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.
