In Situ Characterization of Bak Clusters Responsible for Cell Death Using Single Molecule Localization Microscopy

Area of Science:

• Cell biology
• Molecular biology
• Biophysics

Background:

• Apoptosis is vital for multicellular organism development and tissue homeostasis.
• Bak protein clusters on mitochondria induce apoptosis by releasing pro-apoptotic factors.
• Limitations in light microscopy hinder understanding of Bak cluster characteristics.

Purpose of the Study:

• To quantitatively characterize Bak protein clusters in situ during apoptosis.
• To elucidate the nanoscale properties of Bak clusters, including size and protein density.
• To provide a method applicable to analyzing various protein cluster formations.

Main Methods:

• Development of a novel approach for quantitative characterization of Bak clusters.
• Utilizing single-molecule localization microscopy (SMLM) for nanoscale analysis.
• In situ analysis of Bak protein clusters on the mitochondrial outer membrane.

Main Results:

• Bak proteins form densely packed clusters at the nanoscale on mitochondria during apoptosis.
• Cluster size is highly heterogeneous, while protein density is uniform across clusters.
• The study provides unprecedented quantitative data on Bak cluster characteristics.

Conclusions:

• The developed method allows for precise nanoscale characterization of protein clusters.
• Understanding Bak cluster properties is critical for elucidating the mechanism of mitochondrial outer membrane permeabilization.
• This approach can be applied to study other protein aggregation phenomena in biological systems.