IRSp53 promotes postsynaptic density formation and actin filament bundling

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• IRSp53 (Insulin receptor substrate p53) is a key scaffold protein linking membranes to the actin cytoskeleton in cellular protrusions.
• It is highly expressed in excitatory synapses and plays a vital role in synapse development and plasticity, though its precise mechanisms remain unclear.

Purpose of the Study:

• To elucidate the molecular mechanisms underlying IRSp53's function in excitatory synapses.
• To investigate the role of IRSp53 interactions with PSD-95 and Shank3 in driving phase separation and actin organization.

Main Methods:

• In vitro phase separation assays using purified IRSp53, PSD-95, and Shank3.
• Reconstitution of excitatory postsynaptic density (ePSD) condensates.
• Overexpression of IRSp53 mutants in mouse cortical neurons.
• Analysis of synaptic IRSp53 enrichment and actin filament formation.

Main Results:

• Specific multivalent interactions between IRSp53 and PSD-95/Shank3 induce phase separation, forming ePSD condensates.
• IRSp53 is enriched in these condensates and promotes bundled actin filament formation.
• Disrupting IRSp53-PSD-95 interactions impairs synaptic IRSp53 localization.
• Perturbing IRSp53-actin interactions leads to defects in synaptic maturation.

Conclusions:

• IRSp53-mediated phase separation is a key mechanism for organizing protein complexes and actin at excitatory synapses.
• These findings provide mechanistic insights into IRSp53's essential roles in synapse formation, maturation, and function.