Single molecule kinetic analysis of actin filament capping. Polyphosphoinositides do not dissociate capping proteins

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Actin filaments are crucial for cell structure and motility.
• Capping proteins regulate actin filament length.
• Polyphosphoinositides (PPIs) are key signaling lipids involved in various cellular processes.

Purpose of the Study:

• To investigate the binding and dissociation dynamics of heterodimeric capping proteins (CPs) at the barbed ends of actin filaments.
• To determine the effect of polyphosphoinositides (PPIs) on CP binding and dissociation.
• To compare the properties of mouse CP and fission yeast CP.

Main Methods:

• Total internal reflection fluorescence microscopy to observe single muscle actin filaments.
• Bulk assays to measure rate constants for CP association and dissociation.
• Analysis of homology models to compare CP structures.

Main Results:

• Mouse CP association rate constant: 2.6 x 10^6 M^-1 s^-1; dissociation rate constant: 0.0003 s^-1.
• PPIs (PI(3,4)P2, PI(4,5)P2, PI(3,4,5)P3) prevent CP binding but do not dissociate bound CP.
• Fission yeast CP has a thousandfold lower affinity than mouse CP due to slower association and faster dissociation rates.
• PPIs do not inhibit fission yeast CP binding; it lacks a basic residue patch present in mouse CP.

Conclusions:

• PPIs act as inhibitors of CP binding to actin filament barbed ends, rather than dissociators.
• Structural differences, specifically the absence of a basic residue patch in fission yeast CP, explain its altered binding properties and lack of PPI inhibition.
• These findings provide insights into the regulation of actin dynamics by capping proteins and lipids.