Resolving endogenous protein organization in cells with nanometer resolution

Area of Science:

• Cellular and Molecular Biology
• Advanced Microscopy Techniques
• Biophysics

Background:

• Super-resolution microscopy has advanced significantly, but labeling limitations hinder imaging of nanoscale cellular structures.
• Imaging endogenous multiprotein complexes at the molecular level remains a challenge.

Purpose of the Study:

• To develop an expansion microscopy (ExM) method coupled with direct stochastic optical reconstruction microscopy (dSTORM) for high-resolution imaging of cellular nano-architecture.
• To overcome labeling density limitations in super-resolution microscopy for studying endogenous protein complexes.

Main Methods:

• Developed a novel ExM technique utilizing double-homogenized hydrogels.
• Applied ExM with dSTORM to immunolabeled samples, achieving 7-8 fold expansion.
• Utilized two-color Ex-dSTORM for simultaneous imaging of multiple proteins.

Main Results:

• Achieved ~4-fold increase in labeling density, resolving 8 nm spacing between alpha-tubulin molecules.
• Visualized the polyhedral lattice of clathrin-coated pits and confirmed 8 nm periodicity of alpha/beta-tubulin heterodimers.
• Resolved the organization of RIM and Munc13-1 in 44-48 nm presynaptic ring structures.

Conclusions:

• Ex-dSTORM enables nanometer-resolution imaging of endogenous multiprotein complexes in unmodified cells.
• This versatile technique provides new insights into molecular organization in physiological contexts.
• The method overcomes previous limitations in resolving nanoscale protein interactions.