Distinct Network Morphologies from In Situ Polymerization of Microtubules in Giant Polymer-Lipid Hybrid Vesicles

Abstract

Creating artificial cells with a dynamic cytoskeleton, akin to those in living cells, is a major goal in bottom-up synthetic biology. In this study, we demonstrate the in situ polymerization of microtubules encapsulated in giant polymer-lipid hybrid vesicles (GHVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine and an amphiphilic block copolymer. The block copolymer is comprised of poly(cholesteryl methacrylate-co-butyl methacrylate) as the hydrophobic block and either poly(6-O-methacryloyl-D-galactopyranose) or poly(carboxyethyl acrylate) as the hydrophilic extension. Depending on the concentrations of guanosine triphosphate (GTP) or its slowly hydrolyzable analog, guanosine-5'-[(α,β)-methyleno]triphosphate (GMPCPP), different microtubule morphologies are observed, including encapsulated microtubule networks, spike protrusions, as well as membrane-associated or aggregated microtubules. Overall, this work represents a step forward in mimicking the cellular cytoskeletons and uncovering the influence of membrane composition on microtubule morphologies.

Keywords: artificial cells; giant hybrid vesicles; microtubules; tubulin polymerization.

Scheme 1

a) The molecular structures of the amphiphilic block copolymers used for the assembly of GHVs. i) The hydrophobic block poly(cholesteryl methacrylate‐co‐butyl methacrylate) and the resulting amphiphilic block copolymers BCP1 (poly(cholesteryl methacrylate‐co‐butyl methacrylate)‐block‐poly(carboxyethyl acrylate)) and BCP2 (poly(cholesteryl methacrylate‐co‐butyl methacrylate)‐block‐poly(6‐O‐methacryloyl‐D‐galactopyranose)) after the extension with either 6‐O‐methacryloyl‐D‐galactopyranose or carboxyethyl acrylate. b) A cartoon illustrating the different types of assembled giant vesicles, (i) GUV, constructed from 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) and cholesterol, (ii) GHV1, from DOPC with BCP1 and (iii) GHV2, from DOPC and BCP2, all encapsulating tubulin with GTP or GMPCPP.

Figure 1

GPC traces of the homopolymers p(CMA‐co‐BuMA) (A, orange) and p(proGalacMA) (B, gray) as well as their mixture (C, pink) and BCP2^p (D, turquoise).

Figure 2

GHV characterization. a) Representative SDCM images of (i) ^Rhod+NBDGHV1^2.5 and (ii) ^Rhod+NBDGHV2^2.5 (green: BCPX^f, red: ^RhodPE; Scale bar: 50 µm). b) General polarization values derived from Laurdan spectra of GUV^2.5, GHV1^2.5 and GHV2^2.5. The data are expressed as mean ± SD (n = 2–3). c) i) Representative CLSM of GUV^2.5, GHV1^2.5 and GHV2^2.5 after (i) 5 and (ii) 60 min incubated with 5(6)‐ROX (5 µM). (Red: 5(6)‐ROX; Scale bar: 10 µm). iii) Percentage of 5(6)‐ROX‐filled vesicles as a function of time (N = 35–60).

Figure 3

Representative SDCM images of ^RhodGHV2^2.5 encapsulated with different concentrations of tubulin with 1 mm GTP (a) or 1 mm GMPCPP (b). The concentrations of tubulin were 3 µm (i), 11 µm (ii), and 20 µm (iii) (blue: tubulin^F, red: ^RhodPE; Scale bar: 10 µm; n = 3). iv) Cartoon illustrating the time‐dependent, encapsulated microtubule formation.

Figure 4

a) Representative schematics and SDCM images of the different phenotypes of tubulin‐encapsulating ^RhodGHV2^2.5 (Scale bar: 5 µm). ^RhodGHV2^2.5 were loaded with either b) 0 mm, c) 1 mm, or d) 2.5 mm GMPCPP together with 20 µm tubulin, and they were observed over 24 h. (i) Representative images after 1, 4, 8 and 24 h (Scale bar: 10 µm). (ii) The distribution of the different morphologies in the population after 1 and 24 h. At least 100 vesicles were quantified per experiment (n = 3, ^* p < 0.05, one‐way ANOVA with Šídák's multiple‐comparisons test). c) ii ‐ inset) Representative histogram of vesicle diameters for Group 3 and 5, measured from images of samples incubated for 24 h. A minimum of 100 vesicles were measured for each group, and the resulting histograms were fitted to a Gaussian amplitude function, yielding the mean size and variance (given as mean diameter ± 2σ) as indicated above the histograms. c) iii) Representative super‐resolution images of GHV2^2.5 in Group 3 after 24 h incubation with 1 mm GMPCPP showing different types of deformed GHV2^2.5 (red: ^RhodPE, cyan: tubulin^F; n = 3; Scale bar: 5 µm).

Figure 5

^RhodGHV2⁵ encapsulated with either a) 0 mm, b) 1 mm, or c) 2.5 mm GMPCPP together with 20 µm tubulin were observed over 24 h. (i) Representative confocal microscopy images after 1, 4, 8, and 24 h (red: ^RhodPE, cyan: tubulin^F; n = 3; Scale bar: 10 µm). (ii) The distribution of the different morphologies in the population after 1 and 24 h. At least 100 vesicles were quantified per experiment (n = 3, ^* p < 0.05, one‐way ANOVA with Šídák's multiple‐comparisons test).