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

Stepanyants N, Macdonald PJ, Francy CA, Mears JA, Qi X, Ramachandran R - Mol. Biol. Cell (2015)

Bottom Line: Cardiolipin (CL) is an atypical, dimeric phospholipid essential for mitochondrial dynamics in eukaryotic cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106.

No MeSH data available.


Related in: MedlinePlus

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 (lo, NBD-DHPE–labeled, green), and fluid-phase, liquid-disordered (ld, 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 lo 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.
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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 (lo, NBD-DHPE–labeled, green), and fluid-phase, liquid-disordered (ld, 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 lo 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.

Mentions: We previously found that a high spatial density of CL (>10 mol% CL) is required for the effective self-assembly and stimulation of GTPase activity of Drp1 on synthetic liposomes composed of a ternary mixture of native CL, dioleoylphosphatidylethanolamine (DOPE), and dioleoylphosphatidylcholine (DOPC; Macdonald et al., 2014). Consistent with this observation, in independent studies (Ugarte-Uribe et al., 2014), despite discernible membrane recruitment, very little stimulation of GTPase activity was observed for Drp1 on MOM-like liposomes that contained ∼4 mol% CL (8% by weight). By contrast, the yeast Drp1 homologue Dnm1p was found to be maximally stimulated on MOM-like liposomes composed of a variety of negatively charged phospholipids but containing only ∼3 mol% CL (6% by weight; Lackner et al., 2009). Because the overall CL content of the MOM rarely exceeds 10 mol% under homeostatic conditions (van Meer et al., 2008; Horvath and Daum, 2013; Cosentino and Garcia-Saez, 2014), we first determined whether other negatively charged phospholipids function cooperatively with CL in stimulating Drp1 GTPase activity, as observed for yeast Dnm1p earlier. Surprisingly, under similar conditions (i.e., with only 3 mol% CL in MOM-like liposomes), human Drp1 was not stimulated (Figure 1A). However, when the CL content was increased to 10 mol%, MOM-like liposomes were significantly more potent than a ternary CL/DOPE/DOPC mixture in stimulating Drp1 GTPase activity (Figure 1B). These data suggested that other negatively charged phospholipids, when localized together with CL at a relatively high spatial density in the membrane bilayer, positively promote Drp1 recruitment and stimulation. In this context, we surmised that CL-enriched microdomains that putatively exist at contact sites between the MIM and MOM (Ardail et al., 1990; Sorice et al., 2009; Macdonald et al., 2014) function as “recruitment platforms” to promote stable Drp1 association and self-assembly on the mitochondrial surface. Indeed, Drp1 has been found associated with detergent-insoluble, raft-like microdomains isolated from the mitochondria of mammalian cells upon apoptotic stimulation of fission, suggesting that these sites correspond to sites of mitochondrial division (Ciarlo et al., 2010; Sorice et al., 2012).


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

Stepanyants N, Macdonald PJ, Francy CA, Mears JA, Qi X, Ramachandran R - Mol. Biol. Cell (2015)

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 (lo, NBD-DHPE–labeled, green), and fluid-phase, liquid-disordered (ld, 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 lo 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.
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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 (lo, NBD-DHPE–labeled, green), and fluid-phase, liquid-disordered (ld, 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 lo 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.
Mentions: We previously found that a high spatial density of CL (>10 mol% CL) is required for the effective self-assembly and stimulation of GTPase activity of Drp1 on synthetic liposomes composed of a ternary mixture of native CL, dioleoylphosphatidylethanolamine (DOPE), and dioleoylphosphatidylcholine (DOPC; Macdonald et al., 2014). Consistent with this observation, in independent studies (Ugarte-Uribe et al., 2014), despite discernible membrane recruitment, very little stimulation of GTPase activity was observed for Drp1 on MOM-like liposomes that contained ∼4 mol% CL (8% by weight). By contrast, the yeast Drp1 homologue Dnm1p was found to be maximally stimulated on MOM-like liposomes composed of a variety of negatively charged phospholipids but containing only ∼3 mol% CL (6% by weight; Lackner et al., 2009). Because the overall CL content of the MOM rarely exceeds 10 mol% under homeostatic conditions (van Meer et al., 2008; Horvath and Daum, 2013; Cosentino and Garcia-Saez, 2014), we first determined whether other negatively charged phospholipids function cooperatively with CL in stimulating Drp1 GTPase activity, as observed for yeast Dnm1p earlier. Surprisingly, under similar conditions (i.e., with only 3 mol% CL in MOM-like liposomes), human Drp1 was not stimulated (Figure 1A). However, when the CL content was increased to 10 mol%, MOM-like liposomes were significantly more potent than a ternary CL/DOPE/DOPC mixture in stimulating Drp1 GTPase activity (Figure 1B). These data suggested that other negatively charged phospholipids, when localized together with CL at a relatively high spatial density in the membrane bilayer, positively promote Drp1 recruitment and stimulation. In this context, we surmised that CL-enriched microdomains that putatively exist at contact sites between the MIM and MOM (Ardail et al., 1990; Sorice et al., 2009; Macdonald et al., 2014) function as “recruitment platforms” to promote stable Drp1 association and self-assembly on the mitochondrial surface. Indeed, Drp1 has been found associated with detergent-insoluble, raft-like microdomains isolated from the mitochondria of mammalian cells upon apoptotic stimulation of fission, suggesting that these sites correspond to sites of mitochondrial division (Ciarlo et al., 2010; Sorice et al., 2012).

Bottom Line: Cardiolipin (CL) is an atypical, dimeric phospholipid essential for mitochondrial dynamics in eukaryotic cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106.

No MeSH data available.


Related in: MedlinePlus