自噬的分析<em>产黄青霉</em>通过结合使用饥饿垫荧光显微镜
Summary
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.
Abstract
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 ‘self-eating’) 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 ‘starvation pads’ 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.
Introduction
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'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'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 ）。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Microscope slides with central cavity | Carl Roth GmbH, Karlsruhe, Germany | H884.1 | These can be used multiple times after cleaning. |
| Glass beads | Carl Roth GmbH, Karlsruhe, Germany | A553.1 | Diameter: 0.25 – 0.50 mm |
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