We have demonstrated that efficient membrane curvature generation by dynamin requires, in addition to its scaffolding properties, stable insertion of the relatively hydrophobic PH domain VL1 into the lipid bilayer and that this activity is critical for both membrane fission from SUPER templates in vitro and for dynamin function in endocytic CCV release in vivo.
Shallow membrane penetration utilizing a pair of adjacent, loop-residing hydrophobic residues (I533 and M534) is not unique to Dyn1 and has been demonstrated in various PH domains (Manna et al., 2007
). However, the dynamin PH domain is unlikely to be able to generate membrane curvature on its own. Previous studies have shown that the affinity of an isolated dynamin PH domain for the phosphoinositide headgroup is low, therefore necessitating dynamin oligomerization to sustain multivalent, high-avidity PH domain–membrane interactions (Klein et al., 1998
; Lemmon and Ferguson, 2000
). Indeed, in our own studies with the isolated Dyn1 PH domain site-specifically labeled at position I533C with NBD (Dyn1 PH I533C-NBD), we were unable to detect any membrane binding or insertion (data not shown). We have previously shown that middle domain mutants, Dyn1 R399A and Dyn1 R361S, defective in dynamin self-assembly also exhibit a much reduced, albeit measurable, ability to generate membrane tubules from liposomes with correspondingly reduced liposome-stimulated GTPase activity (Ramachandran et al., 2007
). These mutants are unable to mediate either tubule formation or vesicle release from SUPER templates (Pucadyil & Schmid, 2008
). Finally, like the VL1 mutants, assembly-defective Dyn1 mutants are also dominant-negative inhibitors of clathrin-mediated endocytosis in vivo (Song et al., 2004
). Together, these data demonstrate that both self-assembly and dynamin PH domain membrane insertion are necessary
for the highly efficient membrane curvature induction required for dynamin function in vivo and that neither activity alone is sufficient
We have argued that the dominant-negative phenotype caused by overexpression of the VL1 loop mutations (Dyn1 I533A or I533C) is due to their inability to efficiently generate high membrane curvature. However, in vitro analyses of the Dyn1 I533A and Dyn1 I533C mutant proteins revealed partial defects in liposome binding and liposome-stimulated GTPase activities. The following arguments support our belief that these defects per se do not account for the dominant-negative effects of these mutants on CCV release in vivo. First, the Dyn1 I533C mutation has only a mild defect in assembly-stimulated GTPase assay and liposome binding (≤50% inhibited relative to WT) compared with the I533A mutation (~90% inhibited relative to WT), yet both had equally potent dominant-negative effects. Thus the degree of inhibition did not correlate with these in vitro phenotypes. Second, the in vitro membrane binding and assembly-stimulated GTPase defects could largely be rescued on precurved membrane templates, yet these mutants were unable to mediate CCV release in vivo. Thus, we conclude that the in vivo defect in Dyn1 I533A and Dyn1 I533C lies in their inability to efficiently induce high membrane curvature at the neck of a coated pit. We suggest that the I533A and I533C substitutions when compared with uncharged, bulky hydrophobic residues in I533W and I533L, create smaller “footprints” on the membrane surface and are therefore unable to force membrane curvature or stabilize dynamin-membrane association, both of which also require coincident dynamin self-assembly. In this regard it is important to note that, unlike Dyn1 I533A, the assembly-defective Dyn1 R399A mutant cannot be rescued on precurved lipid nanotubes for either self-assembly or assembly-stimulated GTPase activity (data not shown).
At what stage in CCV formation is dynamin-induced membrane curvature required? Although there is a burst of dynamin recruitment at late stages of CCV formation, it is also present, albeit at lower levels, on newly formed CCPs and an early role for dynamin at the nascent stages of coated pit formation has been well documented (Narayanan et al., 2005
; Macia et al., 2006
; Loerke et al., 2009
). Our observation that the I533A mutant can mediate fission given a sufficiently curved, preconstricted template in vitro, yet cannot support CCV formation in vivo, coupled with our finding that invaginated CCPs accumulate in cells expressing the dominant-negative Dyn1 I533A mutant, argues strongly that the in vivo defect must be immediately upstream of neck constriction. Here, by partially inserting into the lipid bilayer, self-assembled dynamin might function to generate further localized curvature to destabilize the membrane and facilitate fission and vesicle release. Curvature-generating molecules such as epsin, which are sufficient to generate budded CCPs from liposomes and planar lipid bilayers (Takei et al., 1998
; Ford et al., 2002
), together with BAR domain-containing dynamin binding partners, such as sorting nexin 9 and endophilin, could create the neck of the emerging bud and direct localized assembly and further curvature generation by dynamin. Thus, as suggested previously (Yoshida et al., 2004
; Ramachandran and Schmid, 2008
), endocytic accessory factors are likely to function in concert with dynamin at the late stages of coated vesicle formation as curvature generators and/or stabilizers to enhance the potency of dynamin in membrane fission. Importantly, our findings establish that efficient curvature generation by dynamin insertion at the necks of deeply invaginated coated pits plays a critical role in membrane fission leading to endocytic CCV formation in vivo.
While this manuscript was under review, a recent article concluded that the dynamin PH domain might function to cluster PIP2
at CCP necks to create a lipid gradient essential for fission (Bethoney et al., 2009
). Whereas we cannot rule out PH domain–mediated PIP2
clustering during dynamin self-assembly as a passive contributor to membrane fission, our data instead demonstrate an active mechanical role for dynamin via VL1-membrane insertion in effecting vesicle scission. The recent observation that dynamin can catalyze nonleaky fission of membrane tubules lacking PIP2
but containing PS (Bashkirov et al., 2008
) is also consistent with such a membrane-active mechanism for dynamin.