Year-end Sale % OFF 
Abstract
We investigated how heterodimeric capping proteins bind to and dissociate from the barbed ends of actin filaments by observing single muscle actin filaments by total internal reflection fluorescence microscopy. The barbed end rate constants for mouse capping protein (CP) association of 2.6 x 10(6) M(-1) s(-1) and dissociation of 0.0003 s(-1) agree with published values measured in bulk assays. The polyphosphoinositides (PPIs), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), PI(4,5)P(2), and PI(3,4,5)P(3), prevent CP from binding to barbed ends, but three different assays showed that none of these lipids dissociate CP from filaments at concentrations that block CP binding to barbed ends. The affinity of fission yeast CP for barbed ends is a thousandfold less than mouse CP, because of a slower association rate constant (1.1 x 10(5) M(-1) s(-1)) and a faster dissociation rate constant (0.004 s(-1)). PPIs do not inhibit binding of fission yeast CP to filament ends. Comparison of homology models revealed that fission yeast CP lacks a large patch of basic residues along the actin-binding surface on mouse CP. PPIs binding to this site might interfere sterically with capping, but this site would be inaccessible when CP is bound to the end of a filament.
Highlights
We investigated how heterodimeric capping proteins bind to and dissociate from the barbed ends of actin filaments by observing single muscle actin filaments by total internal reflection fluorescence microscopy
PIP2 inhibits budding yeast capping protein (CP) [14], we discovered that PPIs do not inhibit binding of fission yeast CP to filament ends, despite other similarities to budding yeast and vertebrate CPs
Kinetics of Capping and Capping by Mouse Capping Protein— We observed the growth and capping of individual actin filaments by total internal reflection fluorescence (TIRF) microscopy using muscle actin-labeled with Oregon Green (OG-actin)
Summary
The barbed end rate constants for mouse capping protein (CP) association of 2.6 ؋ 106 M؊1 s؊1 and dissociation of 0.0003 s؊1 agree with published values measured in bulk assays. Addition of PIP2 to mixtures of capped filaments, CP, and actin monomers in vitro increases the rate of polymerization [4] These experiments led to the widely accepted view that PPIs inhibit association of CP with barbed ends and dissociate CP from actin filaments. These two effects are postulated to bias the growth of actin filament branches toward the inside of the plasma membrane at the leading edge [11, 12].
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
