Este trabalho foi financiado pelo Instituto Canadense de Pesquisa em Saúde. CSC foi financiado por uma bolsa de pós-doutorado de Ciências Naturais e Engenharia do Canadá. FD é um Tier II Cátedra de Pesquisa do Canadá.

<html> Processo geral de reconstituição O processo de reconstituição é iniciado por mistura da proteína de membrana de andaime (MSP) purificado com o MalFGK complexo 2, na presença de detergente solubilizados fosfolípidos. O passo é seguido pela remoção lenta do detergente por um material adsorvente chamado poliestireno Bio-Beads ou Amberlite (Figura 1). O processo de auto-montagem ocorre provavelmente devido às interacções entre os fosfolípidos apolares hidrofóbicas, o MalFGK dois complexos e a superfície da proteína MSP anfipática. O produto final é uma partícula discóide feito de duas moléculas de invólucro em torno do MSP 2 MalFGK complexo. As partículas são separadas a partir dos aductos e agregados por ultracentrifugação e cromatografia de exclusão de tamanho analítica. As partículas são caracterizadas por electroforese em gel nativa e espectroscopia de espalhamento dinâmico da luz. título &quot;&gt; Preparação 1. Andaime da Membrana Proteínas, MSP <ol><li> A sua marcada com MSP (versão MSP1D1 1) é produzido a partir do plasmídeo em E. pMSP1D1 coli BL21 (DE3), induzida em OD 600 ~ 0,5 com 0,5 mM de isopropil β-D-1-tiogalactopiranósido durante 3 horas a 37 ° C. </li><li> As células são colhidas por centrifugação a 5000 xg durante 10 min a 4 ° C e ressuspensas em tampão contendo 100 TSG10 fluoreto de fenilmetano uM. </li><li> As células são lisadas com uma prensa francesa (3x) a 8000 psi e o material insolúvel é removido por centrifugação a 5000 xg durante 10 min a 4 ° C. A fracção solúvel contendo o MSP é isolado por ultra-centrifugação a 125.000 xg durante 45 min. </li><li> A MSP é purificado por cromatografia de níquel-quelante usando ~ 1,5 ml de Ni Sepharose HP em TSG10 buffer. Os contaminantes são removidos por lavagem com TSG10 tampão contendo 50 mM de imidazole. A MSP é eluída com TSG10 tampão contendo 600 mMimidazole. A proteína purificada é dialisada em TSG10 tampão e armazenado em -70 ° C a uma concentração de proteína de ~ 10-15 mg / ml. </li></ol> 2. Preparação do Complexo 2 MalFGK <ol><li> O MalFGK complexo 2, his-etiquetadas no terminal C de Malk, é expressa a partir do plasmídeo pBAD22-FGK em E. coli BL21 (DE3) e induzido a OD 600 ~ 0,5 com 0,2% de L-arabinose, durante 3 horas a 37 ° C. </li><li> Seguindo a lise das células e centrifugação, tal como no passo 1.3, o sedimento de membrana foi ressuspenso em TSG20 tampão a uma concentração final de 5 mg / ml. O material é solubilizado com 1% w / v de n-dodecil-β-maltopyranoside (DDM), durante 3 horas a 4 ° C com agitação suave. </li><li> O material insolúvel é removido por ultra-centrifugação como descrito no passo 1.3 e o sobrenadante contendo o solubilizado MalFGK complexa 2 é recolhido e purificado como descrito no passo 1.4, mas sem diálise. </li><li> Further purificação é alcançada por cromatografia de filtração em gel em tampão TSGD num Superdex 200 HR 10/300 coluna a uma taxa de fluxo de 0,5 ml / min. </li></ol> 3. Preparação de Fosfolípidos <ol><li> Uma E. coli total de lípidos extrair dissolvido em clorofórmio é separada em alíquotas de 1000 nmol em tubos de microcentrífuga de tampa roscada. O solvente é evaporado sob uma corrente suave de azoto e secou-se ainda mais durante a noite num exsicador de vácuo. </li><li> A película de lípido é dissolvido em tampão TS a 5 nM de concentração final e agitadas vigorosamente e sonicada. Os lípidos dissolvidos aparece ligeiramente opaca, devido à sua insolubilidade em geral em tampão TS aquosa, mas deve permanecer em suspensão. DDM é adicionado a uma concentração final de 0,5% (~ 10 mM), em que a solução se torne clara. </li><li> A mistura lipídica é colocada num banho de água e de impulsos ultra-sons durante 5 vezes para ~ 5 seg. A mistura lipídica é armazenado a 4 ° C por um período máximo de 1 week. </li></ol> 4. Preparação de Bio-Beads <ol><li> Aproximadamente 10-15 ml (volume seco) Bio-Beads é colocado dentro de um tubo de 50 ml. </li><li> As esferas são lavadas, sucessivamente, com 50 ml de metanol a 100%, etanol a 95%, H 2 O milliQ, e finalmente tampão TS (duas vezes cada). </li><li> As pérolas lavadas são armazenadas a 4 ° C em tampão TS ~ 10 ml. </li></ol> 5. Reconstituição Nanodisc <ol><li> A MSP é diluída em tampão TSGD na concentração final de ~ 7 mg / ml (~ 0,3 mM). </li><li> ~ Nmol 2 de purificado MalFGK complexo 2 são misturados numa relação proteína: lípido de 1:3:60 ou 1:3:400 em tampão TSGD: MSP. A concentração final é de 6 uM MalFGK 2, 18 | iM de MSP e lípidos uM 360 (1:3:60) ou 2,4 mM de lípidos (1:3:400). O volume final é de 300 uL. A concentração final de DDM é de 0,08% (~ 1,6 mM). A concentração final de glicerol depende da quantidade de lípido adicionado, mas permanece em torno de 5-10% v / v </li&gt; <li> ~ 50 ul de Bio-Bead suspensão é adicionado ao tubo, e a mistura é incubada durante a noite numa mesa oscilante, a 4 ° C. </li><li> As esferas são sedimentadas por gravidade e a solução é pipetada através de uma ponta estreita para evitar tanto Bio-Beads possível. </li><li> Grande precipitados são removidos por ultra-centrifugação a 100.000 xg durante 20 min. Os discos são purificados por cromatografia de filtração em gel como descrito no passo 2.4 em TSG10 buffer. As fracções contendo os discos são reunidas e uma parte alíquota deve ser re-injectada na mesma coluna de filtração em gel para testar a estabilidade da preparação. </li><li> Os discos purificados podem armazenado a -70 ° C por um período de tempo prolongado. Seguindo o descongelamento em gelo, os discos devem ser submetidas a ultra-centrifugação como descrito no passo 5.5 para remover precipitados potencial. Uma alíquota deve ser analisada por cromatografia de filtração em gel para assegurar que a agregação ou precipitação significativa não ocorreu durante o descongelamentoprocesso. </li></ol> 6. Eletroforese em gel nativo <ol><li> 1 uM de nanodiscs (~ 0,2 mg / ml) são analisadas por PAGE nativa 6, 7 (Tris-HCl pH 8,8, 4-12%) para avaliar a qualidade da reconstituição (Figura 3A). </li><li> A ligação de MalE (1 uM) para o complexo de 2-MalFGK reconstituída em nanodiscs é também avaliada por PAGE-nativo (Figura 3A). </li><li> Após a electroforese, o gel é corado com azul de Coomassie durante 10 min, e descorados durante ~ 1 hora. </li></ol> 7. Espalhamento de luz dinâmico (DLS) <ol><li> Nanodiscs são purificados por cromatografia de filtração em gel como descrito no passo 2.4 em TSG10 tampão utilizando uma coluna Superdex 200 HR 10/300 coluna com um caudal de 0,1 ml / min. As fracções contendo nanodiscs são reunidas e concentradas até ~ 10 mg / ml com um filtro Amicon centrífuga. </li><li> A amostra é filtrada duas vezes (filtro de 0,22 nm) antes da análise por DLS usandoum Dynapro nanostar instrumento (Wyatt Technology), em um volume de 1 ul interior da cuvete de quartzo. Os dados são ajustados usando o software DINÂMICA (Wyatt Technology) para estimar o diâmetro e peso molecular das partículas. </li></ol> 8. Medidas ATPase <ol><li> A actividade de ATPase de MalFGK 2-reconstituídos nanodiscs é determinada utilizando um ensaio colorimétrico 8. </li><li> 1 uM de discos purificadas e 1 mM de ATP são misturados com quantidades crescentes de MalE C e incubado a 37 ° C durante 20 min. A libertação de fosfato inorgânico é medida a 660 nm. </li><li> A quantidade de fosfato libertado é comparada com uma curva padrão gerada com uma solução padrão de fósforo. </li></ol> 9. Resultados representativos Os nanodiscs são purificados por cromatografia de filtração em gel (Figura 2A, à esquerda). O cromatograma mostra que a maioria do d reconstituídoISCs eluir (preto traço) como um único pico, enquanto que os discos feitos com lípidos em excesso (vermelho traço) eluem no volume vazio e como uma série de picos largos. A qualidade dos nanodiscs é ainda analisada por electroforese em gel nativa e espectroscopia de espalhamento dinâmico da luz (DLS). Discos adequadamente reconstituídas migrar como uma banda acentuada no gel enquanto aquelas reconstituída, na presença de um excesso de lípidos migrar como uma mancha (Figura 2A, à direita). A análise por DLS mostra que a população seja homogénea disco com um diâmetro médio de 11,4 nm (Figura 2B). Os discos reconstituídos têm um peso molecular aparente de 215 kDa com base na aproximação DLS. As amostras reconstituídas, na presença de excesso de lipídeos exibem largamente distribuído raios em torno de 100 nm, o que é típico para as amostras não homogéneos. A qualidade da 2 MalFGK complexo é avaliada por electroforese em gel nativa e a sua actividade ATPase pelamedições (Figura 3). A proteína de ligação à maltose MalE liga com uma afinidade maior para o transportador 2 MalFGK 9, 10. Usando a electroforese não desnaturante em gel, é possível detectar um complexo entre o macho ea MalFGK 2 (Figura 3A). A estimulação da actividade de ATPase Malk 2 por MalE é mostrado na Figura 3B. <img src="/files/ftp_upload/3910/3910fig1.jpg" alt="Figura 1"/> Figura 1. Fluxograma típico para o protocolo de reconstituição. <img src="/files/ftp_upload/3910/3910fig2.jpg" alt="Figura 2"/> Figura 2. O controle de qualidade da preparação nanodisc. A. A análise de filtração em gel (Superdex 200 HR 10/300 coluna) das nanodiscs reconstituídos em relação lípido baixa (1/3/60; traço negro) ou a razão de lípidos elevados (1/3/400; traço vermelho). Electroforese em gel nativo de preparação do mesmo disco. MolOs marcadores de peso ecular em kDa é indicado. B. análise dinâmica de espalhamento de luz da preparação mesmo disco. <a href="http://www.jove.com/files/ftp_upload/3910/3910fig2large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img src="/files/ftp_upload/3910/3910fig3.jpg" alt="Figura 3"/> Figura 3. Análise da MalFGK 2 nanodisc-partículas. A. Análise de gel mudança do sexo masculino incubadas com quantidades crescentes de MalFGK 2-nanodisc partículas. B. A actividade ATPase da MalFGK 2-nanodisc partículas em função da concentração de MalE.

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Descreve-se um procedimento simples para a reconstituição do transportador de maltose em nanodiscs. O transportador é ATPase ativa e da interação com o macho parceiro solúvel ligação pode ser recriado (Figura 3). A reconstituição bem sucedida do transportador em nanodiscs abrir o caminho para análise biofísica e bioquímica suplementar. De interesse particular será a análise sistemática da ATPase Malk e maltose a actividade de transporte de detergente, lipossomas e nanodiscs. Transportado...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Amicon Ultra-4 50K filtro centrífugo </td><td> Millipore </td><td> UFC805008 </td><td> Siga o protocolo do fabricante para o uso adequado </td></tr><tr><td> Bio-Beads SM-2 Adsorvente </td><td> Bio-Rad </td><td> 152-3920 </td><td></td></tr><tr><td> E. coli lipídios totais </td><td> Lipídios Avanti Polar </td><td> 100500C </td><td> Dissolvido em clorofórmio, como apropriado para tratar um solvente orgânico </td></tr><tr><td> Ni Sepharose HP resina </td><td> GE Healthcare </td><td> 17-5268-01 </td><td></td></tr><tr><td> Solução padrão de fósforo </td><td>Sigma-Aldrich </td><td> P3869 </td><td></td></tr><tr><td> pMSP1D1 </td><td> Addgene </td><td> 20061 </td><td></td></tr><tr><td> Superdex 200 HR 10/300 </td><td> GE Healthcare </td><td> 17-5172-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> Reagentes tabela I. Específicos. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Nome </td><td> Composição </td><td> Comentários </td></tr><tr><td> DDM estoque </td><td> 10% w / v DDM </td><td> Ressuspender em milliQ água e armazenar a -20 ° C </td></tr><tr><td> MalFGK estoque 2 </td><td> 1-2 mg / ml 50 mM Tris-HCl, pH7.9 100 mM de NaCl Glicerol a 10% v / v 0,03% w / v DDM </td><td> Armazenar a -70 ° C, após purificação </td></tr><tr><td> MSP estoque </td><td> 10-15 mg / ml 50 mM Tris-HCl, pH7.9 100 mM de NaCl Glicerol a 10% v / v </td><td> Armazenar a -70 ° C após purificação em alíquotas de 1 ml &lt;e evitar excessivas de congelamento / descongelamento </td></tr><tr><td> Estoque Phospholipid </td><td> 5 nM E. coli de lipídios totais 0,5% w / v (10 mM) DDM 50 mM Tris-HCl, pH 7,9 50 mM NaCl </td><td> Armazenar a 4 ° C durante 1 semana </td></tr><tr><td> TS tampão </td><td> 50 mM Tris-HCl, pH 7,9 50 mM NaCl </td><td> Armazenar a 4 ° C </td></tr><tr><td> TSG10 tampão </td><td> 50 mM Tris-HCl, pH7.9 100 mM de NaCl Glicerol a 10% v / v </td><td> Armazenar a 4 ° C </td></tr><tr><td> TSG20 tampão </td><td> 50 mM Tris-HCl, pH 8 100 mM de NaCl Glicerol a 20% v / v </td><td> Armazenar a 4 ° C </td></tr><tr><td> Tampão TSGD </td><td> 50 mM Tris-HCl, pH7.9 100 mM de NaCl Glicerol a 10% v / v 0,03% w / v DDM </td><td> Armazenar a 4 ° C e adiciona-DDM imediatamente antes da utilização </td></tr><tr><td colspan="3"> Tabela II. Solução receitas. </td></tr></tbody></table></td></tr></tbody></table>

a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles

O nanodisc é uma partícula discoidal (~ 10-12 nm grande) que as proteínas de membrana armadilha em um pequeno pedaço de bicamada de fosfolipídios. O nanodisc é uma opção particularmente atraente para o estudo de proteínas de membrana, especialmente no contexto de interacção ligando-receptor. O método pioneiro Sligar e colegas baseia-se nas propriedades anfipáticas de uma engenharia altamente helicoidal uma proteína derivada do andaime apolipoproteína A1. As faces hidrofóbicas da proteína andaime interagir com os acilo gordos de cadeias laterais na bicamada lipídica que as regiões polares enfrentam o meio ambiente aquoso. As análises de proteínas de membrana em nanodiscs têm vantagens significativas sobre os lipossomas porque as partículas são pequenas, homogénea e solúvel em água. Além disso, os métodos bioquímicos e biofísicos normalmente reservadas para proteínas solúveis pode ser aplicada, e a partir de ambos os lados da membrana. Neste protocolo visual, apresenta-se uma reconstituição passo-a-passo de uma caracte bemterizada transportador ABC bacteriana, o macho-MalFGK complexo 2. A formação do disco é um processo de auto-montagem, que depende de interacções hidrofóbicas que têm lugar durante a remoção progressiva do detergente. Descrevem-se os passos essenciais e destaca-se a importância de escolher a proporção entre proteína e lípido-correcta, a fim de limitar a formação de agregados maiores e polidispersos lipossoma partículas semelhantes. Qualidade simples controles, tais como cromatografia de filtração em gel, electroforese em gel nativa e espectroscopia de dispersão de luz dinâmica garantir que os discos foram correctamente reconstituído.

Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Nanodiscs são pequenas partículas discóides que incorporam proteínas de membrana em um pequeno pedaço de bicamada de fosfolipídios. Nós fornecemos um protocolo visual que mostra a incorporação passo-a-passo do transportador MalFGK2 em um disco.

Um método passo-a-passo para a reconstituição de um transportador ABC em partículas lipídicas Nanodisc

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

a-step-step-method-for-reconstitution-an-abc-transporter-into

Research

JoVE Journal

Biology

25.3K visualizações.  University of British Columbia. Nanodiscs são pequenas partículas discóides que incorporam proteínas de membrana em um pequeno pedaço de bicamada de fosfolipídios. Nós fornecemos um protocolo visual que mostra a incorporação passo-a-passo do transportador MalFGK2 em um disco.

Vídeo: A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

Expressão, solubilização, detergente e Purificação de um transportador de membrana, as proteínas MexB resistência a múltiplas drogas

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Biologia

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Cromatografia de afinidade lipídica mediada por vesículas com separação de células Magnetic Ativado (LIMACS): um novo método para analisar proteínas lipídios Interação

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Na reconstituição in vitro da atividade T. Telomerase castaneum

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Um método simples para criação de imagens Arabidopsis Folhas Usando Perfluorodecalin como um meio de imagem infiltrativas

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

Reconstituição de um canal de Kv para membranas lipídicas de Estudos estruturais e funcionais

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Reconstituição de uma proteína transmembrana, a voltagem-Ion Channel, KvAP, em Gigante Unilamelares Vesicles para microscopia e de Patch Estudos Grampo

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Procedimento para avaliar a eficiência de floculantes para a Remoção de partículas dispersas de extractos de plantas

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

Um método eficiente em Vitro transposição por um sistema de beleza dormindo Transcriptionally regulado empacotado em um vetor de Lentivirus defeituoso de integração

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Um método de compensação por imagem tridimensional do tecido adiposo tecido

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

Um modelo epitelial de interface ar-líquido para exposição de inalação realista e repetida a partículas aéreas para testes de toxicidade

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...