;&#x4E2D;&#xFF0C;&#x4E5F;&#x8BC1;&#x660E;&#x80FD;&#x591F;&#x52A0;&#x8F7D;&#x3001; &#x4FDD;&#x7559;&#x548C;&#x63D0;&#x4F9B;&#x758F;&#x6C34;&#x67D3;&#x6599;&#x548C;&#x836F;&#x7269;&#x3002;&#x7ED3;&#x679C;&#x4ECE;&#x800C;&#x8BC1;&#x660E;&#x6709;&#x9488;&#x5BF9;&#x6027;&#x5730;&#x5F00;&#x5C55;&#x7684;&#x53EF;&#x884C;&#x6027;&#x7684;&#x805A;&#x5408;&#x7269;&#x8815;&#x866B;&#x80F6;&#x675F;&#x3002;

&#x6709;&#x9488;&#x5BF9;&#x6027;&#x7684;&#x8815;&#x866B;&#x80F6;&#x675F;&#x3002;

Giant and stable worm micelles formed from poly(ethylene glycol) (PEG)-based diblock copolymer amphiphiles have the potential advantage compared to smaller assemblies for delivery of a large quantity of hydrophobic drugs or dyes per carrier. Here we show that worm micelles can be targeted to cells with internalization and delivery of nontoxic dyes as well as cytotoxic drugs. Constituent copolymers are end-biotinylated to mediate high affinity binding of worm micelles to both avidin-bearing surfaces and biotin-specific receptors on smooth muscle cells. Pristine worm micelles, that lack biotin, show much less frequent and nonspecific point attachments to the same surfaces. Biotinylated worm micelles prove stable in aqueous solution for at least a month and also prove capable of loading, retaining, and delivering hydrophobic dyes and drugs. The results thus demonstrate the feasibility of targeted delivery by polymeric worm micelles.

Targeted worm micelles.

Micelles ver g&eacute;ant et stable forment de poly(ethylene glycol) (PEG)-fonction dibloc copolym&egrave;res amphiphiles pr&eacute;sentent l&#39;avantage potentiel par rapport aux petites assembl&eacute;es pour la livraison d&#39;une grande quantit&eacute; de m&eacute;dicaments hydrophobes ou colorants par transporteur. Nous d&eacute;montrons que des micelles de ver peuvent &ecirc;tre cibl&eacute;es aux cellules internalisation et de colorants non toxiques, mais aussi des m&eacute;dicaments cytotoxiques. Copolym&egrave;res constitutifs sont biotinylated-fin &agrave; la m&eacute;diation de fixation de haute affinit&eacute; des micelles de ver aux surfaces portantes-avidine et biotine sp&eacute;cifique des r&eacute;cepteurs sur les cellules musculaires lisses. Les micelles ver immacul&eacute;es, qui manquent de biotine, montrent beaucoup moins fr&eacute;quentes et non sp&eacute;cifique point pi&egrave;ces jointes pour les m&ecirc;mes surfaces. Micelles ver biotinyl&eacute; s&#39;av&eacute;rer stables en solution aqueuse pour au moins un mois et s&#39;av&eacute;rer capables de charger, conservant et offrant des m&eacute;dicaments et des colorants hydrophobes. Les r&eacute;sultats montrent donc la faisabilit&eacute; de l&#39;administration cibl&eacute;e par les micelles polym&egrave;res ver.

Micelles ver cibl&eacute;es.

Wurm Riese und stabilen Micellen gebildet aus Poly(ethylene glycol) (PEG)-basierte Diblock Copolymer Amphiphiles haben den m&ouml;glichen Vorteil im Vergleich zu kleineren Assemblys f&uuml;r die Lieferung einer gro&szlig;en Menge von hydrophoben Drogen oder Farbstoffe pro Tr&auml;ger. Hier zeigen wir, dass Wurm Mizellen auf Zellen mit Verinnerlichung und Lieferung von ungiftige Farbstoffe sowie Zytostatika eingesetzt werden k&ouml;nnen. Konstituierende Copolymere sind End-biotinylierten, hohe Affinit&auml;t Schwerg&auml;ngigkeit des Wurm-Mizellen Avidin-Auflagefl&auml;chen und Biotin-spezifische Rezeptoren auf glatten Muskelzellen zu vermitteln. Unber&uuml;hrte Wurm Mizellen, das Biotin fehlt, zeigen viel weniger h&auml;ufige und unspezifische Punkt Anlagen die gleichen Fl&auml;chen. Biotinylierten Wurm Mizellen beweisen f&uuml;r mindestens einen Monat in w&auml;ssriger L&ouml;sung stabil und beweisen auch f&auml;hig laden, Aufbewahrung und Bereitstellung von hydrophoben Farbstoffe und Medikamente. Die Ergebnisse belegen damit die M&ouml;glichkeit der gezielten Lieferung von Polymeren Wurm Mizellen.

Gezielte Wurm Mizellen.

Micelle verme gigante e stabile formano da poly(ethylene glycol) (PEG)-base diblock copolimero anfifili hanno il potenziale vantaggio rispetto ai pi&ugrave; piccoli assiemi per la consegna di una grande quantit&agrave; di farmaci idrofobici o coloranti al vettore. Qui vi mostriamo che verme micelle possono essere mirati alle cellule con l&#39;interiorizzazione e la consegna di coloranti atossici, come pure i farmaci citotossici. Copolimeri costituenti sono fine-biotinilato per mediare il legame ad alta affinit&agrave; delle micelle worm sia superfici portanti avidina biotina specifici recettori sulle cellule muscolari lisce. Micelle worm incontaminate, che la mancanza di biotina, mostrano molto meno frequente e aspecifico punto allegati alle superfici stesse. Biotinilato worm micelle dimostrano stabili in soluzione acquosa per almeno un mese e anche dimostrano in grado di caricare, mantenendo e consegna di farmaci e coloranti idrofobici. I risultati cos&igrave; dimostrano la fattibilit&agrave; della consegna mirata di micelle polimeriche worm.

Micelle worm mirati.

&#x5DE8;&#x5927;&#x306A;&#x5B89;&#x5B9A;&#x3057;&#x305F;&#x30EF;&#x30FC;&#x30E0;&#x306E;&#x30DF;&#x30BB;&#x30EB;&#x5F62;&#x6210; poly(ethylene glycol) (PEG) &#x304B;&#x3089;-&#x30D9;&#x30FC;&#x30B9; &#x30B8;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x5171;&#x91CD;&#x5408;&#x4F53;&#x4E21;&#x89AA;&#x5A92;&#x6027;&#x7269;&#x8CEA;&#x306F;&#x3001;&#x758E;&#x6C34;&#x6027;&#x533B;&#x85AC;&#x54C1;&#x3084;&#x67D3;&#x6599;&#x30AD;&#x30E3;&#x30EA;&#x30A2;&#x3054;&#x3068;&#x306E;&#x5927;&#x898F;&#x6A21;&#x306A;&#x91CF;&#x306E;&#x914D;&#x4FE1;&#x306E;&#x305F;&#x3081;&#x306E;&#x5C0F;&#x3055;&#x306A;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x3068;&#x6BD4;&#x8F03;&#x3059;&#x308B;&#x3068;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x5229;&#x70B9;&#x3092;&#x6301;&#x3063;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x30EF;&#x30FC;&#x30E0; &#x30DF;&#x30BB;&#x30EB;&#x304C;&#x7D30;&#x80DE;&#x306E;&#x5185;&#x90E8;&#x5316;&#x3068;&#x914D;&#x4FE1;&#x306E;&#x7121;&#x6BD2;&#x306A;&#x8272;&#x7D20;&#x7D30;&#x80DE;&#x6BD2;&#x6027;&#x85AC;&#x3092;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x306B;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x69CB;&#x6210;&#x5171;&#x91CD;&#x5408;&#x4F53;&#x7D42;&#x308F;&#x308A;&#x30D3;&#x30AA;&#x30C1;&#x30F3;&#x6A19;&#x8B58;&#x30EF;&#x30FC;&#x30E0; &#x30DF;&#x30BB;&#x30EB; &#x30A2;&#x30D3;&#x30B8;&#x30F3;&#x8EF8;&#x53D7;&#x306E;&#x8868;&#x9762;&#x3068;&#x30D3;&#x30AA;&#x30C1;&#x30F3;&#x56FA;&#x6709;&#x53D7;&#x5BB9;&#x4F53;&#x5E73;&#x6ED1;&#x7B4B;&#x7D30;&#x80DE;&#x306E;&#x9AD8;&#x89AA;&#x548C;&#x6027;&#x7D50;&#x5408;&#x3092;&#x4EF2;&#x4ECB;&#x3059;&#x308B;&#x3053;&#x3068;&#x3067;&#x3059;&#x3002;&#x30D3;&#x30AA;&#x30C1;&#x30F3;&#x306E;&#x4E0D;&#x8DB3;&#x3001;&#x81EA;&#x7136;&#x306E;&#x307E;&#x307E;&#x306E;&#x30EF;&#x30FC;&#x30E0; &#x30DF;&#x30BB;&#x30EB;&#x3001;&#x305D;&#x308C;&#x307B;&#x3069;&#x983B;&#x7E41;&#x306B;&#x3001;&#x975E;&#x7279;&#x7570;&#x7684;&#x30DD;&#x30A4;&#x30F3;&#x30C8;&#x6DFB;&#x4ED8;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x540C;&#x3058;&#x30B5;&#x30FC;&#x30D5;&#x30A7;&#x30B9;&#x3092;&#x8868;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x30D3;&#x30AA;&#x30C1;&#x30F3;&#x6A19;&#x8B58;&#x30EF;&#x30FC;&#x30E0; &#x30DF;&#x30BB;&#x30EB;&#x6C34;&#x6EB6;&#x6DB2;&#x4E2D;&#x3067;&#x5B89;&#x5B9A;&#x3057;&#x305F;&#x5C11;&#x306A;&#x304F;&#x3068;&#x3082; 1 &#x30AB;&#x6708;&#x3092;&#x8A3C;&#x660E;&#x3057;&#x3066;&#x3082;&#x8AAD;&#x307F;&#x8FBC;&#x307F;&#x3001;&#x4FDD;&#x6301;&#x3001;&#x304A;&#x3088;&#x3073;&#x758E;&#x6C34;&#x6027;&#x8272;&#x7D20;&#x304A;&#x3088;&#x3073;&#x85AC;&#x5264;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x306E;&#x306B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x8A3C;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;&#x7D50;&#x679C;&#x306F;&#x3053;&#x306E;&#x3088;&#x3046;&#x306B;&#x30EF;&#x30FC;&#x30E0;&#x304C;&#x9AD8;&#x5206;&#x5B50;&#x30DF;&#x30BB;&#x30EB;&#x306B;&#x3088;&#x308B;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x914D;&#x4FE1;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3092;&#x7D5E;&#x3063;&#x305F;&#x30EF;&#x30FC;&#x30E0; &#x30DF;&#x30BB;&#x30EB;&#x3002;

Poly(ethylene glycol) (&#xB098;&#xBB34; &#xBABB;)&#xC5D0;&#xC11C; &#xD615;&#xC131; &#xB41C; &#xAC70; &#xB300; &#xD558; &#xACE0; &#xC548;&#xC815; &#xB41C; &#xC6DC; micelles-&#xAE30;&#xBC18;&#xB41C; diblock &#xCF54; &#xD3F4;&#xB9AC;&#xBA38; amphiphiles &#xC18C;&#xC218; &#xC131; &#xC57D;&#xBB3C; &#xC774;&#xB098; &#xCE90;&#xB9AC;&#xC5B4; &#xB2F9; &#xC5FC;&#xB8CC;&#xC758; &#xB300;&#xB7C9; &#xB0A9;&#xD488;&#xC744; &#xC704;&#xD55C; &#xC791;&#xC740; &#xC5B4;&#xC148;&#xBE14;&#xB9AC;&#xC5D0; &#xBE44;&#xD574; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xC774;&#xC810;&#xC774; &#xC788;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; &#xC6DC; micelles &#xAD6D;&#xC81C;&#xD654;&#xC640; &#xBC30;&#xB2EC; cytotoxic &#xC57D;&#xBB3C;&#xC73C;&#xB85C; &#xC11C; &#xBB34; &#xB3C5;&#xC131; &#xC5FC;&#xB8CC;&#xC758; &#xC140;&#xC5D0; &#xD0C0;&#xAC9F;&#xD305; &#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xAD6C;&#xC131; &#xACF5;&#xC911; &#xD569;&#xCCB4;&#xB294; &#xC6DC; micelles avidin &#xBCA0;&#xC5B4;&#xB9C1; &#xD45C;&#xBA74; &#xBC0F; &#xD3C9;&#xD65C; &#xADFC; &#xC138;&#xD3EC;&#xC5D0; biotin &#xD2B9;&#xC815; &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xB192;&#xC740; &#xCE5C; &#xD654;&#xB825; &#xBC14;&#xC778;&#xB529; &#xC911;&#xC7AC; &#xB05D;-biotinylated. Biotin &#xC5C6;&#xB294; &#xAE68;&#xB057; &#xD55C; &#xC6DC; micelles &#xBCF4;&#xAE30; &#xAC19;&#xC740; &#xD45C;&#xBA74;&#xC5D0; &#xD6E8;&#xC52C; &#xB35C; &#xC790;&#xC8FC; &#xD558; &#xACE0; &#xC77C;&#xBC18; &#xD3EC;&#xC778;&#xD2B8; &#xCCA8;&#xBD80; &#xD30C;&#xC77C;. Biotinylated &#xC6DC; micelles &#xC801;&#xC5B4;&#xB3C4; &#xD55C; &#xB2EC;&#xC5D0; &#xC218; &#xC6A9;&#xC561;&#xC5D0;&#xC11C; &#xC548;&#xC815; &#xB41C; &#xC99D;&#xBA85; &#xD558; &#xACE0; &#xB610;&#xD55C; &#xB85C;&#xB4DC;, &#xC720;&#xC9C0;, &#xBC0F; &#xC18C;&#xC218; &#xC131; &#xC5FC;&#xB8CC; &#xBC0F; &#xC57D;&#xD488;&#xC758; &#xB2A5;&#xB825;&#xC744; &#xC99D;&#xBA85;. &#xACB0;&#xACFC; &#xB530;&#xB77C;&#xC11C; &#xACE0;&#xBD84;&#xC790; &#xC6DC; micelles &#xC5EC; &#xD0C0;&#xAC9F;&#xB41C; &#xBC30;&#xB2EC;&#xC758; &#xD0C0;&#xB2F9;&#xC131;&#xC744; &#xC785;&#xC99D;.

&#xD0C0;&#xAC9F;&#xB41C; &#xC6DC; micelles&#xC785;&#xB2C8;&#xB2E4;.

Micelas de gusano gigante y estable forman de poly(ethylene glycol) (PEG)-base diblock copol&iacute;mero Anf&iacute;filos tienen la ventaja potencial comparada con conjuntos m&aacute;s peque&ntilde;os para la entrega de grandes cantidades de drogas hidrof&oacute;bicas o tintes por portador. Aqu&iacute; mostramos que gusano micelas pueden orientarse a las c&eacute;lulas con la internalizaci&oacute;n y entrega de tintes no t&oacute;xicos, as&iacute; como f&aacute;rmacos citot&oacute;xicos. Copol&iacute;meros constituyentes son final-biotinilado para mediar la alta afinidad de uni&oacute;n de micelas de gusano tanto a superficies sustentadoras avidina biotina espec&iacute;ficos receptores en las c&eacute;lulas musculares lisas. Micelas de gusano pr&iacute;stina, que carecen de biotina, Mostrar mucho menos frecuentes e inespec&iacute;ficos anexos de punto a las mismas superficies. Biotinilado gusano micelas ser estables en soluci&oacute;n acuosa para al menos un mes y tambi&eacute;n capaces de carga, retenci&oacute;n y entrega de medicamentos y tintes hidrof&oacute;bicos. As&iacute;, los resultados demuestran la viabilidad de entrega dirigida por micelas polim&eacute;ricas gusano.

Micelas de gusano espec&iacute;ficas.

Members of the actin family of proteins exhibit different biochemical properties when ATP, ADP-P(i), ADP, or no nucleotide is bound. We used molecular dynamics simulations to study the effect of nucleotides on the behavior of actin and actin-related protein 3 (Arp3). In all of the actin simulations, the nucleotide cleft stayed closed, as in most crystal structures. ADP was much more mobile within the cleft than ATP, despite the fact that both nucleotides adopt identical conformations in actin crystal structures. The nucleotide cleft of Arp3 opened in most simulations with ATP, ADP, and no bound nucleotide. Deletion of a C-terminal region of Arp3 that extends beyond the conserved actin sequence reduced the tendency of the Arp3 cleft to open. When the Arp3 cleft opened, we observed multiple instances of partial release of the nucleotide. Cleft opening in Arp3 also allowed us to observe correlated movements of the phosphate clamp, cleft mouth, and barbed-end groove, providing a way for changes in the nucleotide state to be relayed to other parts of Arp3. The DNase binding loop of actin was highly flexible regardless of the nucleotide state. The conformation of Ser14/Thr14 in the P1 loop was sensitive to the presence of the gamma-phosphate, but other changes observed in crystal structures were not correlated with the nucleotide state on nanosecond timescales. The divalent cation occupied three positions in the nucleotide cleft, one of which was not previously observed in actin or Arp2/3 complex structures. In sum, these simulations show that subtle differences in structures of actin family proteins have profound effects on their nucleotide-driven behavior.

Nucleotide-mediated conformational changes of monomeric actin and Arp3 studied by molecular dynamics simulations.

Interaction of spherical particles with cells and within animals has been studied extensively, but the effects of shape have received little attention. Here we use highly stable, polymer micelle assemblies known as filomicelles to compare the transport and trafficking of flexible filaments with spheres of similar chemistry. In rodents, filomicelles persisted in the circulation up to one week after intravenous injection. This is about ten times longer than their spherical counterparts and is more persistent than any known synthetic nanoparticle. Under fluid flow conditions, spheres and short filomicelles are taken up by cells more readily than longer filaments because the latter are extended by the flow. Preliminary results further demonstrate that filomicelles can effectively deliver the anticancer drug paclitaxel and shrink human-derived tumours in mice. Although these findings show that long-circulating vehicles need not be nanospheres, they also lend insight into possible shape effects of natural filamentous viruses.

Shape effects of filaments versus spherical particles in flow and drug delivery.

Actin-related protein 2 and 3 (Arp2/3) complex forms a dendritic network of actin filaments during endocytosis and cellular locomotion by nucleating branches on the sides of preexisting actin filaments. Reconstructions of electron tomograms of branch junctions show how Arp2/3 complex anchors the branch, with Arp2 and Arp3 serving as the first two subunits of the branch. Our aim was to characterize the massive conformational change that moves Arp2 ∼30 Å from its position in crystal structures of inactive Arp2/3 complex to its position in branch junctions. Starting with the inactive crystal structure, we used atomistic-scale molecular dynamics simulations to drive Arp2 toward the position observed in branch junctions. When we applied forces to Arp2 while restraining Arp3, one block of structure (Arp2, subunit ARPC1, the globular domain of ARPC4 and ARPC5) rotated counterclockwise by 30° around a pivot point in an α-helix of ARPC4 (Glu⁸¹-Asn¹⁰⁰) to align Arp2 next to Arp3 in a second block of structure including ARPC3 and the globular domains of ARPC2. This active structure buried more surface area than the inactive conformation. The complex was stable in all simulations. In most simulations, collisions of subdomain 2 of Arp2 with Arp3 impeded the movement of Arp2.

Molecular dynamics simulations of Arp2/3 complex activation.

Nanoscale carriers of active compounds, especially drugs, need not be spherical in shape. Worm micelles as blends of degradable polylactic acid (PLA) and inert block copolymer amphiphiles were prepared for controlled release and initial study of carrier transport through nano-porous media. The loading capacity of a typical hydrophobic drug, Triamterene, and the release of hydrophobic dyes were evaluated together with morphological changes of the micelles. Degradation of PLA by hydrolysis led to the self-shortening of worms and a clear transition towards spherical micelles, correlating with the release of hydrophobic dyes. Perhaps equally important for application is the flexibility of worm micelles, which we show allows them to penetrate nanoporous gels where 100 nm sized vesicles cannot enter. Such gels have served as tissue models, and so the results here collectively suggest a new class of hydrophobic drug nano-carriers that are capable of tissue permeation as well as controlled release.

Polymeric worm micelles as nano-carriers for drug delivery.

We investigated the structure, properties and dynamics of the actin filament branch junction formed by actin-related protein (Arp) 2/3 complex using all-atom molecular dynamics (MD) simulations based on a model fit to a reconstruction from electron tomograms. Simulations of the entire structure consisting of 31 protein subunits together with solvent molecules containing ∼3 million atoms were performed for an aggregate time of 175 ns. One 75-ns simulation of the original reconstruction was compared to two 50-ns simulations of alternate structures, showing that the hypothesized branch junction structure is very stable. Our simulations revealed that the interface between Arp2/3 complex and the mother actin filament features a large number of salt bridges and hydrophobic contacts, many of which are dynamic and formed/broken on the timescale of the simulation. The simulations suggest that the DNase binding loops in Arp3, and possibly Arp2, form stabilizing contacts with the mother filament. Unbiased comparison of models sampled from the MD simulation trajectory with the primary experimental electron tomography data identified regions were snapshots from the simulation provide atomic details of the model structures and also pinpoints regions where the initial modeling based on the electron tomogram reconstruction may be suboptimal.

Key structural features of the actin filament Arp2/3 complex branch junction revealed by molecular simulation.

Cells sequester neutral lipids in bodies called lipid droplets. Thus, the formation and breakdown of the droplets are important for cellular metabolism; unfortunately, these processes are difficult to quantify. Here, we used time-lapse confocal microscopy to track the formation, movement and size changes of lipid droplets throughout the cell cycle in fission yeast Schizosaccharomyces pombe. In theory, the number of lipid droplets in these cells must increase for daughter cells to have the same number of droplets as the parent at a reference point in the cell cycle. We observed stable droplet formation events in G2 phase that were divided evenly between de novo formation of nascent droplets and fission of preexisting droplets. The observations that lipid droplet number is linked to the cell cycle and that droplets can form via fission were both new discoveries. Thus, we scrutinized each fission event for multiple signatures to eliminate possible artifacts from our microscopy. We augmented our time-lapse confocal microscopy with electron microscopy, which showed lipid droplet 'intermediates': droplets shaped like dumbbells that are potentially in transition states between two spherical droplets. Using these complementary microscopy techniques and also dynamic simulations, we show that lipid droplets can form by fission.

Lipid droplet de novo formation and fission are linked to the cell cycle in fission yeast.

Particles that have the potential to deliver imaging agents and drugs to cells and tissue now have many different shapes and sizes. This diversity in particle shape could provide new options for potential treatments of diseases because geometry affects biodistribution. However, the myriad of particle shapes now available increases the number of variables or parameters that must be taken into consideration for the drug delivery field to understand particle-cell interactions. This is especially true when the shape of a particle is a tunable parameter along with particle chemistry, charge, and hydrophobicity. Here we review the impact of shape on particle-cell interactions in vitro and the ramifications of different particle geometries on circulation, biodistribution, localization to tumors, and toxicology in rodents.

Particle shape effects in vitro and in vivo.

One of the key questions in cell biology is how organelles are passed from parent to daughter cells. To help address this question, I used Brownian dynamics to simulate lipid droplets as model organelles in populations of dividing cells. Lipid droplets are dynamic bodies that can form both de novo and by fission, they can also be depleted. The quantitative interplay among these three events is unknown but would seem crucial for controlling droplet distribution in populations of dividing cells. Surprisingly, of the three main events studied: biogenesis, fission, and depletion, the third played the key role in maintaining droplet organelle number-and to a lesser extent volume-in populations of dividing cells where formation events would have seemed paramount. In the case of lipid droplets, this provides computational evidence that they must be sustained, most likely through contacts with the endoplasmic reticulum. The findings also agree with video microscopy experiments over much shorter timescales where droplet depletion in fission yeast cells was not observed. In general, this work shows that organelle maintenance is invaluable and lack thereof cannot necessarily be compensated for by organelle formation. This study provides a time-accurate, physical-based template for long-term cell division studies.

Paul Dalhaimer

Department of Biochemistry and Cellular and Molecular Biology,
Department of Chemical and Biomolecular Engineering

University of Tennessee

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Paul Dalhaimer

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Cellular and Molecular Biology,Department of Chemical and Biomolecular Engineering

University of Tennessee

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Department of Biochemistry and Cellular and Molecular Biology,
Department of Chemical and Biomolecular Engineering