Light-Powered Reactivation of Flagella and Contraction of Microtubule Networks: Toward Building an Artificial Cell

Abstract

Artificial systems capable of self-sustained movement with self-sufficient energy are of high interest with respect to the development of many challenging applications, including medical treatments, but also technical applications. The bottom-up assembly of such systems in the context of synthetic biology is still a challenging task. In this work, we demonstrate the biocompatibility and efficiency of an artificial light-driven energy module and a motility functional unit by integrating light-switchable photosynthetic vesicles with demembranated flagella. The flagellar propulsion is coupled to the beating frequency, and dynamic ATP synthesis in response to illumination allows us to control beating frequency of flagella in a light-dependent manner. In addition, we verified the functionality of light-powered synthetic vesicles in in vitro motility assays by encapsulating microtubules assembled with force-generating kinesin-1 motors and the energy module to investigate the dynamics of a contractile filamentous network in cell-like compartments by optical stimulation. Integration of this photosynthetic system with various biological building blocks such as cytoskeletal filaments and molecular motors may contribute to the bottom-up synthesis of artificial cells that are able to undergo motor-driven morphological deformations and exhibit directional motion in a light-controllable fashion.

Figure 1

Structure of cilia/flagella. (A) Cilia/flagella have a cylindrical architecture composed of axoneme, plasma membrane, and basal body. (B) Axoneme has a microtubule-based structure formed of nine microtubule doublets at the periphery and two microtubule singlets at the center, which are associated with protein complexes such as radial spokes, inner dynein arm, outer dynein arm, and nexin linkers. There is a mechanical feedback from the bending on the regulation of dynein activity, which switches the activity of dyneins on the opposite side of the central pair microtubules. (C) In the presence of nexin proteins which cross-link the neighboring doublets, microtubules are not free to slide and active forces generated by dynein molecular motors bend the cilium/flagellum. Nexin linkers play an important role to convert the action of dynein motors in microtubule bending,^, and radial spokes ensure that dyneins work together in a highly coordinated manner to generate a regular wave pattern.

Figure 2

Light-driven ATP production. (A) Schematic representation of light-driven ATP synthesis in lipid vesicles. EF₀F₁-ATP synthase uses the electrochemical proton gradient generated by bacteriorhodopsin to synthesize ATP from ADP and P_i. (B) Measurement of light-driven ATP production over time. The maximal rate of 4.5 μmol ATP (mgEF₀F₁)⁻¹ min^–1 is determined by linear regression. Experiments are performed at room temperature with the HMDEKP buffer as the inner solution (30 mM HEPES-KOH, 5 mM MgSO₄, 1 mM DTT, 1 mM EGTA, 50 mM potassium acetate, 1% (w/v) PEG, pH = 7.4). The same outer solution was adjusted with 5 mM NaH₂PO₄, 2 mM MgCl₂, 1 mM DTT and 810 μM ADP. [P_i] = 5 mM, [lipid] = 0.022 mg/mL, [EF₀F₁] = 2.6 nM, [bR] = 160 nM, and ΔΨ = 143 mV is the membrane potential (the outer potential minus the inner potential). Proteins were reconstituted with 0.8% Triton.

Figure 3

Experiments with pure commercial ATP. (A) Snapshot of a C. reinhardtii cell with its two flagella. (B) Isolated and demembranated flagella are reactivated with pure ATP. (C) Swimming trajectory of an exemplary axoneme beating at 18 Hz with [ATP] = 80 μM (see Videos S1–S2). (D) Color-coded time projection of the axoneme in panel C shows the circular swimming path. (E) Mean beat frequency as a function of ATP concentration. Solid red line is a least-square fit to the modified Michaelis–Menten relation (see text). The critical minimum ATP concentration required to observe axonemal beating was [ATP]_critical = 60 μM. (F) Beat frequency decays over time at a rate that depends on ATP concentration. In the presence of 1 mM ATP (blue line) in a 10 μL channel, axonemes beat for 32 min at a decreasing beating frequency that allows us to estimate the averaged ATP consumption rate of 0.31 nmol/min. This rate depends on ATP concentration and decreases to 0.25 nmol/min for [ATP] = 0.1 mM (yellow line). Error bars are mean ± standard deviation (N = 7).

Figure 4

Integration of the motility module with the light-switchable energy module. (A) Schematic presentation of isolated flagella mixed with energy module. (B) Functionalized liposomes are illuminated for different times, generating ATP concentrations up to 213 μM under illumination with a 5 W microscope lamp. (C) Higher ATP concentrations up to 330 μM was produced under illumination with a 50W LED lamp. Both light sources are located 25 cm away from the sample. Inset shows ATP production in the time interval 0 to 5 min of illumination. (D) Axonemes beat faster at higher ATP concentrations produced by longer illumination of energy module under microscope light. Inset shows that axonemes beat even at small ATP concentrations below 10 μM. (E) Static curvature of the axonemes, defined as the curvature of the mean shape averaged over one beating cycle (arc-shaped filament with cyan color), does not significantly depend on ATP concentration. The black line shows a linear fit with the offset of ∼0.16 μm^–1 and slope of zero. For each data point in panels D and E, frequencies of 10 axonemes are measured to calculate the mean and standard deviation.

Figure 5

Activating role of ADP. (A,B) A hypothetical mechanism introduced in refs (57, 63) illustrating the regulatory effect of ADP on the binding affinity of dynein to the B-subtubule of the outer MT doublets. Hence, in the presence of ADP which binds to a noncatalytic site, dynein is more efficient in generating the sliding force. (C) Pure ATP experiments supplemented with 1.6 mM ADP confirm the activating role of ADP at low ATP concentrations. Note that axonemes are reactivated even at a very low ATP concentration of 0.1 μM. (D) Comparison of two sets of experiments with and without ADP at fixed ATP concentration of 60 μM. While without ADP, axonemes stop beating after 2 min, with 1.6 mM ADP, axonemes beat with higher frequencies and are active for a longer time.

Figure 6

Light-driven reactivation of axonemes with and without calcium. (A) An actively beating axoneme fueled by 213 μM ATP produced during preillumination of the energy module for 45 min by microscope light (SI, Video S4). (B) As axoneme beats with frequency of 72 Hz, it rotates CCW with a slower frequency of 2 Hz. Magenta and green trajectories show the traces of distal and basal ends of the axoneme, respectively. (C) Color-coded time projections of the beating axoneme showing the circular swimming path. (D) Curvature waves propagate along the contour length from the basal toward the distal end. (E) Power spectrum of curvature waves shows dominant peaks at f₀ = 72 Hz and at second harmonic 2f₀. (F) Configurations of the axoneme at different time points are translated and rotated such that the basal end is at (0, 0) and the orientation of the tangle vector at the basal end is in the x̂ direction. Static curvature of this axoneme is ∼0.2 μm^–1. (G–L) A separate experiment with 1 mM CaCl₂ which reduces the static curvature of the axoneme to 0.01 μm^–1 (compare filaments with cyan color in panels F and L). Thus, the axoneme swims in a straight trajectory (compare panels C and I) utilizing ∼1 μM ATP synthesized by energy of microscope light without 45 min preillumination step. At such a low ATP concentration, axoneme beats at a slower frequency of 19 Hz (SI, Video S7).

Figure 7

Cell-like confinement of MTs, kinesin-1 molecular motors, and light-switchable energy module. Kinesin-1 is a dimer with two heads which forms cluster through streptavidin. (A) Schematic representation of the MTs/kinesin-1 network coencapsulated with functionalized vesicles inside water-in-oil droplets. Upon illumination, synthesized ATP provides fuel for the kinesin-1 molecular motors, which are plus-end directed motors that exert contractile stresses by sliding MTs relative to each other. (B) MTs/kinesin-1 network shows a relatively uniform distribution shortly after encapsulation. (C) Snapshots of network contraction after 40 min. The yellow arrows indicate network contraction. (D) Rotational flows observed in some of the droplets during network contracts. (E) Snapshots of contractile active network at two different time points. The yellow circle shows the area covered by the network after ∼5 min. (F) Space-time plot showing network contraction along the red dashed line in panel E. (G) Relative reduction of network area over time for three droplets shown in panel E. A₀ is the initial area of the network before contraction, as marked with a black circle in panel D.

Figure 8

Photostimulated contraction of MTs/kinesin-1 network inside a millifluidic device (SI, Video S10). (A) Schematic representation of the millifluidic device highlighting the area illuminated with discontinuous microscope light. Taxol-stabilized MTs are mixed with kinesin-1 motors and a preilluminated energy module prior to injection into a millifluidic channel. (B,C) Over time, as available ATP in the energy module is consumed by force-generating kinesin-1 motors, discontinuous microscope illumination compensates for ATP consumption and filamentous network contracts up to 38%. Quantitative analysis of the width of the contracted network in the illuminated region shows an exponential decay over time. The initial width of the network is W = 1.5 mm. (D) Space-time plot demonstrating the network contraction along the white dashed line drawn in part C. (E) In the nonilluminated areas, where ATP is only consumed but not replenished, we monitored a reduced contractility of up to 10% after 1 h.