Este trabalho foi financiado pelo NIH concede # DA15169 a HEM

Resumo procedimento: Usando essa abordagem, proteínas de superfície celular são covalentemente marcado com biotina na extracelular resíduos de lisina disponível usando um impermeant membrana, dissulfeto-coupled reagente Biotinilação (sulfo-NHS-SS-biotina), sob o tráfico de condições restritivas (ou seja, de baixa temperatura) (ver Fig. 1. para ilustração). Um conjunto de células é deslocado para o tráfico de condições permissivas (37 ° C) e proteínas biotiniladas internalizar. O outro conjunto de células são mantidos em baixas temperaturas como controles para 1) proteína de superfície do total em tempo = 0, e 2) controle de stripping. Após um curto período de interiorização, as células são transferidas de volta para baixa temperatura para parar de internalização, biotina e qualquer superfície residual é retirado por tratar células com um agente redutor, que cliva a biotina dissulfeto-coupled. Proteínas biotiniladas que surgiu a partir da superfície da célula e foram internalizadas são protegidos da etapa de decapagem, e será a única proteínas biotiniladas que permanecem. Após a lise celular, proteínas biotiniladas são isolados por cromatografia de afinidade e estreptavidina a proteína de interesse é detectada por immunoblotting quantitativa. Para determinar a taxa endocítica, a quantidade de proteína internalizada é comparado com o controle de superfície total rotulados em tempo = 0. Temos utilizado com sucesso esta abordagem para medir a taxa de internalização da norepinefrina e dopamina neuronal 1 1-4 transportadores. Protocolo detalhado: Dia 1: <ol><li> Células em placa de 6 placas bem como de que eles serão confluentes ~ 80% no Dia 2. Alternativamente, se as células transfectadas estão sendo usados, transfecção com uma densidade de tal forma que eles serão confluentes ~ 80% no momento a taxa de internalização será medido. Se as células não são fortemente aderente, utensílios de cultura de tecidos devem ser tratados com um substrato de adesão celular (por exemplo, poli-D-lisina) para evitar a perda de células durante as etapas de lavagem extensiva. </li><li> Para cada proteína que está sendo testado, placa de 2 poços em uma placa para ser usada como a proteína de superfície total (t = 0) e controles de stripping. Em uma segunda placa, placa de um poço para cada condição de interiorização que está sendo testado (taxa basal ou seja endocítica vs tratados com o fármaco). </li><li> Prepare as seguintes soluções e armazenar à temperatura indicada para uso no dia 2: <ul><li> PBS 2 +: Solução tampão de fosfato (pH 7,4) suplementado com 1,5 mM MgCl 2, 0,2 mM CaCl 2, (4 ° C) </li><li> Biotinilação Quench Solução: 2 + PBS suplementado com 100 mM de glicina, (4 ° C) </li><li> NT buffer: 150 mM NaCl, 1,0 mM EDTA, 0,2% BSA, 20 mM Tris, pH 8,6, (4 ° C) </li><li> RIPA buffer: 10 mM Tris, pH 7,4, 150 mM NaCl, 1,0 mM EDTA, 0,1% SDS, 1,0% Triton X 100, desoxicolato de sódio 1,0%, (4 ° C) </li><li> Sulfo-NHS-SS-biotina solução estoque: dissolver em dimetilsulfóxido (DMSO) a 200 mg / ml, (-20 ° C) </li><li> Tris (2-Carboxietil) Cloridrato de fosfina solução-mãe (TCEP): 500 mm de H 2 O, (-20 ° C, coberto de papel alumínio para bloquear a luz) </li></ul></li></ol> Dia 2: <ol><li> Prepare PBS 2 + suplementado com glucose 0.18g/ml, 0,2% IgG / protease livre de albumina sérica bovina (PBS 2 + / g / BSA). Pré-aquecer esta solução a 37 ° C em banho-maria. </li><li> Descongelar a solução estoque sulfo-NHS-SS-biotina na bancada para derreter o DMSO. Imediatamente antes de usar, preparar solução sulfo-NHS-SS-biotina frescos (2,5 mg / ml em PBS gelado 2 +, suficientes para 0,75 ml / poço). Vortex vigorosamente a solução para solubilizar o DMSO. Note-se que o reagente NHS-biotina é facilmente hidrolisado em solução aquosa. Portanto, todas as soluções devem ser preparadas imediatamente antes do uso. </li><li> Biotinilação: placas Coloque em um banho de gelo na sala fria e enxágüe 3 x 2 ml com PBS gelado 2 +. Esteja certo de que as placas são ligeiramente inclinado para permitir a drenagem completa e remoção da solução tampão. Adicionar 0,75 ml / poço da solução sulfo-NHS-SS-biotina fresco a cada poço. Incubar x 15 &quot;, 4 ° C em banho de gelo com agitação vigorosa. Após a incubação é completa, prepare outra solução sulfo-NHS-SS-biotina fresco. Substituir a solução antiga com a nova solução e incubar x 15 &#39;, 4 ° C. </li><li> Têmpera: É fundamental que todos os não-reagir NHS-biotina moléculas são extinto, de modo que eles não vão reagir com e biotinylate proteínas intracelulares uma vez que as células são lisadas. Lave as células 3 x 2 ml com solução de têmpera e incubar duas vezes em 2 ml solução saciar x 15 &quot;, 4 ° C, agitando suavemente. </li><li> Interiorização: Se os tratamentos de drogas estão sendo testadas, adicione a concentração de fármaco adequado para PBS 2 + / g / BSA. Manter controle de placa a 4 ° C e trazer placa internalização fora da sala fria. Lavar 3 x 2 ml com PBS pré-aquecido 2 + / g / BSA (+ / - drUGS) e deixar em soluções de mesma (2 ml / poço). Preencher qualquer vazio, com poços pré-aquecido solução para garantir até mesmo a temperatura ao longo da placa. Transferência das células para a 37 ° C incubadora por 10 &#39;. Imediatamente antes da final de 37 ° C de incubação, preparar solução TCEP fresco 50 mM em tampão NT a ser utilizado para a etapa de decapagem e armazenar no gelo. Preparar a quantidade suficiente para 1,0 ml / poço. </li><li> Stripping: Imediatamente placa de transferência (s) para banho de gelo e voltar para o quarto frio. Lavar rapidamente as células com gelado NT buffer, 3 x 2 ml para parar endocitose. Também lavar faixa de controle de poços 3 x 2 ml com tampão NT. Adicionar 1,0 ml solução fresca stripping a cada poço. Incubar no gelo, 15 &#39;, 4 ° C, agitando suavemente. Substituir os poços com solução de stripping fresco e incubar mais 15 &quot;, 4 ° C no gelo. </li><li> Lise: Lave todos os poços que foram expostas a solução de decapagem, 3 x 2 ml NT buffer. Em seguida, lavar os poços (incluindo controles total) com 3 x 2 ml PBS 2 +. Lyse em 300μl/well RIPA buffer (tampão de lise ou outros compatíveis para a proteína de interesse) contendo inibidores da protease frescos (1,0 mM PMSF, 1,0 mg / ml cada leupeptin, aprotinina e pepstatin). Lyse agitando x 20 &quot;, 4 ° C. Transferência para tubos de microcentrífuga e restos celulares claro por centrifugação 18.000 xg, 10 &#39;, 4 ° C. </li><li> Determinação da concentração de proteína: Use um ensaio de proteína compatível com suas condições de lise (por exemplo, ensaio de proteína DC, Bio-Rad) para determinar a concentração de proteína do lisados ​​em comparação com uma curva padrão de BSA. </li><li> Estreptavidina cromatografia de afinidade: Prepare os tubos de microcentrífuga com quantidades equivalentes de proteína para cada amostra. Adicionar tampão de lise para trazer volume final de cada amostra de 200μl. Vortex as contas estreptavidina agarose vigorosamente para levar a uma suspensão mesmo. Usando um pipetador 200μl com a ponta cortada, contas pipeta em cada tubo. Recomenda 20μl lisado mg beads/50. Incubar overnight, a 4 ° C em um tubo de rotator. </li></ol> Dia 3: <ol><li> Lavar talão: Centrifugar amostras, 18.000 xg, 2 &#39;para coletar pérolas. Aspirar off lisado, tomando cuidado para não se aproximar talão pellet com aspirador. Use uma ponteira de plástico na ponta do aspirador para um melhor controle. Adicionar 0,75 ml de tampão de lise cada tubo, e vortex para lavar contas. Amostras centrífuga, 18.000 xg, 2 &#39;para coletar pérolas e aspiração e lavagem repetir duas vezes (três lavagens em total). Após a lavagem final, aspirar o máximo do buffer quanto possível, sem perturbar o sedimento talão. Tombamento do tubo em direção ao aspirador ajuda com esta etapa. </li><li> Eluição da amostra: Adicionar 20-25μl tampão de amostra Laemmli 2x (reduzir) a cada tubo. O agente redutor irá decompor a ligação dissulfeto-SS NHS-biotina, liberando as proteínas isoladas em solução. A maioria das proteínas da membrana são altamente vulneráveis ​​à agregação quando cozidos, e não deve ser aquecida antes de carregar em gel de SDS-PAGE. Determine sensibilidade ao calor de sua proteína de interesse antes de se realizar esses experimentos. Se a proteína não pode tolerar aquecimento ebulição /, incubar em um rotador, 30 &#39;, temperatura ambiente antes da análise por SDS-PAGE. Isto é suficiente para decompor o dissulfeto de títulos e eluir as proteínas. </li><li> SDS-PAGE e immunoblotting: proteínas separadas por SDS-PAGE. Para cada amostra, é mais fácil executar os exemplos na seguinte ordem: superfície total (tempo = 0), tiras, condição internalização # 1, # 2 Condição, etc Transferência para membrana de blot e immunoblotting com anticorpo apropriado para sua proteína de interesse. Captura de bandas imunorreativas usando uma câmera CCD-sistema de documentação de gel, sendo certo que não existem pixels saturados. Quantificar cada densidade banda usando software de análise de gel / densitometria. </li></ol> Resultados representativos: Um resultado immunoblot representativa é mostrado na Figura 2A. O sinal mais forte é na pista &quot;total&quot; (T), que é a quantidade total de proteínas da superfície antes de internalização. O &quot;strip&quot; de controle (S) devem, idealmente, estar perto de branco, o que demonstra que a faixa foi eficiente para o experimento. A eficiência tira é calculada comparando a densidade do &quot;strip&quot; para que a pista da pista &quot;total&quot; (proteína por exemplo, que foi biotinilado em paralelo com o strip, mas não era nem aquecido a 37 ° C, nem exposta a uma solução stripping). A seguinte fórmula é usada: [1 - (tira / total)] * 100% Usando esta fórmula, a tira na Figura 2 foi 99,8% eficiente. Finalmente, você vai ver bandas de menor intensidade do que o total na pista internalização (s) (I). No exemplo (Figura 2), as células tratadas foram tratados com veículo ou acetato de forbol miristato 1 Hm (PMA), durante a 10 &#39;taxas de internalização internalização e transportador de dopamina foram medidos para a 10 &quot;período de interiorização inicial. A taxa de interiorização é calculado comoseguinte forma: internalizada / total * 100 Como visto na Figura 2B, 10,4% da superfície DAT internalizados ao longo de 10 &#39;sob o veículo-tratados condições. PMA tratamento aumentou as taxas de internalização DAT para 23,2% de DAT superfície total. <img src="/files/ftp_upload/1669/1669fig1.jpg" alt="Figura 1" /> Figura 1. Células protocolo de ilustração. Biotinilado são a 4 ° C a etiqueta exclusivamente a população de superfície, e são transferidas para 37 ° C para iniciar internalização. Após a internalização, as células são rapidamente resfriados para parar processos endocítica e biotina superfície residual é retirado por tratar células com um agente redutor. As proteínas só biotinilado que permanecem são aquelas que surgiram a partir da superfície em t = 0 e foram internalizadas, protegendo-os do tratamento stripping. Proteínas biotiniladas são isolados por lote cromatografia de afinidade com miçangas e estreptavidina a proteína de interesse é detectada por immunoblotting. <img src="/files/ftp_upload/1669/1669fig2.jpg" alt="Figura 2" /> Figura 2. Ativação da PKC aumenta a taxa de DAT endocítica. Internalização ensaio. PC12 expressando estavelmente células DAT foram biotinilado, 4 ° C, conforme descrito no &quot;Protocolo detalhado&quot;. As células foram rapidamente aquecidas a 37 ° C ± 1 mM PMA e incubadas 10 &#39;, 37 ° C. Biotina residual foi despojado, reduzindo, as células foram lisadas e as proteínas biotiniladas foram isoladas por cromatografia de afinidade da estreptavidina. (A) immunoblot Representante mostrando DAT superfície total em t = 0 (T), faixa de controle (S), e internalizada DAT (I), nas condições indicadas. (B) Bandas foram capturadas com uma câmera CCD e quantificadas com quantidade de dados software (Bio-Rad). Dados estão expressos em min% total internalized/10 DAT. <img src="/files/ftp_upload/1669/1669fig3.jpg" alt="Figura 3" /> Figura 3. Immunoblot exemplo que descreve um strip biotina pobres. Internalização ensaio. PC12 expressando estavelmente células DAT foram biotinilado, 4 ° C, conforme descrito no &quot;Protocolo detalhado&quot;. As células foram rapidamente aquecidas a 37 ° C e incubados 10 &#39;, 37 ° C. Biotina residual foi despojado, reduzindo, as células foram lisadas e as proteínas biotiniladas foram isoladas por cromatografia de afinidade da estreptavidina. O immunoblot mostra DAT superfície total em t = 0 (T), faixa de controle (S), e internalizada DAT (I). Observe a banda visível na faixa de controle strip, indicativo de eficiência strip pobres.

<ol>
	<li>Holton, K. L., Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+distinct+endocytic+signals+dictate+constitutive+and+PKC-regulated+neurotransmitter+transporter+internalization.">Nonclassical distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization.</a> Nat Neurosci. 8, 881-888 (2005).</li><li>Boudanova, E., Navaroli, D. M., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+decreases+in+dopamine+transporter+surface+expression+are+protein+kinase+C-independent.">Amphetamine-induced decreases in dopamine transporter surface expression are protein kinase C-independent.</a> Neuropharmacology. 54, 605-612 (2008).</li><li>Boudanova, E., Navaroli, D. M., Stevens, Z., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+transporter+endocytic+determinants:+carboxy+terminal+residues+critical+for+basal+and+PKC-stimulated+internalization.">Dopamine transporter endocytic determinants: carboxy terminal residues critical for basal and PKC-stimulated internalization.</a> Mol Cell Neurosci. 39, 211-217 (2008).</li><li>Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopamine+transporter+constitutively+internalizes+and+recycles+in+a+protein+kinase+C-regulated+manner+in+stably+transfected+PC12+cell+lines.">The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines.</a> J Biol Chem. 278, 22168-22174 (2003).</li></ol>

Problemas comuns: O problema mais comum que surge nesses experimentos é a eficiência strip pobres. A eficiência da tira é fundamental para ser capaz de interpretar os resultados. A menos que a faixa foi altamente eficiente, não é possível concluir que todas as proteínas biotiniladas nas pistas internalização foram, de fato, internalizados a partir da superfície. Tiras ≥ 90% de eficiência são ótimas, e descartar qualquer resultado, se a tira cair abaixo deste nível. Um exemplo de uma tira de pobre...

measuring plasma membrane protein endocytic rates by reversible biotinylation

Proteínas da membrana plasmática são um grupo grande e diverso de proteínas composta de receptores, canais iônicos, transportadores e bombas. Atividade dessas proteínas é responsável por uma série de eventos-chave de celulares, incluindo a entrega de nutrientes, excitabilidade celular e sinalização química. Muitas proteínas da membrana plasmática são dinamicamente reguladas, por tráfico endocítica, que modula a função das proteínas, alterando a expressão de proteínas de superfície. Os mecanismos que facilitam a endocitose de proteínas são complexos e não são totalmente compreendidos por proteínas da membrana muitos. A fim de compreender totalmente os mecanismos que controlam o tráfico endocítica de uma determinada proteína, é fundamental que a taxa de endocítica a proteína s ser medido com precisão. Para muitos receptores, direta medições taxa endocítica são freqüentemente alcançados utilizando ligantes receptores rotulados. No entanto, para muitas classes de proteínas da membrana, como os transportadores, bombas e canais iônicos, não há ligante conveniente que pode ser usado para medir a taxa de endocítica. No presente relatório, nós descrevemos um método reversível Biotinilação que nós empregamos para medir o transportador de dopamina (DAT) taxa de endocítica. Este método fornece uma abordagem simples para medir as taxas de internalização, e pode ser facilmente empregada para estudos de tráfico de proteínas mais de membrana.

Watch this Scientific Journal Video about Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation at JoVE.com

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Endocitose regulada governa o celular níveis de expressão de superfície da maioria das proteínas da membrana. Aqui nós utilizamos redutível, membrana reagentes Biotinilação impermeant para medir a taxa endocítica do transportador de dopamina (DAT), uma proteína de membrana politópica. O método facilita uma abordagem simples para medir a taxa de proteínas mais endocítica membrana plasmática.

Taxas de medição de plasma Proteína de membrana endocítica por Biotinilação reversível

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

measuring-plasma-membrane-protein-endocytic-rates-reversible

Research

JoVE Journal

Biology

16.9K visualizações.  University of Massachusetts Medical School. Endocitose regulada governa o celular níveis de expressão de superfície da maioria das proteínas da membrana. Aqui nós utilizamos redutível, membrana reagentes Biotinilação impermeant para medir a taxa endocítica do transportador de dopamina (DAT), uma proteína de membrana politópica. O método facilita uma abordagem simples para medir a taxa de proteínas mais endocítica membrana plasmática.

Vídeo: Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Medição Perto membrana plasmática e global Dynamics cálcio intracelular em astrócitos

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Biologia

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by &Delta;&Phi;, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of &Delta;&Phi; on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of &Delta;&Phi;. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of &Delta;&Phi;.

External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (&Delta;&Phi;). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the &Delta;&Phi; noninvasively. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS.

Medir a tensão de membrana induzida com Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Medição quantitativa do GLUT4 translocação para a membrana plasmática por citometria de fluxo

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

Mapeamento de Difusão Molecular na membrana plasmática por Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Colheita e Cryo-resfriamento cristais de proteínas de membrana crescida no mesofases lipídicas para a determinação da estrutura de Cristalografia Macromolecular

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

Em tempo real, análises de Transporte Retinol pelo receptor de membrana da proteína de Retinol Binding

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

Imagem Plasma Membrane Deformações Com pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrane Transport processos analisados ​​por um Sistema Nanopore Chip altamente paralelos na resolução Protein Individual

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

Determinação da membrana plasmática de particionamento para proteínas associadas perifericamente

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...