Single Cell-like Systems Reveal Active Unidirectional and Light-Controlled Transport by Nanomachineries

Abstract

Cellular life depends on transport and communication across membranes, which is emphasized by the fact that membrane proteins are prime drug targets. The cell-like environment of membrane proteins has gained increasing attention based on its important role in function and regulation. As a versatile scaffold for bottom-up synthetic biology and nanoscience, giant liposomes represent minimalistic models of living cells. Nevertheless, the incorporation of fragile multiprotein membrane complexes still remains a major challenge. Here, we report on an approach for the functional reconstitution of membrane assemblies exemplified by human and bacterial ATP-binding cassette (ABC) transporters. We reveal that these nanomachineries transport substrates unidirectionally against a steep concentration gradient. Active substrate transport can be spatiotemporally resolved in single cell-like compartments by light, enabling real-time tracking of substrate export and import in individual liposomes. This approach will help to construct delicate artificial cell-like systems.

Keywords: ABC transporter; giant liposomes; membrane transport; single-liposome analysis; synthetic biology.

Figure 1

Reconstitution of ABC transporters in giant liposomes. (a) SulfoCy5-labeled TmrAB was reconstituted in GUVs at a protein-to-lipid ratio of 1:20_w/w. A line profile displays the mean gray values (mgv). (b) GUVs with and without reconstituted TmrAB were site-specifically labeled via the C-terminal His-tag of TmrA by trisNTA^Alexa647. For dual-color visualization, the fluorescence gain was enhanced in the lower left and right image. The lipid bilayer was stained by DOPE^ATTO390. Line profiles demonstrate the colocalization of TmrAB and the lipid bilayer. (c) ATPase activity of TmrAB in isolated GUVs after detergent solubilization. The mean ± SD (n = 3) is shown.

Figure 2

Functional reconstitution and active uphill transport of human TAPL in giant liposomes. (a) CoreTAPL^mVenus incorporation and ATTO⁶⁵⁵ encapsulation in giant liposomes. (b) Peptide transport of coreTAPL^mVenus for 2 h at 37 °C, after addition of ATP, ARS, and C4F peptides. Scale bars, 10 μm. All images are processed by ImageJ for better visualization.

Figure 3

Transport against a concentration gradient. (a) Transport assay of TmrAB in giant liposomes. The membrane-impermeable Oyster⁶⁴⁷ dye was encapsulated in proteoGUVs to control membrane integrity. GUVs were tethered to functionalized glass slides. ATP was added to drive the transport of fluorescent C4F peptides. (b) Time-lapse recordings demonstrate the transport activity of TmrAB. GUVs were filled with the Oyster⁶⁴⁷ fluorophore, and transport was induced by addition of ATP, ARS, and C4F peptides. Scale bar, 10 μm. (c) Representative analysis of two time-lapse traces. The accumulation of C4F peptides in giant liposomes was normalized to background fluorescence. (d) Rate constants (C4F per TmrAB per min) were determined for different C4F concentrations. Each dot represents the transport rate for an individual giant liposome. The median is given for each box plot (red line) and is illustrated in the inset for various peptide concentrations; 25–75% of the mean are shown as boxes and error bars as SD. The data were obtained from more than nine independent experiments (biological replicates).

Figure 4

Light-controlled unidirectional export and sequential import. (a) Light-controlled export of TmrAB in giant liposomes. Caged-ATP was activated in situ by illumination at 405 nm using a confocal laser-scanning microscope. (b) Time-lapse images of the light-controlled export assay. Scale bar, 10 μm. (c) Time-dependent change in fluorescence intensity of C4F peptide. The inset illustrates the fluorescence signal of an exemplary GUV loaded with caged-ATP before photoactivation. (d) Export efflux constants for nine individual GUVs were summarized in a histogram. (e) Export and import of C4F peptides for the same giant liposome. The experiment was performed under identical conditions to those in (a), followed by addition of ATP and ARS driving peptide import. The focus was readjusted after addition of ATP/ARS, and the brightness of the red channel was enhanced by 6% for better visualization. Normalized and background-corrected fluorescence intensities (%) for the dyes inside the GUVs’ lumen are given below the images. Scale bar, 10 μm.