DMIB is a typical long-tail class-I myosin with a short non-helical tail that contains a membrane-binding region, an ATP-insensitive actin-binding site (in addition to the ATP-sensitive actin-binding site in its motor domain) and a Src-homology 3 (SH3) domain. A short region of basic-hydrophobic amino acids (BH-site) within the membrane-binding region-site is responsible for the association of DMIB with the plasma membrane. 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). 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 or formation of pseudopodia, for example to form phagocytic cups. 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 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 their dynamic localization by TIRF microscopy. We find that DMIB is not required for wave formation, i.e. DMOB-null cells form waves, possibly because there are two other long-tailed myosins Is in Dictyostelium. We find that the BH-site in the DMIB tail is essential for binding DMIB to waves, the actin-binding site in the motor domain contributes to wave-binding, but neither the actin-binding site in the tail or the SH3-domain in the tail make any contribution to the association of DMIB with actin waves

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National Heart, Lung, and Blood Institute
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