highly-multiplexed-super-resolution-imaging-of-t-cells-using-madstorm

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

National Institutes of Health

Enduring modification of synapses is central to long-lasting neural circuit plasticity. Such adaptations include rapid posttranslational modification of existing synaptic proteins over periods of minutes and persisting changes in the abundance of synaptic proteins over hours to days. Recently, ubiquitination and protein degradation have emerged as additional mechanisms for modifying the function and molecular composition of synapses. These recent findings raise intriguing questions as to how enduring changes at synapses are accomplished in the face of robust, ongoing molecular turnover.

Ubiquitin and protein turnover in synapse function.

Alterations in cellular structure and synapse composition are central to proper nervous system function. Recent work has identified the ubiquitin-proteasome system (UPS) as a key regulator of neuronal biology. The UPS is essential for the growth and development of immature neurons and is a critical mediator of synaptic adaptability in mature neurons. Furthermore, proteinaceous deposits that accumulate in diverse neurodegenerative disorders are enriched in components of the UPS, suggesting that UPS dysfunction may be pivotal for pathogenesis. Here, we summarize existing knowledge about the role of the UPS in brain function, highlighting recent work delineating its importance in neuronal development, plasticity, and degeneration.

Emerging roles for ubiquitin and protein degradation in neuronal function.

Pyramidal neurons of the hippocampus and cortex have polarized dendritic arbors, but little is known about the cellular mechanisms distinguishing apical and basal dendrites. We used morphometric analysis and time lapse imaging of cultured hippocampal neurons to show that glutamatergic neurons develop progressive dendritic asymmetry in the absence of polarized extrinsic cues. Thus, pyramidal neurons have a cellular program for polarized dendrite growth independent of tissue microenvironment.

Cell type-specific dendritic polarity in the absence of spatially organized external cues.

In the mammalian brain, the specification of a single axon and multiple dendrites occurs early in the differentiation of most neuron types. Numerous intracellular signaling events for axon specification have been described in detail. However, the identity of the extracellular factor(s) that initiate neuronal polarity in vivo is unknown. Here, we report that transforming growth factor beta (TGF-beta) initiates signaling pathways both in vivo and in vitro to fate naive neurites into axons. Neocortical neurons lacking the type II TGF-beta receptor (TbetaR2) fail to initiate axons during development. Exogenous TGF-beta is sufficient to direct the rapid growth and differentiation of an axon, and genetic enhancement of receptor activity promotes the formation of multiple axons. Finally, we show that the bulk of these TGF-beta-dependent events are mediated by site-specific phosphorylation of Par6. These results define an extrinsic cue for neuronal polarity in vivo that patterns neural circuits in the developing brain.

TGF-beta signaling specifies axons during brain development.

&#x7EE7;&#x7EED;&#x7684;&#x6DFB;&#x52A0;&#x65B0;&#x7684;&#x795E;&#x7ECF;&#x5143;&#x6210;&#x719F;&#x55C5;&#x7535;&#x8DEF;&#x8868;&#x793A;&#x7EC6;&#x80DE;&#x548C;&#x7ED3;&#x6784;&#x6027;&#x8111;&#x53EF;&#x5851;&#x6027;&#x7684;&#x5353;&#x8D8A;&#x6A21;&#x5F0F;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x65B0;&#x6210;&#x7ACB;&#x7684;&#x7535;&#x8DEF;&#x3001; &#x7C7B;&#x578B;&#x548C;&#x5F62;&#x6210;&#x65B0;&#x7684;&#x7A81;&#x89E6;&#x8FDE;&#x63A5;&#x7684;&#x795E;&#x7ECF;&#x7EC6;&#x80DE;&#x7684;&#x6570;&#x76EE;&#x548C;&#x7A81;&#x89E6;&#x8FDE;&#x63A5;&#x5728;&#x55C5;&#x7403;&#x5BF9;&#x611F;&#x5B98;&#x4F53;&#x9A8C;&#x7684;&#x5F71;&#x54CD;&#x7684;&#x89E3;&#x5256;&#x5B66;&#x914D;&#x7F6E;&#x4ECD;&#x7136;&#x77E5;&#x4E4B;&#x751A;&#x5C11;&#x3002;&#x4F7F;&#x7528;&#x4F53;&#x5185;&#x7535;&#x7A7F;&#x5B54;&#x548C;&#x5355;&#x7A81;&#x89E6;&#x7684;&#x8DDF;&#x8E2A;&#xFF0C;&#x6211;&#x4EEC;&#x663E;&#x793A;&#x8BE5;&#x4EA7;&#x540E;&#x51FA;&#x751F;&#x7684;&#x9897;&#x7C92;&#x7EC6;&#x80DE;&#x5F62;&#x6210;&#x7A81;&#x89E6;&#x8FDE;&#x63A5;&#x79BB;&#x5FC3;&#x6295;&#x5165;&#x4E0E;&#x4E8C;&#x5C16;&#x74E3;/&#x7C07;&#x7ED2;&#x7EC6;&#x80DE;&#x5C0F;&#x9F20;&#x55C5;&#x7403;&#x4E2D;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x521A;&#x51FA;&#x751F;&#x7684;&#x9897;&#x7C92;&#x7EC6;&#x80DE;&#x4ECE;&#x5F53;&#x5730;&#x6291;&#x5236;&#x77ED;&#x8F74;&#x7A81;&#x7684;&#x5355;&#x5143;&#x683C;&#x4E2D;&#xFF0C;&#x77E5;&#x4E4B;&#x751A;&#x5C11;&#x7684;&#x5355;&#x5143;&#x683C;&#x4EBA;&#x53E3;&#x5F97;&#x5230;&#x4E86;&#x5927;&#x91CF;&#x6295;&#x5165;&#x3002;&#x77ED;&#x8F74;&#x7A81;&#x7EC6;&#x80DE;&#x7684;&#x8FDE;&#x63A5;&#x663E;&#x793A;&#x805A;&#x96C6;&#x7684;&#x7EC4;&#x7EC7;&#xFF0C;&#x548C;&#x4ED6;&#x4EEC;&#x5230;&#x65B0;&#x751F;&#x9897;&#x7C92;&#x7EC6;&#x80DE;&#x7684;&#x7A81;&#x89E6;&#x6295;&#x5165;&#x6781;&#x5927;&#x5730;&#xFF0C;&#x5E76;&#x6709;&#x9009;&#x62E9;&#x6027;&#x5730;&#x6269;&#x5C55;&#x4E0E;&#x6C14;&#x5473;&#x523A;&#x6FC0;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x611F;&#x5B98;&#x7ECF;&#x9A8C;&#x4FC3;&#x8FDB;&#x65B0;&#x7684;&#x795E;&#x7ECF;&#x5143;&#x7A81;&#x89E6;&#x878D;&#x5165;&#x7EC6;&#x80DE;&#x7279;&#x5B9A;&#x4E8E;&#x7C7B;&#x578B;&#x7684;&#x55C5;&#x89C9;&#x7535;&#x8DEF;&#x3002;

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The continued addition of new neurons to mature olfactory circuits represents a remarkable mode of cellular and structural brain plasticity. However, the anatomical configuration of newly established circuits, the types and numbers of neurons that form new synaptic connections, and the effect of sensory experience on synaptic connectivity in the olfactory bulb remain poorly understood. Using in vivo electroporation and monosynaptic tracing, we show that postnatal-born granule cells form synaptic connections with centrifugal inputs and mitral/tufted cells in the mouse olfactory bulb. In addition, newly born granule cells receive extensive input from local inhibitory short axon cells, a poorly understood cell population. The connectivity of short axon cells shows clustered organization, and their synaptic input onto newborn granule cells dramatically and selectively expands with odor stimulation. Our findings suggest that sensory experience promotes the synaptic integration of new neurons into cell type-specific olfactory circuits.

Activity-induced remodeling of olfactory bulb microcircuits revealed by monosynaptic tracing.

L&#x26;#39;ajout continu de nouveaux neurones matures &#xE0; circuits olfactifs repr&#xE9;sente un mode remarquable de la plasticit&#xE9; du cerveau cellulaire et structurale. Toutefois, la configuration anatomique de circuits nouvellement cr&#xE9;&#xE9;es, les types et le nombre de neurones qui forment de nouvelles connexions synaptiques, et l&#x26;#39;effet de l&#x26;#39;exp&#xE9;rience sensorielle sur la connectivit&#xE9; synaptique dans le bulbe olfactif sont encore mal compris. En utilisant &#xE9;lectroporation in vivo et monosynaptique tra&#xE7;age, nous montrons que les cellules granulaires postnatale-n&#xE9;s forment des connexions synaptiques avec les entr&#xE9;es centrifuges et mitrale / tuft&#xE9; cellules dans le bulbe olfactif de la souris. En outre, les cellules granulaires nouveau-n&#xE9;s re&#xE7;oivent de nombreux commentaires de locaux inhibiteurs des cellules axones courts, une population de cellules mal compris. La connectivit&#xE9; des cellules axones courts montre l&#x26;#39;organisation en cluster, et leur entr&#xE9;e synaptique sur les cellules granulaires nouveau-n&#xE9;s de fa&#xE7;on spectaculaire et s&#xE9;lective augmente avec une stimulation odeur. Nos r&#xE9;sultats sugg&#xE8;rent que l&#x26;#39;exp&#xE9;rience sensorielle favorise l&#x26;#39;int&#xE9;gration synaptique de nouveaux neurones dans la cellule de type circuits sp&#xE9;cifiques olfactifs.

Activit&#xE9; induite par remodelage de Microcircuits bulbe olfactif R&#xE9;v&#xE9;l&#xE9; par monosynaptique Tracing

Die fortgesetzte Addition neuer Nervenzellen ausreifen olfaktorische Schaltungen darstellt einen bemerkenswerten Modus der zellul&auml;ren und strukturellen Gehirns Plastizit&auml;t. Die anatomische Konfiguration des neu gegr&uuml;ndeten Schaltungen, die Typen und die Anzahl an Neuronen, die neue synaptische Verbindungen bilden und die Wirkung der sensorischen Erfahrung auf synaptischen Konnektivit&auml;t in den Riechkolben bleiben jedoch weitgehend unverstanden. Mit in-vivo Electroporation und monosynaptischen Ablaufverfolgung, zeigen wir, dass postnatale geborener Untermodul Zellen synaptischen Verbindungen mit Radial ein- und Mitral-/getuftet Zellen in den Riechkolben Maus. Dar&uuml;ber hinaus erhalten neu geboren Untermodul Zellen umfangreiche Eingabe von lokalen hemmenden kurzen Axon Zellen, einer Verst&auml;ndnism&auml;ngel Zellpopulation. Die Anbindung von kurzen Axon Zellen zeigt gruppierten Organisation und synaptische Eingaben auf Neugeborene Untermodul Zellen drastisch und selektiv erweitert mit Geruch-Stimulation. Unsere Einsch&auml;tzungen zufolge f&ouml;rdert die sinnliche Erfahrung die synaptische Integration neuer Nervenzellen in Zelle typspezifische olfaktorischen Schaltungen.

Aktivit&auml;t induziert Umbau der Riechkolben Mikroschaltungen von monosynaptischen Ablaufverfolgung offenbart.

La continua aggiunta di nuovi neuroni maturi circuiti olfattivi rappresenta una modalit&agrave; di notevole della plasticit&agrave; cellulare e strutturale del cervello. Tuttavia, la configurazione anatomica della neonata circuiti, i tipi e il numero di neuroni che formano nuove connessioni sinaptiche e l&#39;effetto dell&#39;esperienza sensoriale sulla connettivit&agrave; sinaptica nel bulbo olfattivo restano scarsamente comprensibile. Usando elettroporazione in vivo e l&#39;analisi systemseizures, mostriamo quel granello postnatale nato connessioni sinaptiche di cellule forma con ingressi centrifughi e cellule mitral/trapuntati nel bulbo olfattivo del mouse. Inoltre, le cellule granulo appena nato ricevano input estesa da cellule locali assone breve inibitorio, una popolazione di cellule scarsamente comprensibile. La connettivit&agrave; delle cellule assone breve Mostra organizzazione in cluster, e loro ingresso sinaptico su cellule granulo neonato si espande drasticamente e in modo selettivo con stimolazione di odore. I nostri risultati suggeriscono che l&#39;esperienza sensoriale favorisce l&#39;integrazione sinaptica di nuovi neuroni in circuiti olfattive specifiche per il tipo di cella.

Attivit&agrave;-indotta da rimodellamento del bulbo olfattivo microcircuiti rivelati dall&#39;analisi systemseizures.

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&#xC131;&#xC219; &#xD6C4; &#xAC01; &#xD68C;&#xB85C;&#xC5D0; &#xC0C8;&#xB85C;&#xC6B4; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; &#xC9C0;&#xC18D;&#xC801;&#xC778;&#xB41C; &#xCD94;&#xAC00; &#xC140;&#xB8F0;&#xB7EC; &#xBC0F; &#xAD6C;&#xC870;&#xC801; &#xB1CC;&#xAC00; &#xC18C;&#xC131;&#xC758; &#xB180;&#xB77C;&#xC6B4; &#xBAA8;&#xB4DC;&#xB97C; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xC0C8;&#xB85C; &#xC124;&#xB9BD;&#xB41C; &#xB41C; &#xD68C;&#xB85C;, &#xC720;&#xD615; &#xBC0F; &#xC0C8;&#xB85C;&#xC6B4; &#xC2DC; &#xB0C5; &#xC2A4; &#xC5F0;&#xACB0;&#xC744; &#xD615;&#xC131; &#xD558;&#xB294; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; &#xC22B;&#xC790;&#xC640; &#x55C5; &#xC2DC; &#xB0C5; &#xC2A4; &#xC5F0;&#xACB0;&#xC5D0; &#xAC10;&#xAC01; &#xACBD;&#xD5D8;&#xC758; &#xC601;&#xD5A5;&#xC758; &#xD574; &#xBD80; &#xAD6C;&#xC131; &#xB0A8;&#xC544; &#xC81C;&#xB300;&#xB85C; &#xC774;&#xD574;. In vivo electroporation &#xBC0F; monosynaptic &#xCD94;&#xC801;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;, &#xC6B0;&#xB9AC;&#xB294; &#xCD9C;&#xC0DD; &#xD6C4; &#xCD9C;&#xC0DD;&#xACFC; &#xB9BD; &#xC138;&#xD3EC; &#xC6D0;&#xC2EC; &#xC785;&#xB825;&#xACFC; mitral/&#xC220; &#xC138;&#xD3EC;&#xC640; &#xC2DC; &#xB0C5; &#xC2A4; &#xC5F0;&#xACB0;&#xC5D0;&#xC5D0;&#xC11C; &#xD45C;&#xC2DC; &#xB9C8;&#xC6B0;&#xC2A4; &#x55C5;. &#xB610;&#xD55C;, &#xC0C8;&#xB85C; &#xD0DC;&#xC5B4;&#xB09C;&#xB41C;&#xACFC; &#xB9BD; &#xC138;&#xD3EC; &#xC9C0;&#xBC29; &#xC5B5;&#xC81C; &#xC9E7;&#xC740; &#xCD95; &#xC0AD; &#xC138;&#xD3EC;, &#xC81C;&#xB300;&#xB85C; &#xC774;&#xD574; &#xC140; &#xC778;&#xAD6C;&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; &#xAD11;&#xBC94;&#xC704; &#xD55C; &#xC785;&#xB825;&#xC744; &#xBC1B;&#xC744;. &#xC2E0;&#xC0DD;&#xC544;&#xACFC; &#xB9BD; &#xC138;&#xD3EC;&#xC5D0; &#xC2DC; &#xB0C5; &#xC2A4; &#xC785;&#xB825; &#xB0C4;&#xC0C8; &#xC790;&#xADF9;&#xC73C;&#xB85C; &#xD655;&#xC7A5; &#xD558;&#xB294; &#xADF9;&#xC801;&#xC73C;&#xB85C; &#xADF8;&#xB9AC;&#xACE0; &#xC120;&#xD0DD;&#xC801;&#xC73C;&#xB85C; &#xBC0F; &#xC9E7;&#xC740; &#xCD95; &#xC0AD; &#xC138;&#xD3EC;&#xC758; &#xC5F0;&#xACB0; &#xD074;&#xB7EC;&#xC2A4;&#xD130; &#xB41C; &#xC870;&#xC9C1;, &#xBCF4;&#xC5EC;&#xC900;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xAC10;&#xAC01; &#xACBD;&#xD5D8; &#xC140; &#xC720;&#xD615;&#xBCC4; &#xD6C4; &#xAC01; &#xD68C;&#xB85C;&#xC5D0; &#xC0C8;&#xB85C;&#xC6B4; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; &#xC2DC; &#xB0C5; &#xC2A4; &#xD1B5;&#xD569;&#xC744; &#xCD09;&#xC9C4; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xD65C;&#xB3D9; &#xC720;&#xB3C4; &#x55C5; microcircuits monosynaptic &#xCD94;&#xC801; &#xD558; &#xC5EC; &#xBC1D;&#xD600; &#xB9AC; &#xBAA8;&#xB378;&#xB9C1;.

La adici&#xF3;n continuada de nuevas neuronas maduras a circuitos olfativos representa un modo notable la plasticidad del cerebro celular y estructural. Sin embargo, la configuraci&#xF3;n anat&#xF3;mica de los circuitos de reciente creaci&#xF3;n, los tipos y el n&#xFA;mero de neuronas que se forman nuevas conexiones sin&#xE1;pticas, y el efecto de la experiencia sensorial en la conectividad sin&#xE1;ptica en el bulbo olfatorio, siguen siendo poco conocidos. Gracias al uso de la electroporaci&#xF3;n in vivo y monosin&#xE1;ptico trazado, nos muestran que despu&#xE9;s del parto nacidos en las c&#xE9;lulas granulares forman conexiones sin&#xE1;pticas con las entradas de centr&#xED;fugas y las c&#xE9;lulas mitrales / mo&#xF1;udo en el bulbo olfatorio del rat&#xF3;n. Adem&#xE1;s, los reci&#xE9;n nacidos c&#xE9;lulas granulares reciben una amplia contribuci&#xF3;n de las c&#xE9;lulas inhibidoras locales ax&#xF3;n corto, una poblaci&#xF3;n de c&#xE9;lulas poco conocidos. La conectividad de las c&#xE9;lulas de ax&#xF3;n corto muestra la organizaci&#xF3;n en cl&#xFA;ster, y su entrada sin&#xE1;ptica en las c&#xE9;lulas granulosas del reci&#xE9;n nacido de manera espectacular y se expande de forma selectiva con estimulaci&#xF3;n olor. Nuestros hallazgos sugieren que la experiencia sensorial promueve la integraci&#xF3;n sin&#xE1;ptica de las neuronas nuevas en el tipo de c&#xE9;lulas espec&#xED;ficas de los circuitos del olfato.

Actividad inducida Remodelaci&#xF3;n de microcircuitos bulbo olfatorio revelado por monosin&#xE1;ptico de seguimiento

Actin retrograde flow and actomyosin II contraction have both been implicated in the inward movement of T cell receptor (TCR) microclusters and immunological synapse formation, but no study has integrated and quantified their relative contributions. Using Jurkat T cells expressing fluorescent myosin IIA heavy chain and F-tractin-a novel reporter for F-actin-we now provide direct evidence that the distal supramolecular activation cluster (dSMAC) and peripheral supramolecular activation cluster (pSMAC) correspond to lamellipodial (LP) and lamellar (LM) actin networks, respectively, as hypothesized previously. Our images reveal concentric and contracting actomyosin II arcs/rings at the LM/pSMAC. Moreover, the speeds of centripetally moving TCR microclusters correspond very closely to the rates of actin retrograde flow in the LP/dSMAC and actomyosin II arc contraction in the LM/pSMAC. Using cytochalasin D and jasplakinolide to selectively inhibit actin retrograde flow in the LP/dSMAC and blebbistatin to selectively inhibit actomyosin II arc contraction in the LM/pSMAC, we demonstrate that both forces are required for centripetal TCR microcluster transport. Finally, we show that leukocyte function-associated antigen 1 clusters accumulate over time at the inner aspect of the LM/pSMAC and that this accumulation depends on actomyosin II contraction. Thus actin retrograde flow and actomyosin II arc contraction coordinately drive receptor cluster dynamics at the immunological synapse.

Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells.

T cells rapidly reposition their centrosome to the center of the immunological synapse (IS) to drive polarized secretion in the direction of the bound target cell. Using an optical trap for spatial and temporal control over target presentation, we show that centrosome repositioning in Jurkat T cells exhibited kinetically distinct polarization and docking phases and required calcium flux and signaling through both the T cell receptor and integrin to be robust. In "frustrated" conjugates where the centrosome is stuck behind the nucleus, the center of the IS invaginated dramatically to approach the centrosome. Consistently, imaging of microtubules during normal repositioning revealed a microtubule end-on capture-shrinkage mechanism operating at the center of the IS. In agreement with this mechanism, centrosome repositioning was impaired by inhibiting microtubule depolymerization or dynein. We conclude that dynein drives centrosome repositioning in T cells via microtubule end-on capture-shrinkage operating at the center of the IS and not cortical sliding at the IS periphery, as previously thought.

Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage.

We describe an approach to selectively activate a kinase in a specific protein complex or at a specific subcellular location within living cells and within minutes. This reveals the effects of specific kinase pathways without time for genetic compensation. The new technique, dubbed rapamycin-regulated targeted activation of pathways (RapRTAP), was used to dissect the role of Src kinase interactions with FAK and p130Cas in cell motility and morphodynamics. The overall effects of Src activation on cell morphology and adhesion dynamics were first quantified, without restricting effector access. Subsets of Src-induced behaviors were then attributed to specific interactions between Src and the two downstream proteins. Activation of Src in the cytoplasm versus at the cell membrane also produced distinct phenotypes. The conserved nature of the kinase site modified for RapRTAP indicates that the technique can be applied to many kinases.

Dissecting motility signaling through activation of specific Src-effector complexes.

Optogenetic control of endogenous signaling can be an important tool for probing cell behavior. Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1, we developed analogues of kinase inhibitors whose activity is light dependent. Inhibitory peptides were appended to the Jα helix, where they potently inhibited kinases in the light but were sterically blocked from kinase interaction in the dark. Photoactivatable inhibitors for cyclic-AMP dependent kinase (PKA) and myosin light chain kinase (MLCK) are described, together with studies that shed light on proper positioning of the peptides in the LOV domain. These inhibitors altered endogenous signaling in living cells and produced light-dependent changes in cell morphodynamics.

Manipulation of endogenous kinase activity in living cells using photoswitchable inhibitory peptides.

Ectonucleotidases are membrane-bound or secreted proteins that hydrolyze extracellular nucleotides.  Recently, we identified three ectonucleotidases that hydrolyze extracellular adenosine 5'-monophosphate (AMP) to adenosine in primary somatosensory neurons.  Currently, it is unclear which ectonucleotidases hydrolyze ATP and ADP in these neurons.  Ectonucleoside triphosphate diphosphohydrolases (ENTPDs) comprise a class of enzymes that dephosphorylate extracellular ATP and ADP.  Here, we found that ENTPD3 (also known as NTPDase3 or CD39L3) was located in nociceptive and non-nociceptive neurons of the dorsal root ganglion (DRG), in the dorsal horn of the spinal cord, and in free nerve endings in the skin.  To determine if ENTPD3 contributes directly to ATP and ADP hydrolysis in these tissues, we generated and characterized an Entpd3 knockout mouse.  This mouse lacks ENTPD3 protein in all tissues examined, including the DRG, spinal cord, skin, and bladder.  However, DRG and spinal cord tissues from Entpd3 (-/-) mice showed no reduction in histochemical staining when ATP, ADP, AMP, or UTP were used as substrates.  Additionally, using fast-scan cyclic voltammetry (FSCV), adenosine production was not impaired in the dorsal spinal cord of Entpd3 (-/-) mice when the substrate ADP was applied.  Further, Entpd3 (-/-) mice did not differ in nociceptive behaviors when compared to wild-type mice, although Entpd3 (-/-) mice showed a modest reduction in β-alanine-mediated itch.  Taken together, our data indicate that deletion of Entpd3 does not impair ATP or ADP hydrolysis in primary somatosensory neurons or in dorsal spinal cord.  Moreover, our data suggest there could be multiple ectonucleotidases that act redundantly to hydrolyze nucleotides in these regions of the nervous system.

Deletion of ENTPD3 does not impair nucleotide hydrolysis in primary somatosensory neurons or spinal cord.

Deletion of UBE3A causes the neurodevelopmental disorder Angelman syndrome (AS), while duplication or triplication of UBE3A is linked to autism. These genetic findings suggest that the ubiquitin ligase activity of UBE3A must be tightly maintained to promote normal brain development. Here, we found that protein kinase A (PKA) phosphorylates UBE3A in a region outside of the catalytic domain at residue T485 and inhibits UBE3A activity toward itself and other substrates. A de novo autism-linked missense mutation disrupts this phosphorylation site, causing enhanced UBE3A activity in vitro, enhanced substrate turnover in patient-derived cells, and excessive dendritic spine development in the brain. Our study identifies PKA as an upstream regulator of UBE3A activity and shows that an autism-linked mutation disrupts this phosphorylation control. Moreover, our findings implicate excessive UBE3A activity and the resulting synaptic dysfunction to autism pathogenesis.

An Autism-Linked Mutation Disables Phosphorylation Control of UBE3A.

Microvilli set the stage for T-cell activation.

Investigation of heterogeneous cellular structures using single-molecule localization microscopy has been limited by poorly defined localization accuracy and inadequate multiplexing capacity. Using fluorescent nanodiamonds as fiducial markers, we define and achieve localization precision required for single-molecule accuracy in dSTORM images. Coupled with this advance, our new multiplexing strategy, madSTORM, allows accurate targeting of multiple molecules using sequential binding and elution of fluorescent antibodies. madSTORM is used on an activated T-cell to localize 25 epitopes, 14 of which are on components of the same multimolecular T-cell receptor complex. We obtain an average localization precision of 2.6 nm, alignment error of 2.0 nm, and <0.01% cross-talk. Combining these technical advances affords the ability to move beyond obtaining superresolved structures to defining spatial relationships among constituent molecules within structures. Probing the molecular topology of complex signaling cascades and other heterogeneous networks is feasible with madSTORM.

madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy.

Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1). Using structured-illumination microscopy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments generated by one or more formins present at the IS distal edge. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs. Three-dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs. Consistently, disrupting actin arc formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T-B cell adhesion, and diminished TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport.

Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse.

The adapter molecule linker for activation of T cells (LAT) plays a crucial role in forming signaling complexes induced by stimulation of the T cell receptor (TCR). These multi-molecular complexes are dynamic structures that activate highly regulated signaling pathways. Previously, we have demonstrated nanoscale structure in LAT-based complexes where the adapter SLP-76 (also known as LCP2) localizes to the periphery of LAT clusters. In this study, we show that initially LAT and SLP-76 are randomly dispersed throughout the clusters that form upon TCR engagement. The segregation of LAT and SLP-76 develops near the end of the spreading process. The local concentration of LAT also increases at the same time. Both changes require TCR activation and an intact actin cytoskeleton. These results demonstrate that the nanoscale organization of LAT-based signaling complexes is dynamic and indicates that different kinds of LAT-based complexes appear at different times during T cell activation.

Development of nanoscale structure in LAT-based signaling complexes.

Single-molecule localization microscopy (SMLM) comprises methods that produce super-resolution images from molecular locations of single molecules. These techniques mathematically determine the center of a diffraction-limited spot produced by a fluorescent molecule, which represents the most likely location of the molecule. Only a small cohort of well-separated molecules is visualized in a single image, and then many images are obtained from a single sample. The localizations from all the images are combined to produce a super-resolution picture of the sample. Here we describe the application of two methods, photoactivation localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM), to the study of signaling microclusters in T cells.

Super-resolution Analysis of TCR-Dependent Signaling: Single-Molecule Localization Microscopy.

UBE3A is a HECT domain E3 ubiquitin ligase whose dysfunction is linked to autism, Angelman syndrome, and cancer. Recently, we characterized a de novo autism-linked UBE3A mutant (UBE3AT485A) that disrupts phosphorylation control of UBE3A activity. Through quantitative proteomics and reporter assays, we found that the UBE3AT485A protein ubiquitinates multiple proteasome subunits, reduces proteasome subunit abundance and activity, stabilizes nuclear β-catenin, and stimulates canonical Wnt signaling more effectively than wild-type UBE3A. We also found that UBE3AT485A activates Wnt signaling to a greater extent in cells with low levels of ongoing Wnt signaling, suggesting that cells with low basal Wnt activity are particularly vulnerable to UBE3AT485A mutation. Ligase-dead UBE3A did not stimulate Wnt pathway activation. Overexpression of several proteasome subunits reversed the effect of UBE3AT485A on Wnt signaling. We also observed that subunits that interact with UBE3A and affect Wnt signaling are located along one side of the 19S regulatory particle, indicating a previously unrecognized spatial organization to the proteasome. Altogether, our findings indicate that UBE3A regulates Wnt signaling in a cell context-dependent manner and that an autism-linked mutation exacerbates these signaling effects. Our study has broad implications for human disorders associated with UBE3A gain or loss-of-function, and suggest that dysfunctional UBE3A might affect additional proteins and pathways that are sensitive to proteasome activity.

Article	Year
Ubiquitin and protein turnover in synapse function. Neuron\| PubMed ID: 16129392	2005
Emerging roles for ubiquitin and protein degradation in neuronal function. Pharmacological reviews\| PubMed ID: 17329546	2007
Cell type-specific dendritic polarity in the absence of spatially organized external cues. Brain cell biology\| PubMed ID: 17940911	2006
TGF-beta signaling specifies axons during brain development. Cell\| PubMed ID: 20603020	2010
Activity-induced remodeling of olfactory bulb microcircuits revealed by monosynaptic tracing. PloS one\| PubMed ID: 22216277	2011
Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells. Molecular biology of the cell\| PubMed ID: 22219382	2012
Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage. The Journal of cell biology\| PubMed ID: 23979719	2013
Dissecting motility signaling through activation of specific Src-effector complexes. Nature chemical biology\| PubMed ID: 24609359	2014
Manipulation of endogenous kinase activity in living cells using photoswitchable inhibitory peptides. ACS synthetic biology\| PubMed ID: 24905630	2014
Deletion of ENTPD3 does not impair nucleotide hydrolysis in primary somatosensory neurons or spinal cord. F1000Research\| PubMed ID: 25717362	2014
An Autism-Linked Mutation Disables Phosphorylation Control of UBE3A. Cell\| PubMed ID: 26255772	2015
Microvilli set the stage for T-cell activation. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 27663734	2016
madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy. Molecular biology of the cell\| PubMed ID: 27708141	2016
Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse. The Journal of cell biology\| PubMed ID: 27799367	2016
Development of nanoscale structure in LAT-based signaling complexes. Journal of cell science\| PubMed ID: 27875277	2016
Super-resolution Analysis of TCR-Dependent Signaling: Single-Molecule Localization Microscopy. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28255704	2017
The autism-linked UBE3A T485A mutant E3 ubiquitin ligase activates the Wnt/β-catenin pathway by inhibiting the proteasome. The Journal of biological chemistry\| PubMed ID: 28559284	2017

Jason Yi

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