A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms
- Eikon Therapeutics Inc, United States
- University of California, Berkeley, United States
Figures
Figure 1 with 3 supplements
Benchmarking a high-throughput single-molecule tracking platform.
(A) Diffusion state probability distributions from three cell lines expressing Histone H2B-Halo, Halo-CaaX, or Halo alone. Shaded bins represent the diffusive states characteristic of each cell …
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Figure 1—source data 1
TIFF versions of the images in Figure 1.
- https://cdn.elifesciences.org/articles/93183/elife-93183-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Overview of the high-throughput single-molecule tracking (htSMT) workflow.
(A) Schematic representation of the end-to-end automated htSMT process, beginning with the physical preparation of the sample; image collection on a custom-assembled super-resolution microscope; …
Figure 1—figure supplement 2
Characterization of high-throughput single-molecule tracking (htSMT) system performance.
(A) Distribution of localization error measured across multiple independent wells using Histone H2B-Halo cells. Median localization error of 39 nm. (B) Number of measurable nuclei in a 94 µm by 94 …
Figure 1—video 1
Example SMT field of view as shown in Figure 1.
A representative mixture of H2B-Halo, Halo-CaaX, and free Halo expressing cells. Video is played back at 10 frames per second, 10× slower than real time.
Figure 2 with 1 supplement
Chromatin binding by steroid hormone receptors (SHRs) is affected by compound treatment.
(A) Distribution of diffusive states for Halo-AR, Halo-ER, Halo-GR, and Halo-PR in U2OS cells before and after stimulation with an activating ligand. The area in the shaded region is fbound. Shaded …
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Figure 2—source data 1
Tabular data to generate plots from Figure 2 and related figure supplements.
- https://cdn.elifesciences.org/articles/93183/elife-93183-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Activation of steroid hormone receptors (SHRs) changes free diffusion and impacts downstream gene expression.
(A) Cartoon illustration of SHR function. Under basal conditions, SHRs are sequestered in complex with HSP90 and other co-factors. Upon ligand binding, the receptor dissociates from the inactive …
Figure 3 with 4 supplements
Bioactive screen results of change in estrogen receptor fbound for two biological replicates, each with 2 well replicates per compound of 5067 compounds.
Select inhibitors are grouped and uniquely colored by pathway or target. Error bars are SEM of all replicates.
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Figure 3—source data 1
List of compounds as part of the Sellek L-1700 bioactive set along with the ‘Target’ and ‘Pathway’ annotations as identified by Selleck.
- https://cdn.elifesciences.org/articles/93183/elife-93183-fig3-data1-v1.csv
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Figure 3—source data 2
Tabular data to generate plots from Figure 3 and related figure supplements.
- https://cdn.elifesciences.org/articles/93183/elife-93183-fig3-data2-v1.xlsx
Figure 3—figure supplement 1
Experimental design for bioactive molecule screen.
(A) Cumulative number of trajectories as a function of imaging time for DMSO-treated cells labeled with 20 pM JF549. Shaded error bars are 1 SD. (B) Median of the jump length distribution, reported …
Figure 3—figure supplement 2
Screen of bioactive molecules produces robust data with good assay performance.
(A) Change in fbound repeatability of screening results assessed over two biological replicates. Compounds in magenta were identified as active if their magnitude of change in fbound when averaged …
Figure 3—figure supplement 3
Contributions of different sources of variability of jump length in the high-throughput single-molecule tracking (htSMT) bioactive compound screen against ER.
(A) Change in fbound for 30 known estrogen receptor (ER) interaction molecules circled in (B), ordered by the magnitude of effect. Error bars are the SEM. (B) Variance in mean jump length after …
Figure 3—figure supplement 4
Antagonists of estrogen receptor (ER).
(A), progesterone receptor (PR) (B), androgen receptor (AR) (C), and glucocorticoid receptor (GR) (D) show distinct effects on target protein dynamics. Change in fbound after addition of 1 µM …
Figure 4 with 1 supplement
Selective estrogen receptor (ER) modulators and degraders induce DNA-binding measurable through high-throughput single-molecule tracking (htSMT).
(A) Diffusion state probability distribution for ER treated with 100 nM of exemplified selective ER modulators (SERMs) and selective ER degraders (SERDs). Each state distribution is generated from …
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Figure 4—source data 1
Tabular data to generate plots from Figure 4 and related figure supplements.
- https://cdn.elifesciences.org/articles/93183/elife-93183-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders (SERDs) decrease free diffusion and increase fbound rapidly after addition.
(A) Normalized occupation of diffusive states (diffusion coefficient 0.2–100 µm2/s) for 100 nM SERD- or SERM-treated samples compared with DMSO. Histograms are normalized to integrate to 1. Shaded …
Figure 5 with 1 supplement
High-throughput single-molecule tracking (htSMT) can be used to determine chemical structure–activity relationships.
(A) Correlation of potency measured by estrogen receptor (ER) degradation and cell proliferation in MCF7 cells (black) and T47d cells (magenta) for compounds in the GDC-0927 structural series. Cell …
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Figure 5—source data 1
PDF report of western blot produced by Jess system (Protein Simple).
- https://cdn.elifesciences.org/articles/93183/elife-93183-fig5-data1-v1.zip
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Figure 5—source data 2
TIFF versions of the images in Figure 5.
- https://cdn.elifesciences.org/articles/93183/elife-93183-fig5-data2-v1.csv
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Figure 5—source data 3
Images to generate plots from Figure 5 and related figure supplements.
- https://cdn.elifesciences.org/articles/93183/elife-93183-fig5-data3-v1.zip
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Figure 5—source data 4
Tabular data to generate plots from Figure 5 and related figure supplements.
- https://cdn.elifesciences.org/articles/93183/elife-93183-fig5-data4-v1.xlsx
Figure 5—figure supplement 1
GDC-0927 structural variants characterized by estrogen receptorER degradation or cell proliferation assays.
(A) Western blot of the ER-expressing breast cancer cell lines MCF7 and T47d compared to ER expression in ER-null lines SK-BR-3 or U2OS. Samples were treated with fulvestrant for 24 hr prior to …
Figure 6 with 3 supplements
Bioactive molecules targeting pathways associated with estrogen receptor (ER) affect its dynamics.
(A) Bioactive screen results with select inhibitors grouped and uniquely colored by pathway. Change in fbound across a 12-point dose titration of three representative compounds targeting each of …
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Figure 6—source data 1
Tabular data to generate plots from Figure 6 and related figure supplements.
- https://cdn.elifesciences.org/articles/93183/elife-93183-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Deeper investigation of bioactive screening data identifies cutoff for active molecules.
(A) Examples of compound effects on estrogen receptor (ER) fbound from a dose titration experiment. Ninety-two compounds from the primary screen are ranked based on the magnitude of their effect. …
Figure 6—figure supplement 2
Some pathway inhibitors modulating estrogen receptor (ER) dynamics are specific to ER.
(A) Change in fbound of ER following treatment with inhibitors of HSP90, the proteasome, mTOR, and cyclin-dependent kinase (CDK) from the initial bioactive screen. CDK inhibitors were further …
Figure 6—figure supplement 3
Dose titration plots of ER(S104A/S106A/S118A) with mTOR and CDK9 compounds.
Each point represents the mean and SEM of three biological replicates.
Videos
Video 1
Example field of view of single-molecule tracking (SMT) from Halo-ER cells.
Video is played back at 10 frames per second, 10× slower than real time.
Video 2
Example field of view of single-molecule tracking (SMT) from Halo-ER cells treated with 25 nM estradiol for 1 hr prior to imaging.
Video is played back at 10 frames per second, 10× slower than real time.
