helixCAM: A platform for programmable cellular assembly in bacteria and human cells

Abstract

Interactions between cells are indispensable for signaling and creating structure. The ability to direct precise cell-cell interactions would be powerful for engineering tissues, understanding signaling pathways, and directing immune cell targeting. In humans, intercellular interactions are mediated by cell adhesion molecules (CAMs). However, endogenous CAMs are natively expressed by many cells and tend to have cross-reactivity, making them unsuitable for programming specific interactions. Here, we showcase "helixCAM," a platform for engineering synthetic CAMs by presenting coiled-coil peptides on the cell surface. helixCAMs were able to create specific cell-cell interactions and direct patterned aggregate formation in bacteria and human cells. Based on coiled-coil interaction principles, we built a set of rationally designed helixCAM libraries, which led to the discovery of additional high-performance helixCAM pairs. We applied this helixCAM toolkit for various multicellular engineering applications, such as spherical layering, adherent cell targeting, and surface patterning.

Keywords: cell aggregation; cell interaction; cell patterning; cell targeting; image processing; mammalian synthetic biology; membrane protein; protein engineering; rational design; tissue engineering.

Figure 1.. Overview of helixCAM design and applications and demonstration with Z17/Z18.

A. Schematic of the helixCAM concept and potential applications. B. Design of helixCAMs in E. coli: the coiled-coil domain is fused to the EhaA autotransporter protein for surface presentation. C. helixCAM-induced E. coli aggregation, with Z17 cells co-expressing mCherry, and Z18 cells co-expressing eGFP. Representative views are shown from several fields of view. Images were taken at 60X magnification and cropped (uncropped images and more fields of views in S2). D. Design of mammalian helixCAMs: the coiled-coil domain is fused to the PDGFR transmembrane domain. E. helixCAM-induced K562 aggregation, with Z17 cells co-expressing mCherry, and Z18 cells co-expressing eGFP. Representative views are shown from several fields of view. Image was taken at 20X magnification with 3x3 tiling and cropped (uncropped images in S3). F. 3D reconstruction of helixCAM-induced K562 aggregates of various sizes, demonstrating heterodimeric three-dimensional patterning. Images were taken at 40X magnification using a spinning disc confocal system as a Z-stack and reconstructed using ImageJ.

Figure 2.. Characterization of additional helixCAM pairs and three-pair interaction orthogonality.

A. K562 cell aggregates formed by P3/AP4 interactions. P3 cells co-express iRFP670 and AP4 cells co-express eBFP2. Image was taken at 20X magnification and cropped (uncropped image in S3). B. K562 cell aggregates formed by P9/AP10 interaction. P9 cells co-express mPlum, and AP10 cells co-express mOrange. Image was taken at 20X magnification and cropped (uncropped image in S3). C. Z17^mCherry, Z18^eGFP, P3^iRFP670, AP4^eBFP2, P9^mPlum, and AP10^mOrange cells were induced to express helixCAMs in a single mixed culture. Clear sub-clusters can be observed. In this image, mCherry appears more orange and mOrange appears more yellow due to the filter sets used. Image was taken at 20X with a 6x6 tile across five channels and cropped to show regions of interest (uncropped image in S8). D. Interaction frequency table was derived from the uncropped three-pair co-culture image. Analysis pipeline detailed in S9.

Figure 3.. Rational Design of helixCAM-optimized Coiled-Coil library and two-stage screen for helixCAM performance.

A. Table of amino acid substitutions for each of four template-derived helixCAM libraries. Each template consists of five heptads, within which either the “g”, “a” and “e” position or the “g”, “d”, and “e” positions are varied to form new electrostatic and hydrophobic interactions. B. Design of the tripartite split-GFP assay for CC affinity. One CC library was fused to β-strand 10 and another to β-strand 11 of the tripartite split-GFP. Interaction between CCs stabilizes a fluorescent GFP protein. C. Graph of CC candidate pairs’ frequency (in percent) in the population versus their pair score (graph uses W=0.1). The pair score represents a pair’s specificity to each other and is adjusted with a weight parameter W (Detailed in methods). The top 30 hits using three different W’s, along with several high-frequency pairs (totaling 102 individual CCs) were selected for subsequent screening. D. Design of modified yeast SynAg mating assay for helixCAM-compatible CC selection. Haploid yeast cells MATa or MATα expressing surface-presented CC candidates were mixed. CC binding induces haploid cells to mate, creating a diploid cell that survives dual auxotrophic selection. The fusion of cells also leads to the expression of the Cre recombinase (orange), which integrates the two CC constructs and their barcodes into the same DNA strand. E. Stage 2 CC candidate pairs’ enrichment versus their orthogonality. Enrichment is the log of the observed frequency of the pair as a ratio to their individual frequency in the pre-mated populations, whereas orthogonality is the log of the frequency of the pair divided by the total observations of each of the two CCs in the pair. Two pairs, sg30/sg61 and sg83/sg88, were selected from the red group for helixCAM use. F. Large aggregates of sg30^mCherry and sg61^eGFP K562 cells. Image was taken at 20X magnification with a 3x3 tile and subsequently cropped. G. Aggregates of sg83^iRFP670 and sg88^eBFP2 K562 cells. Image was taken at 20X magnification and presented without cropping.

Figure 4.. HCSRA measurement of helixCAM affinity and optimized three-pair aggregation.

A. Cartoon of Human Cell Sedimentation Rate Assay (HCSRA). B. Heatmap showing Δt₅₀ values from HCSRA for helixCAM affinity for self, pairwise, and wild-type conditions. Each square represents the mean of three replicates. C. Comparison of Δt₅₀ values from HCSRA to pairwise interaction frequencies from Figure 2D. A linear correlation was observed, with an R² of 0.624 and p-value of 0.0005, using the F-statistic. D. Bar graph of Δt₅₀ for complementary helixCAM pairs, demonstrating a range of affinity. Error bars represent S.D., N=3. E. Dose curve of helixCAM-induced binding strength for the top four helixCAM pairs. Δt₅₀ for each pair is normalized to each pair’s maximum measured Δt₅₀ value. Positions along the x-axis are slightly shifted for ease of interpretation. Error bars represent S.D., N=4. Absolute Δt₅₀ values and statistical comparison are in S15. F. P9^mPlum, AP10^mOrange, sg30^mCherry, sg61^eGFP, sg83^iRFP670, and sg88^eBFP2 cells were induced to express helixCAM within a single mixed culture. This subset of helixCAMs was selected as the most orthogonal set from HCSRA results. Clear sub-clusters can be observed. Widefield image was taken at 20X with a 5x5 tile across five channels and cropped to show regions of interest (Uncropped image in S17). Confocal image was taken at 40X magnification with a spinning disc confocal system as a Z-stack and reconstructed using Nikon Elements. Due to slight movement of cells during Z-stack acquisition across channels, mOrange cells can appear half yellow and half red. G. Interaction frequency table derived from the optimized three-pair co-culture image. Designed co-localization can be observed in all three helixCAM pairs. Analysis pipeline detailed in S9.

Figure 5.. helixCAMs enable additive construction of sophisticated three-dimensional cell structures

A. Schematic of spherical layering workflow. Five K562 lines were built, expressing either one or two helixCAMs and an identifying fluorescent protein. Layer 1 cells were mixed with core cells at an 8:1 ratio, and subsequent layers were then mixed at a 2:1 ratio. B. Three select aggregates resembling the desired patterning are shown. Arrows overlaid on Aggregate 1 indicate the location of each cell type. Aggregates were imaged at 20X magnification and cropped for emphasis. C. Confocal imaging of patterned spherical structures. Core and layer cells are visible and near intended locations. Confocal images were taken at 40X magnification with a spinning disc confocal system as a Z-stack and reconstructed using Nikon Elements. D. Interaction frequency of cells within Aggregate 1. Analysis pipeline detailed in S9.

Figure 6.. helixCAMs enable targeting of suspension cells to adherent cells and can impact adherent cell morphology.

A. Workflow for targeting suspended HEK293 cells to adherent HEK293 cells. After cells were grown to confluency, one population is trypsinized and added to the other population, after which unbound cells are washed off. Bound cells are then allowed to re-establish an adherent morphology. B. Panel images show three helixCAM pairs uninduced, induced for 48 hours and immediately post-wash, or induced for 48 hours and then incubated for an additional 24 hours post-wash. The 24h post-wash condition is shown as slice view, with horizontal cross-sections of a z-stack shown on the bottom and right panels. Uninduced and induced 0h conditions were imaged using a widefield microscope at 20X magnification with 2x2 tiling, and induced 24h images were taken using a confocal microscope as a Z-stack, also at 20X magnification with 2x2 tiling. C. Workflow for joint seeding of complementary helixCAM-expressing HEK293 cells. Cells were seeded and induced, followed by trypsinization and re-seeding as a single mixture. D. Panels show the distribution and morphology of co-cultured helixCAM HEK293 cells for three helixCAM pairs. Uninduced cells establish normal cell morphology, whereas induced cells form long, stretched bundles.

Figure 7.. helixCAMs enable tunable and simultaneous patterning of multiple cell types onto CC-patterned surfaces.

A. Workflow for CC surface patterning across a gradient and downstream automated image segmentation and analysis. Wells are coated with His-tagged CCs or His-tagged CC-GFP fusion protein solutions, then cells are added. Unbound cells were washed off, and the entire well was imaged at 4X magnification as a 2x2 tile. Images were segmented and cells were counted by CellProfiler. B. Normalized bound cell count is plotted against CC coating concentration for each of four CC-His peptides tested. Bound cell counts were normalized to the maximum in each channel to reduce variance from segmentation (raw counts in S21). Reported values are medians with N=4 and error bars are S.D. C. Normalized bound cell count is plotted against CC coating concentration for each of four CC-GFP-His peptides tested. All four proteins demonstrated strong capability for binding complementary helixCAM cells. Reported values are medians with N=4 and error bars are S.D. D. Workflow for simultaneous patterning of two cell types within one well. Two distinct CC-GFP-His protein solutions were added in a “G” or a “C” pattern. The two complementary helixCAM cell populations are added as a single mixture to cover the well surface. Unbound cells were then washed off and the bound cells were imaged at 4X magnification as a 2x2 tile. E. Dual-CC-patterned wells with two cell populations pre- and post-wash. Pre-wash, cell populations are evenly distributed and fully cover the plate surface. Post-wash, helixCAM cells complementary to the CC patterned at the G or C locations remain bound, but the non-complementary cells are not bound to those locations, and outside of patterned regions, few cells are observed (quantification of cells in each region in S22).