Flippase-Mediated Hybrid Vesicle Division

Abstract

The assembly of synthetic systems with the ability for protein-mediated division remains a challenge in bottom-up synthetic biology. Here, the reconstitution of an active Drs2p-Cdc50p lipid flippase in polymer lipid hybrid vesicles (HVs) made from phospholipids and 1 or 2.5 mol% amphiphilic block copolymers, with poly(carboxyethyl acrylate) or poly(6-O-methacryloyl-d-galactopyranose) as the hydrophilic extension and either cholesteryl methacrylate or butyl methacrylate or combinations thereof as the hydrophobic blocks is demonstrated. The reconstitution of Drs2p-Cdc50p in HVs flip 2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) lipids from the inner to the outer leaflet, leading to transmembrane asymmetry. Importantly, the chemical nature of the hydrophobic block in the amphiphilic block copolymers used to assemble the HVs is crucial to support changes in the spontaneous curvature of the bilayers due to translocation of DOPS lipids that results in HV constriction and division. Taken together, this effort is a step forward in imitating cell division in synthetic assemblies toward potentially bottom-up assembled self-replicating units.

Keywords: hybrid vesicle; lipid flippase; membrane constriction; transmembrane asymmetry; vesicle division.

Scheme 1

a) Cartoon showing HVs with reconstituted Flippase (HVX_Flip) that undergoes constriction and division when exposed to ATP due to the translocation (flipping) of the DOPS lipids. b) i) Schematic illustration of three different amphiphilic block copolymers (BCPs), with different hydrophobic blocks and either poly(carboxyethyl acrylate) or poly(6‐O‐methacryloyl‐d‐galactopyranose) hydrophilic extension. ii) The hydrophobic block in poly(cholesteryl methacrylate)‐block‐poly(2‐carboxyethyl acrylate) (BCP1) is poly(cholesteryl methacrylate), poly(cholesteryl methacrylate‐co‐butyl methacrylate) in poly(cholesteryl methacrylate‐co‐butyl methacrylate)‐block‐poly(2‐carboxyethyl acrylate) (BCP2) as well as poly(cholesteryl methacrylate‐co‐butyl methacrylate)‐block‐poly(6‐O‐methacryloyl‐D‐galactopyranose) (BCP4), and poly(butyl methacrylate) in poly(butyl methacrylate)‐block‐poly(2‐carboxyethyl acrylate) (BCP3). iii) BCPX can be employed to assemble small or giant hybrid vesicles (sHVX or GHVX) together with phospholipids DOPS and 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) as well as1,2‐dioleoyl‐sn‐glycero‐3‐phospho‐(1′‐myo‐inositol‐4′‐phosphate) (PI4P) for reconstitution with Flippase. iv) Illustration of the Flippase activity in the hybrid membrane using DOPS as substrate.

Figure 1

Flippase reconstitution in sHVXs. a) Illustration showing the Flippase reconstitution process in sHVXs. Representative negative‐stain TEM (top) and cryoEM (bottom) images of b) sHV1, c) sHV2, and d) sHV3 only exposed to CHAPS, sHVX_C (left) or exposed to CHAPS to reconstituted Flippase, sHVX_Flip (right). Insets in the upper right corners show a 2.5× zoom (scale bar: 500 nm (top)/50 nm (bottom)).

Figure 2

a) i) ATP consumption of sHVX_Flip in comparison to sHVX_C. sHVX_Flip without ATP addition did not give any signal. ATP consumption of sHV2_Flip ii) without PI4P, and iii) without DOPS lipids. The data were normalized to the value of fluorescence intensity of 1 mM ATP (final concentration) and presented as normalized fluorescence intensity (nFI). b) Repeated ATP consumption of sHVX_Flip over 3 h. The arrows indicate the ATP addition (n = 3; the data are expressed as mean ± standard deviation (SD)).

Figure 3

a) Schematic illustration showing the ATP‐dependent DOPS lipid redistribution to the outer leaflet in sHVX_Flip followed by the immobilization of Annexin^F. Representative super‐resolution images of b) sHV1, c) sHV2, and d) sHV3, (i) sHVX_C − ATP, ii) sHVX_C + ATP, iii) sHVX_Flip − ATP, and iv) sHVX_Flip + ATP (orange: Annexin^F; scale bar: 5 µm)), to and v) the corresponding pixel quantification of Annexin^F expressed as Annexin^F area in %, indicating the area above the threshold (n = 2; the data are expressed as mean ± SD * p < 0.1; ** p < 0.05, one‐way analysis of variance (ANOVA) with Šídák's multiple‐comparisons test, the * above a plot indicates a significant difference from all other groups).

Figure 4

Flippase reconstitution in GHVs. i) Representative overview CLSM images of a) GHV2_Flip, b) GHV3_Flip, c) GHV4_Flip, and d) $\text{GHV}{\mathrm{4}}_{\mathrm{Flip}}^{2.5}$ (green: Oregon Green‐labeled BCPX, red: Rho‐PE, blue: Flippase^F) (scale bar: 20 µm). CLSM images of a ii) representative GHVX_Flip vesicle and a iii) representative lipid only based giant vesicle from the same population split in images with the signal originating from Rho‐PE (top), ^OGBCPX (middle), and Flippase^F (bottom). iv) ATP consumption of GHVX_Flip and pristine GHVX_C in comparison to GHVX_Flip in the absence of ATP (n = 3; the data are expressed as mean ± SD).

Figure 5

a) Schematic illustration of the assessment of the transmembrane distribution of NBD‐PS in GHV2_Flip when preincubated with ATP before exposure to dithionite. b) Representative CLSM images of i) GHV2_Flip + ATP, ii) GHV2_Flip − ATP, iii) GHV2_C + ATP, and iv) GHV2_C ‐ ATP. c) Whisker plot of the fluorescence intensity of the membrane of GHV2_c or GHV2_Flip before (black) and after (gray) dithionite exposure depending on the ATP preincubation (green: NBD‐PS; scale bar: 10 µm; n = 2, N = 400).

Figure 6

Transmembrane distribution of DOPS lipids in GHV2_Flip. a) Schematic illustration showing the ATP‐dependent DOPS lipid redistribution to the outer leaflet in GHV2_Flip followed by the binding of Annexin^F. b) i) Representative CLSM images and ii) the corresponding whisker plot of the vesicles fluorescence intensities of GHV2_C ‐ ATP, GHV2_C + ATP, GHV2_Flip − ATP, and GHV2_Flip + ATP after incubation with Annexin^F. iii) Pie charts showing the ATP‐dependent distribution of constricted (“snowman‐” like) and spherical GHV2_C and GHV2_Flip observed in CLSM images. c) i) Representative CLSM and ii) the corresponding whisker plot of the vesicles fluorescence intensities of GHV3_C ‐ ATP, GHV3_C + ATP, GHV3_Flip − ATP, and gHV3_Flip + ATP. d) i) Representative CLSM images and ii) the corresponding whisker plot of the vesicles fluorescence intensities of GHV4_C ‐ATP, GHV4_C + ATP, GHV4_Flip − ATP, and GHV4_Flip + ATP. e) i) Representative CLSM images and ii) the corresponding whisker plot of the vesicles fluorescence intensities of $\text{GHV}{\mathrm{4}}_{\mathrm{C}}^{2.5}$ ‐ ATP, $\text{GHV}{\mathrm{4}}_{\mathrm{C}}^{2.5}$ + ATP, $\text{GHV}{\mathrm{4}}_{\mathrm{Flip}}^{2.5}$ − ATP, and $\text{GHV}{\mathrm{4}}_{\mathrm{Flip}}^{2.5}$ + ATP (orange: Annexin^F; scale bar: 10 µm) (n = 2, N = 400, * p < 0.05, one‐way ANOVA with Šídák's multiple‐comparisons test, the * above a plot indicates a significant difference from all other groups).

Figure 7

a) Schematic of GHV2_Flip exposed to ATP leading to constriction (top) or constriction followed by division (bottom). b) Representative CLSM image series of constricted GHV2_Flip after 20 to 60 min exposure to ATP, and of (c) dividing gHV2_Flip 30 to 60 min after ATP addition. d) Illustration of GHV4_Flip in the presence of ATP. e) Representative CLSM image series of constricted GHV4_Flip after 10 to 15 min exposure to ATP (red: Rho‐PE, scale bars: 10 µm).