Abstract
Spatial and temporal control of actin polymerization is fundamental for many cellular processes, including cell migration, division, vesicle trafficking, and response to agonists. Many actin-regulatory proteins interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and are either activated or inactivated by local PI(4,5)P2 concentrations that form transiently at the cytoplasmic face of cell membranes. The molecular mechanisms of these interactions and how the dozens of PI(4,5)P2-sensitive actin-binding proteins are selectively recruited to membrane PI(4,5)P2 pools remains undefined. Using a combination of biochemical, imaging, and cell biologic studies, combined with molecular dynamics and analytical theory, we test the hypothesis that the lateral distribution of PI(4,5)P2 within lipid membranes and native plasma membranes alters the capacity of PI(4,5)P2 to nucleate actin assembly in brain and neutrophil extracts and show that activities of formins and the Arp2/3 complex respond to PI(4,5)P2 lateral distribution. Simulations and analytical theory show that cholesterol promotes the cooperative interaction of formins with multiple PI(4,5)P2 headgroups in the membrane to initiate actin nucleation. Masking PI(4,5)P2 with neomycin or disrupting PI(4,5)P2 domains in the plasma membrane by removing cholesterol decreases the ability of these membranes to nucleate actin assembly in cytoplasmic extracts.
Highlights
Spatial and temporal control of actin polymerization is fundamental for many cellular processes, including cell migration, division, vesicle trafficking, and response to agonists
The uneven distribution of assembled actin seen by fluorescent phalloidin staining (Fig. 1D) and its corresponding quantification showed that the amount of polymerized actin per unit area within the Lo domains was 80% less than that measured in the Ld phase (Fig. 1E)
The salient findings include the following. (a) The presence of cholesterol enhances formin binding to PI[4,5]P2 resulting in lower effective Kd values for large unilamellar vesicles (LUVs) A compared with LUVs B; this lowering of the effective Kd is consistent with the enhancement in multivalent interactions observed in the molecular dynamics simulations when cholesterol was included in the bilayers. (b) The change in Kd with increase in formin, PI[4,5]P2, and cholesterol concentration can be explained by a two-step reaction mechanism of formin recruitment to the membrane and multivalent binding with PI[4,5]P2; the parameters of the kinetic model are consistent with the findings of molecular simulations. (c) Our results suggest that as formin concentration increases, the number of filament nucleation sites Ns can saturate at a lower concentration of PI[4,5]P2
Summary
Spatial and temporal control of actin polymerization is fundamental for many cellular processes, including cell migration, division, vesicle trafficking, and response to agonists. Masking PI[4,5]P2 with neomycin or disrupting PI[4,5]P2 domains in the plasma membrane by removing cholesterol decreases the ability of these membranes to nucleate actin assembly in cytoplasmic extracts It constitutes less than 1% of the total phospholipid of the cell, phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) is. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Because the same pool of cytoplasmic actin needs to be arranged differently to support the spectrum of its cellular functions, a dynamic lipid-based regulation at the cytoplasm/membrane interface provides a unique mechanism to control and modify actin assembly with spatial and temporal specificity
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