In Situ Characterization of Bak Clusters Responsible for Cell Death Using Single Molecule Localization Microscopy.
Apoptosis plays a pivotal role in development and tissue homeostasis in multicellular organisms. Clustering of Bak proteins on the mitochondrial outer membrane is responsible for the induction of apoptosis by evoking a release of pro-apoptotic proteins from mitochondria into cytosol. However, how the protein cluster permeabilizes the mitochondrial membrane remains unclear because elucidation of the cluster characteristics such as size and protein density has been hampered by the diffraction-limited resolution of light microscopy. Here, we describe an approach to quantitatively characterize Bak clusters in situ based on single molecule localization. We showed that Bak proteins form densely packed clusters at the nanoscale on mitochondria during apoptosis. Quantitative analysis based on the localization of each Bak protein revealed that the density of Bak protein is uniform among clusters although the cluster size is highly heterogeneous. Our approach provides unprecedented information on the size and protein density of Bak clusters possibly critical for the permeabilization and is applicable for the analysis of different cluster formations.
Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Yusuke
Y
Nasu
Laboratory of Experimental Biophysics, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Alexander
A
Benke
Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 103-5802, Japan.
Satoko
S
Arakawa
Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 103-5802, Japan.
Go J
GJ
Yoshida
Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Genki
G
Kawamura
Laboratory of Experimental Biophysics, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Suliana
S
Manley
Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 103-5802, Japan.
Shigeomi
S
Shimizu
Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Takeaki
T
Ozawa
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D016923:Cell Death / Q000502:physiology*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D051379:Mice; D008853:Microscopy / Q000379:methods; D008928:Mitochondria / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism*
C494772:BAK1 protein, human; D050998:bcl-2 Homologous Antagonist-Killer Protein