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.

P.の調製飢餓実験用クリソゲヌム <ol><li> P.の場合関心のクリソゲヌム株は米（「緑米&#39;）に保管され、1.5ミリリットルマイクロ遠心チューブに菌糸体を胞子形成に覆わ2-3米粒を配置。 500μlのYGG（10g / lのKClを、20g / lのグルコース、10g / lの酵母窒素塩基、5グラム/リットルのK 2 HPO 4、20g / lの酵母エキス）で塗りつぶす。 <ol><li>胞子が効率的にお米から切り離すことができるように、30秒間チューブをボルテックス。 </li><li>室温で1日（ 例えば 、20〜25℃）のためにチューブをインキュベートする。 </li><li>無菌水道水におけるこの胞子懸濁液の1/50希釈液を調製、よく混ぜる。 </li></ol></li><li> P.の場合関心のクリソゲナム菌株は、400μlの滅菌水道水及び約100mgのglのを含む1.5 mlのマイクロ遠心チューブに菌糸体の小片を移送、寒天プレート（ 例えば 、YGG寒天プレート）上に保持されている無菌の歯のピックまたはピペットチップを使用して、お尻のビーズ。 <ol><li> 2分間ボルテックスチューブを菌糸体が断片化になるように。すぐにチューブの内容を使用してください。 </li></ol></li></ol> Pの栽培2.準備手順飢餓パッド上クリソゲヌム <ol><li>加熱プレートのスイッチを入れ、約60℃に設定します。 </li><li>電子レンジで調理することにより、溶液を100ミリリットルの水道水にアガロースを2gを溶解する。このソリューションは、加熱後には完全には明らかであることに注意してください。そうでない場合、アガロースが完全に溶解するまで、再びそれを調理。アガロース溶液は、使用前に、約60℃まで冷却してみましょう。 </li><li>各無菌水道水400μlの（サンプル数に応じて、2-4、）を1.5 mlマイクロチューブを埋める（追加の化合物を添加しない）、または400-XμL（ステップ2.5における化合物溶液のμLxの追加）、それを配置少なくとも1分間、加熱板上になるようワットER内部の暖かい取得します。 </li><li>少なくとも1分間加熱プレート上に中央の空洞が含まれている顕微鏡スライドを配置します。 </li><li>簡潔に1.5ミリリットルのマイクロチューブとボルテックスで溶液にアガロース400μlの2％を追加します。必要に応じて、このステップの後、追加の化合物（ すなわち、1μMのラパマイシン）を追加します。これが行われた場合、追加の各物質の後に簡単に渦がチューブにピペットで。時期尚早の凝固を防ぐために、加熱用テーブルの上に戻ってチューブを入れてください。 注：マイクロ遠心チューブ内の総容積は800μLでなければなりません。 </li></ol>飢餓パッドを含む顕微鏡用スライドガラスの調製<ol><li> （依然として加熱板上に配置されている）顕微鏡スライドのキャビティ内にアガロース溶液をピペットで140μlの。可能な場合は、気泡を避けるようにしてください。 </li><li>すぐにアガロース溶液上にカバースリップを配置。 注：これは平らな表面になります。 </li><li>マイクロを入れてアガロースパッドが凝固することを可能にするために約4分間の実験台の上にスコープスライド。 </li><li>慎重に（汚染を避けるために手袋を着用！）親指または人差し指の助けを借りてそれをスライドさせて、アガロースパッドからカバースリップを削除します。 </li><li>乾燥を防ぐために、湿ったチャンバー内にパッドを備えた顕微鏡スライドを置きます。推奨：まだピペットチップを保持するためのインサートを持って空の先端ボックスを使用します。底部が覆われるようにボックスに滅菌水を追加します。インサート上に顕微鏡スライドを配置し、ボックスを閉じます。 </li></ol> 4. Pと飢餓パッドに接種クリソゲヌムおよびインキュベーション<ol><li>ピペット1/50胞子希釈液5μl（培養物は、ご飯の上に成長させた場合）、または菌糸断片を含む原液の5μL（培養物を寒天プレート上で成長させた場合）飢餓パッドの中心へ。 </li><li>これは、お楽しみいただけます（湿ったチャンバーを閉じ、ビニール袋に包む飢餓パッドの乾燥に対する追加の保護）。あまりにも多くの他の方法で接種滴が乱されるためボックスを移動しないように注意してください。 </li><li>サンプルを分析する前に、少なくとも20時間室温で包まれた箱に保管してください。 </li></ol>サンプルの5。顕微鏡分析<ol><li>インキュベーションの20時間後、試料を顕微鏡分析のために準備ができている。乾燥したティッシュペーパー上で顕微鏡スライドを配置（下湿潤なる傾向がある）。 </li><li>飢餓パッド上に少量の水（約50μl）をピペット。パッド上にカバーガラスを入れ、余剰水を絞り出す。ティッシュペーパーで余剰水を除去。 </li><li>蛍光顕微鏡の観察台に顕微鏡スライドを置く。菌糸体の成長の概要を取得するには、20X目標を使用しています。菌糸にGFPの局在を研究するための63Xまたは100Xの目的を使用してください。のために試料を分析しないサンプルの段階的な乾燥を防ぐためにより長い15分のウェット室に戻って、それらを入れる前。 </li><li> （40時間と成長の60時間後に、 例えば ）一定の間隔で5.3を繰り返します。 注：サンプルは、湿ったチャンバー内に戻すことができます。それは、菌糸体の成長に影響を与えないようにカバースリップを除去する必要がない。 </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.

ここで紹介する方法はPにオートファジーの便利で再現可能な研究を可能にするクリソゲヌム 。例えば、それらはこの菌かのオートファジー応答を調節することができるかどうかを、種々の化合物の有効性をスクリーニングするために使用することができる。ラパマイシンを用いた結果は、TORシグナル伝達の阻害は、Pにおけるオートファジーの顕著な誘導を導くことを証?...

糸状菌は、発達過程の研究のための優れたモデル系である。彼らは安い栽培、子孫と遺伝のアクセシビリティの高い番号のようないくつかの実験的な利点を提供します。最後の点は、様々な細胞メカニズムのそれまで未同定の遺伝子の重要性を調査できるように、形質転換体の構築のために特に重要である。糸状菌は、剰余金の除去および/または機能不全の細胞成分のための維持のためにミトコンドリアの完全性およびオートファジーを維持するため、異常なタンパク質の分解のためのプロテアーゼ活性のような細胞の品質管理経路のいくつかの要素とメカニズムの解明のためにミトコンドリアのダイナミクス尽力されてきた飢餓1,2,3の時代に、細胞生存率。 糸状菌2におけるオートファジーの研究のために利用可能ないくつかの実験技術があります：液胞の（I）の調査私がプロテアーゼは、透過型電子顕微鏡4によって阻害されたときfはそれらが密なオートファジー体を含むこと、（ii）蛍光色素モノダンシルカダベリを使用して、蛍光顕微鏡5,6酸性化オートファゴソーム構造の（iii）の検出によりGFP-Atg8病巣を監視することによりオートファゴソームの可視化7。 ここで、オートファジー研究のために、ペニシリウム·クリソゲヌムを成長させる新規な方法が提示される。主な要素は、単にアガロース滅菌水道水に溶解した1％からなる「飢餓パッド」である。追加の化合物（ 例えば、ストレス因子、スカベンジャー、オートファジー変調器）は、それらが自己蛍光を表示しないように、パッドに追加することができます。パッドは浅い中央の空洞が含まれている顕微鏡スライドに位置しています。このパッドに胞子懸濁液または小さな菌糸断片のいずれかで接種した。興味のある歪がsporulatに失敗した場合、後者が推奨される効率的に電子（ 例えば、ΔのATG1株 8）。スライドは、試料の乾燥を防ぐために（これらは容易に空のピペットチップボックスを使用して構成することができる）湿潤チャンバー内に配置し、室温でインキュベートする。P.クリソゲヌムは、これらの条件下で、数日間成長させることができる。オートファジーは、真菌、オートファジーのための肯定的なマーカーである液胞の拡大によって顕微鏡的に観察することができる。この貢献、Pにクリソゲナム菌株 （ウィスコンシン州54から1255）は、そのC末端&#39;SKL&#39;配列9によってペルオキシソームに標的化された緑色蛍光タンパク質を形成するために使用される。従って、ペルオキシソームの劣化を監視することが可能である。適切な局在化シグナルを使用してそれらの分解を分析することにより、細胞（ 例えば、ミトコンドリア）の他の区画を標識することが可能である。 Pからデータもののクリソゲヌム Ws54-1255（GFP-SKLが）それは確かに可能であり、ここで提示されている他の糸状菌（ 例えば、アカパンカビ、Sordaria macrospora、アスペルギルス種、 など ）にも「飢餓パッド&#39;メソッドを使用します。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
	</tbody>
</table>

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.

Pにペルオキシソームの劣化の上に詳細なプロトコルの有用性を実証するために、 クリソゲナム菌株 Ws54-1255（GFP-SKL）を分析した。この株ではGFP-SKLは通常、ペルオキシソーム9にインポートされます。サンプルは、蛍光顕微鏡によって分析されている場合に、複数の球状の外観をもたらす。オートファジーが発生した場合は、空胞を拡大。 GFP-SKLは、オートファジー（pexop...

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.

オートファジーの解析で<em&gt;ペニシリウム·クリソゲヌム</em&gt;蛍光顕微鏡と組み合わせて飢餓パッドを使用した

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 ビュー.  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.

ビデオ: 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.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

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

生物学

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.

高度な3D蛍光顕微鏡を用いたオートファジーの定量分析

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.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（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.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

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.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

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.

蛍光顕微鏡でストロミュール頻度を可視化

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.

マウスで有酸素運動によってオートファジーのアクティブ化

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.

VirusMapperと超解像顕微鏡のためのオープンソースの単一粒子分析

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.

蛍光生細胞顕微鏡によるミトティック・マイオシス酵母核ダイナミクスの検討

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.

数と明るさの分析と組み合わせた全内部反射蛍光顕微鏡による生細胞における細胞表面受容体のオリゴマー化ダイナミクス

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