Abstract
Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (Schmid and Frolov, 2011; Ferguson and De Camilli, 2012). Dynamin fission reaction is strictly dependent on GTP hydrolysis, but how fission is mediated is still debated (Antonny et al., 2016): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We show that in fibroblasts, dynamin GTP hydrolysis occurs as stochastic bursts, which are randomly distributed relatively to the peak of dynamin assembly. Thus, dynamin disassembly is not coupled to GTPase activity, supporting that the GTP energy is primarily spent in constriction.
Highlights
There are three mammalian variants of dynamin that share 80% sequence identity
Dynamins are constituted of 5 domains: the GTPase (G) domain, the stalk – a rigid coiled-coil, which mediates assembly into helical polymers through 3 structurally conserved interfaces (Faelber et al, 2011; Ford et al, 2011; Reubold et al, 2015), the Bundle Signaling Element (BSE), which connects the GTPase domain to the stalk (Chappie et al, 2010; Chappie et al, 2011), a Pleckstrin Homology (PH) domain, which binds to phosphatidylinositol 4,5-bisphosphate (PI (4,5)P2) (Salim et al, 1996) and the proline-rich domain (PRD), which binds partners such as Bin Amphiphysin Rvs (BAR)-domain proteins (Raimondi et al, 2011)
Pull-downs were performed with dynamin assembled onto SUPported bilayers with Excess membrane Reservoir (SUPER) templates (Pucadyil and Schmid, 2008; Neumann et al, 2013) adapted for pull-down assays: magnetic silica beads were coated with lipid membranes, from which dynamin-coated membrane tubules can be formed, as dynamin binding is lost when membranes are dissolved with saponin (Figure 1—figure supplement 2)
Summary
There are three mammalian variants of dynamin that share 80% sequence identity. Dynamin can assemble into a helical coil around membrane necks and tubes. The constriction of the coil is required for fission and the most constricted state of dynamin is observed in presence of GTP (Antonny et al, 2016). When GTP is added to pre-assembled dynamin tubes in vitro, rapid torsion and constriction are observed (Roux et al, 2006). When GTP is added along with dynamin to membranes in vitro, highly constricted helices are observed, suggesting that GTP-bound dynamin assembles directly into a highly constricted state (Sundborger et al, 2014).
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