Photoreceptor-Like Signal Transduction Between Polymer-Based Protocells

Abstract

Deciphering inter- and intracellular signaling pathways is pivotal for understanding the intricate communication networks that orchestrate life's dynamics. Communication models involving bottom-up construction of protocells are emerging but often lack specialized compartments sufficiently robust and hierarchically organized to perform spatiotemporally defined signaling. Here, the modular construction of communicating polymer-based protocells designed to mimic the transduction of information in retinal photoreceptors is presented. Microfluidics is used to generate polymeric protocells subcompartmentalized by specialized artificial organelles. In one protocell population, light triggers artificial organelles with membrane-embedded photoresponsive rotary molecular motors to set off a sequence of reactions starting with the release of encapsulated signaling molecules into the lumen. Intercellular communication is mediated by signal transfer across membranes to protocells containing catalytic artificial organelles as subcompartments, whose signal conversion can be modulated by environmental calcium. Signal propagation also requires selective permeability of the diverse compartments. By segregating artificial organelles in distinct protocells, a sequential chain of reactions mediating intercellular communication is created that is further modulated by adding extracellular messengers. This connective behavior offers the potential for a deeper understanding of signaling pathways and faster integration of proto- and living cells, with the unique advantage of controlling each step by bio-relevant signals.

Keywords: artificial organelles; cell mimics; molecular motors; protocell communication and signaling.

Figure 1

Schematic of light‐triggered, organelle‐based chain of reactions mediating intercellular communication between sender and receiver protocells, each with a distinct set of functional AOs. One type of AO contains light‐responsive rotary molecular motors in their membranes, enabling the controlled release of “signaling” molecules in sender protocells upon illumination. Signaling molecules are transduced through membrane pores in both sender and receiver protocells to the second type of AO with catalytic function inside receiver protocells, where they act as substrates that are converted into reporting molecules. Signal transduction in receiver protocells can be modulated by the addition of environmental CaCl₂.

Figure 2

Formation and characterization of compartmentalized protocells. a) Bioactive cargo molecules are encapsulated in self‐assembled artificial organelles by film rehydration, forming functional AOs. Subsequently, stimuli‐responsive AOs are encapsulated into GUVs using double emulsion microfluidics. b,c) Representative fluorescence micrographs of ATTO488‐loaded AOs (green) in protocells (cyan) at inner aqueous phase concentrations of b) 3.4 × 10¹⁰ AO and c) 3.4 × 10¹¹ AO mL⁻¹. Scale bars, 10 µm. d) 3d reconstruction of AO_A488 distribution (green) within a protocell stained with BODIPY 630/650 (red). e) Experimentally determined loading numbers of AO models per protocell (black) from 3D reconstructed protocells (n > 3) and theoretical loading values (blue) determined by input AO concentration and protocell volume (n = 3). Data is expressed as a mean ± SD. f) 3D AO distribution within a 2 µm segment of a protocell (concentration of 3.4 × 10¹⁰ AO mL⁻¹) visualized through a 2D Kernel Density Estimation plot with 20 levels along the x and y axes. Histograms represent the AO density distribution along the x and y axes. The symmetric and broad density contours suggest a uniform particle distribution in the sphere's volume, with a higher concentration at the core (n = 3). g) FCS autocorrelation curves for AO_A488 encapsulated in the cavity of protocells at different concentrations (n = 30). Dotted lines represent raw data averages and solid lines represent fitted curves.

Figure 3

Light‐triggered intracellular signaling between AOs. a) Schematic representation of stimuli responsive AOs containing molecular motors (MM) in their membranes. Upon illumination with light (hv) at 430 nm, the molecular motors rotate, thereby destabilizing the polymer membrane and releasing the cargo from the AO lumen. Release of ATTO488 dye from the AO lumen upon exposure at 430 nm. Fluorescence measured from fluorescence micrographs (n = 3). b) Release of ATTO488 dye from the intracellular AO upon exposure at 430 nm for up to 20 min with 2 min intervals. Fluorescence measured in the lumen of protocells from fluorescence micrographs (n = 3). c) Schematic representation of stimuli‐responsive intracellular signaling cascade between AOs in a protocell. Upon irradiation, MM‐AO release FDG that can diffuse through melittin pores into a second, βGal‐encapsulating AO. The βGal hydrolyzes the non‐fluorescent substrate FDG to the fluorescent fluorescein product. Normalized fluorescence inside protocells encapsulating AO_MM_FDG and AO_βGal with and without melittin pores after 1 h of incubation (n = 5). Significance levels: p > 0.05 (n.s.), p < 0.05 (*), p < 0.005 (**), and p < 0.0005 (***).

Figure 4

Intercellular signaling involving organelles spatially confined in separate protocells. a) Schematic overview of light‐triggered signaling cascade from sender to receiver protocell. Irradiation at 430 nm causes of FDG‐encapsulating AOs to rupture, releasing non‐fluorescent FDG. FDG diffuses via DNA nanopores from the sender protocell to the receiver protocell containing AOs encapsulating the enzyme βGal. Inside the receiver protocells, FDG enters AO_mel_βGal via melittin pores, and finally gets hydrolyzed to its fluorescent product fluorescein by the confined enzyme. b) Normalized fluorescence inside receiver protocells encapsulating AO_mel_βGal with and without DNA nanopores after 1 h of co‐incubation with sender protocells encapsulating free FDG (n ≥ 5). c) Normalized fluorescence inside receiver protocell encapsulating AO_mel_βGal with and without DNA nanopores after 1 h of co‐incubation with sender protocells encapsulating FDG in photolabile AOs (AO_MM_FDG) (n ≥ 5). d) Fluorescence micrographs of representative single receiver protocells from (c) showing fluorescein fluorescence (white) without (left) and with (right) DNA nanopores after 1 h co‐incubation with AO_MM_FDG‐encapsulating sender protocells. Scale bar, 10 µm.

Figure 5

Sensitivity modulation of intercellular communication. a) The external addition of CaCl₂ decreases the signal transmission in the receiver protocell, thereby decreasing the gain of the system. b) Calcium has an inhibitory effect on βGal encapsulated in the receiver protocell, thereby modulating the overall photosensitivity of the receiver protocell. c). Normalized fluorescein intensity in receiver protocells encapsulating AO_mel_βGal in the presence or absence of CaCl₂ after 1 h of incubation (n ≥ 5). Significance levels: p > 0.05 (n.s.), p < 0.05 (*), p < 0.005 (**), and p < 0.0005 (***).