Os autores agradecem René Scriwanek pela ajuda com a preparação da Figura. FR é suportado pelo ECHO (700.59.003), ALW Programa Open (821.02.017 e 822.02.014), a cooperação DFG-NWO (DN82-303) e ZonMW VICI (016.130.606) subvenções.

1. Esferoplastos Preparação <ol><li> Incubar a levedura durante a noite a 30 ° C em 10 ml do meio apropriado determinado pela concepção da experiência. </li><li> No dia seguinte, medir a densidade óptica da cultura a 600 nm (OD600) usando um fotómetro, Diluir as células no mesmo meio de cultura para uma DO 600 de 0,2-0,4 e crescem-los a uma fase de crescimento exponencial, a menos que o desenho do experimento requer uma condição diferente. As células estão na fase exponencial quando a cultura tem uma OD 600 de 1-2. </li><li> Recolha 10 OD 600 equivalentes de células por centrifugação a 3500 xg durante 5 min num tubo de 50 ml. Após a centrifugação, desprezar o sobrenadante. </li><li> Ressuspender o sedimento de células em 5 ml de 100 mM de PIPES (pH 9,6), ditiotreitol 10 mM e incubou-se a 30 ° C durante 10 min. </li><li> Recolha de novo as células por centrifugação a 3500 xg durante 5 min. Desprezar o sobrenadante. </li><li> Ressuspender as células in 5 ml de meio (determinados pelo desenho do experimento) contendo 1 M de sorbitol e 5 mg de enzima lítica, e incubam-se a mistura a 30 ° C com agitação suave durante 30 min. </li><li> Centrifugar a suspensão de células a 300 xg durante 5 minutos para recolher a fracção de sedimento, que corresponde aos esferoplastos. Desprezar o sobrenadante. </li><li> Ressuspender os esferoplastos em 960 ul de meios de gelo frio (média determinada pelo desenho do experimento) contendo 1 M de sorbitol e transfere-se a mistura em um gelado de 2 ml tubo de microcentrífuga. </li></ol> 2. Nanogold Absorção <ol><li> Com uma pipeta, misture delicadamente o esferoplasto com 4 nmol de partículas carregadas positivamente nanogold ressuspendido em 40 mL de água. O volume final da mistura deve ser de 1 ml. </li><li> Colocar a suspensão de células obtidas em gelo durante 15 min. </li><li> Transferir a suspensão à temperatura ambiente e incubam-lo durante o tempo necessário para permitir a internalização por e nanogoldndocytosis. NOTA: A 5 min a captação vai, principalmente, para a rotulagem de vesículas de endocitose e compartimentos endossomais iniciais, enquanto a 15 min captação permitirá marcar todo o sistema endosomal (precoce e endosomes final). Tempos de incubação mais longos (mais de 30 min) também vai permitir a rotular o vacúolo. NOTA: experiências de marcação de pulso de perseguição não pode ser realizado com este método. Portanto, quando a rotulagem para a análise de LE, nanogold também serão encontrados no PM e INEE. </li></ol> 3. Fixação e Seccionamento <ol><li> Pare a absorção nanogold por adição de 1 ml de fixador força dupla [4% de paraformaldeído (PFA), 0,4% de glutaraldeído (GA) em tampão de 0,1 M PHEM (PIPES 20 mM, HEPES 50 mM, pH 6,9, EGTA 20 mM, MgCl 4 mM 2)] contendo 1 M de sorbitol à suspensão de células. Mantido o tubo à temperatura ambiente. </li><li> Inverter cuidadosamente manualmente os tubos micocentrifuge várias vezes durante 30 minutos (isto irá permitir manter os esferoplastos emsuspensão) e em seguida, centrifugar duas vezes em 1700 xg por 25 seg. </li><li> Substituir o fixador, descartando-se o sobrenadante e adicionando 1 ml de fixador fresco força padrão (2% de PFA, a 0,2% em tampão de GA PHEM 0,1 M) contendo 1 M de sorbitol e incubar durante 2 horas à temperatura ambiente em uma roda de rotação lenta. </li><li> Processar as células de crio-seccionamento, tal como descrito anteriormente 6. NOTA: Para os procedimentos de aumento de absorção nanogold e prata, o tratamento com ácido periódico descrito no protocolo indicado não tem de ser executada. O tratamento com ácido periódico é melhor permeabilizar a parede da célula 26, mas esta estrutura tem sido eliminado durante a geração de esferoplastos. </li><li> Cortar 50 criossecções delgadas Nm a -120 ° C com faca de diamante seco usando uma UCT ultramicrótomo como descrito anteriormente 8 e colocá-los em carbono Formvar revestido 50 malhas de redes de níquel. </li></ol> 4. Prata Enhancement para Nanogold Particle Visualização NOTA: O procedimento para preparar as misturas de reação é, basicamente, a indicada pelo fabricante. Adaptações práticas que utilizam este protocolo, torna o procedimento de aumento de prata eficaz e confiável em criossecções. <ol><li> Retirar o kit de melhoria de prata do congelador e descongelar em uma incubadora a 37 ° C ou banho de água. Quando descongelado, coloque o kit em uma incubadora de 24 ° C colocado em uma sala escura até a sua utilização (ver 4.7). </li><li> Colocar a placa de aquecimento na câmara escura e de aquecê-lo até a temperatura final de 24 ° C. </li><li> Cubra o topo da placa de aquecimento com o protetor de superfície, o lado brilhante para cima, e prenda-o com uma fita. </li><li> Coloque o Parafilm na Benchkote e marcam as arestas com um marcador preto para ser capaz de ver as bordas Parafilm no escuro. </li><li> Tape um termómetro no topo da Benchkote para monitorizar a temperatura da placa de aquecimento. Ajustar gradualmente a temperatura se não for 24 ° C. </li><li> Place o tubo de 50 ml com água bidestilada e do kit acessório de prata no interior. </li><li> Encha as pequenas placas de Petri com água duplamente destilada, pré-aquecido a 37 ° C. Coloque as grades na água, espécime lado de baixo, por 30 min. </li><li> Repita este passo 4.7 mais uma vez. </li><li> Transfira as grades na caixa de armazenamento e trazê-los para a sala escura. </li><li> Lavar as grelhas de novo, passando-os (com o lado da amostra para baixo) em várias gotas de água bi-destilada a 24 ° C colocados no parafilme que foi imobilizado na placa de aquecimento. </li><li> Certifique-se que nove duplas gotas de água destilada adicionais estão prontos na Parafilm, para o enxágüe após a reação aprimoramento prata. </li><li> Desligar a luz e certifique-se de que todas as luzes estão apagadas. Desligue a luz vermelha. </li><li> Retire das soluções A e B do estojo de prata reforço da incubadora 24 ° C. </li><li> Coloque primeiros seis gotas de solução A, e depois 6 gotas de solução B em um 1,5 mtubo de microcentrífuga de l, e misturar bem com uma pipeta de Pasteur de vidro evitando fazer bolhas. A solução é bastante espesso e tem que ser misturada manualmente com uma pipeta, antes de finalmente vortex brevemente. </li><li> Coloque as soluções A e B de volta na incubadora 24 ° C e retire a solução C. </li><li> Adicionar 6 gotas da solução de C para a mistura A / B. Em seguida, misture novamente em primeiro lugar com uma pipeta Pasteur e, em seguida, em vórtice, evite fazer bolhas. </li><li> Utilizar imediatamente a mistura final colocando gotas (~ 20 ul / gota) no Parafilm. </li><li> Com uma limpeza, pinças anti-magnéticos, colocar as grades na mistura (por exemplo,., Reacção prata reforço) para 6 a 15 minutos, dependendo do acessório (ou seja,. Tamanho das partículas de ouro), que pretendia ser obtido. </li><li> Retirar as grelhas a partir da mistura e passa-os no topo da água bi-destilada, gotas, a 24 ° C durante as lavagens. Em primeiro lugar, usar em rápida sucessão 6 gotas e, em seguida, 3 gotas com 7 min tempo de incubação por gota, Antes de virar as luzes acesas. </li><li> Vá para a próxima etapa, ou rotulagem imuno-ouro ou coloração da membrana 27, a fim de visualizar o resultado de uma investigação EM. </li></ol>

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The authors have nothing to disclose.

Imuno-microscopia de electrões é uma técnica que permite a combinação de localização de proteínas com a resolução ultra-estrutural das transportadoras e organelos onde estas proteínas residem. Isto é particularmente importante quando se estuda o sistema de levedura endosomal porque seus compartimentos aparecem como estruturas pontuar em microscopia de fluorescência. Por isso, é difícil distingui-los. Por esta razão, o uso de uma sonda detectável por EM e entrar na rota endocítica numa forma dependente ...

O sistema endossomal é um grande aparelho de separação de membrana, que desempenha várias funções celulares importantes incluindo o tráfico de enzima lisossomal receptores de triagem e a reciclagem de membrana plasmática (PM) de receptores de 1,2. Endossomos são divididos em três compartimentos diferentes, isto é,. os primeiros endossomas (EE), os endossomas tardios (LE) e os endossomas de reciclagem. Esta classificação baseia-se o tempo que leva para o material de endocitose para os alcançar, em proteínas marcadoras específicas e à sua morfologia. Membranas, ou seja, proteínas e lipídios bicamadas, internalizadas do PM pode ser entregue a lisossomos via endossomos para a degradação ou ser reciclado. As membranas são também transportadas para os endossomas de Golgi e da mesma forma, ou continuar a lisossomas ou ser recuperado de volta para o Golgi. Além disso, as proteínas podem ser classificados em vesículas luminais brotamento para dentro a partir da limitação da membrana endossomal, um processo que leva à formação deuma subcategoria de LE, os corpos multivesiculares. O sistema endossomal levedura é relativamente menos complexo do que o de células eucarióticas elevados. Endosomes fúngicas são divididas em EE e LE. Em contraste com células de mamíferos que não contêm endossomas de reciclagem, mas também organelas relacionadas com lisossoma específicos de tecidos. Conseqüentemente, eles têm uma rede menos complexo de rotas de tráfico endossomais 3,4. Portanto levedura representou e ainda representa um sistema experimental vantajoso para estudar alguns dos princípios tráfego membrana subjacente no sistema endosomal. Esta vantagem é realçada pelo facto de que numerosos genes envolvidos nas vias endossomais foram inicialmente isolado com telas genéticas na levedura 5. Enquanto o sistema endossomal levedura no tipo selvagem e mutantes de células tem sido extensamente estudada utilizando abordagens bioquímicas e de microscopia de fluorescência, a investigação ao nível ultra-estrutural tem sido somentemínima. As análises morfológicas são particularmente relevantes na levedura, porque a maioria das organelas endossomais são detectados como estruturas pontuar por microscopia de fluorescência, o que torna difícil a sua identificação inequívoca 6. Infelizmente, apenas um número limitado de anti-soros reconhecer marcadores de proteína de levedura endossomais está trabalhando em imuno-elétron-microscópio (IEM) preparações 7-10. Para algumas proteínas, este problema tem sido contornado pela marcação endógena do gene de interesse e a utilização de um anticorpo que reconhece a marca para detectar 7,11,12. Muitas vezes, no entanto, as proteínas são indetectáveis ​​pelo IEM por causa dos seus baixos níveis de expressão. Sua superexpressão não é uma solução porque esta abordagem pode induzir mal-localizações e / ou alterações nas organelas de morfologia / funções. Assim, a rotulagem dos compartimentos endocíticas com uma sonda detectável por microscopia eletrônica de EM é uma opção eficaz. Esta é uma solução óptima, especialmente se o probe está a entrar no percurso de endocitose de uma maneira dependente do tempo, o que permite saber quando se vai marcar um organelo específico 6. A absorção de nanogold carregado positivamente por esferoplastos de levedura (ou seja. Levedura em que a parede celular foi removida enzimaticamente) tem sido utilizado com sucesso para identificar o endossomal levedura compartimentos 10. Estas partículas ligam-se fortemente com os lípidos carregados negativamente que compõem as membranas biológicas. Assim, os associados nanogold carregados positivamente com o PM, penetra na célula por endocitose, e passa através do EE e LE antes de atingir o vacúolo. Estas pequenas partículas de ouro, no entanto, não têm um tamanho adequado para ser visto por EM. Para torná-los visíveis, seu tamanho pode ser ampliada por meio de reações químicas que levam à deposição de prata ou de ouro ao redor da sonda ouro 13-15. Nós desenvolvemos e aplicado com sucesso uma abordagem IEM com base no método de realizar Tokuyasu subcelularlocalização estuda 8,16. Este método permite realizar imunomarcação em preparações de levedura com uma excelente resolução da morfologia 8,17-24. Temos também estabeleceu um procedimento que combina este protocolo IEM com a rotulagem nanogold do sistema de levedura endosomal compartimentos 6. Usando essa abordagem, temos caracterizados morfologicamente diferentes subclasses de endosomes e ultraestruturalmente examinado mutantes com um tráfico endosomal defeito 6,25. Além disso, foi demonstrado que esta rotulagem nanogold pode ser combinado com rotulamentos imunomarcação proporcionando a possibilidade de explorar a distribuição de uma proteína de interesse nas diferentes subpopulações de endossoma. Aqui apresentamos como a rotulagem do sistema de levedura endosomal com nanogold carga positiva está praticamente realizado.

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nanogold labeling of the yeast endosomal system for ultrastructural analyses

Endossomos são um dos principais membrana triagem checkpoints em células eucarióticas e que regulam a reciclagem ou destruição de proteínas principalmente a partir da membrana plasmática e do Golgi. Como resultado, o sistema endossomal desempenha um papel central na manutenção da homeostase celular, e mutações em genes pertencentes a esta rede de organelos interligadas por transporte vesicular, causar patologias graves, incluindo o cancro e desordens neurobiológicos. Por isso, é de relevância primordial para compreender os mecanismos subjacentes à biogênese e organização do sistema endosomal. A levedura Saccharomyces cerevisiae tem sido fundamental nessa tarefa. Para identificar e analisar especificamente em nível ultra-estrutural do sistema endosomal deste organismo modelo, apresentamos aqui um protocolo detalhado para a captação nanogold carregada positivamente por esferoplastos seguido pela visualização dessas partículas através de uma reação aprimoramento prata. Este método também é muito valiosol para o exame morfológico de mutantes com defeitos de tráfico endosomal. Além disso, não é aplicável apenas para exames ultraestruturais, mas também pode ser combinado com rotulamentos imunomarcação para investigações de localização de proteínas.

De acordo com o protocolo apresentado, a morfologia do sistema de levedura do endossoma podem ser acesso por EM transmissão. Figura 1 mostra diferentes tipos de compartimentos endossomais que foram alcançados e, por conseguinte, rotulados com nanogold. O nanogold reforçada-prata pode ser visto claramente como electrões partículas densas. As configurações óptimas estabelecidas para a reacção de reforço prata permite que possui partículas de ouro numa gama de tamanho homogéneo entre 5 e 15 nm...

Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

A levedura Saccharomyces cerevisiae, tem sido um organismo chave modelo para identificar e estudar genes que regulam a biogênese e funções do sistema endosomal. Aqui apresentamos um protocolo detalhado para a rotulagem específica dos compartimentos endossomais para estudos ultra-estruturais.

Nanogold Labeling do Sistema de levedura Endossomal de Análises ultraestruturais

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

nanogold-labeling-yeast-endosomal-system-for-ultrastructural

Research

JoVE Journal

Biology

10.2K visualizações.  University Medical Center Utrecht.  A levedura Saccharomyces cerevisiae, tem sido um organismo chave modelo para identificar e estudar genes que regulam a biogênese e funções do sistema endosomal. Aqui apresentamos um protocolo detalhado para a rotulagem específica dos compartimentos endossomais para estudos ultra-estruturais. 

Vídeo: Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

Uma técnica rápida para a visualização de células vivas de levedura Imobilizado

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Biologia

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

Análises bioelétrica de um Sistema de Design Inteligente osseointegrados Implant para Amputados

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Labeling fluorescentes de Drosophila Estruturas Coração

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

Evisceração de Vítreo Mouse e Retina de Análises Proteômica

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Modificado Sistema Yeast-Two-Hybrid para identificar proteínas Interagindo com o Fator de Crescimento progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

O Processo de monstro verde para a Geração de linhagens de levedura de transporte deleções de genes múltiplos

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Brotando Yeast extração e purificação de proteínas para o estudo da função, interações e modificações pós-traducionais

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Aplicações de pHluorin para Quantitative, Kinetic e de alta capacidade: Análise de endocitose em brotamento Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

This manuscript describes an easy-to-use and low-cost cryofixation method for visualizing suspension cells by transmission electron microscopy.

Congelamento Plunge: Uma ferramenta para os estudos ultra-estruturais e de imunolocalização de células em suspensão em Microscopia Eletrônica de Transmissão

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Protocolo do laboratório para análises genéticas de conteúdo intestino de rDNA usando de macroinvertebrados aquáticos grupo-específicos Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...