SuperResNET - single-molecule network analysis detects changes to clathrin structure induced by small-molecule...

Area of Science:

• Cell biology
• Biophysics
• Microscopy

Background:

• Clathrin-mediated endocytosis is crucial for cellular processes.
• Understanding the precise mechanisms of endocytosis inhibitors is vital.
• Single-molecule localization microscopy (SMLM) provides high-resolution insights into molecular dynamics.

Purpose of the Study:

• To apply the SuperResNET pipeline to analyze SMLM dSTORM data.
• To investigate the effects of pitstop 2, dynasore, and latrunculin A on clathrin-coated pit morphology.
• To characterize distinct stages of clathrin-coated pit formation and maturation.

Main Methods:

• Network analysis of point cloud data using SuperResNET.
• Application of SMLM direct stochastic optical reconstruction microscopy (dSTORM).
• Analysis of clathrin structures in HeLa and Cos7 cells treated with inhibitors.

Main Results:

• Identified three classes of clathrin structures: oligomers, pits/vesicles, and clusters.
• Pitstop 2 and dynasore induced distinct class II structures, suggesting different endocytosis arrest points.
• Latrunculin A induced large, heterogeneous structures, indicating effects independent of actin depolymerization.
• Ternary analysis revealed pitstop 2-treated cells had planar clathrin profiles, resembling early pits.

Conclusions:

• SuperResNET effectively analyzes SMLM data to reveal inhibitor effects on clathrin structures.
• Pitstop 2 arrests clathrin pit maturation at early stages of formation.
• This approach allows in situ detection of small molecule effects on cellular targets from SMLM datasets.