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Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics

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Sci. Signal.  19 Apr 2016:
Vol. 9, Issue 424, pp. rs3
DOI: 10.1126/scisignal.aad4149
  • Fig. 1 Determination of the molar concentrations of actin regulators in 5C.C7 T cells.

    (A and B) 5C.C7 T cells that were left untransduced (−) or were retrovirally transduced to express Arp3-GFP (+) were lysed, resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) together with the indicated quantities of purified Arp3 protein, and analyzed by Western blotting with antibody against Arp3 (A). Band intensities from the Arp3 Western blot were quantified and normalized to those corresponding to actin, which was a loading control for the cell extracts (B). Arp3 amounts in the cell extracts were determined from the calibration curve. Data are from a single experiment and are representative of three experiments. Black vertical line indicates noncontiguous lanes. Raw data for other actin regulators are shown in fig. S6. (C) For each of the indicated actin regulators, protein abundance in 5C.C7 T cells was determined from three measurements, the average endogenous protein amount in nontransduced cells (black line in the middle of the colored box) as derived from experiments such as those shown in (A) and (B), the 5 and 95% percentile of expression (colored bars, fig. S6), and the average combined amount of endogenous and GFP-tagged protein in retrovirally transduced cells [colored bar in the samples labeled OE (overexpression)], as derived from experiments such as those shown in (A) and (B). For actin regulators that were largely excluded from the nucleus (all but cofilin; Fig. 2), cytoplasmic volume was used in the conversion of the amounts of actin regulator in T cells to molar concentrations.

  • Fig. 2 Representative imaging data of actin regulators.

    (A to I) Representative interactions of 5C.C7 T cells expressing the indicated GFP fusion proteins with CH27 cells loaded with 10 μM MCC at the indicated times relative to the formation of a tight cell-cell conjugate. Top: DIC images. Bottom: Top-down maximum projections of 3D fluorescence data are shown in a rainbow-like false-color intensity scale (increasing from blue to red). Scale bars, 5 μm. Images are representative of 49 to 134 cells per protein from four to seven experiments.

  • Fig. 3 Illustration of the image analysis pipeline and resulting spatiotemporal models.

    (A) Image 1: Brightfield image centered on a T cell–APC conjugate. Images 2 to 8: Single slices of 3D images that are approximately perpendicular to the immunological synapse, which is shown facing upward in images 5 to 8. Image 2: False-colored raw coronin 1A–GFP fluorescence image. Image 3: Cell shape extracted by the segmentation algorithm. Image 4: Segmented intensity image. Image 5: Aligned segmentation (immunological synapse facing approximately upward). Image 6: Aligned segmented intensity image. Image 7: Standard template shape. Image 8: Segmented intensity image deformed into the shape of a standardized cell. (B) Illustrations of spatiotemporal models for cofilin (top), MRLC (middle), and WAVE2 (bottom), which have distinct subcellular distributions at different times. Each panel contains slices perpendicular to the immunological synapse of the full model at 0 (left) or 180 s (right) after synapse formation for each sensor. Within a panel, slices are shown starting at the upper left corner and moving horizontally to the upper right, then wrapping to the lower left corner and continuing to move horizontally toward the lower right; these correspond to z positions from one side to the other relative to the midline of the cell. The models were constructed from 49 to 134 cells from four to seven experiments (see table S1).

  • Fig. 4 Hierarchical clustering of full models constructed for nine sensors under full stimulus and costimulation blockade conditions.

    Left: The dendrogram shows the results of hierarchical clustering of all sensors for both conditions using the Euclidean distance between the concatenated maps of all voxels for all time points as the distance metric. The hierarchical clustering groups together sensors that have similar distributions across all time points, such as the group from FullStim - CPα1 through FullStim - WAVE2. Right: Each image shows a single slice from the 3D model of the distribution of the indicated sensor at the time shown on the horizontal axis. The slices are perpendicular to the synapse and through the middle of the model (the synapse is facing upward). The cophenetic coefficient of the dendrogram is 0.856.

  • Fig. 5 Kinetics of protein enrichment in the immunological synapse region.

    (A to C) To compute the enrichment of a sensor in the core volume of actin turnover, we defined the core region (A) that approximates the immunological synapse as described in Materials and Methods and calculated the ratio of the amount of the sensor in the region to the average amount across the whole cell. This was done for all time points under conditions of either full stimulus (B) or costimulation blockade (C). An asterisk in the legend indicates a sensor that had at least one time point with a statistically significant difference between the two different conditions. (D to G) Detailed comparisons of enrichment for full stimulus (solid lines) and costimulation blockade (dashed lines) for WAVE2 (D), cofilin (E), HS1 (F), and WASP (G). Asterisks indicate time points with statistically different values between the two conditions. Detailed statistics are provided in table S4.

  • Fig. 6 Active Rac and cofilin restore actin dynamics at the immunological synapse during costimulation blockade.

    (A and B) Enrichment of WAVE2 (A) and actin (B) in the immunological synapse region (calculated as described in Fig. 5) is shown for full stimulus conditions (green), costimulation blockade (red), and costimulation blockade in cells transduced with 250 nM cofilinca and 1 μM Rac1ca (blue). Note that the recruitment of WAVE2 was restored by active cofilin and Rac1. (C) Representative electron microscopy micrographs of cell-cell conjugates formed between a 5C.C7 T cell and a CH27 cell (APC) in the presence of 10 μM MCC peptide under the indicated conditions. The T cell membranes at the interface are highlighted in red. Micrographs are representative of 41 to 51 cells from four to five experiments. (D) Ratios of interface length [that is, the red outline in (C)] to diameter (that is, the straight line across) are given at an early undulation-rich time point (<2 min after tight cell coupling) and a late time point (3 to 5 min). T cells were treated with buffer-only (control), 250 nM active cofilinca, 1 μM Rac1ca, or both. APCs were treated with buffer-only (control) or with anti-CD80 (10 μg/ml) and anti-CD86 (10 μg/ml) antibodies (B7 blockade). On average, 24 cell couples were analyzed per condition, for a total of 244.

  • Fig. 7 Active Rac and cofilin restore defective LAT localization upon costimulation blockade.

    (A) Representative images of interactions between 5C.C7 T cells expressing LAT-GFP and CH27 cells loaded with 10 μM MCC at the indicated times relative to the formation of a tight cell conjugate are given for cells subjected to full stimulus, cells subjected to costimulation blockade with anti-CD80 antibody (10 μg/ml) and anti-CD86 antibody (10 μg/ml) (B7 blockade), and cells subjected to costimulation blockade and in which actin dynamics were restored with 250 nM cofilinca and 1 μM Rac1ca, as indicated. Top: DIC images. Bottom: Top-down maximum projections of 3D fluorescence data are shown in a rainbow-like false-color intensity scale (increasing from blue to red). Scale bars, 5 μm. (B) Comparison of the 4D distributions of LAT between the three conditions. The dendrogram measures the overall similarity of the distributions across all time points, indicating that the costimulation blockade condition is different from the other two conditions (the cophenetic coefficient of the dendrogram is 0.987). The maps shown are in the same order as in the dendrogram. (C) Comparison of the 3D distributions of LAT at each time point for different pairs of conditions. The Euclidean distance is shown between full stimulus and costimulation blockade (red triangles), full stimulus and reconstitution (green squares), and costimulation blockade and reconstitution (blue circles). (D) Enrichment of LAT in a cylindrical region at the center of the immunological synapse as a function of time for full stimulus (green squares), costimulation blockade (red triangles), and reconstitution (blue circles).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/424/rs3/DC1

    Fig. S1. Example images from the segmentation, alignment, and morphing pipeline.

    Fig. S2. Illustrations of the SDs of the spatiotemporal models.

    Fig. S3. Spatiotemporal models for actin distribution using two-point annotation.

    Fig. S4. Analysis of radial distributions confirms differences between myosin and actin.

    Fig. S5. Comparison of LAT spatiotemporal dynamics reveals restoration of costimulation by active Rac and cofilin.

    Fig. S6. Quantification of actin regulatory proteins in primary mouse T cells.

    Fig. S7. Volume measurements of primary mouse T cells.

    Fig. S8. Variation in cell shape and morphing across time points and conditions.

    Table S1. The number of imaging fields and manually marked immunological synapses used to construct the spatiotemporal model of each condition-sensor combination.

    Table S2. The manually tuned parameters used for both stages of segmentation by the active contour method.

    Table S3. Average amount of morphing required for each sensor at each time point.

    Table S4. Statistical testing of differences in sensor enrichment in the immunological synapse region between the full stimulus condition and the costimulation blockade.

    Movie S1. Four-dimensional maps of three representative sensors.

    Data file

  • Supplementary Materials for:

    Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics

    Kole T. Roybal, Taráz E. Buck, Xiongtao Ruan, Baek Hwan Cho, Danielle J. Clark, Rachel Ambler, Helen M. Tunbridge, Jianwei Zhang, Paul Verkade, Christoph Wülfing,* Robert F. Murphy*

    *Corresponding author. E-mail: christoph.wuelfing{at}bristol.ac.uk (C.W.); murphy{at}cmu.edu (R.F.M.)

    This PDF file includes:

    • Fig. S1. Example images from the segmentation, alignment, and morphing pipeline.
    • Fig. S2. Illustrations of the SDs of the spatiotemporal models.
    • Fig. S3. Spatiotemporal models for actin distribution using two-point annotation.
    • Fig. S4. Analysis of radial distributions confirms differences between myosin and actin.
    • Fig. S5. Comparison of LAT spatiotemporal dynamics reveals restoration of costimulation by active Rac and cofilin.
    • Fig. S6. Quantification of actin regulatory proteins in primary mouse T cells.
    • Fig. S7. Volume measurements of primary mouse T cells.
    • Fig. S8. Variation in cell shape and morphing across time points and conditions.
    • Table S1. The number of imaging fields and manually marked immunological synapses used to construct the spatiotemporal model of each condition-sensor combination.
    • Table S2. The manually tuned parameters used for both stages of segmentation by the active contour method.
    • Table S3. Average amount of morphing required for each sensor at each time point.
    • Table S4. Statistical testing of differences in sensor enrichment in the immunological synapse region between the full stimulus condition and the costimulation blockade.
    • Legend for movie S1

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    Technical Details

    Format: Adobe Acrobat PDF

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Four-dimensional maps of three representative sensors.
    • Data file

    Citation: K. T. Roybal, T. E. Buck, X. Ruan, B. H. Cho, D. J. Clark, R. Ambler, H. M. Tunbridge, J. Zhang, P. Verkade, C. Wülfing, R. F. Murphy, Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics. Sci. Signal. 9, rs3 (2016).

    © 2016 American Association for the Advancement of Science