Este trabalho é apoiado pela Universidade de Takasaki da saúde e bem-estar.

1. cultivo de células e adição de antígenos exógenos <ol><li>preparar bOVA usando uma rotulagem de biotina-proteína kit seguindo o fabricante &#39; protocolo s. 
	Nota: Normalmente, bOVA contém biotina 2 M por 1 M de óvulos em média.</li>
	<li>DC2.4 crescer células em RPMI-1640 suplementado com 2 mM L-glutamina, piruvato de sódio 1 mM, aminoácidos não essenciais de 0,1 mM, 100 U/mL de penicilina-estreptomicina, 55 mM 2-Mercaptoetanol, 10 mM HEPES (pH 7,5) e soro fetal bezerro de 10% (doravante RPMI) a 37 ° C em 5 % CO 2 em uma incubadora umidificada (doravante sem menção, as células são incubadas a essa condição). Também é possível usar DMEM suplementado com 10% de soro fetal bezerro, 3,7 g/L NaHCO 3 (doravante DMEM) no lugar de RPMI.</li>
	<li>Um dia antes da purificação, dividir as células em RPMI a 1 x 10 5 células/mL em uma placa de cultura de tecido. Evite manter as células em um estado confluente. DC2.4 células podem altamente incorporar antígenos exógenos até um estado semi confluente, mas rapidamente perdem a capacidade depois de atingir um estado de excesso confluente.</li>
	<li>Antes do DC2.4 células são semi confluentes, adicionar 250 µ g/mL de bOVA para 1 x 10 6 células/mL e incubar durante 2-4 h. 
	Nota: Durante a jusante experimentos, todos os buffers e os reagentes são mantidos a 4 ° C a menos que indicado de outra forma.</li>
</ol> 2. Preparação de Microsomes <ol><li>colher as DC2.4 células pipetando suave da placa de tecido em um novo tubo cónico de 50 mL.</li>
	<li>Centrifugar 1.000 x g por 5 min, 4 ° C e remova cuidadosamente o meio com bOVA por aspiração.</li>
	<li>Lavar as células DC2.4 duas vezes com 40 mL de tampão PBS (1,37 mM NaCl, 8,1 mM Na 2 HPO 4, 2,68 mM KCl, 1,47 mM KH 2 PO 4) por centrifugação a 1.000 x g, durante 5 min, 4 ° C e descartar o sobrenadante de cada vez por aspiração.</li>
	<li>Ressuspender o pellet em passo 2.3 em 1-2 mL (1/20-1/10 volume de meio de cultura) do meio de homogeneização (0,25 M de sacarose, 1 mM EDTA, 10 mM HEPES-NaOH [pH 7,4]) com volume de 1/1.000 de coquetéis de inibidor de protease e transferência a ressuspensão DC2.4 células em um gelada homogenizacao homogenizador.</li>
	<li>Interromper as células DC2.4 ressuspensa suavemente por 10-20 pancadas com o gelado homogenizacao homogenizador. 
	Nota: Durante a etapa de perturbação, a homogenizacao homogeneizador é refrigerado no gelo</li>
	<li>Transferir a suspensão celular-interrompido para um novo tubo cônico de 15 mL.</li>
	<li>Adicionar 8-9 mL de meio de homogeneização total 10 mL e centrifugar o tubo cônico durante 10 minutos a 2.000 x g e 4 ° C.</li>
	<li>Transferir o sobrenadante para um novo tubo cônico de 15 mL para remover células ininterrupta e núcleos e centrifugar o tubo cônico novamente por 10 min a 2.000 x g e 4 ° C.</li>
	<li>Transferir o sobrenadante para um novo tubo de ultracentrifugação e centrifugar por 45 min em 100.000 x g e 4 ° C.</li>
	<li>Aspirar o sobrenadante e ressuspender o cuidadosamente em 1-2 mL de meio de homogeneização com volume de 1/1.000 de cocktails de inibidor de protease pipetando a solução acima e para baixo várias vezes para fazer uma fração do microsome para jusante experiências.</li>
	<li>Transferir a fração do microsome para um novo tubo de fundo redondo de 5 mL. 
	Nota: Após esta etapa, é possível enriquecer microssomas objetivas por iodixanol centrifugação gradiente de densidade de acordo com o fabricante &#39; protocolo de s, remover microssomas específico. As frações de pico para antígenos exógenos são recolhidas e em seguida, submetidas à etapa seguinte de purificação (etapa 3).</li>
</ol> 3. Purificação de Microsomes com bOVA ERAD passando por <ol><li>volume de adicionar 1/100 de fresco grânulos SA-magnético para a fração do microsome da etapa 2.11 no 5 mL tubo de fundo redondo. 
	Nota: Antes desta etapa, é possível pre-limpar a fração do microsome por grânulos controle magnético para reduzir contaminações de específico microsomes.</li>
	<li>Delicadamente, misture bem e gire o tubo de fundo redondo, lentamente por 30 min a 4 ° C.</li>
	<li>Adicionar 2-3 mL de meio de homogeneização total 4 ml para o tubo de fundo redondo e misture suavemente bem.</li>
	<li>Colocar o tubo de fundo redondo num suporte magnético e incubar durante 10 minutos a 4 ° C. Desde que os grânulos ligados para as vesículas são atraídos para o ímã, microgrânulos marrons gradualmente acumularão na parede do tubo mais próximo ao ímã.</li>
	<li>Com o tubo restante no suporte magnético, cuidadosamente descartar os sobrenadantes por aspiração para remover as vesículas desacopladas.</li>
	<li>Lavagem os grânulos magnéticos vinculados as vesículas duas vezes com 5 mL de meio de homogeneização, a magnética defende a 10 min a 4 ° C e descartar o sobrenadante cada vez por cuidadosamente aspiração. 
	Nota: Sem o suporte magnético, purificação por centrifugações a 2.000 x g por 10 min, 4 ° C também está disponível.</li>
	<li>Ressuspender os grânulos magnéticos vinculados as vesículas cuidadosamente em meio de homogeneização 100 µ l a pipetagem a solução acima e para baixo várias vezes.</li>
	<li>Transferir as ressuspensa vesículas em um microtubo de novo como do microsome purificado para jusante experiências. 
	Nota: Normalmente, fração do microsome 50 µ l contendo 5-20 µ g proteínas de 1 x 10 7 células pode ser isolados.</li>
</ol> 4. Análise do Microsomes purificado grânulos <ol><li>Ressuspender o magnético ligados para as vesículas de passo 3.8 a 100-200 µ l de tampão TNE (pH de 20 mM Tris-HCl 7.4, 150 mM de NaCl, EDTA 0,5 M, 1% Nonidet P-40) com volume de 1/1.000 de cocktails de inibidor de protease em vez de meio de homogeneização.</li>
	<li>Transferir o lisado de passo 4.1 para um novo microtubo de 1,5 mL.</li>
	<li>Determinar a concentração de proteína da etapa 4.2 usando um kit de ensaio do BCA pelo fabricante &#39; protocolo de s. 
	Nota: Normalmente, 5-10 µ l lisado da etapa 4.2 é suficiente para determinar a concentração de proteína.</li>
	<li>Transferir o lisado de passo 4.2 (2 µ g de proteína para coloração de prata e 10 µ g de proteína para a mancha ocidental) em microtubos novos.</li>
	<li>Colocar os microtubos em um bloco de calor em proteínas de 95 ° C e ferver em tampão de carregamento de gel de SDS 1x (pH 100 mM Tris-HCl 6,8, azul de bromofenol 200mm ditiotreitol, SDS de 4%, 0,2%, 20% de glicerol) 5 min.</li>
	<li>Centrifugar os microtubos para 10 min a 21.500 x g e 4 ° C. coletar os sobrenadantes em microtubos novos para remover as frações insolúveis.</li>
	<li>Analisar as proteínas resolvidas na etapa 4.6 (2 µ g) por padrão SD-PAGE.</li>
	<li>Visualize as bandas de proteína pela prata coloração usando coloração prata kit após o fabricante &#39; protocolo de s.</li>
	<li>Analisar as proteínas resolvidas na etapa 4.6 (10 µ g) por padrão SD-PAGE e Western Bloting. Use o reagente (SA-HRP) pelo fabricante &#39; s de protocolo e Visualizar por fluorography.</li>
</ol> 5. In Vitro Reconstituição da ERAD de bOVA usando purificado Microsomes <ol><li>transferência o purificado microssomas (5-10 µ g de proteína) da etapa 3.8 com um volume de 50% de reticulócitos lisado (RL), em tampão de reação (50 mM Tris pH 7,4, 3mm ATP, 0.5 mM MgCl 1x 2) e 12:02 bandeira-tag ubiquitin em um microtubo de novo. O volume final deste experimento é 20-40 µ l.</li>
	<li>Incubar o microtubo para 2 h a 37 ° C. 
	Nota: Se a quantidade de ubiquitinated bOVA na etapa 5.12 é muito pequena para detectar pela mancha ocidental, adicionar 10 µ m de MG132 ou 2 µM de lactacystin para inibir o processamento do bOVA ubiquitinated por proteasomes.</li>
	<li>Parar a reação, colocando o microtubo na Ice.</li>
	<li>Resolver as microssomas adicionando buffer de TNE 500 µ l com volume de 1/1.000 de cocktails de inibidor de protease.</li>
	<li>o microtubo de centrifugação durante 10 minutos a 21,500 x g e 4 ° C. recolher os sobrenadantes para um microtubo novo para remover as frações insolúveis, que contêm ubiquitinated bOVA em DC2.4 antes do ensaio em vitro ubiquitination.</li>
	<li>Transferir o sobrenadante para um microtubo de novo e adicionar o volume de 1/100 de novas contas SA-magnético (geralmente 5 µ l para 500 µ l de sobrenadantes).</li>
	<li>Delicadamente, misture bem e girar o microtubo lentamente por 30 min a 4 ° C.</li>
	<li>Centrifugar o microtubo para 10 min a 21.500 x g e 4 ° C. descartar o sobrenadante por aspiração para recuperar o bOVA e ubiquitinated bOVA vinculado com os grânulos SA-magnético.</li>
	<li>Lavar duas vezes os grânulos SA-magnética coletados com 1 mL de tampão TNE por centrifugação durante 10 min a 21.500 x g e 4 ° C. descartar o sobrenadante de cada vez por aspiração.</li>
	<li>Ferver os grânulos SA-magnético em 1x do gel de SDS carregando buffer para min 5 a 95 ° C, em um bloco de calor para resolver as proteínas purified pelos grânulos magnético-SA.</li>
	<li>Centrifugar o microtubo para 10 min a 21.500 x g e 4 ° C. recolher os sobrenadantes para um microtubo novo para remover as frações insolúveis.</li>
	<li>Analisar as contas SA-magnéticos ligadas às proteínas por padrão SD-PAGE e Western Bloting. O uso de anticorpos (anti-Flag, anti-multi-Ub e anti-mouse IgG HRP) e o reagente (SA-HRP) é pelo fabricante &#39; protocolo s e visualizado por fluorography.</li>
</ol>

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L., Kyritsis, C., Tamp&#233;, R., Cresswell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+phagosomes+in+dendritic+cells+form+a+cellular+compartment+sufficient+for+cross+presentation+of+exogenous+antigens.">Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens.</a> Proc Natl Acad Sci U S A. 100 (22), 12889-12894 (2003).</li><li>Saveanu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRAP+identifies+an+endosomal+compartment+required+for+MHC+class+I+cross-presentation.">IRAP identifies an endosomal compartment required for MHC class I cross-presentation.</a> Science. 325 (5937), 213-217 (2009).</li><li>Zehner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translocon+protein+Sec61+mediates+antigen+transport+from+endosomes+in+the+cytosol+for+cross-presentation+to+CD8(+)+T+cells.">The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.</a> Immunity. 42 (5), 850-863 (2015).</li></ol>

Os autores não têm nada para divulgar.

Em estudos anteriores de CP, os antígenos exógenos incorporados acumularam na área restrita do atrasado endosome ou ER por microscopia de imunofluorescência16,30,31,32. Estima-se que ERAD, como transporte e processamento de antígenos exógenos são realizados nestas áreas especializadas da ER ou endossomo atrasado, como o compartimento celular foi identificado pela sacarose ou iodixanol u...

O MHC moléculas são expressas na superfície de todas as células nucleadas, com curtos peptídeos antigênicos derivados de antígenos endógenos, que são processados pelo sistema ubiquitina-Proteassoma no citosol1. Após o processamento, peptídeos antigênicos são transportados para o lúmen do retículo endoplasmático (ER) pelo transportador peptídeo TAP. No lúmen do ER, uma série de acompanhantes específicos auxiliar o carregamento do peptide e o dobramento correto do MHC complexo eu. Esta série de moléculas é chamado o complexo peptídeo-carregamento (PLC), indicando que o ER é um compartimento central para peptídeo carregando em cima do MHC eu2. Depois de peptídeo de carregamento, o MHC moléculas são transportadas à superfície da célula e desempenhar um papel fundamental no sistema imune adaptativo como marcadores auto e habilita o CD8+ linfócitos T citotóxicos (CTLs) para detectar células cancerosas ou agentes infecciosos por antigênica peptídeos de proteínas não-auto3.
Em células apresentadoras de (APC), peptídeos antigênicos de antígenos exógenos são também apresentados em MHC eu4,5,6,7,8 através do CP, que é realizada principalmente por DCs9,10,11. CP é essencial tanto para a ativação do ingênuo CD8+ T memória CD8 e células+ T células em anti-infecciosos e anti-tumoral CTLs12,13e na manutenção da tolerância imunológica pela inactivar de auto interino ingênuo T células14,15. O CP desempenha muitos papéis importantes no sistema imune adaptativo, porém os mecanismos moleculares de CP tem ainda que ser descrita em pormenor. Estudos anteriores de CP revelaram que antígenos exógenos foram localizados no pronto-socorro e o endossomo e foram processados por ERAD, sugerindo que antígenos exógenos são transportados desde o endossomo ao pronto-socorro para a ERAD, como processamento e peptídeo carregando16 . No entanto, acumula evidências indicam que o carregamento do peptide do CP é realizado, não no PS, mas sim em compartimentos endocítica não-clássica, que também têm características distintivas do ER (Figura 1)17,18 ,19,20,21. Para evitar a degradação dos precursores de peptídeo antigênico pela alta atividade de aminopeptidase22 no citosol, processamento e peptídeo carregando no CP ocorre na região proximal desses compartimentos endocítica não-clássica (Figura 1). Embora as caracterizações desses compartimentos endocítica são controversas, não há nenhuma molécula de específica existente além de antígenos exógenos neste compartimento.
ERAD é uma via celular, que especificamente remove proteínas misfolded da emergência. Na via da ERAD, misfolded proteínas retrogradely são transportadas através da membrana de ER para o citoplasma e processadas pelo sistema da ubiquitina-Proteassoma23,24,25. Quando as grandes moléculas, tais como proteínas, são transportadas através da bicamada lipídica, essas moléculas passam por um aparato molecular chamado um translocon, tais como o complexo de Sec61 e Derlin complexo em ER26e o complexo de Tom e Tim complexo na mitocôndrias,27. Quando adicionado exogenamente antígenos são transportados através da membrana de ER, eles devem penetrar a bicamada lipídica em complexo com translocons, tais como o complexo de Sec61. O método descrito aqui purificado da vesícula alvo utilizando estas moléculas de membrana-penetrante como marcadores para os compartimentos endocítica.
O método descrito aqui é um novo protocolo de purificação de vesículas usando o DC, como célula linha DC2.428 e biotinilado ovalbumina (bOVA) como um antígeno exógeno. Os compartimentos endocítica foram purificados por streptavidin (SA)-grânulos magnéticos usando o bOVA penetrar a membrana como um criador. Neste do microsome purificado, alguns adicionados exogenamente bOVA ainda foi preservado em frações de membrana, mas foram transportado para a parte externa do microsome e então ubiquitinated e processada em vitro29. Este microsome purificado contidos não só endocítica compartimento específico proteínas, mas também proteínas ER-residente para ERAD e o peptídeo carregamento complexo; sugerindo que o compartimento celular é o compartimento endocítica prospectivo para CP29. Este protocolo não é dependente do tipo de antígenos exógenos e também é aplicável para outros subconjuntos de DC e outros tipos de células, como macrófagos, células B e células endoteliais, para esclarecer o mecanismo molecular preciso de DCs para CP proficiente.

<table><tbody><tr><td>RPMI 1640</td><td>gibco by life technologies</td><td>11875-093</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Equitech bio</td><td>SFB30</td><td></td></tr><tr><td>Sodium pyruvate</td><td>gibco by life technologies</td><td>11360-070</td><td></td></tr><tr><td>MEM non-essential amino acids</td><td>gibco by life technologies</td><td>11140-050</td><td></td></tr><tr><td>HEPES</td><td>gibco by life technologies</td><td>15630-080</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>gibco by life technologies</td><td>21985-023</td><td></td></tr><tr><td>L-glutamine</td><td>gibco by life technologies</td><td>25030-164</td><td></td></tr><tr><td>Penisicillin-Sreptomycin</td><td>gibco by life technologies</td><td>15140-122</td><td></td></tr><tr><td>DMEM</td><td>gibco by life technologies</td><td>12100-46</td><td></td></tr><tr><td>OVA</td><td>SIGMA</td><td>A5503</td><td></td></tr><tr><td>Biotin-protein labelling kit</td><td>Thermo Fisher Scientific</td><td>F6347</td><td></td></tr><tr><td>MG-132</td><td>Santa Cruz Biotechnology</td><td>201270</td><td></td></tr><tr><td>lactacystin</td><td>SIGMA</td><td>L6785</td><td></td></tr><tr><td>Dounce homogenizer</td><td>IUCHI</td><td>131703</td><td></td></tr><tr><td>protease inhibitor cocktails</td><td>SIGMA</td><td>P8340</td><td></td></tr><tr><td>iodixanol</td><td>Cosmo bio</td><td>1114542</td><td></td></tr><tr><td>SA-magnetic beads</td><td>New England Biolabs</td><td>201270</td><td></td></tr><tr><td>control magnetic beads</td><td>Chemagen</td><td>M-PVA012</td><td></td></tr><tr><td>magnetic stand</td><td>BD Biosciences</td><td>552311</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>silver staining kits</td><td>Cosmo bio</td><td>423413</td><td></td></tr><tr><td>Reticulocyte Lysate</td><td>Promega</td><td>1730714</td><td></td></tr><tr><td>Flag-tagged ubiquitin</td><td>SIGMA</td><td>U5382</td><td></td></tr><tr><td>anti-ovalbumin (OVA,mouse)</td><td>Antibody Shop</td><td>HYB 094-06</td><td></td></tr><tr><td>ant-multi-ubiquitin (mouse)</td><td>MBL</td><td>D058&amp;minus;3</td><td></td></tr><tr><td>anti-Flag (mouse)</td><td>SIGMA</td><td>F3165</td><td></td></tr><tr><td>trypsin</td><td>SIGMA</td><td>85450C</td><td></td></tr></tbody></table>

purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation

Células dendríticas (DCs) são altamente capazes de processar e apresentar antígenos exógenos interiorizados a classe principal de histocompatibilidade (MHC) moléculas também conhecido como Cruz-apresentação (CP). CP desempenha um papel importante não só na estimulação de ingênuo CD8+ T memória CD8 e células+ T células para infecciosas e imunidade de tumor, mas também na inativação de auto-agente ingênuo T células por células T anergy ou supressão de células T. Embora o mecanismo molecular crítico da CP continua a ser elucidado, acumula evidências indicam que antígenos exógenos são processados através de retículo endoplasmático associado degradação (ERAD) após a exportação do não-clássica endocítica compartimentos. Até recentemente, caracterizações desses compartimentos endocítica limitavam-se porque não havia nenhum marcadores moleculares específicos além de antígenos exógenos. O método descrito aqui é um novo protocolo de isolamento de vesículas, que permite a purificação desses compartimentos endocítica. Usando esta purificada do microsome, nós reconstituído a ERAD, como transporte, ubiquitination e processamento do antígeno exógeno em vitro, sugerindo que o sistema ubiquitina-Proteassoma processado o antígeno exógeno após exportação deste compartimento celular. Este protocolo pode ser mais aplicado a outros tipos de células para esclarecer o mecanismo molecular da CP.

Para elucidar o mecanismo molecular da CP, é necessário identificar os compartimentos celulares, onde se submeter a antígenos exógenos ERAD, como transporte e processamento. Enquanto observações por microscopia de imunofluorescência ou microscopia eletrônica identificaram o compartimento celular onde antígenos exógenos acumularam16,17,18,1<sup class="x...

Watch this Scientific Journal Video about Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation at JoVE.com

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

O método descrito aqui é um novo protocolo de isolamento de vesículas, que permite a purificação dos compartimentos celulares onde os antígenos exógenos são processados pelo retículo endoplasmático associado a degradação em cruz-apresentação.

Purificação do compartimento da membrana retículo endoplasmático associado a degradação de antígenos exógenos em cruz-apresentação

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

purification-membrane-compartment-for-endoplasmic-reticulum

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.2K visualizações.  Takasaki University of Health and Welfare. O método descrito aqui é um novo protocolo de isolamento de vesículas, que permite a purificação dos compartimentos celulares onde os antígenos exógenos são processados pelo retículo endoplasmático associado a degradação em cruz-apresentação.

Vídeo: Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Purificação e Agregação do Domínio proteína precursora de amilóide intracelular

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Medicina

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Mitocôndrias associadas Membranas ER (MAMs) e glicoesfingolipídeos microdomínios enriquecidos (GEMS): Isolamento de cérebro de rato

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Neurociência

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

Reconstituição da Atividade de Extração de Msp1 com Componentes Totalmente Purificados

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Bioquímica

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

Formação espontânea e rearranjo das redes de nanotubo lipídica Artificial como um modelo de baixo para cima para o retículo endoplasmático

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Bioengenharia

Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific physicochemical conditions during purification to obtain them with high purity and structural quality and in a catalytically active form. The disaccharide trehalose is a compatible solute that is synthesized and accumulated in high concentrations in the yeast cytoplasm to stabilize the membranes and proteins. The use of trehalose as an additive in the protocol for the purification of plasma membrane H+-ATPase results in a high-quality preparation, the hexameric structure of which is shown by biochemical analytical methods. Trehalose can, therefore, be used as a stabilizing additive for the purification of membrane proteins (P-ATPases). This protocol describes the modification of the classical protocol for SERCA purification by subjecting SERCA to centrifugation on a trehalose concentration gradient. The inclusion of this carbohydrate led to the purification of SERCA in a catalytically active form with high purity and, importantly, in a stable form. Partial biochemical characterization of the purified SERCA (SDS-PAGE, enzyme kinetics, FITC labeling, circular dichroism spectroscopy) showed that the enzyme is suitable for functional and structural studies. The use of trehalose in the purification protocol of P-type ATPases and other labile membrane (and cytosolic) proteins is suggested.

This protocol describes an improved SERCA purification method, which includes the disaccharide trehalose in the final centrifugation step. This carbohydrate stabilizes proteins under harsh conditions. The purified SERCA was catalytically active and displayed high purity, making it suitable for structural and functional studies.

Purificação do retículo sarco-endoplasmático Ca2+-ATPase do músculo de coelho

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualização de mRNAs retículo endoplasmático localizados em células de mamíferos

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Biologia

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualização do retículo endoplasmático Subdomains em células cultivadas

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

Cicloheximida Análise perseguição de degradação da proteína em<em&gt; Saccharomyces cerevisiae</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

The Endoplasmic Reticulum

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary protein and lipid synthesis factory for most cell organelles, such as the Golgi apparatus, lysosomes, secretory vesicles, and the plasma membrane. Despite being the most extensive and functionally complex subcellular organelle, ER was the last to be discovered. After years of deliberation, Keith Porter and George Palade in the year 1954, produced the first high-resolution electron microscope images to affirm the presence of ER in the eukaryotic cell.
The ER membranes were identified as sites for protein synthesis and crucial for intracellular transport using radio-labeled and fluorescent-labeled amino acids. The isolation of ER is a tricky task as it forms an intricate mesh with other subcellular organelles. However, on cell homogenization, the disrupted ER membranes reseal into small closed vesicles called microsomes. These vesicles form a microsystem capable of sustaining all ER-related functions like protein and lipid synthesis, calcium signaling, and glycosylation. Subcellular fractionation is the best and often-used technique for the purification of these membranes. When separating using a sucrose gradient, the rough ER microsomes sediments at a higher density than smooth ER microsomes.
The ER network in a cell is dynamic. It is constantly shape-shifting along with the cytoskeleton to bolster mechanical support for the cell structure. Despite the distinct cisternae and tubule morphologies of the ER membrane, interconversion between the two is possible and is governed by the expression of membrane proteins. The ER network rearranges by tubule growth, retraction, and fusion of adjacent ER-ER membranes.
As mentioned above, the ER network is required for optimal overall cellular health. Disruption of ER morphology is linked to pathological conditions, including neurological disorders like Alzheimer&#39;s disease, hereditary spastic paraplegia, and viral infections like hepatitis C virus, and dengue virus.

O Retículo Endoplasmático

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary ...

Education

JoVE Core

Biologia Celular

Unidade 1

Transporte Transmembranar no Retículo Endoplasmático e Peroxissomos

TERMOS E CONCEITOS CHAVE

ER Retrieval Pathway

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct location. Occasionally, misfolded proteins and incorrect proteins escape their original compartments, and a retrieval pathway is used to return the escaped proteins to their original compartment.
The ER uses many checkpoints to prevent the entry of incorrectly folded or a resident protein as cargo onto a transport vesicle. These mechanisms include cargo selectivity of the receptors, aggregation of functionally similar proteins, and retention sequences on resident proteins.
Biochemical studies characterizing the cargo-sorting receptors have shown that they are ligand-specific and bind only at specific pH (pH 5.0 in yeast and mammals). Functionally similar ER-resident can proteins aggregate to form large complexes that cannot be loaded onto transport vesicles, thus avoiding the accidental escape of these proteins from the organelle. Additionally, the membrane-spanning domain of several ER and Golgi proteins contain a retention sequence that marks these proteins for staying back in the ER, so receptors do not bind them.
If an ER-resident protein escapes these checkpoints and gets loaded onto a Golgi-bound vesicle, a specific amino acid sequence called a retrieval signal helps the protein get recognized and transported back to the ER. The receptors involved in retrieval mechanisms recognize the retrieval signals at the carboxyl terminus of soluble proteins of the ER and in the cytoplasmic domain of some ER and Golgi membrane proteins. The receptors selectively capture such proteins and package them in vesicles that transport them back to the ER.
Two well-characterized retrieval signals are the carboxy-terminal tetrapeptide KDEL and KKXX found in many ER lumen resident proteins. BiP, a molecular chaperone, plays a vital role in preventing the aggregation of misfolded proteins. Interaction between BiP and unfolded proteins is mediated by a substrate-binding domain and a nucleotide-binding domain for ATPase activity. KDEL receptor in the post-ER compartments such as ERGIC and Golgi binds with the carboxyl-terminal retrieval signal of BiP and returns BiP to the ER via COPI vesicles.

Via de Recuperação do ER

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct ...

Purificação do compartimento da membrana retículo endoplasmático associado a degradação de antígenos exógenos em cruz-apresentação

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