In situ imaging of mitochondrial outer-membrane pores using atomic force microscopy

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Mitochondrial membrane integrity is crucial for cellular health.
• Understanding pore formation is key to studying mitochondrial dysfunction.
• Existing imaging techniques have limitations in visualizing dynamic pore formation in situ.

Purpose of the Study:

• To develop and apply a novel atomic force microscopy (AFM) technique for imaging mitochondrial outer membrane contours.
• To visualize and quantify mitochondrial membrane pore formation during toxic or metabolic stress.
• To achieve high-resolution, in situ imaging of mitochondrial membrane dynamics.

Main Methods:

• Utilized atomic force microscopy (AFM) for imaging mitochondrial membranes.
• Applied AFM in fluid imaging conditions.
• Subjected mitochondria to metabolic (glucose) and toxic (1,3-dinitrobenzene - DNB) challenges.
• Correlated AFM 3-D surface features with electron microscopy (EM) data.

Main Results:

• Successfully imaged outer contours of mitochondrial membranes with high resolution.
• Observed and quantified pore formation in mitochondria under glucose and DNB challenge.
• Captured potentially the first in situ images of protein clusters during mitochondrial membrane transition pore formation.
• Extended AFM application to fluid imaging of mitochondrial membranes.

Conclusions:

• The developed AFM technique offers unprecedented high-resolution imaging of mitochondrial membrane pore formation in situ.
• This method allows for the quantification of individual mitochondrial membrane pore events.
• The findings provide new insights into the dynamics of mitochondrial membrane transitions during stress.
• The study bridges the gap between AFM and EM for mitochondrial membrane analysis.