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. Préparation de P. chrysogenum pour les expériences de famine <ol><li> Si l&#39;P. chrysogenum souche d&#39;intérêt est maintenu sur le riz (&#39;de riz vert »), placez 2-3 grains de riz recouvertes de sporulation mycélium dans un tube de 1,5 ml. Remplir avec 500 ul YGG (10 g / l de KCl, 20 g / l de glucose, 10 g / l de base azotée de levure, 5 g / l K 2 HPO 4, 20 g / l d&#39;extrait de levure). <ol><li> Vortex le tube pendant 30 secondes afin que les spores peuvent se détacher du riz efficacement. </li><li> Incuber le tube pendant 1 jour à la température ambiante (par exemple entre 20 et 25 ° C). </li><li> Préparer une dilution 1/50 de cette suspension de spores dans l&#39;eau du robinet stérile, bien mélanger. </li></ol></li><li> Si l&#39;P. chrysogenum souche d&#39;intérêt est maintenu sur une plaque de gélose (par exemple, YGG plaque de gélose), transférer de petits morceaux de mycélium dans un tube de 1,5 ml contenant 400 pi d&#39;eau du robinet stérile et environ 100 mg glperles de cul en utilisant un cure-dent stérile ou pointe de la pipette. <ol><li> Vortex le tube pendant 2 min afin que le mycélium est fragmenté. Utilisez le contenu du tube immédiatement. </li></ol></li></ol> 2. Les étapes préparatoires pour la culture de P. chrysogenum sur Pads de famine <ol><li> Mettre sur une plaque chauffante et mettez-le à environ 60 ° C. </li><li> Dissoudre 2 g d&#39;agarose dans 100 ml d&#39;eau du robinet par cuisson de la solution dans un four à micro-ondes. Veillez à ce que cette solution est tout à fait clair après chauffage. Si ce ne est pas, faire cuire à nouveau jusqu&#39;à ce que l&#39;agarose est complètement dissous. Laisser la solution d&#39;agarose à refroidir à environ 60 ° C avant utilisation. </li><li> Remplir tubes de 1,5 ml de microcentrifugation (4/2, en fonction du nombre d&#39;échantillons) avec 400 pi d&#39;eau du robinet stérile chacune (pas de composés additionnels ajoutés) ou 400-x pi (addition de x ul de solution de composé à l&#39;étape 2.5) et placez-les sur la plaque chauffante pendant au moins 1 min de sorte que le water obtient l&#39;intérieur chaud. </li><li> Placer des lames de microscope contenant une cavité centrale sur la plaque chauffante pendant au moins 1 min. </li><li> Ajouter 400 ul de 2% d&#39;agarose à la solution 1,5 ml dans des tubes de microcentrifugation et brièvement vortex. Après cette étape, si désiré, ajouter des composés supplémentaires (ce est à dire, une uM rapamycine). Si cela est fait, vortex brièvement après chaque substance supplémentaire est introduit à la pipette dans le tube. Mettez les tubes de retour sur la table de chauffage pour éviter la solidification prématurée. NOTE: Le volume total dans le tube à centrifuger devrait être de 800 pi. </li></ol> 3. Préparation de microscopiques contenant Pads famine <ol><li> Introduire à la pipette 140 ul de la solution d&#39;agarose dans la cavité de la lame de microscope (qui se trouve encore sur la plaque chauffante). Essayez d&#39;éviter de faire des bulles si possible. </li><li> Immédiatement placer une lamelle sur la solution d&#39;agarose. NOTE: Cela se traduira par une surface plane. </li><li> Mettez le microportée glisser sur le banc de laboratoire pendant environ quatre minutes pour permettre le pad agarose se solidifier. </li><li> Retirez délicatement la lamelle du tampon agarose en le faisant glisser à l&#39;aide d&#39;un pouce ou l&#39;index (porter des gants pour éviter la contamination!). </li><li> Placer la lame de microscope avec le patin dans une chambre humide pour éviter la dessiccation. Recommandation: Utilisez une boîte vide de la pointe qui a encore l&#39;insert de maintien des embouts de pipette. Ajouter de l&#39;eau stérile à la boîte de sorte que le fond est recouvert. Placer les lames de microscope sur l&#39;insert et fermer la boîte. </li></ol> 4. inoculation les Pads famine avec P. chrysogenum et incubation <ol><li> Introduire à la pipette 5 ul de la dilution 1/50 de spores (si les cultures ont été cultivées sur du riz) ou 5 ul de la solution non dilué contenant des fragments de mycélium (si les cultures ont été cultivées sur une plaque de gélose) sur le centre de la plaquette de famine. </li><li> Fermez la chambre humide et l&#39;envelopper dans un sac en plastique (cela sertune protection supplémentaire contre la dessiccation des plots de famine). Veillez à ne pas déplacer la boîte trop car sinon les gouttes d&#39;inoculation seront perturbées. </li><li> Rangez la boîte enveloppée à température ambiante pendant au moins 20 h avant d&#39;analyser les échantillons. </li></ol> 5. Analyse microscopique des échantillons <ol><li> Après 20 h d&#39;incubation, les échantillons sont prêts pour analyse microscopique; mis la lame de microscope sur papier de tissu sec (le fond tend à devenir humide). </li><li> Introduire à la pipette un petit volume d&#39;eau (environ 50 pi) sur le bloc de la famine. Mettez une lamelle sur le tampon et presser le surplus d&#39;eau. Retirer le surplus d&#39;eau avec un mouchoir en papier. </li><li> Mettre la lame de microscope sur la table d&#39;observation du microscope à fluorescence. Pour obtenir un aperçu de la croissance du mycélium, utilisez un objectif 20X. Utilisez 63X ou 100X objectifs pour étudier la localisation de la GFP dans les hyphes. Afin d&#39;éviter la dessiccation graduelle de l&#39;échantillon ne ont pas analyser les échantillons pourplus de 15 min avant de les remettre dans la chambre humide. </li><li> Répétez 5,3 à intervalles réguliers (par exemple, après 40 h et 60 h de la croissance). NOTE: L&#39;échantillon peut être remis dans la chambre humide. Il ne est pas nécessaire d&#39;enlever la lamelle couvre-objet en tant que cela ne affecte pas la croissance du mycélium. </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.

La méthode présentée ici permet l&#39;étude pratique et reproductible de l&#39;autophagie dans P. chrysogenum. Par exemple, il peut être utilisé pour le criblage de l&#39;efficacité des différents composés se ils sont capables de moduler la réponse de l&#39;autophagie de ce champignon ou non. Les résultats démontrent que la rapamycine avec inhibition de la signalisation TOR conduit à une induction marquée de l&#39;autophagie dans P. chrysogenum qui a également été démontré pour d&#39...

Les champignons filamenteux sont d&#39;excellents systèmes modèles pour l&#39;étude des processus de développement. Ils offrent plusieurs avantages expérimentaux comme la culture pas cher, un grand nombre de descendants et l&#39;accessibilité génétique. Le dernier point est particulièrement important pour la construction de transformants qui permettent enquête l&#39;importance des gènes so-far non caractérisées pour divers mécanismes cellulaires. Les champignons filamenteux ont joué un rôle pour l&#39;élucidation de plusieurs éléments et les mécanismes de voies cellulaires de contrôle de la qualité comme l&#39;activité de la protéase de la dégradation des protéines aberrantes, la dynamique mitochondriale pour maintenir l&#39;intégrité mitochondriale et l&#39;autophagie pour l&#39;élimination de l&#39;excédent et / ou des composants cellulaires dysfonctionnelles et pour le maintien la viabilité des cellules en temps de famine 1,2,3. Il existe plusieurs techniques expérimentales disponibles pour l&#39;étude de l&#39;autophagie dans champignons filamenteux 2: (i) l&#39;étude des vacuoles if ils contiennent des corps autophagiques denses lorsque proteases sont inhibées par microscopie électronique à transmission 4, (ii) visualisation de autophagosomes en surveillant GFP-Atg8 foyers par microscopie de fluorescence 5,6 et (iii) la détection des structures de autophagosomal acidifiés à l&#39;aide du colorant fluorescent monodansyl cadavérine 7. Ici, une nouvelle méthode de Penicillium chrysogenum croissante pour les études de l&#39;autophagie est présenté. L&#39;élément principal est le «pad de famine», qui consiste simplement à 1% d&#39;agarose dissous dans l&#39;eau du robinet stérilisée. Des composés supplémentaires (par exemple, les facteurs de stress, des charognards, des modulateurs de l&#39;autophagie) peuvent être ajoutés au pad tant qu&#39;ils ne se affichent pas auto-fluorescence. Le tampon est situé dans des lames de microscope qui contiennent une cavité centrale peu profonde. Ce coussin en inoculé soit avec une suspension de spores ou de petits fragments de mycélium. Ce dernier est conseillé si la souche d&#39;intérêt ne sporulate efficacement (par exemple, les souches de ATG1 Δ 8). Les lames sont placées dans des chambres humides (ceux-ci peuvent être facilement construits en utilisant des boîtes vides pointe de pipette) pour empêcher la dessiccation de l&#39;échantillon et on incube à la température ambiante. P. chrysogenum est capable de croître pendant quelques jours dans ces conditions. L&#39;autophagie peut être observée au microscope par l&#39;élargissement vacuolaire qui est un marqueur positif pour l&#39;autophagie fongique. Dans cette contribution, un P. chrysogenum souche (Wisconsin 54-1255) est utilisé qui forme une protéine fluorescente verte qui est ciblé vers les peroxysomes par sa séquence C-terminale &#39;SKL «9. Par conséquent, il est possible de surveiller la dégradation des peroxysomes. Il est possible de marquer également d&#39;autres compartiments de la cellule (par exemple, les mitochondries) en utilisant des signaux de localisation appropriés et pour analyser leur dégradation. Bien que les données de P. chrysogenum Ws54-1255 (GFP-SKL) est présenté ici, il est certainement possibled&#39;utiliser la méthode du «tapis de famine» aussi pour d&#39;autres champignons filamenteux (par exemple, Neurospora crassa, Sordaria macrospora, les espèces d&#39;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>
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			<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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			<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.

Pour démontrer l&#39;utilité du protocole détaillé ci-dessus dégradation des peroxysomes dans le P. chrysogenum souche Ws54-1255 (GFP-SKL) a été analysée. Dans cette souche GFP-SKL est généralement importé dans les peroxysomes neuf. Cela se traduit par l&#39;apparition de multiples formes sphériques quand l&#39;échantillon est analysé par microscopie à fluorescence. Si l&#39;autophagie se produit, vacuoles agrandir. GFP-SKL est incorporé dans des vacuoles par autophagie (pexophagy). E...

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.

Analyse de l&#39;autophagie dans<em&gt; Penicillium chrysogenum</em&gt; À l&#39;aide de famine Pads en association avec microscopie de fluorescence

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 ...

analysis-autophagy-penicillium-chrysogenum-using-starvation-pads

Research

JoVE Journal

Biology

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

8.5K vues.  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.

Vidéo: 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.

Imagerie time-lapse de la mitose après transfection de siRNA

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

Recherche

Biologie

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.

Analyse quantitative de l&#39;autophagie en utilisant avancée Fluorescence Microscopy 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.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<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 suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

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 glucocorticoïdes Analyse: Surveillance surrénale non invasive en Equidés

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.

Visualisation Fréquence Stromule avec 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 ...

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.

Activation autophagie par exercice aérobie chez la souris

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.

Open Source Analyse unique pour particules super-résolution Microscopie avec 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.

Examen de la dynamique nucléaire de levure de fission mitotique et méiotique par fluorescence Microscopie à cellules vivantes

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 ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

Dynamique d'oligomerisation des récepteurs de surface cellulaire dans les cellules vivantes par microscopie de fluorescence interne totale combinée à l'analyse du nombre et de la luminosité

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...