Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors

Abstract

The Notch protein is one of the most mechanistically direct transmembrane receptors-the intracellular domain contains a transcriptional regulator that is released from the membrane when engagement of the cognate extracellular ligand induces intramembrane proteolysis. We find that chimeric forms of Notch, in which both the extracellular sensor module and the intracellular transcriptional module are replaced with heterologous protein domains, can serve as a general platform for generating novel cell-cell contact signaling pathways. Synthetic Notch (synNotch) pathways can drive user-defined functional responses in diverse mammalian cell types. Because individual synNotch pathways do not share common signaling intermediates, the pathways are functionally orthogonal. Thus, multiple synNotch receptors can be used in the same cell to achieve combinatorial integration of environmental cues, including Boolean response programs, multi-cellular signaling cascades, and self-organized cellular patterns. SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues.

Figure 1. Modular Configuration of Synthetic Notch (SynNotch) Receptors

(A) Conceptual design of synNotch receptor systems. Left: wild-type Notch has a large extracellular domain that binds to its ligand, Delta, expressed on opposing partner cells, and an intracellular transcriptional regulatory domain that is released by ligand induced cleavage. Arrows indicate the multiple proteolytic cleavage sites. Middle: Notch reporters have been built in which the intracellular domain is replaced by an orthogonal transcription factor. Right: in synNotch receptors both the extracellular and intracellular domains have been completely replaced, leaving only the small central regulatory region of Notch. Both novel inputs and outputs can be defined by using the synNotch architecture. (B) Modularity of the synNotch platform: the input and output domains from Notch can be swapped with diverse domains. On the extracellular side, diverse recognition domains can be used (antibody based, or peptide tags are shown) and on the intracellular side, diverse effector can be used (transcriptional activators with different DNA-binding domains are shown, as well as a transcriptional repressor). See also Figure S1.

Figure 2. SynNotch Receptors can be used to Program Contact-Dependent Transcriptional Regulation

(A) Synthetic Notch receptors can be used to detect endogenous disease antigens and induce the expression of a reporter gene. Mouse fibroblasts (L929 line) expressing anti-CD19/tTA synNotch are cultivated with K562 sender cells expressing Delta, CD19, or CD19 in the presence of the gamma-secretase inhibitor DAPT. FACS plots of the resulting GFP reporter intensity in receiver cells are shown. Inset shows an image of MDCK cells expressing the anti-CD19→ GFP synNotch, when co-cultivated for 24h with MDCK sender cells expressing CD19 (constitutively labeled with tagBFP). Only receiver cells in contact with (blue) sender cells activate the reporter and turn green. (B) Mouse fibroblasts (L929 line) with anti-CD19 synNotch with a transcriptional repressor intracellular domain (Gal4-KRAB) are co-cultivated with K562 sender cells. The receiver cells constitutively express GFP downstream of a SV40/UAS combined promoter. FACS plot of receiver cells is shown, in presence of K562 sender cells with or without CD19 expression, as indicated in figure. (C) Stimulation of mouse fibroblasts expressing anti-GFP synNotch with ligands in different formats. Anti-GFP synNotch receiver cells are stimulated for 1h with GFP either in soluble form, presented on a K562 sender-cell, or cis-presented on the receiver cell itself. The receiver cells show activation only when the ligand is present on an opposing surface and if they do not express the ligand in cis. The FACS data are recorded at 24h after the beginning of stimulation. See also Figure S2D for similar data on the anti-CD19 synNotch. FACS histograms include at least 10,000 cells for each condition.

Figure 3. SynNotch Receptors Function in Diverse Cell Types, Including Neurons and Lymphocytes

(A) Neurons. Primary hippocampal neurons were dissociated from E18 rat embryos and are nucleofected to express an anti-CD19 synNotch → GFP receptor and reporter. Neurons were plated on glass-bottom 35mm culture dish coated with Poly-D-Lysine and Laminin. 2 hours after neuron plating, sender cells (K562s) are added to the culture. Images are taken from live cells at day 4 after plating. On the right, representative images for neurons that are co-cultured with plain K562 cells (upper panel) or with CD19+ K562 sender cells (bottom panel) are shown. Neurons co-cultured with ligand presenting sender cells strongly induce GFP expression. See Figure S3 for quantification. (B) T cell line. Jurkat T clonal cell line engineered to stably express an anti-CD19→ GFP synNotch receptor system. Data on the right show fluorescence of clonal Jurkat cell population upon stimulation with CD19+ or CD19- sender cells (K562s) at t=24h. T cells are activated only when they encounter cell with the cognate ligand. FACS histograms include at least 10,000 cells for each condition.

Figure 4. SynNotch Receptors Yield Spatial Control of Diverse Cellular Behaviors

(A) Boundary detection in epithelial monolayer. Epithelial cells (MDCKs) are engineered as follows: sender cells express an extracellular GFP linked to a transmembrane domain; receiver cells are a clonal population that express the anti-GFP synNotch with LaG17 anti-GFP nanobody as extracellular domain, and Gal4-VP64 as intracellular domain, along with a UAS→BFP reporter construct. Sender cells are seeded into the receiver cell monolayer at a 1:50 ratio. Confocal images are taken 48h after plating of confluent monolayer. Representative pictures of high magnification and low magnification are shown, alongside with a representative line of intensity of BFP fluorescence over distance. Only receiver cells that are in contact with the green sender cells turn on the blue reporter, forming a ring around the sender cells. Receiver cells away from sender cells remain uninduced. Scale bars: higher magnification 20um; lower magnification 200um. (B) SynNotch activation of a myogenesis master regulator (myoD) in fibroblasts induces transdifferentiation in a spatially controlled manner. C3H mouse fibroblasts are engineered as follows: sender cells express extracellular CD19 linked to a transmembrane domain, plus a tagBFP marker; receiver cells express the anti-CD19 synNotch with tTA intracellular domain, along with a TRE→myoD cassette and a constitutive mCherry marker. Sender fibroblasts (blue) are plated first in a limited region of the plate and allowed to adhere to the plate; after 1h the receiver cells (red) are plated to uniformly cover the entire glass plate. Images show a large area of the co-culture and are still-frames from a movie that span the first 48h after co-plating (See also Movie 1). GFP channel shows the induction of myoD-GFP in received cells in a region that overlaps with the blue channel (sender cells). Receiver cells away from sender cells remain uninduced, and provide an internal control for the experiment (See also Figure S4A). A higher magnification of the field for the green channel is shown on the right, showing the induction of multinucleate myotubes (scale bar = 50um). (C) SynNotch can induce epithelial to mesenchymal transition in cultured epithelial cells. Epithelial cells (MDCKs) are engineered as follows: receiver cells express the anti-GFP synNotch with LaG17 anti-GFP nanobody as extracellular domain, and tTA as intracellular domain, along with a TRE→Snail-ires-BFP effector construct. Sender cells are GFP-expressing K562 cells. Representative bright field microscope images of epithelial cells before and after 48h from the addition of sender cells are shown (sender cells were removed before imaging). Scale bar = 20um. See Figure S4B for quantification and controls.

Figure 5. SynNotch Pathways are Orthogonal to One Another and Can be Used for Combinatorial Regulation

(A) SynNotch and wild-type Notch activate orthogonal signaling pathways. L929 mouse fibroblasts receivers are engineered to express (i) the wild-type Notch receptor with a tTA intracellular domain and a TRE→GFP reporter, and (ii) a synNotch receptor with anti-CD19 extracellular domain and Gal4-VP64 intracellular domain, and a UAS→tagBFP reporter. The graph on the right shows the clonal population of receiver cells fluorescence signal for the BFP and the GFP reporters in different conditions: black - untreated; blue - stimulated with CD19 expressing senders; orange - stimulated with delta senders; and red - stimulated with sender cells expressing both CD19 and delta. Sender cells are mouse L929 fibroblasts. See Figure S5A for quantification. (B) Multiple synNotch receptors are orthogonal to one another. L929 mouse fibroblast receiver cells are engineered to express (i) the anti-CD19 synNotch receptor with a tTA intracellular domain and a TRE→BFP reporter; and also (ii) the synNotch receptor with anti-CD19 extracellular domain and Gal4-VP64 intracellular domain, and a UAS→mCherry reporter. The graph on the right shows the receiver cell (clonal population) fluorescence signal for the BFP and the mCherry reporters in different conditions: black - untreated cells; red - stimulated with CD19 expressing sender cells; green - stimulated with GFP sender cells; and blue - stimulated with sender cells expressing both GFP and CD19. Sender cells are K562 cells. See Figure S5B for quantification. (C) Cells engineered with two synNotch AND gate respond only when both the inputs are present. L929 mouse fibroblasts receivers are engineered to express: (i) the anti-CD19 synNotch receptor with a tTA intracellular domain and a TRE promoter that drives the expression of the DNA-binding domain (DBD) of Gal4 fused to a leucine zipper domain, and (ii) the synNotch receptor with anti-GFP extracellular domain and the VP64 transcriptional activation domain fused to a complementary leucine zipper as intracellular domain, and (iii) a Gal4-responsive promoter driving a red fluorescent protein (mCherry). The graph on the right shows the normalized mCherry fluorescence collected from a clonal population of receiver cells in co-culture with different sender cells (K562), that express either the two ligands alone (GFP or CD19), or both ligands together. Activation occurs only in the presence of both the inputs. Data shown are the median and coefficient of variance of at least 10,000 cells per condition.

Figure 6. Multiple SynNotch Receptors can be used to Generate Multi-Layered Self-Organizing Epithelial Patterns

Epithelial cells (MDCKs) are engineered as follows: sender cells express extracellular GFP linked to a transmembrane domain; receiver cells express two synNotch receptors: (i) the anti-GFP synNotch with tTA intracellular domain, along with a TRE→CD19-mCherry effector cassette; (ii) the anti-CD19 receptor with Gal4-VP64 intracellular domain, and a UAS→tagBFP reporter. Here, the first synNotch receptor, when stimulated, induced the expression of the ligand for the second synNotch receptor. (A) Representative images are shown for the epithelial layer of sender cells and a clonal population of receiver cells co-cultivated at a 1:50 ratio for 10h (START) 34h (DAY 1) and 58h (DAY2). Scale bar = 20um. (B) Multiple images of different fields of view of the co-culture at day 2. Scale bar = 50um (C) Representative quantification of the fluorescence signal as calculated from the fluorescence images for a pattern around sender cells at day 2. Scale bar = 20um.

Figure 7. Modularity of SynNotch Receptors Expands Sensing/Response Engineering of Mammalian Cells

(A) Putative mechanism of activation of the synNotch receptors. As shown by Blacklow and others (Gordon et al., 2007), in native Notch, the LNR domains mask the protease cleavage site in the unbound conformation (left). When the ligand engages the receptor, mechanical force is thought to expose this protease site, initiating the multi-step process leading to release of the intracellular transcriptional domain (right). We propose that when the extracellular domain is replaced by a novel recognition domain (here an anti-GFP nanobody is shown), then proper cell-cell engagement of the cognate ligand (GFP on the opposing cell) can exert a similar force to expose the Notch core to proteolysis. (B) Alignment of LNR-containing molecules from different metazoan and pre-metazoan species shows that full length Notch (with delta binding domain) is only found in metazoans, but that proto-Notch genes that lack the delta binding domain are found in pre-metazoans such as choanoflagellates and capsaspora (Gazave et al., 2009; King et al., 2008). (C) The modularity of the synNotch receptor platform allows the user to specify the extracellular cues the cells now respond to, as well as the cellular responses that are induced downstream of receptor activation.