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The neuronal dynamin1 functions in the release of synaptic vesicles by orchestrating the process of GTPase-dependent membrane fission. Dynamin1 associates with the plasma membrane-localized phosphatidylinositol-4,5-bisphosphate (PIP 2 ) through the centrally located pleckstrin homology domain (PHD). The PHD is dispensable as fission (in model membranes) can be managed, even when the PHD-PIP 2 interaction is replaced by a generic polyhistidine- or polylysine-lipid interaction. However, the absence of the PHD renders a dramatic dampening of the rate of fission. These observations suggest that the PHD-PIP 2 -containing membrane interaction could have evolved to expedite fission to fulfill the requirement of rapid kinetics of synaptic vesicle recycling. Here, we use a suite of multiscale modeling approaches to explore PHD-membrane interactions. Our results reveal that 1) the binding of PHD to PIP 2 -containing membranes modulates the lipids toward fission-favoring conformations and softens the membrane, and 2) PHD associates with membrane in multiple orientations using variable loops as pivots. We identify a new loop (VL4), which acts as an auxiliary pivot and modulates the orientation flexibility of PHD on the membrane-a mechanism that we believe may be important for high-fidelity dynamin collar assembly. Together, these insights provide a molecular-level understanding of the catalytic role of PHD in dynamin-mediated membrane fission.

Myosin 1s have critical roles in linking membranes to the actin cytoskeleton via direct binding to acidic lipids. Lipid binding may occur through PIP3/PIP2-specific PH domains or nonspecific ionic interactions involving basic-hydrophobic (BH) sites but the mechanism of myosin 1s distinctive lipid targeting is poorly understood.  Now we show that PH domains occur in all Dictyostelium myosin 1s and that the BH sites of Myo1A, B, C, D, and F are in conserved positions near the β3/β4 loops of their PH domains. In spite of these shared lipid-binding sites, we observe significant differences in myosin 1s highly dynamic localizations. All myosin 1s except Myo1A are present in macropinocytic structures but only Myo1B and Myo1C are enriched at the edges of macropinocytic cups and associate with the actin in actin waves.  In contrast, Myo1D, E, and F are enclosed by the actin wave.  Mutations of BH sites affect localization of all Dictyostelium myosin 1s. Notably, mutation of the BH site located within the PH domains of PIP3-specific Myo1D and Myo1F completely eradicates membrane binding. Thus, BH sites are important determinants of motor targeting and may have a similar role in the localization of other myosin 1s.

Classical dynamins bind the plasma membrane-localized phosphatidylinositol-4,5-bisphosphate using the pleckstrin-homology domain (PHD) and engage in rapid membrane fission during synaptic vesicle recycling. This domain is conspicuously absent among extant bacterial and mitochondrial dynamins, however, where loop regions manage membrane recruitment. Inspired by the core design of bacterial and mitochondrial dynamins, we reengineered the classical dynamin by replacing its PHD with a polyhistidine or polylysine linker. Remarkably, when recruited via chelator or anionic lipids, respectively, the reengineered dynamin displayed the capacity to constrict and sever membrane tubes. However, when analyzed at single-event resolution, the tube-severing process displayed long-lived, highly constricted prefission intermediates that contributed to 10-fold reduction in bulk rates of membrane fission. Our results indicate that the PHD acts as a catalyst in dynamin-induced membrane fission and rationalize its adoption to meet the physiologic requirement of a fast-acting membrane fission apparatus., (© 2017 Dar and Pucadyil. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)