Single-molecule displacement mapping unveils nanoscale heterogeneities in intracellular diffusivity

Area of Science:

• Cellular Biology
• Biophysics

Background:

• Intracellular diffusion is essential for cellular functions.
• Existing methods struggle to visualize protein diffusion with high spatial resolution and sensitivity.

Purpose of the Study:

• To develop and apply a super-resolution strategy for nanoscale mapping of intracellular protein diffusivity.
• To investigate the nanoscale heterogeneity of protein diffusion in mammalian cells.
• To explore the influence of protein charge and subcellular environment on diffusion.

Main Methods:

• Introduced single-molecule displacement/diffusivity mapping (SMdM), a super-resolution technique.
• Analyzed local statistics of instantaneous displacements of freely diffusing single molecules.
• Applied SMdM to average-sized and differently charged proteins in cytoplasm and nucleus.

Main Results:

• Demonstrated nanoscale spatial heterogeneity in protein diffusion within the cytoplasm and nucleus.
• Correlated diffusion variations with actin cytoskeleton ultrastructure and genome organization.
• Revealed that positive net charges significantly impede protein diffusion, while negative charges do not.
• Showed that diffusion rates are dependent on specific subcellular environments.

Conclusions:

• Intracellular diffusion is highly heterogeneous at the nanoscale.
• Actin cytoskeleton and genome organization significantly influence protein mobility.
• Protein net charge, particularly positive charge, plays a critical role in modulating diffusion.
• SMdM provides unprecedented insights into nanoscale intracellular transport dynamics.