CQS receives a fellowship from the LOEWE Excellence Cluster for Integrative Fungal Research (IPF). The author would like to thank Ida J. van der Klei for the P. chrysogenum strains used in this work and Andreas S. Reichert for the gift of rapamycin.

1. Preparação de P. chrysogenum para os experimentos de fome <ol><li> Se o P. chrysogenum estirpe de juros é mantida em arroz (&#39;arroz verde &quot;), coloque 2-3 grãos de arroz cobertos com esporulação micélio em um tubo de microcentrífuga de 1,5 ml. Preenchê-lo com 500 ul YGG (10 g / l de KCl, 20 g / l de glucose, 10 g / l de base azotada de levedura, 5 g / l de K 2 HPO 4, 20 g / l de extracto de levedura). <ol><li> Vortex o tubo durante 30 segundos de modo a que os esporos podem separar de forma eficiente o arroz. </li><li> Incubar o tubo durante 1 dia a temperatura ambiente (por exemplo, entre 20 e 25 ° C). </li><li> Preparar uma diluição dessa suspensão de esporos na água da torneira estéril 1/50, misture bem. </li></ol></li><li> Se o P. chrysogenum estirpe de interesse é mantida numa placa de agar (por exemplo, YGG placa de agar), transferir pequenos pedaços de micélio em um tubo de microcentrífuga de 1,5 ml contendo 400 ul de água de torneira esterilizada e cerca de 100 mg glgrânulos burro usando um palito estéril ou ponteira. <ol><li> Vortex o tubo durante 2 minutos de modo que se torna o micélio fragmentado. Utilizar o conteúdo do tubo imediatamente. </li></ol></li></ol> 2. Passos preparatórios para o cultivo de P. chrysogenum em Pads de fome <ol><li> Ligue a placa de aquecimento e configurá-lo para cerca de 60 ° C. </li><li> Dissolve-se 2 g de agarose em 100 ml de água da torneira por cozimento a solução num forno de microondas. Tome cuidado para que esta solução é completamente claro após o aquecimento. Se não for, cozinhá-lo novamente até que a agarose é dissolvido completamente. Deixar a solução arrefecer de agarose a cerca de 60 ° C antes da utilização. </li><li> Encher tubos de 1,5 ml de microcentrífuga (2-4, dependendo o número de amostras) com 400 ul de água estéril (sem toque em cada um adicionados compostos adicionais) ou 400 ul-x (x adição de uL de solução do composto no passo 2.5) e colocá-los sobre a placa de aquecimento durante pelo menos 1 minuto, de modo que o water dentro fica quente. </li><li> Colocar as lâminas de microscópio que contêm uma cavidade central sobre a placa de aquecimento durante pelo menos 1 min. </li><li> Adicionar 400 ul de 2% de agarose para a solução nos tubos de microcentrífuga de 1,5 ml e vortex brevemente. Após este passo, se desejado, adicionar compostos adicionais (ou seja, 1 uM de rapamicina). Se isso for feito, vortex brevemente depois de cada substância adicional é pipetado para o tubo. Colocar os tubos de volta para a mesa de aquecimento para evitar a solidificação prematura. NOTA: O volume total no tubo de microcentrífuga deverá ser de 800 uL. </li></ol> 3. Preparação de microscópicas Contendo Inanição Pads <ol><li> Pipetar 140 uL da solução de agarose para dentro da cavidade da lâmina de microscópio (ainda que está localizado na placa de aquecimento). Tente evitar fazer bolhas, se possível. </li><li> Imediatamente coloque uma lamela para a solução de agarose. NOTA: Este vai resultar em uma superfície plana. </li><li> Coloque o microâmbito deslizante na bancada do laboratório para aproximadamente 4 min para permitir a almofada de agarose para solidificar. </li><li> Remova cuidadosamente a lamela do pad agarose, deslizando-a com a ajuda de um polegar ou o dedo indicador (usar luvas para evitar a contaminação!). </li><li> Coloque a lâmina de microscópio com a almofada em uma câmara úmida para evitar a dessecação. Recomendação: Use uma caixa de ponta vazia que ainda tem o insert para manter as pontas de pipeta. Adicionar água estéril para a caixa de modo que o fundo é coberta. Coloque as lâminas de microscópio para a inserção e fechar a caixa. </li></ol> 4. inoculação da inanição Pads com P. chrysogenum e incubação <ol><li> Pipetar 5 ul de uma diluição 1/50 de esporos (se as culturas foram cultivadas em arroz) ou 5 ul da solução não diluída contendo fragmentos de micélio (se as culturas foram cultivadas numa placa de agar) para o centro da almofada de inanição. </li><li> Feche a câmara úmida e envolvê-la em um saco plástico (isso serve comoproteção adicional contra a dessecação das almofadas de fome). Tenha cuidado para não mover a caixa muito, porque de outra forma as gotas de inoculação será perturbado. </li><li> Armazenar a caixa embrulhada à temperatura ambiente durante pelo menos 20 h, antes de analisar as amostras. </li></ol> 5. microscópica análise das amostras <ol><li> Após 20 horas de incubação, as amostras estão prontas para análise microscópica; colocar a lâmina de microscópio em papel tecido seco (o fundo tende a ficar molhado). </li><li> Pipetar um pequeno volume de água (cerca de 50 mL) para a almofada de inanição. Coloque uma lamela sobre o bloco e esprema a água excedente. Retire o excesso de água com um lenço de papel. </li><li> Colocar a lâmina do microscópio sobre a mesa de observação de um microscópio de fluorescência. Para obter uma visão geral de crescimento micelial, use um objetivo 20X. Uso objectivos 63x ou 100x para estudar a localização de GFP nas hifas. A fim de evitar a dessecação progressiva da amostra não analisar as amostras durantemais de 15 min antes de colocá-los de volta para a câmara úmida. </li><li> Repita 5,3 em intervalos regulares (por exemplo, depois de 40 horas e 60 horas de crescimento). NOTA: A amostra pode ser colocado de volta na câmara úmida. Não é necessário retirar as lamelas uma vez que não afecta o crescimento do micélio. </li></ol>

<ol>
	<li>Fischer, F., Hamann, A., Osiewacz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+quality+control:+an+integrated+network+of+pathways.">Mitochondrial quality control: an integrated network of pathways.</a> Trends Biochem. Sci. 37 (7), 284-292 (2012).</li><li>Pollack, J. K., Harris, S. D., Marten, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+filamentous+fungi.">Autophagy in filamentous fungi.</a> Fungal Genet. Biol. 46 (1), 1-8 (2009).</li><li>Scheckhuber, C. Q., Osiewacz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podospora+anserina:+a+model+organism+to+study+mechanisms+of+healthy+ageing.">Podospora anserina: a model organism to study mechanisms of healthy ageing.</a> Mol. Genet. Genomics. 280 (5), 365-374 (2008).</li><li>Pinan-Lucarr&#233;, B., Iraqui, I., Clav&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podospora+anserina+target+of+rapamycin.">Podospora anserina target of rapamycin.</a> Curr. Genet. 50 (1), 23-31 (2006).</li><li>Kikuma, T., Ohneda, M., Arioka, M., Kitamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+the+ATG8+homologue+Aoatg8+and+role+of+autophagy+in+differentiation+and+germination+in+Aspergillus+oryzae.">Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae.</a> Eukaryot. Cell. 5 (8), 1328-1336 (2006).</li><li>Pinan-Lucarr&#233;, B., Balguerie, A., Clav&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+cell+death+in+Podospora+autophagy+mutants.">Accelerated cell death in Podospora autophagy mutants.</a> Eukaryot. Cell. 4 (11), 1765-1774 (2005).</li><li>Veneault-Fourrey, C., Barooah, M., Egan, M., Wakley, G., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagic+fungal+cell+death+is+necessary+for+infection+by+the+rice+blast+fungus.">Autophagic fungal cell death is necessary for infection by the rice blast fungus.</a> Science. 312 (5773), 580-583 (2006).</li><li>Bartoszewska, M., Kiel, J. A., Bovenberg, R. A., Veenhuis, M., van der Klei, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+deficiency+promotes+beta-lactam+production+in+Penicillium+chrysogenum.">Autophagy deficiency promotes beta-lactam production in Penicillium chrysogenum.</a> Appl. Environ. Microbiol. 77 (4), 1413-1422 (2011).</li><li>Meijer, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomes+are+required+for+efficient+penicillin+biosynthesis+in+Penicillium+chrysogenum.">Peroxisomes are required for efficient penicillin biosynthesis in Penicillium chrysogenum.</a> Appl. Environ. Microbiol. 76 (17), 5702-5709 (2010).</li><li>Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding,+improving+and+using+green+fluorescent+proteins.">Understanding, improving and using green fluorescent proteins.</a> Trends Biochem. Sci. 20 (11), 448-455 (1995).</li><li>Jung, C. H., Ro, S. H., Cao, J., Otto, N. M., Kim, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTOR+regulation+of+autophagy.">mTOR regulation of autophagy.</a> FEBS Lett. 584 (7), 1287-1295 (2010).</li><li>Hickey, P. C., Read, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+living+cells+of+Aspergillus+in+vitro.">Imaging living cells of Aspergillus in vitro.</a> Med. Mycol. 47, S110-S119 (2009).</li></ol>

The author has nothing to disclose.

O método aqui apresentado permite o estudo conveniente e reprodutível de autofagia em P. chrysogenum. Por exemplo, ele pode ser usado para o rastreio de a eficácia de vários compostos se eles são capazes de modular a resposta autofagia deste fungo ou não. Os resultados obtidos com rapamicina demonstram que a inibição da sinalização de TOR conduz a uma indução de autofagia pronunciada em P. chrysogenum que foi também demonstrada para outros organismos 11. <p class="jove_conte...

Os fungos filamentosos são excelentes sistemas de modelo para o estudo dos processos de desenvolvimento. Eles oferecem vários benefícios experimentais como cultivo barato, um elevado número de descendentes e acessibilidade genética. O último ponto é de particular relevância para a construção de transformantes que permite que se investigue a importância de genes tão longe descaracterizados para vários mecanismos celulares. Os fungos filamentosos têm sido fundamentais para a elucidação de vários elementos e mecanismos de controlo de qualidade das vias celulares, como a actividade de protease para a degradação de proteínas anormais, dinâmica mitocondrial para manter a integridade mitocondrial e autofagia para a remoção de excesso e / ou componentes celulares e para a manutenção disfuncionais viabilidade celular em tempos de fome 1,2,3. Existem várias técnicas experimentais disponíveis para o estudo da autofagia em fungos filamentosos 2: (i) a investigação das vacúolos if eles contêm corpos autophagic densos quando as proteases são inibidas por microscopia electrónica de transmissão 4, (ii) a visualização de autofagossomas por monitorização focos GFP-Atg8 através de microscopia de fluorescência de 5,6 e (iii) detecção de estruturas autophagosomal acidificadas utilizando o corante fluorescente monodansyl cadaverina 7. Aqui, um novo método para o crescimento de Penicillium chrysogenum para estudos autofagia é apresentado. O elemento principal é o &#39;pad fome &quot;, que consiste simplesmente de agarose a 1% dissolvido em água de torneira esterilizada. Compostos adicionais (por exemplo, factores de stress, sequestrantes, moduladores autofagia) pode ser adicionado à almofada, enquanto eles não exibem auto-fluorescência. A almofada está localizada em lâminas de microscópio que contêm uma cavidade central raso. Esta almofada em inoculados com uma suspensão de esporos ou com fragmentos de micélio pequenos. O último é aconselhável se a estirpe de interesse não sporulate eficiente (por exemplo, estirpes atg1 Δ 8). As lâminas estão posicionadas em câmaras húmidas (estes podem ser facilmente construídas usando caixas de ponta de pipeta vazios) para evitar a secagem da amostra e incubou-se à temperatura ambiente. P. chrysogenum é capaz de crescer durante alguns dias sob estas condições. Autophagy pode ser observada microscopicamente por alargamento vacuolar que é um marcador positivo para autofagia fúngica. Nesta contribuição, a P. chrysogenum estirpe (Wisconsin 54-1255) é usado, que forma a proteína verde fluorescente que é direcionado para peroxisomes pela sua sequência C-terminal &#39;SKL «9. Por conseguinte, é possível monitorizar a degradação dos peroxissomas. É possível rotular também outros compartimentos da célula (por exemplo, mitocôndrias) usando os sinais de localização adequados e analisar a sua degradação. Embora os dados de P. chrysogenum Ws54-1255 (GFP-SKL) é apresentado aqui, é certamente possívelpara usar o método de &#39;almofada de fome &quot;, também para outros fungos filamentosos (por exemplo, Neurospora crassa, Sordaria macrospora, espécies de Aspergillus, etc.).

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			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Microscope slides with central cavity</td>
			<td style="width:71px;">Carl Roth GmbH, Karlsruhe, Germany</td>
			<td style="width:119px;">H884.1</td>
			<td>These can be used multiple times after cleaning.</td>
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		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Glass beads</td>
			<td style="width:71px;">Carl Roth GmbH, Karlsruhe, Germany</td>
			<td style="width:119px;">A553.1</td>
			<td>Diameter: 0.25 - 0.50 mm</td>
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analysis of autophagy in penicillium chrysogenum by using starvation pads in combination with fluorescence microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Para demonstrar a utilidade do protocolo detalhado acima degradação de peroxissoma no P. chrysogenum estirpe Ws54-1255 (GFP-SKL) foi analisada. Nesta estirpe GFP-SKL é geralmente importada para peroxisomes 9. Isto resulta no aparecimento de múltiplas formas esféricas quando a amostra é analisada através de microscopia de fluorescência. Se ocorrer a autofagia, vacúolos ampliar. GFP-SKL é incorporado vacúolos por autofagia (pexophagy). Devido ao facto de que a GFP é resistente à degradaç?...

Watch this Scientific Journal Video about Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy at JoVE.com

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

Análise de Autophagy em<em&gt; Penicillium chrysogenum</em&gt; Por a inanição Pads em combinação com microscopia de fluorescência

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

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JoVE Journal

Biology

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

8.5K visualizações.  Senckenberg Research Institute. A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

Vídeo: Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Lapso de tempo de imagens de Mitose Depois de Transfection siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Biologia

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Electrosoldável célula visualizado com microscopia de fluorescência

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Análise Quantitativa de Autophagy usando microscopia de fluorescência avançada 3D

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualizando Frequency Stromule com microscopia de fluorescência

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Ativando Autophagy pelo exercício aeróbio em ratos

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Análise de partícula única open-source para Microscopia Super-resolução com VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Examinação da dinâmica nuclear do fermento mitotic e Meiotic da fissão pela microscopia viva-pilha da fluorescência

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...