フリーズエッチ電顕法による薬剤・遺伝子導入用ナノ粒子の細胞内取込み機構の解明

2015 Fiscal Year Annual Research Report

• Project/Area Number: 25253004
• Research Institution: Kyoto University
• Principal Investigator: Heuser John 京都大学, 物質-細胞統合システム拠点, 教授 (40571815)
• Co-Investigator(Kenkyū-buntansha): 村上 達也 京都大学, 物質-細胞統合システム拠点, 准教授 (90410737)
• Project Period (FY): 2013-05-31 – 2016-03-31
• Keywords: 電子顕微鏡 / ドラッグデリバリー / エンドサイトーシス / トランスフェクション

Outline of Annual Research Achievements

The basic purpose of this project was to visualize with the electron microscope (the EM) the mechanism of entry of the important cell-penetrating therapeutic agents available today, with the hypothesis that this entry is via endosome-rupture after cells have “endocytosed” the agents.
The agents we obtained from different laboratories all totally failed to provide any tools that would be visible by electron microscopy, or potent enough to penetrate cells and thus be used for drug delivery.
As a last resort, we finally fell back on simply trying to achieve just one of the desired characteristics of drug delivery agents. Among the different endosome-disrupting peptides we sought to characterize, we chose the HA2 peptide, derived from Influenza virus, and the GALA peptide, a de novo-designed peptide. However, such endosome disrupting peptides are of small molecular weight, and thus are totally invisible in the electron microscope. Therefore, in order to study their mechanism of action, we had to combine the use of these peptides with probes that we could actually see by electron microscopy, and then follow the uptake of these 'markers' at different stages of the internalization-mechanism.
We did all of this by intensive use of classical thin section electron microscopy, as well as by our own very special and unique procedures for quick-freeze, freeze-etch electron microscopy. Sadly, we utterly failed to achieve any success in these efforts, and learned absolutely nothing about how or why or when any of these cell-penetrating peptides ultimately reach the cytoplasm.

Research Progress Status

27年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

27年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] NIH(米国)
  • Country Name: U.S.A.
  • Counterpart Institution: NIH
• [Journal Article] The modeling of Alzheimer's disease by the overexpression of mutant Presenilin 1 in human embryonic stem cells. (2016)
  • Author(s): Honda M, Minami I, Tooi N, Morone N, Nishioka H, Uemura K, Kinoshita A, Heuser JE, Nakatsuji N, Aiba K.
  • Journal Title: Biochem Biophys Res Commun. Volume: 469 Pages: 587-92.
  • DOI: 10.1016/j.bbrc.2015.12.025.
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Mitochondrial fission protein Drp1 regulates mitochondrial transport and dendritic arborization in cerebellar Purkinje cells. (2015)
  • Author(s): Fukumitsu K, Hatsukano T, Yoshimura A, Heuser J, Fujishima K, Kengaku M.
  • Journal Title: Mol Cell Neurosci. Volume: 71 Pages: 56-65.
  • DOI: 10.1016/j.mcn.2015.12.006.
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Eisosome Ultrastructure and Evolution in Fungi, Microalgae, and Lichens. (2015)
  • Author(s): Lee JH, Heuser JE, Roth R, Goodenough U.
  • Journal Title: Eukaryot Cell. Volume: 14 Pages: 1017-42.
  • DOI: 10.1128/EC.00106-15.
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Synergistic action of dendritic mitochondria and creatine kinase maintains ATP homeostasis and actin dynamics in growing neuronal dendrites. (2015)
  • Author(s): Fukumitsu K, Fujishima K, Yoshimura A, Wu YK, Heuser J, Kengaku M.
  • Journal Title: J Neurosci. Volume: 35 Pages: 5707-23.
  • DOI: 10.1523/JNEUROSCI.4115-14.2015.
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Role of the clathrin adaptor PICALM in normal hematopoiesis and polycythemia vera pathophysiology. (2015)
  • Author(s): Ishikawa Y, Maeda M, Pasham M, Aguet F, Tacheva-Grigorova SK, Masuda T, Yi H, Lee SU, Xu J, Teruya-Feldstein J, Ericsson M, Mullally A, Heuser J, Kirchhausen T, Maeda T.
  • Journal Title: Haematologica. Volume: 100 Pages: 439-51.
  • DOI: 10.3324/haematol.2014.119537.
  • Peer Reviewed / Int'l Joint Research
• [Presentation] Discovering the cause of adult-onset muscular dystrophy by electron microscopy of mouse genetic models. (2016)
  • Author(s): John Heuser
  • Organizer: 第１２１回日本解剖学会総会・学術集会
  • Place of Presentation: 福島、日本
  • Year and Date: 2016-03-28 – 2016-03-30
  • Invited
• [Presentation] Profound plasma membrane changes caused by acute cholesterol elevation (2015)
  • Author(s): Azumi Yoshimura, Silvia Pujals, Nobuhiro Morone, Tanya Tenkova and John Heuser
  • Organizer: Biochemistry and Molecular Biology 2015
  • Place of Presentation: Kobe, Japan
  • Year and Date: 2015-12-01 – 2015-12-04
  • Invited