Communication and quorum sensing in non-living mimics of eukaryotic cells

Abstract

Cells in tissues or biofilms communicate with one another through chemical and mechanical signals to coordinate collective behaviors. Non-living cell mimics provide simplified models of natural systems; however, it has remained challenging to implement communication capabilities comparable to living cells. Here we present a porous artificial cell-mimic containing a nucleus-like DNA-hydrogel compartment that is able to express and display proteins, and communicate with neighboring cell-mimics through diffusive protein signals. We show that communication between cell-mimics allows distribution of tasks, quorum sensing, and cellular differentiation according to local environment. Cell-mimics can be manufactured in large quantities, easily stored, chemically modified, and spatially organized into diffusively connected tissue-like arrangements, offering a means for studying communication in large ensembles of artificial cells.

Fig. 1

Formation of cell-mimics with artificial nuclei capable of gene expression. a Optical micrographs (top) of a cell-mimic with GelRed stained hydrogel nucleus (brightfield, red fluorescence, merge) and scanning electron microscopy of porous cell-mimic membrane (bottom). b Microfluidic production of double emulsion droplets encapsulating a pre-hydrogel in a photocurable middle layer, and schematic of subsequent processing steps. IA: Inner aqueous, MO: middle organic, OA: Outer aqueous phase. c Schematic and timelapse images d of expression and capture of TetR-sfGFP in hydrogel nuclei (green, merged with brightfield images). TX-TL reagents were added at 0 h. e Dynamics of fluorescence signal increase in the hydrogel nuclei of 100 cell-mimics with average shown in bold

Fig. 2

Protein exchange between neighboring and distant cell-mimics. a Schematic of diffusive TetR-sfGFP exchange between neighboring sender and receiver cell-mimics and timelapse images of neighboring senders (rhodamine B stained membranes) and receivers (unstained membranes). Merge of brightfield and fluorescence channels (sender membranes, magenta; TetR-sfGFP, green). b Distribution of TetR-sfGFP (green) in a dense droplet of receivers and sparse senders (magenta) after 3 h of expression. A small region around a sender (white box) is magnified and spreading of fluorescence is shown at different time points. c Inhomogeneous mix of two types of cell-mimics producing and binding different color reporter proteins. tetR-sfGFP / tetO (green) and tetR-mCherry / tetO cell-mimics (magenta) were distributed in a channel to stay separate at the sides and mix in the center. Bottom image shows the distribution of sfGFP and mCherry fluorescence after 5 h. Merge of the two channels results in a white signal (middle). Magnified images from indicated positions along the channel are shown above. Merged image with cell-mimic types indicated by colored, dashed circles (top), and brightfield, sfGFP and mCherry signals shown separately (below)

Fig. 3

Communication between cell-mimics via a diffusive genetic activator. Schematic of the two types of cell-mimics communicating through a distributed genetic activation cascade. Micrographs show a merge of brightfield images with rhodamine B fluorescence in the membranes of activators (magenta) and fluorescence of TetR-sfGFP (green) in the hydrogel nuclei of reporters directly after addition of TX-TL and after 2 h of expression

Fig. 4

Density sensing in populations of cell-mimics. a Artificial quorum sensing cell-mimics contain T3 activation cascade DNA templates and 240 × tetO plasmids. b Micrographs of cell-mimics in 4.5 µl droplets of TX-TL (left). The number of cell-mimics is indicated. Enlarged regions (indicated by white boxes) show presence and absence of fluorescence (green) in hydrogel nuclei after 3 h of expression. c Scatter dot plot of fluorescence intensities in individual cell-mimics at different densities. Each density category combines data in increments of 50 cell-mimics per droplet and contains data from at least 156 cell-mimics (Methods). Black bars show average fluorescence. d A 2-color response to density is achieved by adding a constitutively expressed reporter (pT7-tetR-mCherry), which is on independent of density. e 2-color density sensors were spread at increasing density in an elongated chamber (brightfield image, bottom). Panels above show magnified fluorescence images of indicated regions (mCherry fluorescence: magenta, left; sfGFP fluorescence: green, middle; merge of fluorescence channels: right). Images in each channel are window leveled to the same values and have a width of 70 µm. f Ratio between sfGFP to mCherry fluorescence in individual hydrogel nuclei along the chamber. Positions correspond to tile regions of the image above