Investigating the inner structure of focal adhesions with single-molecule localization microscopy

TL;DR

This study uses single-molecule localization microscopy to analyze the detailed substructure of focal adhesions, revealing their size, shape, and composition at a nanoscale level, advancing understanding of cellular adhesion mechanisms.

Contribution

Developed a Gaussian mixture-based method to quantify the substructure of focal adhesions from SMLM data, enabling detailed analysis of their nanoscale features.

Findings

Focal adhesion substructures typically range from 0.01 to 1 μm² in area.

FAs contain 10 to 100 localizations, indicating their molecular complexity.

Substructures can exhibit substantial eccentricity, reflecting diverse shapes.

Abstract

Cells rely on focal adhesions (FAs) to carry out a variety of important tasks, including motion, environmental sensing, and adhesion to the extracellular matrix. Although attaining a fundamental characterization of FAs is a compelling goal, their extensive complexity and small size, which can be below the diffraction limit, have hindered a full understanding. In this study we have used single-molecule localization microscopy (SMLM) to investigate integrin $\beta$3 and paxillin in rat embryonic fibroblasts growing on two different extracellular matrix-representing substrates (i.e. fibronectin-coated substrates and specifically bio-functionalized nano-patterned substrates). To quantify the substructure of FAs, we developed a method based on expectation maximization of a Gaussian mixture that accounts for localization uncertainty and background. Analysis of our SMLM data indicates that the structures within FAs, characterized as a Gaussian mixture, typically have areas between 0.01 and 1 $\mu$m$^2$, contain 10 to 100 localizations, and can exhibit substantial eccentricity. Our approach based on SMLM opens new avenues for studying structural and functional biology of molecular assemblies that display substantial varieties in size, shape, and density.