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.准备P.的黄青霉的饥饿实验<ol><li>如果P.感兴趣的产黄青霉株保持对水稻（“绿色大米”），将布满菌丝孢子到1.5 ml离心管2-3米粒。它用500μlYGG（10克/升氯化钾20克/升葡萄糖，10g / l酵母氮碱，5克/升K 2 HPO 4 20克/升酵母提取物）填充。 <ol><li>涡管30秒，使孢子可从水稻分离效率。 </li><li>孵育管中1天，在室温（ 例如 ，20至25℃）。 </li><li>准备一个1/50稀释这种孢子悬浮液中无菌自来水，搅拌均匀。 </li></ol></li><li>如果P.感兴趣chrysogenum菌株被保持在琼脂平板上（ 例如 ，YGG琼脂板），转移小块菌丝到含有400微升无菌自来水和大约100毫克GL 1.5ml微量离心管屁股珠用无菌牙签或枪头。 <ol><li>涡管2分钟，使菌丝体变得支离破碎。立即使用该管的内容物。 </li></ol></li></ol>为培育P. 2预备步骤黄青霉对饥饿垫<ol><li>接通加热板，并将其设定为约60℃。 </li><li>溶解2克琼脂糖在100毫升由烹调在微波炉中的溶液自来水。请注意，这个解决方案是加热后完全清楚。如果不是，再次煮直到琼脂糖完全溶解。让琼脂糖溶液冷却至约60℃，使用前。 </li><li>填充的1.5 ml离心管（2-4，这取决于样品的数量）用400μl无菌自来水（不添加额外的化合物），每个或400-X微升（除了x的微升在步骤2.5化合物溶液），并把它们到加热板为至少1分钟，以使笏呃内得到温暖。 </li><li>放置显微镜载玻片包含一个中心腔到加热板至少1分钟。 </li><li>琼脂加入400微升2％至1.5 ml离心管和涡简要的解决方案。在此步骤之后，如果需要的话，加入另外的化合物（ 即，1μM的雷帕霉素）。如果这样做，涡旋后短暂每个附加物质被吸向管。放管放回加热表，以防止过早固化。 注：在离心管中的总量应该是800微升。 </li></ol> 3.准备显微镜载玻片的含饥饿垫<ol><li>吸管140微升琼脂糖溶液到显微镜载玻片的腔（其仍位于加热板）。尽量避免，如果可能，气泡。 </li><li>立即将盖玻片上的琼脂糖溶液。 注意：这将导致在一个平面上。 </li><li>把微范围滑动到实验台上大约4分钟，使琼脂糖垫巩固。 </li><li>小心地用拇指或食指的帮助下滑动它关闭删除琼脂糖垫盖玻片（戴手套，以避免污染！）。 </li><li>放置与垫显微镜载玻片成湿腔室，以防止干燥。建议：使用一个空的提示框，仍然有插入举办的枪头。加入无菌水到盒使得底部被覆盖。放置在显微镜载玻片上的插入件并关闭对话框。 </li></ol> 4.接种饥饿垫与P.黄青霉和孵化<ol><li>吸移管将5μl的1/50稀释孢子（如果培养物生长在水稻）或5微升含有片段的菌丝体（如培养物生长在琼脂平板上）上饥饿垫的中心的未稀释溶液组成。 </li><li>关闭湿室，把它包在一个塑料袋（这作为额外的保护，防止饥饿垫干燥）。要小心，不要将箱子太大，否则接种滴会受到干扰。 </li><li>分析样品之前，存储在RT包装的盒子进行至少20小时。 </li></ol>该样品5显微分析<ol><li> 20小时温育后，将样品准备显微镜分析;放在干燥的纸巾显微镜载玻片（底部趋于湿）。 </li><li>吸取小体积的水（约50微升）到饥饿垫。把盖玻片上垫挤掉多余的水分。用纸巾去除多余的水。 </li><li>把载玻片上的荧光显微镜的观察表。为了让菌丝生长的概述，用20倍的目标。使用63X或100X目标研究GFP的本地化的菌丝。为了防止试样的逐渐干燥不分析样品为前长于15分钟把它们放回湿室。 </li><li>重复5.3定期（ 例如 ，40小时和60小时生长后）。 注：样品可以放回湿室中。这是没有必要除去盖玻片，因为它不影响菌丝生长。 </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.

这里介绍的方法允许自噬在P.方便和可重复的研究黄青霉 。例如，它可用于筛选各种化合物的功效它们是否能够调节这种真菌与否的自噬反应。雷帕霉素的研究结果表明，抑制TOR信令导致明显的诱导自噬在体育黄青霉这也被证明对其他生物体11。 它可以使用在更复杂的显微镜（ 例如 ，共聚焦激光扫描显微镜）上生长饥饿焊盘用于分析菌丝?...

丝状真菌是优秀的模型系统的发育过程的研究。他们提供了几个实验的优惠，如廉价的种植，高数量的后代遗传的可访问性。最后一点是转化的允许调查迄今为止在非特异基因的各种细胞机制的重要建设特别重要的。丝状真菌是有助的几个元件和蜂窝质量控制通路像为异常蛋白，线粒体动力学的降解蛋白酶活性的机制的阐明对维持线粒体的完整性和自噬用于去除过剩和/或不正常的细胞成分和用于维持细胞活力在饥饿1,2,3次。 有可用于自噬在丝状真菌2该研究的几个实验技术：空泡（ⅰ）调查我˚F它们含有致密的自噬体时的蛋白酶是由透射电子显微镜4的抑制作用，（ⅱ）自噬体通过经由荧光显微镜5,6和（iii）检测酸化autophagosomal结 ​​构监测GFP-ATG8灶通过使用荧光染料monodansyl尸胺可视7。 这里，一个新的增长的产黄青霉菌对自噬的研究方法。主要元素是&#39;饥饿垫“，这只是由琼脂糖溶解在无菌自来水中的1％。另外的化合物（ 例如，压力源，清除剂，自噬调制器）可以使用，只要它们不显示自发荧光加至垫。该垫位于显微镜载玻片包含一个浅中心空腔。此衬垫在接种要么用孢子悬浮液，或以小的菌丝体片段。后者是可取的，如果所关注的菌株不能sporulatË有效（ 例如，ΔATG1株8）。将载玻片放置在湿室（这些可以很容易地构造使用空枪头框），以防止样品的干燥和在室温下孵育。P.黄青霉能够生长在这些条件下几天。自噬可以通过显微镜液泡增大其是阳性标志物的真菌自噬被观察到。在这方面的贡献，一个P. chrysogenum菌株（威斯康星54-1255）用于形成是受它的C-末端“SKL笔顺9靶向过氧化物酶体绿色荧光蛋白。因此，可以监测过氧化物酶的降解。它是通过使用适当的定位信号也以标记细胞（ 例如，线粒体）的其他车厢，并分析它们的降解是可行的。虽然从数据P.黄青霉 Ws54-1255（GFP-SKL）在这里介绍，这当然是可能的使用&#39;饥饿垫“的方法也可用于其它丝状真菌（ 例如，粗糙脉孢菌，Sordaria macrospora，曲霉菌属等 ）。

<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.

为了证明在P.上述过氧化物酶降解详细介绍了协议的效用chrysogenum菌株Ws54-1255（GFP-SKL）进行了分析。在这株GFP-SKL通常被导入到过氧化物酶体9。这导致多个球形的外观，当样品通过荧光显微镜进行分析。如果发生自噬，空泡放大。 GFP-SKL成为自噬（pexophagy）并入空泡。由于这一事实，即GFP是通过液泡蛋白酶降解抗性它所标记这些细胞器10。因此这两个参数，观察自噬（p...

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.

自噬的分析<em&gt;产黄青霉</em&gt;通过结合使用饥饿垫荧光显微镜

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 次观看.  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.

视频: 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.

有丝分裂的siRNA转染后的时间推移成像

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

科研

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.

采用先进的3D荧光显微镜定量分析自噬

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.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（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.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

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.

糖皮质激素粪分析：在马属动物无创监测肾上腺

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.

可视化Stromule频率与荧光显微镜

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.

小鼠中自噬激活有氧运动

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.

与VirusMapper超分辨率显微镜开源单粒子分析

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.

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

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.

通过总内部反射荧光显微镜与数量和亮度分析相结合，活细胞中细胞表面受体的显微化动力学

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