Multiscale imaging and quantitative analysis of plasma membrane protein-cortical actin interplay

Area of Science:

• Cell Biology
• Biophysics
• Molecular Imaging

Background:

• The spatiotemporal organization of cell surface receptors is crucial for cell signaling and is influenced by cortical actin (CA).
• Previous studies using actin perturbation experiments have limitations, including nonspecific effects and difficulty in quantifying CA architecture and dynamics under unperturbed conditions.
• Directly correlating CA properties with plasma membrane (PM) protein organization remains challenging.

Approach:

• Developed a multiscale imaging and computational analysis framework integrating single-molecule imaging (SMI) of PM proteins and fluorescent speckle microscopy (FSM) of CA in live cells (SMI-FSM).
• Applied SMI-FSM to simultaneously visualize and quantify the dynamics of PM proteins and the architecture of CA within the same cell.
• Utilized computational statistical analysis to establish direct relationships between CA characteristics and PM protein spatiotemporal organization.

Key Points:

• SMI-FSM revealed differential relationships between PM proteins and CA, dependent on protein actin-binding ability, diffusion type, and local CA density.
• Highlighted the complexity of actin perturbation experiments, showing that global actin changes do not always reflect local CA properties near PM proteins.
• Demonstrated that PM protein behavior changes upon perturbation vary based on the local CA environment.

Conclusions:

• The SMI-FSM framework provides a direct method to study the influence of CA architecture and dynamics on PM protein organization.
• This versatile approach is expected to be widely applicable for investigating actin-dependent cellular processes, such as cell migration.
• Offers a quantitative basis for understanding how CA influences cell surface receptor organization and cellular behaviors.