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Systematic comparison of unilamellar vesicles reveals that archaeal core lipid membranes are more permeable than bacterial membranes [1]

['Urszula Łapińska', 'Living Systems Institute', 'Biosciences', 'University Of Exeter', 'Exeter', 'United Kingdom', 'Georgina Glover', 'Zehra Kahveci', 'Nicholas A. T. Irwin', 'Merton College']

Date: 2023-04

Microfluidic screening to explore membrane permeability characteristics

Here, we report a system to enable the capture and individual placement of unilamellar vesicles obtained via electroformation of synthetic lipids (see Methods) in multiple parallel arrays of tens of vesicles using microfluidics (Fig 1).

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TIFF original image Download: Fig 1. Schematics illustrating the microfluidic approach used to study membrane permeability. (A) Vesicles (green circles) loaded into the fluidic device (at t = −20 min) display an intracellular carboxyfluorescein (CF) concentration that is similar to the extracellular CF concentration; thus, vesicles confined in the fluidic coves appear as bright as the extracellular environment, as illustrated by the micrograph. Free CF molecules are removed from the microfluidic environment around the vesicles by applying a flow rate of 25 μL/h from the washing buffer inlet for 20 min. (B) At t = 0 min, vesicles appear bright on a dark background as shown in the micrograph. The metabolite (small blue circles) under investigation is delivered to the vesicles by applying a flow rate of 25 μL/h from the metabolite inlet for 3 min. (C) The metabolite accumulates within the vesicles if their membrane is permeable to the metabolite. https://doi.org/10.1371/journal.pbio.3002048.g001

This approach enabled us to precisely control the chemostatic fluid environment of the vesicles [35]. The diameter of these vesicles is in the range of 5 to 15 μm. Although such diameter range is large for prokaryotic cell sizes, these dimensions were chosen to aid imaging and manipulation. In order to measure metabolite permeation into the unilamellar vesicles, we loaded the vesicles with a neutral pH buffer and carboxyfluorescein (CF). This fluorescent dye allows direct assessment of metabolite uptake by variations in fluorescent properties in response to changes in intra-vesicle metabolite concentration [16,36]. In fact, the introduction of metabolites in the vesicle reduces the self-quenching properties of CF, resulting in increased vesicle fluorescence. Therefore, a relative increase in intra-vesicle fluorescence indicates membrane permeability to the target metabolite when delivered via continuous flow through the microfluidic device (Fig 1 and Methods). Individual metabolites were delivered into the extra-vesicle environment (also containing a neutral pH buffer) at a concentration of 1 mM, while imaging the changes in fluorescence levels of multiple individually trapped vesicles (Fig 1 and Methods). By using this experimental approach, we conducted parallel controlled experiments exploring how cellular metabolites can cross membranes of different phospholipid chemical composition; we used relative changes in CF fluorescence as a reporter for relative permeability to each metabolite of the different lipid membranes investigated.

We first studied the permeability of single vesicles composed of synthetic lipids with isoprenoid chains containing methyl branches bonded to a G1P backbone via ether bonds (archaeal-like membrane phospholipids—abbreviated here as “archaeal 4ME diether G1PC”) (lipid 1 in S1 Table) or synthetic lipids with fatty acids bonded to a G3P backbone via ester bonds (bacterial-like membrane phospholipids—ternary lipid mixture abbreviated here as “bacterial diester G3PE-PG-CA”, lipid 2 in S1 Table) to 18 small metabolites (S2 Table). Notably, synthetic G1P diether lipids with methyl branches are not commercially available, to our knowledge, and were therefore synthesised de novo for the purpose of this study (see Methods). We chose metabolites with different molecular weight, hydrophobicity, and number of rotatable bonds (S2 Table).

Our single-vesicle measurements revealed heterogeneity in the permeability of each synthetic lipid type to each metabolite: Some vesicles of each lipid type displayed a decrease in intracellular fluorescence during the delivery of each metabolite, while other vesicles displayed an increase in intracellular fluorescence (temporal dependence of single-vesicle fluorescence for archaeal 4ME diether G1PC lipids and bacterial diester G3PE-PG-CA lipids are reported with dashed magenta lines and dashed-dotted blue lines in S1 Fig and Data A in S1 File). For example, the coefficient of variations in the permeability of archaeal 4ME diether G1PC vesicles and of bacterial diester G3PE-PG-CA vesicles to aspartic acid, glyceraldehyde, and adenine were 118% and 103%, 60% and 198%, 76% and 108% (S1D, S1G and S1Q Fig, respectively), in accordance with the proposition that lipid membranes are a system with heterogeneous functions [37–42].

Therefore, in order to compare the permeability of different synthetic lipid types to the same metabolite, we carried out Mann–Whitney two-tailed statistical comparisons between the distributions of single intra-vesicle fluorescence after 3 min of delivery of each metabolite for each synthetic lipid type. Temporal dependence of fluorescence distribution means and standard deviations are reported in Fig 2 (magenta triangles and dashed lines for archaeal 4ME diether G1PC vesicles, blue squares and dashed-dotted lines for bacterial diester G3PE-PG-CA vesicles) together with single-vesicle fluorescence distributions and Mann–Whitney two-tailed statistical comparisons at t = 3 (further details about Mann–Whitney two-tailed statistical comparisons are reported in S2 File).

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TIFF original image Download: Fig 2. Archaeal 4ME diether G1PC vesicles are consistently more permeable to a range of metabolites than bacterial diester G3PE-PG-CA vesicles. Temporal dependence of average CF fluorescence in archaeal 4ME diether G1PC (magenta triangles) and bacterial diester G3PE-PG-CA vesicles (blue squares) during the exposure to 1 mM of variant metabolites delivered to the microfluidic coves. Mean (symbols) and standard deviation (error bars) were calculated from at least 10 single-vesicle measurements across three independent experiments. Lines are guides for the eye. N is the number of single vesicles investigated for each metabolite exposure and each type of vesicles (magenta and blue for archaeal 4ME diether G1PC and bacterial diester G3PE-PG-CA vesicles, respectively). N varies across different metabolite experiments investigated due to technical constraints (see Methods). However, care has been taken to obtain the same N for each metabolite experiment across the two different types of vesicles to ensure reliable statistical comparisons. Such comparisons have been carried out via two-tailed Mann–Whitney tests between the distributions of CF fluorescence values at t = 3 min for archaeal 4ME diether G1PC and bacterial diester G3PE-PG-CA vesicles. ****: p-value < 0.0001, ***: p-value < 0.001, **: p-value < 0.01, *: p-value < 0.05 and are shown with corresponding violin plots next to each time-course graph. Numerical values of CF fluorescence in individual vesicles for each lipid type during the delivery of each metabolite are provided in Data A in S1 File. The lipids used for creating archaeal 4ME diether G1PC and bacterial diester G3PE-PG-CA vesicles are lipids 1 and 2, respectively, in S1 Table. https://doi.org/10.1371/journal.pbio.3002048.g002

We found that archaeal 4ME diether G1PC vesicles were significantly more permeable to the amino acids glycine, alanine, leucine, aspartic acid, tryptophan, and glutamine compared to bacterial diester G3PE-PG-CA vesicles (Figs 2A–2F and S1A-S1F and Data A in S1 File). Consistent with the amino acid findings, archaeal 4ME diether G1PC vesicles were also more permeable to the sugars glyceraldehyde, glycerol, deoxyribose, ribose, and arabinose compared to bacterial diester G3PE-PG-CA vesicles (Figs 2G–2K and S1G-S1K and Data A in S1 File), whereas we did not measure a significant difference in the permeability to dihydroxyacetone (Figs 2L and S1L and Data A in S1 File). Interestingly, the difference in permeability was strongly distinct for three relatively large sugar types, deoxyribose, ribose, and arabinose, the two former sugars including primary constituents of the hereditary materials DNA and RNA, respectively.

Finally, archaeal 4ME diether G1PC vesicles were also more permeable to the amide urea, and the nucleobases cytosine, uracil, guanine, and adenine compared to bacterial diester G3PE-PG-CA vesicles (Figs 2M–2Q and S1M-S1Q and Data A in S1 File). These data suggest that important nitrogen sources and components of DNA and RNA can permeate archaeal 4ME diether G1PC vesicles. The phosphonate 2-aminoethyl phosphonic acid (Figs 2R and S1R and Data A in S1 File) showed no significant difference in permeability characteristics between the two types of vesicles.

Pooling together the data for the 18 metabolites investigated, we did not find a significant correlation between permeability and lipid molecular weight or hydrophobicity (Pearson coefficient r = −0.22, 0.36, respectively, for archaeal 4ME diether G1PC vesicles; r = 0.09, 0.24, respectively, for bacterial diester G3PE-PG-CA vesicles; none were significant at p = 0.05; S2 Table). These data suggest that the metabolic selectivity of these membrane mimics is complex and does not rely solely on basic molecular properties, such as molecular weight or hydrophobicity. Taken together with previous findings, demonstrating that solute hydrophobicity correlates poorly with permeability coefficients of eukaryotic fatty acid or phospholipid membranes [36] and that amino acid residue hydrophobicity might have not been key for the emergence of the genetic code [43], our data corroborate the hypothesis that subtle variations in the metabolite atomic structure could contribute to differences in membrane permeability [36,37,44]. In fact, we found a significant negative correlation between permeability and rotatable bond number for archaeal 4ME diether G1PC vesicles (Pearson coefficient r = −0.49, *), but not for bacterial diester G3PE-PG-CA vesicles (r = −0.32, nonsignificant at p = 0.05). It is also conceivable that other physicochemical properties such as the partition energy of amino acids (i.e., the propensity of amino acid residues to be exposed to water [45]), which has recently been proposed to be the most optimized property of amino acids in the genetic code [46], could contribute to differences in membrane permeability.

The striking difference in CF fluorescence between archaeal 4ME diether G1PC and bacterial diester G3PE-PG-CA vesicles was confirmed when we extended the duration of our permeability experiments from 3 to 6 min (S2 Fig and Data A in S1 File). Moreover, archaeal 4ME diether G1PC and bacterial diester G3PE-PG-CA vesicles displayed similar dimensions in range 5 to 15 μm, and the dimensions of each vesicle did not significantly change during metabolite delivery (S3 Fig and Data A in S3 File). These data confirm that vesicle deformation did not occur in our experiments and that neither differences in vesicle curvature nor deformation play a role in the observed differences in permeability traits between archaeal 4ME diether G1PC vesicles and bacterial diester G3PE-PG-CA vesicles. We cannot exclude, however, the possibility that differences in curvature between bacteria and archaea, with dimensions in range 1 to 5 μm, could play a role in permeability to metabolites.

Such differences were also not attributable to differential metabolite accumulation within the lipid bilayers, since (i) CF has very low affinity for the lipid hydrophobic chains [16], so interactions with substrates within the membrane are unlikely; (ii) CF fluorescence intensity was uniform across whole vesicles for both lipid types and the full range of metabolites investigated, consistent with previous data obtained on fatty acid and phospholipid liposomes [16,36]. Our data do not allow us to infer detailed kinetics of the permeation of each metabolite and could not be complemented via pulse-chase experiments requiring faster fluidic exchanges. Therefore, we did not attempt to extract absolute kinetic parameters, such as the permeability coefficient, but centred this current work on directly comparing relative changes in CF fluorescence (as a proxy for permeability) between archaeal 4ME diether G1PC and bacterial diester G3PE-PG-CA vesicles. However, we were able to observe differences in terms of both the uptake onset and slope for different metabolites. For example, glycine, ribose, and uracil displayed a steep uptake during the first minute of their delivery to archaeal 4ME diether G1PC vesicles (Fig 2A, 2J and 2O, respectively), whereas the uptake of aspartic acid, glutamine, and dihydroxyacetone started only after the first minute of their delivery to archaeal 4ME diether G1PC vesicles (Fig 2D, 2F and 2L). These data demonstrate that metabolites are not passing across vesicles via puncture holes generated during vesicle formation but by genuine diffusion through the lipid bilayers in a metabolite-specific manner.

As discussed in the introduction, natural archaeal membranes are formed from heterogenous mixtures of lipids some with tetraether bipolar lipids (e.g., caldarchaeol), which act to directly connect the membrane bilayers, a function that is likely to increase the stiffness of the membrane and reduce permeability [47]. Such mixtures might have different properties than the homogenous membranes studied here. To explore this possibility, we attempted to use lipids extracted from Haloferax volcanii, predominantly containing diether lipids with head group derivatives of phosphatidylglycerol [48], and lipids extracted from Sulfolobus acidocaldarius, predominantly containing bipolar tetraether lipids with head group derivatives of phosphatidylhexose [49]. However, we could not obtain mechanically stable vesicles via electroformation for either of these lipid mixtures. It is therefore important to mention that our experiments do not reveal the permeability traits of extant prokaryotic membrane mixtures but rather identify the contrasting permeability traits of the common and core building blocks of the archaeal and bacterial membranes.

Finally, we wanted to rule out that the relatively lower permeability of bacterial diester G3PE-PG-CA vesicles could be due to interactions between different lipids within the ternary lipid mixture that we employed to mimic more closely bacterial membranes (lipid 2 in S1 Table). To do so, we measured and contrasted permeability to urea, glycine, ribose, deoxyribose, glycerol, and phosphonate in vesicles made of bacterial ternary-lipid mixtures (G3PE-PG-CA, lipid 2 in S1 Table) and vesicles made of single lipids (G3PE, lipid 6 in S1 Table) and found that these two different bacterial mimics displayed comparably low permeabilities to all the metabolites tested (S4 Fig and Data B in S1 File). We selected these six metabolites because archaeal 4ME diether G1PC vesicles and bacterial diester G3PE-PG-CA vesicles display different patterns of permeabilities to these metabolites (Fig 2).

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002048

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