Protocells Capable of Generating a Cytoskeleton‐Like Structure from Intracellular Membrane‐Active Artificial Organelles

Abstract

The intricate nature of eukaryotic cells with intracellular compartments having differences in component concentration and viscosity in their lumen provides (membrane‐active) enzymes to trigger time‐ and concentration‐dependent processes in the intra‐/extracellular matrix. Herein, membrane‐active, enzyme‐loaded artificial organelles (AOs) are capitalized upon to develop fluidic and stable proteinaceous membrane‐based protocells. AOs in protocells induce the self‐assembly of oligopeptides into an artificial cytoskeleton that underlines their influence on the structure and functionality of protocells. A series of microscopical tools is used to validate the intracellular assembly and distribution of cytoskeleton, the changing protocells morphology, and AOs inclusion within cytoskeletal growth. Thus, the dynamics, diffusion, and viscosity of intracellular components in the presence of cytoskeleton are evaluated by fluorescence tools and enzymatic assay. Membrane‐active alkaline phosphatase in polymersomes as AOs fulfills the requirements of biomimetic eukaryotic cells to trigger intracellular environment, mobility, viscosity, diffusion, and enzymatic activity itself as well as high mechanical stability and high membrane fluidity of protocells. Thus membrane‐active AOs in protocells provide a variable reaction space in a changing intracellular environment and underline their regulatory role in the fabrication of complex protocell architectures and functions. This study contributes significantly to the effective biomimetics of cell‐like structures, shapes, and functions.

Construction of Membraneless and Multicompartmentalized Coacervate Protocells Controlling a Cell Metabolism-like Cascade Reaction

“…The use of ALP-Psomes offers a crucial steric hindrance, preventing phosphorylated polyesters from accessing ALP within Psomes. In our study, it is mandatory to retain ALP functions in a protected environment, using it as membrane-integrated enzymes in Psomes (also attributed to nonreleasing properties of ALP from Psomes , ) with promising metabolism characteristics (e.g., higher activity over time compared to free ALP and pH-triggered activity of ALP) . To understand the influence of the organelles SnR and AOs on the system reactivity (Figure ), the ALP-catalyzed hydrolysis of the FDP into fluorescent F was performed at pH 5.5 and 7.5 in 10 mM MOPS by using SnR (FDP-NPs) as the substrate as well as the native ALP and AOs (ALP-Psomes) as the catalyst (see details in the Supporting Information, Sections 2.12 and 5.7).…”

“…Moreover, they should be able to perform a metabolism-like enzymatic cascade reaction mediated by the pH-driven substrate nanoreservoir responsiveness triggered by the addition of an exogenous fuel (Figure ). For this purpose, four cell-like structures are combined: (i) substrate nanoreservoir (SnR), whose function is to protect the substrate under acidic conditions and release it as a response to a pH change from acidic to physiological conditions (around 7.4); (ii) pH-sensitive artificial organelles with membrane-integrated active enzyme (AOs), which is “inactive” (low enzyme activity) under acidic conditions and “active” (high enzyme activity) at pH close to physiological conditions; , (iii) pH- and salt-stable cytosol-like scaffold, responsible for the incorporation and feeding of AOs as well as free-enzymes; , and (iv) signal transduction component (enzyme), which converts the exogenous fuel into an endogenous signal (pH increase). This endogenous signal is then transmitted over the cytosol-like scaffold to trigger the metabolism-like process within the protocell.…”