Geometric catalysis of membrane fission driven by flexible dynamin rings

Abstract

Biological membrane fission requires protein-driven stress. The guanosine triphosphatase (GTPase) dynamin builds up membrane stress by polymerizing into a helical collar that constricts the neck of budding vesicles. How this curvature stress mediates nonleaky membrane remodeling is actively debated. Using lipid nanotubes as substrates to directly measure geometric intermediates of the fission pathway, we found that GTP hydrolysis limits dynamin polymerization into short, metastable collars that are optimal for fission. Collars as short as two rungs translated radial constriction to reversible hemifission via membrane wedging of the pleckstrin homology domains (PHDs) of dynamin. Modeling revealed that tilting of the PHDs to conform with membrane deformations creates the low-energy pathway for hemifission. This local coordination of dynamin and lipids suggests how membranes can be remodeled in cells.

Fig. 1

Metastable scaffolds formed by wild type dynamin (WT) under conditions of constant GTP turnover. A. In the absence of GTP, dynamin polymers (dyn-Alexa488 fluorescence, green) constrict lipid nanotubes (Rh-DOPE fluorescence, red). B. Membrane (red) and lumenal (3kDa dextran fluorescein, blue) markers show similar axial profiles marking constricted zones. C. Kymograph showing development of the dynamin scaffolds (dark regions) constricting lipid NT in the presence of 1 mM GTP. The selected profiles (1–3) illustrate formation of initial isolated scaffolds (2) and membrane bulging at high protein coverage (3). D. Kymographs showing the steady-state constriction of the NT without and with GTP as indicated. The variable lengths (L) of the constricted parts (blue-violet) is measured as shown. E. Variability of the length (L, see D) of constricted parts (normalized to mean length for each scaffold (L_n)) in the steady-state. F. Distribution of pixel intensities for kymographs showing the development of NT constriction by dynamin (as in C). Two peaks, corresponding to bare and constricted parts, are detected both in the absence and presence of GTP. G, H. Rapid disassembly of the dynamin scaffold (green) upon fission, the frame sequence and the graph illustrate nucleation (white arrowhead) followed by slow accumulation and fast disappearance of the dyn-Alexa488 fluorescence in the NT (pulled from a GUV seen above) region. The insert shows images illustrating the moment of fission (100ms between frames). I. Without GTP the dynamin scaffold prevents shortening of the NT when a pipette holding the NT end approaches the GUV.

Fig. 2

Short metastable dynamin scaffolds catalyze hemifission. A-C. Patterns of slow NT constriction (monotonic (A), stepwise (B) and reversible (C)) are observed by measuring the conductance of lipid NTs. Conductance is normalized to the value prior to dynamin addition. The cartoons in A and B illustrate the growth of the dynamin scaffold on NT; the initial (R₀) and the final (R_Dyn) radii of the nanotube are determined from the NT conductance using Ohm’s law, assuming that at the final stage NT is uniformly covered by dynamin; the length of the dynamin scaffold in the intervening states (L_s) is determined from changes in G_n assuming that each scaffold constricts NT to the same radius R_dyn. Lower (0.2 mM GTP) and upper (1 mM GTP) panels in C show that GTP accelerates disassembly of the dynamin scaffold. D. Characteristic examples of the L_s behavior without (left) and with (right) GTP. E. Cumulative distribution functions of the length of the dynamin scaffold recalculated from the conductance steps (ΔG_n) measured as shown in B (- GTP, red arrow) and C (+ GTP, red arrows). Fission corresponds to the conductance of the NT prior to fission as shown in C (ΔG_n^fission, blue arrow). The insert shows the beginning of the main graph. F. Reversible changes of the NT conductance (“flicker”) preceding complete fission, red line illustrates the “open” state; insert shows an example of the short-living “closed” state.

Fig. 3

Membrane wedging plays a critical role in the hemifission catalysis. A. Constriction of a long NT, seen as a decrease in conductance (G_n, normalized to the value prior to dynamin addition) by WT dynamin (pink), I533A dynamin (red) and I533A in the presence of GTP (blue). B. The behavior of G_n upon I533A addition to short NTs with (blue) and without (red) GTP. C. The mean length of protein scaffolds formed by WT (black) and I533A (blue) dynamin in the presence of GTP is similar (the length is measured from ΔG_n as in Fig. 2G). D. Stationary NT constriction (measured in G_n units) produced by WT and I533A dynamin under conditions indicated. E. Frame sequence illustrating constriction and fission of NT pulled from a GUV by I533A, with 5mol% PI(4, 5)P2. Membrane fission occurs within 20s (3 trials, (32)), while at lower PIP2 concentrations (F) tubes remain stable at 100s time scale (1mol%PIP2 n=10, 2mol%PIP2 n=4).

Fig. 4

Role of membrane wedging by pleckstrin homology domain (PHD) of dynamin in the catalysis of fission. A. Energy diagram for dynamin-lipid complex illustrating the hemifission catalysis. Dynamin polymerization leads to formation of a highly stressed “reactant” state (2) that remains stable in the absence of GTP. GTP hydrolysis causes partial “melting” of dynamin rings resulting in a new metastable intermediate around the constricted neck (3) where the PHDs on the rings can tilt following changes in the geometry of lipid bilayer until the hemifission stage (4) is achieved. The PHD mobility is characterized by the tilt angle (α) between dynamin subunits assembled in adjacent rings. Although each PHD can tilt independently, we consider the mirror-symmetric tilt as the simplest approximation. Complete fission is stochastically coupled to disassembly of the metastable dynamin scaffold and is accelerated by membrane tension. B. The membrane wedging module of dynamin is formed by adjacent PHDs interacting with PI(4,5)P2 lipids (red) and inserting small hydrophobic regions into the lipid monolayer; the membrane wedging is approximated by a shallow hydrophobic inclusion (H_i) imposing stresses on the neighboring lipids (purple, see also Fig. S3). C. Dependence of the energy barrier (ΔW) between the constricted neck (3) and hemifission intermediate (4) on α (red) and H_i (black). The black curve was calculated assuming α as a free parameter, the energy barrier for H_i=0.7nm is indicated by the dashed line. The red curve was calculated using fixed H_i=0.7nm.