Cardiolipin's propensity for phase transition and its reorganization by dynamin-related protein 1 form a basis for mitochondrial membrane fission

Abstract

Cardiolipin (CL) is an atypical, dimeric phospholipid essential for mitochondrial dynamics in eukaryotic cells. Dynamin-related protein 1 (Drp1), a cytosolic member of the dynamin superfamily of large GTPases, interacts with CL and functions to sustain the balance of mitochondrial division and fusion by catalyzing mitochondrial fission. Although recent studies have indicated a role for CL in stimulating Drp1 self-assembly and GTPase activity at the membrane surface, the mechanism by which CL functions in membrane fission, if at all, remains unclear. Here, using a variety of fluorescence spectroscopic and imaging approaches together with model membranes, we demonstrate that Drp1 and CL function cooperatively in effecting membrane constriction toward fission in three distinct steps. These involve 1) the preferential association of Drp1 with CL localized at a high spatial density in the membrane bilayer, 2) the reorganization of unconstrained, fluid-phase CL molecules in concert with Drp1 self-assembly, and 3) the increased propensity of CL to transition from a lamellar, bilayer arrangement to an inverted hexagonal, nonbilayer configuration in the presence of Drp1 and GTP, resulting in the creation of localized membrane constrictions that are primed for fission. Thus we propose that Drp1 and CL function in concert to catalyze mitochondrial division.

FIGURE 1:

Drp1 preferentially remodels membranes containing a high spatial density of fluid-phase CL. (A–D) Stimulated GTPase activity of Drp1 WT (0.5 μM final) preassembled on liposomes of defined lipid composition (150 μM total lipid) containing varying mole fractions (mol%) of native CL plotted as specific activity (min⁻¹) ± SD (n = 3). (E) Confocal fluorescence images of surface-immobilized Rh-DOPE–labeled GUVs containing 10 mol% native CL (red; left) in the presence of BODIPY-FL-labeled Drp1 WT (0.5 μM protein final; green; middle). Right, merged images. (F) Confocal fluorescence images of GUVs phase-separated into raft-phase, liquid-ordered (l_o, NBD-DHPE–labeled, green), and fluid-phase, liquid-disordered (l_d, Rh-DOPE–labeled, red) membrane regions before (left) and after (right) addition of unlabeled Drp1 WT (0.5 μM final). Only merged images are shown. Arrow points to Drp1-generated membrane tubules originated from Rh-DOPE–labeled, fluid-phase membrane regions. (G) Same as F, but containing unlabeled, dark, raft-phase l_o regions before (left) and after (right) addition of BODIPY-FL–labeled Drp1 WT. Arrow points to Drp1-decorated membrane tubules originating from fluid-phase membrane regions. Only merged images are shown. Scale bar, 5 μm.

FIGURE 2:

Drp1 does not remodel membranes containing constrained, raft-phase CL. (A) Confocal fluorescence images of phase-separated, TMCL-containing GUVs containing coexisting Rh-DOPE–labeled fluid (red) and unlabeled, raft-like (dark; arrow) membrane regions before (left) and after (right) addition of BODIPY-FL–labeled Drp1 WT (0.5 μM protein final). (B) Stimulated GTPase activity of Drp1 WT (0.5 μM final) preassembled on liposomes of defined lipid composition containing 25 mol% of either native CL or TMCL (150 μM total lipid) plotted as specific activity (min⁻¹) ± SD (n = 3). (C) Same as A, but with EggPC replacing both DOPC and DOPE. (D) Same as B, but on liposomes composed of a binary mixture of either DOPC or POPC and native CL or TMCL (25 mol% CL species) as indicated. (E) Schematic illustration of BODIPY-FL–Drp1−Rh-DOPE FRET on liposomes. (F) FRET-sensitized increase in rhodamine emission intensity as a function of CL concentration (in mol%) plotted as F/F₀, where F₀ and F represent the initial and at-time-t rhodamine emission intensities upon BODIPY–FL-Drp1 WT (0.1 μM final) addition to Rh-DOPE–labeled liposomes (10 μM total lipid). (G) The final extents (top) and the initial rates (bottom) of the FRET-sensitized rhodamine emission intensity increase upon stable BODIPY-FL–labeled Drp1 WT association plotted as a function of CL concentration. F_f represents the final emission intensity, and k₁ represents the major, apparent rate constant for the emission intensity increase. (H) FRET-sensitized increase in rhodamine emission intensity upon BODIPY-FL–Drp1 WT addition to TMCL- or native-CL–containing liposomes plotted as in G. Buffer contained 2 mM MgCl₂, which markedly reduces the affinity of Drp1 for CL-containing membranes as described in Materials and Methods. Scale bar, 5 μm.

FIGURE 3:

Membrane fluidity influences Drp1-induced CL reorganization. (A) Relative FRET-sensitized increase in Rh-DOPE emission intensity upon stable membrane association of BODIPY-FL–labeled Drp1 WT (0.1 μM final) on membrane templates of varying average diameter (liposomes) or fluidity (LTs; 10 μM total lipid) plotted as F_f/F₀, where F_f and F₀ represent the final and initial emission intensities, respectively. (B) Comparison of the stimulated GTPase activities of Drp1 WT (0.5 μM final) on the various membrane templates used (150 μM total lipid; n = 3). The data are plotted as percentage of maximum activity as indicated above. (C) Same as B, but with 25 mol% TMCL instead of native CL. (D) Same as A, but comparing native CL- and TMCL-containing membrane templates.

FIGURE 4:

Drp1 reorganizes CL into condensed membrane regions. (A) Emission spectra of TopFluor-CL in native CL-containing liposomes (150 μM total lipid) before and after addition of Drp1 WT (0.5 μM final). (B) Extent of TopFluor-CL self-quenching as a function of Drp1 WT or mutant concentration is plotted as percent quenching. (C–F) Confocal fluorescence images of TopFluor-CL distribution in GUVs before (C) and after addition of 0.5 μM (final) Drp1 WT (D) or mutants (E, F). Slender arrows point to membrane tubules, and block arrows point to highly intense, condensed membrane regions. Scale bar, 5 μm.

FIGURE 5:

Drp1 induces apparent GTP- and B-insert–dependent CL phase transition. (A) NBD emission intensity change in liposomes containing either 25 mol% native CL or TMCL in the presence of DOPC and DOPE (50 μM total lipid) upon addition of Drp1 WT (0.5 μM final). Relative extent of lamellar-to-H_II membrane phase transition induced by Ca²⁺ ions (5 mM CaCl₂ final; Supplemental Figure S2A) is shown for comparison. (B) NBD emission intensity change upon addition of Drp1 WT (0.5 μM final) to 25 mol% native CL-containing liposomes (50 μM total lipid) in the constant presence of various nucleotides (nucl; 1 mM final). (C) NBD emission intensity increase upon addition of various nucleotides (1 mM final) to Drp1 WT (0.5 μM final) preassembled on 25 mol% native CL-containing liposomes (50 μM total lipid) before nucleotide addition. (D, E) Comparison of the extent of NBD emission intensity increase observed for Drp1 WT on 25 mol% native CL-containing liposomes (Lipo) in the constant presence of nucleotides as in B with that of GTPase domain mutants (D) or B-insert mutants (E).

FIGURE 6:

GTP-dependent apparent CL phase transition causes membrane constriction. (A) Time course of Rh-DOPE–labeled GUV membrane morphology changes upon BODIPY-FL–labeled Drp1 WT (0.5 μM final) addition in the constant presence of GTP (1 mM). (B) Same as A, but in the constant presence of GMP-PCP. (C) Endpoint confocal fluorescence image of Drp1 WT-induced GUV membrane remodeling as before but in the constant presence of GDP. (D) same as C, but for various Drp1 mutants in the constant presence of GTP. Slender arrow in C points to Drp1-decorated membrane tubules. In the D panels, slender arrows point to constricted “membrane buds,” whereas triangular, block arrows point to condensed regions of the membrane that presumably mark sites of extensive CL lamellar-to-H_II phase transition. Scale bar, 5 μm.

FIGURE 7:

Drp1–CL interactions generate membrane constriction for fission both in vitro and in vivo. (A) Representative negative-stain EM images of Drp1-decorated membrane tubules displaying time-dependent membrane morphology changes upon GTP hydrolysis. Arrows point to highly localized, narrowly constricted regions of the membrane tubule that are predisposed to fission. Far right, inset, enlarged portion of the image. Scale bars, 200 nm. (B) Confocal fluorescence images of mitochondrial morphology (left; green) in Drp1 KO MEFs expressing either Myc-tagged Drp1 WT or Drp1 4KA (red; grayscaled for clarity; right). Arrows point to either fragmented mitochondria (in the case of Drp1 WT) or hyperfused mitochondrial networks (in the case of Drp1 4KA) localized to the perinuclear region of the cell. (C) Quantification of mitochondrial fragmentation in Drp1 KO MEFs cells expressing either Myc-tagged Drp1 WT or Drp1 4KA. (D) Representative Western blot showing expression levels of Myc-tagged Drp1 WT and Drp1 4KA in transfected Drp1 KO MEFs. Untransfected cells (Ctrl) served as negative control, and actin was used as loading control for total protein. (E) Model of Drp1-induced CL reorganization and phase transition at the MOM surface that presumably leads to membrane fission and mitochondrial division. H_II phase, inverted, hexagonal phase; MIM, mitochondrial inner membrane; MOM, mitochondrial outer membrane.