A light-gated transcriptional recorder for detecting cell-cell contacts
- Cancer Biology Program, Stanford University, United States
- Department of Genetics, Stanford University, United States
- Department of Neurology and Neurological Sciences, Stanford University, United States
- BridgeBio, United States
- Department of Pathology, Stanford University, United States
- Department of Pediatrics, Stanford University, United States
- Department of Neurosurgery, Stanford University, United States
- Howard Hughes Medical Institute, Stanford University, United States
- Department of Biology, Stanford University, United States
- Department of Chemistry, Stanford University, United States
- Chan Zuckerberg Biohub, United States
Figures
Figure 1
Design of TRACC (Transcriptional Readout Activated by Cell-cell Contacts).
(A) Schematic of TRACC. A ligand is presented on sender cells; a GPCR specifically activated by the selected ligand is expressed in receiver cells. The GPCR is fused to the …
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Figure 1—source data 1
Primary data for luminescence graphs in Figure 1C.
- https://cdn.elifesciences.org/articles/70881/elife-70881-fig1-data1-v1.xlsx
Figure 2 with 2 supplements
Using TRACC (Transcriptional Readout Activated by Cell-cell Contacts) to detect cell-cell contacts in HEK293T culture.
(A) Experimental design for co-plating sender and receiver cells. (B) Luciferase assay using the CCR6-CCL20 GPCR-ligand pair in trans. HEK293T cells transfected with the CCL20 sender construct were …
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Figure 2—source data 1
Primary data for luminescence and cell count graphs in Figure 2.
- https://cdn.elifesciences.org/articles/70881/elife-70881-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Additional quantification of HEK293T imaging in trans.
(A) Distribution of V5 intensity in V5-positive cells in Figure 2E. The distributions of sender-contacting cells and non-contacting cells are similar, suggesting that mCherry activation is …
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Figure 2—figure supplement 1—source data 1
Primary data for graphs in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/70881/elife-70881-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Evaluation of TRACC (Transcriptional Readout Activated by Cell-cell Contacts) in HEK293T cells using lentiviral transduction.
Confocal fluorescence imaging of sender cells co-plated with receiver cells using UAS-mCherry. TRACC components were introduced by lentivirus transduction. Approximately 8 hr after 10 min blue light …
Figure 3 with 1 supplement
Using TRACC (Transcriptional Readout Activated by Cell-cell Contacts) to detect contacts in neuron culture and HEK293T-neuron co-culture.
(A) Constructs used in TRACC in neuron culture. CCR6 is the GPCR and CCL20 is its activating peptide ligand. For expression in neurons, the transcription factor (TF) is changed from Gal4 to tTA and …
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Figure 3—source data 1
Primary data for luminescence graphs in Figure 3.
- https://cdn.elifesciences.org/articles/70881/elife-70881-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Additional characterization of TRACC (Transcriptional Readout Activated by Cell-cell Contacts) constructs in neuron culture.
(A) Confocal imaging of receiver constructs expressed in primary rat cortical neurons. Neurons were fixed and immunostained with anti-VP16 and anti-V5 to detect the GPCR and arrestin components, …
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Figure 3—figure supplement 1—source data 1
Primary data for colocalization and luminescence graphs in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/70881/elife-70881-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 1 supplement
Using TRACC (Transcriptional Readout Activated by Cell-cell Contacts) to detect contacts in DIPG (diffuse intrinsic pontine glioma) culture.
(A) Optimization of TRACC components in transposon-integrated SU-DIPG-VI stable cell lines. We compared TRACC constructs containing eLOV or hLOV, and WT TEVp, uTEV1, or uTEV2. Cells were plated and …
Figure 4—figure supplement 1
Quantification of activation in diffuse intrinsic pontine glioma (DIPG) stable lines.
(A) Quantification of mCherry activation upon recombinant CCL20 addition and light stimulation for each transposon-integrated DIPG stable line. 97–208 cells were analyzed for each condition. (B) …
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Figure 4—figure supplement 1—source data 1
Primary data for cell count graphs in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/70881/elife-70881-fig4-figsupp1-data1-v1.xlsx
