Molecular Basis of Dynamic Localization of Class-I Myosins

Dictyostelium myosin IB （DMIB）是一种典型的长尾i类肌球蛋白，与传统的II类肌球蛋白相比，它具有短的非螺旋尾，包含一个碱性疏水膜结合区（BH-site）、一个结合F-actin的Gly-Pro-Gln区域和一个src同源3 （SH3）结构域。与其他肌球蛋白i优先结合磷脂酰肌醇二磷酸（PIP2）相比，bh位点与酸性磷脂集中的膜区域结合，仅基于净负电荷，而不考虑磷脂的组成。Gly-Pro-Gln区域在ATP存在或不存在的情况下与f -肌动蛋白结合，这与马达结构域中ATP敏感的肌动蛋白结合位点不同。

Dictyostelium myosin IB (DMIB) is a typical long-tail class-I myosin with a short (compared to conventional Class II myosins) non-helical tail that contains a basic-hydrophobic membrane-binding region (BH-site), a Gly-Pro-Gln region that binds F-actin, and a Src-homology 3 (SH3) domain. The BH-site binds to membrane regions where acidic phospholipids are concentrated based solely on net negative charge, irrespective of the composition of the phospholipids, in contrast to other myosin-Is that bind preferentially to phosphatidylinositol bisphosphate (PIP2). The Gly-Pro-Gln region binds F-actin in the presence or absence of ATP unlike the ATP-sensitive actin-binding site in the motor domain. Actin waves are self-propagating, membrane-associated F-actin complexes that move from the rear to the front of Dictyostelium cells providing the actin required to advance the leading edge of motile cells and formation of pseudopodia. DMIB, CARMIL (a scaffolding protein that binds Arp2/3) and Arp2/3 (which initiates actin filament branching) are known components of actin waves. The Gerisch lab has postulated that DMIB may be involved in the association of actin waves with the plasma membrane and, through its SH3-domain, bind to CARMIL which would bind Arp2/3. We have investigated the molecular basis of the association of DMIB with actin waves by co-expressing GFP-tagged wild-type and mutant DMIB constructs with RFP-tagged lifeact in DMIB-null cells and monitoring the dynamic localization of DMIB and F-actin by fluorescence microscopy. We find that DMIB is not required for wave formation (i.e. DMIB-null cells form waves) possibly because there are two other long-tailed myosins Is in Dictyostelium. We find that both the membrane-binding BH-site and the actin-binding Gly-Pro-Gln region in the DMIB tail are required for binding DMIB to waves. The motor domain (head) of DMIB is not required for association of DMIB with actin waves but the actin-binding site in the head strengthens the association and stabilizes waves. We conclude that DMIB contributes to anchoring actin waves to the plasma membrane by the binding of the BH-site to acidic phospholipids in the plasma membrane and the binding of the Gly-Pro-Gln region to F-actin in the wave.