Project Explorer

Dynamics of perturbagen responses in living single cells

Somponnat Sampattavanich1,2 and Peter K. Sorger1

1 HMS LINCS Center, Harvard Medical School, Boston, MA; 2 Current address: Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

(This dataset is unpublished. Please see our terms of use.)

Many signal transduction networks exhibit pulsatile behavior whose amplitude and dynamics regulate downstream processes (see Levine and Elowitz, 2013 1) In this unpublished dataset, human mammary epithelial TCT cells expressing a FRET-based reporter of ERK activity were treated with EGF and various small molecule inhibitors targeted against the ErbB kinases.

Interactive lookup table

Drug
concentration
Kinase inhibitor Kinase inhibitor
+ Batimastat @ 10.0 μM
Gefitinib Lapatinib Canertinib Gefitinib Lapatinib Canertinib
0.1 μM Gefitinib @ 0.1 μM Lapatinib @ 0.1 μM Canertinib @ 0.1 μM Gefitinib @ 0.1 μM  + Batimastat @ 10.0 μM Lapatinib @ 0.1 μM  + Batimastat @ 10.0 μM Canertinib @ 0.1 μM  + Batimastat @ 10.0 μM
0.25 μM Gefitinib @ 0.25 μM Lapatinib @ 0.25 μM Canertinib @ 0.25 μM Gefitinib @ 0.25 μM  + Batimastat @ 10.0 μM Lapatinib @ 0.25 μM  + Batimastat @ 10.0 μM Canertinib @ 0.25 μM  + Batimastat @ 10.0 μM
0.5 μM Gefitinib @ 0.5 μM Lapatinib @ 0.5 μM Canertinib @ 0.5 μM Gefitinib @ 0.5 μM  + Batimastat @ 10.0 μM Lapatinib @ 0.5 μM  + Batimastat @ 10.0 μM Canertinib @ 0.5 μM  + Batimastat @ 10.0 μM
1.0 μM Gefitinib @ 1.0 μM Lapatinib @ 1.0 μM Canertinib @ 1.0 μM Gefitinib @ 1.0 μM  + Batimastat @ 10.0 μM Lapatinib @ 1.0 μM  + Batimastat @ 10.0 μM Canertinib @ 1.0 μM  + Batimastat @ 10.0 μM
2.5 μM Gefitinib @ 2.5 μM Lapatinib @ 2.5 μM Canertinib @ 2.5 μM Gefitinib @ 2.5 μM  + Batimastat @ 10.0 μM Lapatinib @ 2.5 μM  + Batimastat @ 10.0 μM Canertinib @ 2.5 μM  + Batimastat @ 10.0 μM
5.0 μM Gefitinib @ 5.0 μM Lapatinib @ 5.0 μM Canertinib @ 5.0 μM Gefitinib @ 5.0 μM  + Batimastat @ 10.0 μM Lapatinib @ 5.0 μM  + Batimastat @ 10.0 μM Canertinib @ 5.0 μM  + Batimastat @ 10.0 μM
10.0 μM Gefitinib @ 10.0 μM Lapatinib @ 10.0 μM Canertinib @ 10.0 μM Gefitinib @ 10.0 μM  + Batimastat @ 10.0 μM Lapatinib @ 10.0 μM  + Batimastat @ 10.0 μM Canertinib @ 10.0 μM  + Batimastat @ 10.0 μM

Purpose and dataset details

The purpose of this dataset was to delineate the differences between clinical grade inhibitors of ErbB signal transduction that have fundamentally different chemistry. Gefitinib binds reversibly to the active kinase conformation, Lapatinib binds “irreversibly” to the inactive kinase conformation (that is, it dissociates very slowly2) and Canertinib is a covalent modifier. Batimastat is a matrix metalloproteinase inhibitor (MMPI) that blocks autocrine shedding of ligand.

TCT cells expressing EKAREV, a FRET-based reporter of ERK activity3, were serum-starved overnight and at the same time treated with Batimastat to inhibit autocrine secretion of EGFR ligands or left untreated. They were then stimulated with EGF at 100 ng/ml and treated 15 minutes later with inhibitors of ErbB receptor tyrosine kinases at varying concentrations. Images were acquired approximately every 7 minutes over a time course of 20 hours. To track cells through time, the nucleus and cytoplasm were segmented using a nuclear-localized mCherry fluorescent protein. To determine the level of ERK activity, we calculated the mean intensity of the EKAREV FRET signal in the cytoplasm divided by the CFP signal.

The individual .csv files available through the lookup table above have one row per cell per time point, with the following 5 columns:

  1. cell_id: A unique integer for each distinct cell detected by the segmentation and tracking algorithm.
  2. time: Time in minutes since the beginning of the image acquisition.
  3. x: Cell x coordinate in pixels within the image frame, counting from the left side.
  4. y: Cell y coordinate in pixels within the image frame, counting down from the top.
  5. erk_signal: The calculated value of the ERK signal — mean intensity of the EKAREV FRET signal divided by the CFP signal across the cytoplasm.

Note that cell coordinates of (-1, -1) indicate that the tracking algorithm failed to detect a given cell at the corresponding time point, or that the cell is a sister cell whose mother cell divides at a later time. (-2, -2) indicates that the cell was detected but classified as dead.

Available data and software

Data Interactive lookup table across all conditions that provides access to computed trajectory plots, movies of cells showing ERK activity, and a .csv file of the position and ERK signal across time for each cell analyzed. View table
Data Computed ERK trajectories with image segmentation information. Download (2.5 GB zip)
Data Raw image data via our OMERO server. View data
Software A link to our HMS LINCS GitHub for access to the code for the interactive lookup table presented on this project exploration website. hmslincs at GitHub

References

  1. Levine, J. H., Lin, Y. and Elowitz, M. B. (2013) Functional Roles of Pulsing in Genetic Circuits. Science. 342(6163), 1193-1200. doi:10.1126/science.1239999 PMID:24311681 PMCID:PMC4100686
  2. Kleiman, L. B., Maiwald, T., Conzelmann, H., Lauffenburger, D. A. and Sorger, P. K. (2011) Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding. Mol Cell. 43(5), 723-737. doi:10.1016/j.molcel.2011.07.014 PMID:21884975 PMCID:PMC3222462
  3. Komatsu, N., Aoki, K., Yamada, M., Yukinaga, H., Fujita, Y., Kamioka, Y., Matsuda, M. (2011) Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol Biol Cell. 22(23), 4647-4656. doi:10.1091/mbc.E11-01-0072 PMID:21976697 PMCID:PMC3226481