Real-time detection reveals that effectors couple dynamin's GTP-dependent conformational changes to the membrane - PubMed (original) (raw)

Real-time detection reveals that effectors couple dynamin's GTP-dependent conformational changes to the membrane

Rajesh Ramachandran et al. EMBO J. 2008.

Abstract

The GTPase dynamin is a mechanochemical enzyme involved in membrane fission, but the molecular nature of its membrane interactions and their regulation by guanine nucleotides and protein effectors remain poorly characterized. Using site-directed fluorescence labeling and several independent fluorescence spectroscopic techniques, we have developed robust assays for the detection and real-time monitoring of dynamin-membrane and dynamin-dynamin interactions. We show that dynamin interacts preferentially with highly curved, PIP2-dense membranes and inserts partially into the lipid bilayer. Our kinetic measurements further reveal that cycles of GTP binding and hydrolysis elicit major conformational rearrangements in self-assembled dynamin that favor dynamin-membrane association and dissociation, respectively. Sorting nexin 9, an abundant dynamin partner, transiently stabilizes dynamin on the membrane at the onset of stimulated GTP hydrolysis and may function to couple dynamin's mechanochemical conformational changes to membrane destabilization. Amphiphysin I has the opposite effect. Thus, dynamin's mechanochemical properties on a membrane surface are dynamically regulated by its GTPase cycle and major binding partners.

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Figures

Figure 1

A fluorescence-based assay for dynamin–membrane interactions. (A) Schematic illustration of dynamin domain arrangement and ribbon representation of the human dynamin 1 PH domain crystal structure (PDB ID: 1dyn). GED, GTPase effector domain; PH, pleckstrin homology; PRD, proline and arginine-rich domain. Positions of the Cys residues replaced with Ser are indicated and variable loops 1–3 (VL1–3) in the PH domain are labeled. The location of I533 in VL1 that was replaced with a Cys (I533C) in this study is shown in stick representation. (B) Representative NBD emission spectra for 0.5 μM NBD-dynamin before and after incubation with either 15 mol% PIP2-containing liposomes or 100 mol% DOPC liposomes (150 μM total lipid). (C) Time-dependent emission intensity profile for 0.1 μM NBD-dynamin before and after addition of 15 mol% PIP2-containing liposomes (30 μM total lipid). For this and all subsequent figures, unless otherwise stated, _F_0 is the initial intensity of NBD-dynamin before addition of lipid (at time=0) and F is the intensity at time t. Representative data from at least three independent experiments are indicated as F/_F_0 and plotted versus time. (D) Quenching of NBD fluorescence by nitroxide moieties located at different depths in the membrane bilayer. Representative NBD emission spectra for 0.5 μM NBD-dynamin before and after incubation with either 15 mol% PIP2-containing liposomes (no quencher) or with 15 mol% PIP2-containing liposomes that also contain 10 mol% of the indicated nitroxide-labeled phospholipid (150 μM total lipid).

Figure 2

Dynamin–membrane interaction is highly sensitive to PIP2 spatial density and membrane curvature. (A) Binding of 0.1 μM NBD-dynamin to increasing concentration of liposomes containing either 2 mol% (○) or 15 mol% PIP2 (•). The average membrane-dependent intensity increase is indicated as F/_F_0. (B) Binding of 0.1 μM NBD-dynamin to a fixed concentration of liposomes (30 μM total lipid) with increasing content of PIP2 (0–15 mol%). (C) Normalized time-dependent NBD emission intensity profiles for 0.1 μM NBD-dynamin upon addition of either 2 mol% (○) or 15 mol% (•) PIP2-containing liposomes (30 μM total lipid). To allow direct comparison of the rate of change of NBD fluorescence, each intensity profile was normalized to _F_f, the final intensity at _t_=17 min after liposome addition, and plotted as the total fractional intensity change as a function of time. Data were best fit to either a one-phase (for 15 mol% PIP2) or a two-phase (for 2 mol% PIP2) association model as described in Supplementary data to obtain the indicated rate constants. (Inset) Binding of NBD-dynamin to 15 mol% PIP2-containing liposomes is plotted against the log of time in minutes. (D) Binding of 0.1 μM NBD-dynamin to 15 mol% PIP2-containing liposomes (30 μM total lipid) of varying diameter. The average membrane-dependent intensity increase of three independent experiments is indicated as F/_F_0.

Figure 3

Guanine nucleotides regulate dynamin–membrane interactions. (A) Representative time-dependent emission-intensity profiles for 0.1 μM NBD-dynamin before and after sequential additions (see arrows) of 15 mol% PIP2-containing liposomes (30 μM total lipid) at 3 min and the indicated nucleotides (1 mM final) at 6 min; GMPPCP (•), GDP (⋄), GTP (○). (Insets) Sedimentation analysis of 0.5 μM NBD-dynamin after incubation for 20 min at 25°C with 15 mol% PIP2-containing liposomes (150 μM total lipid) and the indicated nucleotide (1 mM final) was performed as described under Methods. The supernatant (S) and pellet (P) fractions were subjected to SDS–PAGE and visualized by Coomassie blue staining. See Supplementary Figure 3C for quantification of data from three independent experiments. (B) Representative time-dependent emission-intensity profiles for 0.1 μM NBD-dynamin were acquired as in (A) except that the indicated nucleotides (1 mM final) now were added before (at 3 min) the addition of PIP2-containing liposomes.

Figure 4

GTP hydrolysis triggers a major conformational rearrangement in membrane-associated dynamin. (A) Representative time-dependent emission-intensity profiles for 0.1 μM BODIPY-dynamin upon sequential additions of 15 mol% PIP2-containing liposomes (30 μM total lipid) at 3 min and the indicated nucleotides (1 mM final) at 6 min. (B) Same as in (A) except using 0.1 μM of either the lipid binding-defective BODIPY-DynK535A (only liposomes were added) or BODIPY-Dyn1S45N, a dynamin mutant defective in GTP binding. (C) Direct comparison of the rate of dynamin–membrane dissociation and the rate of dynamin disassembly. Profiles for NBD emission-intensity decrease (dynamin–membrane dissociation; Figure 3A) and BODIPY emission-intensity increase (dynamin disassembly; (A)) upon addition of 1 mM GTP were normalized and plotted as the total fractional intensity change as a function of time as in Figure 2C. Rate constants for dynamin–membrane dissociation and dynamin disassembly were determined as described in Supplementary data.

Figure 5

SNX9 stabilizes dynamin–membrane interactions. (A) Representative time-dependent emission-intensity profiles for 0.05 μM NBD-dynamin before and after sequential additions (arrows) of 15 mol% PIP2-containing liposomes (15 μM total lipid) and 0.05 μM SNX9 (○) or vice versa (•), and upon addition of GTP (1 mM final; arrow). (B) SNX9 SH3 domain is necessary but not sufficient for enhanced dynamin–membrane recruitment. Experiments were performed as in (A), except using the SNX9 SH3 domain alone or the SNX9ΔSH3 truncation mutant as indicated. (C) Both PX and BAR domains are required for SNX9-mediated stabilization of dynamin–membrane interactions. Same as in (A) except that SNX9 mutants defective either in PX domain (RYK; □) or BAR domain (mutBAR2; ⋄) functions or both (RYK-mutBAR2; ○) as described by Yarar et al (2007) were used and compared to WT SNX9 (•). For these experiments, 0.4 μm diameter liposomes containing 62.5 mol% DOPC, 15 mol% DOPE, 15 mol% DOPS and 7.5 mol% PIP2 were used. (D, E). To directly compare the rates of dynamin–membrane dissociation upon addition of either GTP (D) or GDP (E) in the presence or absence of SNX9, each intensity profile was normalized and plotted as the total fractional intensity change as a function of time as in Figure 2C. Rate constants for dissociation in the presence of GTP (D) or GDP (E) in the presence or absence of SNX9 were determined as described under Supplementary data.

Figure 6

Amphiphysin destabilizes dynamin–membrane interactions. Representative time-dependent emission-intensity profiles for 0.05 μM NBD-dynamin before and after sequential additions of 15 mol% PIP2-containing liposomes (15 μM total lipid) and 0.05 μM AmphI (○) or vice versa (•), and upon addition of GTP (1 mM final).

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Real-time detection reveals that effectors couple dynamin's GTP-dependent conformational changes to the membrane - PubMed (original) (raw)