論文

国際誌
2014年11月

Metallothionein labeling for CLEM method for identification of protein subunits.

Microscopy (Oxford, England)
  • Ryutaro Yamanaka
  • ,
  • Yuka Hirasaka
  • ,
  • Mingyue Jin
  • ,
  • Haruaki Yanagisawa
  • ,
  • Takuo Yasunaga

63 Suppl 1
開始ページ
i32
終了ページ
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1093/jmicro/dfu065

CLEM (correlative light and electron microscopy) is one of the powerful techniques to elucidate the localization and structure of the target proteins or their complexes in cell. First, target proteins labeled fluorescently can be searched using a fluorescence microscope, i.e., due to its low resolution (200nm), it is used as rough searching of target proteins. After rough detection of the localization of target proteins, they can be easily observed by electron microscopy with a high resolution and processed into fine structure, especially 3D structure. On the other hand, in the case of only electron microscopy, it is difficult for researchers to detect their localization due to a narrow range of views and no labeling of them.Thus, CLEM normally needs fluorescent labels for fluorescence microscopy but a label for electron microscopy is also expectedly for easier detection. Thus we focused on metallothionein. Metallothionein binds to cadmium ions, i.e., heavy atoms with strong density in electron microscopy [1]; in addition, cadmium ions and selenium ions are known to form Qdot-like nanoparticles induced by metallothionein [2]. These are 2 ∼ 5nm in size, fluorescent wavelength changes depending on the size of nanoparticles. Thus, target proteins fused with metallothionein could be observed by both of fluorescence microscopy and electron microscopy.We here used Chlamydomonas reinhardtii, single cell green algae with two flagella. Flagella are used for bending motion and motility. Flagella contain FAP20 (Flagellar Asociate Protein 20) and PACRG (PArkin Co-Regulated gene), which are related to composing axoneme architecture. If Chlamydomonas reinhardtii doesn't have FAP20 or PACRG, they can't generate bending motion. It is considered that FAP20 and PACRG locate on the root of the radial spoke. Recently the location of FAP20 was reported by Yanagisawa et al.[3]. First, we also focus on detecting localization of FAP20 and then will do so on that of PACKRG.We could observe fluorescence of metallothionein fused with FAP20 to form nanoparticle. We are now trying to observe larger electron density from metallothionein with cadmium for CLEM.

リンク情報
DOI
https://doi.org/10.1093/jmicro/dfu065
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/25359836
ID情報
  • DOI : 10.1093/jmicro/dfu065
  • PubMed ID : 25359836

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