Supramolecular Assembly in Live Cells Mapped by Real-Time Phasor-Fluorescence Lifetime Imaging

Area of Science:

• Biophysics
• Cell Biology
• Nanotechnology

Background:

• Understanding molecular assembly in living systems is challenging due to complex dynamics.
• Current technologies lack real-time tracking capabilities for crucial early assembly events.

Purpose of the Study:

• To map the real-time transformation of a pro-assembling molecule into nanofibers and their interaction with cellular endosomes.
• To correlate molecular assembly events with biological impacts, specifically metabolic dysfunction.

Main Methods:

• Utilized a chemically designed pro-assembling molecule.
• Employed phasor-fluorescence lifetime imaging (phasor-FLIM) in L929, A549, and MDA-MB 231 epithelial cells.
• Integrated correlative light-electron microscopy and tomography (CLEM) for spatiotemporal analysis.

Main Results:

• Successfully mapped the formation of nanofibers and their fusion with endosomes, creating hollow fiber clusters.
• Observed spatiotemporal correlation between assembly events and metabolic dysfunction.
• Demonstrated assembly-induced endosomal disruption leading to reduced glucose transport and impaired mitochondrial respiration.

Conclusions:

• Phasor-FLIM and CLEM provide critical real-time insights into molecular assembly dynamics.
• Molecular assembly can trigger cellular metabolic dysfunction through endosomal disruption and impaired respiration.
• This study establishes a link between nanoscale assembly processes and cellular bioenergetics.