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. 2019 Aug 28;5(8):1360-1365.
doi: 10.1021/acscentsci.9b00345. Epub 2019 Jul 3.

Mimicking Cellular Compartmentalization in a Hierarchical Protocell through Spontaneous Spatial Organization

Affiliations

Mimicking Cellular Compartmentalization in a Hierarchical Protocell through Spontaneous Spatial Organization

Alexander F Mason et al. ACS Cent Sci. .

Abstract

A systemic feature of eukaryotic cells is the spatial organization of functional components through compartmentalization. Developing protocells with compartmentalized synthetic organelles is, therefore, a critical milestone toward emulating one of the core characteristics of cellular life. Here we demonstrate the bottom-up, multistep, noncovalent, assembly of rudimentary subcompartmentalized protocells through the spontaneous encapsulation of semipermeable, polymersome proto-organelles inside cell-sized coacervates. The coacervate microdroplets are membranized using tailor-made terpolymers, to complete the hierarchical self-assembly of protocells, a system that mimics both the condensed cytosol and the structure of a cell membrane. In this way, the spatial organization of enzymes can be finely tuned, leading to an enhancement of functionality. Moreover, incompatible components can be sequestered in the same microenvironments without detrimental effect. The robust stability of the subcompartmentalized coacervate protocells in biocompatible milieu, such as in PBS or cell culture media, makes it a versatile platform to be extended toward studies in vitro, and perhaps, in vivo.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Scheme 1
Scheme 1. Formation of a Hierarchical Protocell
Through the spontaneous sequestration of polymersomal proto-organelles by a coacervate microdroplet (A); subsequent membranization with a synthetic terpolymer (B) provides stability to the overall construct, which was evaluated to demonstrate the advantageous properties of a spatially organized, subcompartmentalized system (C) that mimics the advanced properties of a eukaryotic cell.
Figure 1
Figure 1
Hierarchical protocell sequestering three distinct subpopulations of polymersomal proto-organelles loaded with fluorescently labeled proteins. (A) Confocal micrograph of multicompartmentalized protocell (containing FITC-, RITC-, and Cy5-labeled succinylated bovine serum albumin (BSA) in separate vesicles) encapsulated within membranized coacervate protocells—depicted in (B), which is not drawn to scale.
Figure 2
Figure 2
Coencapsulation of enzymes in the multicompartment protocell leads to an enhancement in the overall rate of reaction. (A) The enzyme cascade utilized. Glucose is oxidized by glucose oxidase to form gluconolactone and H2O2, which then diffuses to horseradish peroxidase, catalyzing the formation of fluorescent resorufin from amplex red. (B) Overall enzymatic rates of reaction as determined by bulk resorufin fluorescence in a plate reader. Coencapsulation of enzymes in polymersomes results in a rate increase compared to separately encapsulated enzymes. (C) Confocal images showing resorufin production over time inside a protocell containing coencapsulated GOx/HRP polymersomes (scale bars = 5 μm).
Figure 3
Figure 3
Spatial organization and isolation of macromolecules leads to control over their degradation. (A) Overlabeling of BSA with FITC leads to self-quenching, until ProtK digestion. (B) Bulk fluorescence emission spectroscopy of self-assembled systems. When free in solution, this model reaction progresses rapidly (black curve); however, BSA-FITC can be protected from degradation via both encapsulation in coacervate protocells (red curve) and subcompartmentalization of ProtK in polymersomes (blue curve). Please note: at 50 s, the protease K solution was added to the FITC-BSA protocell experiment (red), and this accounts for the high error observed. (C) Confocal micrograph demonstrating coencapsulation of both FITC-BSA substrate (false colored blue) with RITC-labeled ProtK polymersomes (false colored yellow) within coacervate protocells (scale bars = 20 μm).
Figure 4
Figure 4
Confocal microscopy image of coacervate protocells stained with Nile red (A) formed in VCBM and (B) after 24 h of dialysis (scale bars = 20 μm).

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