Research Article

Visualizing dynamic microvillar search and stabilization during ligand detection by T cells

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Science  12 May 2017:
Vol. 356, Issue 6338, eaal3118
DOI: 10.1126/science.aal3118
  • Dynamics of T cell antigen search.

    (A) Microvilli distribute with fractal organization on T cell surfaces and efficiently scan their surroundings. Microvilli are stabilized in the immunological synapse upon antigen recognition. (B) Overlays of an isolated T cell at three time points indicate the dynamic nature of microvilli. (C) The en face view of an immune synapse at three time points highlights a microvillus stabilized upon antigen recognition. Scale bar, 5 μm.

  • Fig. 1 Effective surface scanning by T cell protrusions.

    (A) Surface projection rendering of a mouse T cell imaged by LLS (left). Isolated tracks for individual microvilli (right). See also Movie 1 and movie S1. Tracking was assisted in some cases by image stabilization, to account for modest cell drift (fig. S1A). (B) Track speeds, (C) turning angles, and (D) MSD for microvilli (n > 232 microvilli for all time points, across three cells). The curve follows the power law of MSD ~ tα for t < 15 s, α = 1.13 and for t > 15 s, α = 0.85. Error bar, SD. (E) Three-color overlay of a subsection of an isolated T cell at three time points. Scale bar, 1 μm. (F) Fractal analysis: plot of the number of boxes needed to cover the active area of a T cell versus length of the box (L). The slope of the fit line is used to determine the fractal dimension (Fd). (Inset) Fd over time for a single T cell. (G) Representation of the masking and threshold method used to calculate the instantaneous and cumulative coverage of T cell surfaces and IS ROIs. Antibody to CD45 was used to label cell surfaces in this example. Scale bar, 5 μm. See also movie S2. (H) (Left axis) Percentage of surface coverage (cumulative in blue, instantaneous in red) of an isolated T cell throughout time from LLS in vitro. (Right axis) Percentage of T-APC contacts remaining versus time, from two-photon imaging data in vital lymph nodes.

  • Fig. 2 Altered microvillar cumulative coverage in response to ligand detection.

    (A) (Left) Image of a T cell in interaction with a peptide-loaded APC (unlabeled). The nonsynaptic (non-IS) plane of interest is outlined in blue; the synapse (IS) is outlined in green. (See also Movie 2 for another example of an immunological synapse formed between a T cell and an APC with both cells labeled.) (Right) Thresholded images of the non-IS (blue) and IS (green) contact face at a single time point and a cumulative image when coverage had reached 75%. (B) (Left) Percent surface coverage throughout time for a non-IS and IS region of a single T cell. (Right) The distribution of average surface coverage for non-IS regions and IS regions taken from multiple T cells. Error bar, SD. Data are pooled from four separate experiments. (C) Percent cumulative coverage comparing the non-IS and IS region of a single Tcell. Line indicates 75% surface coverage. (D) Comparison between the distributions of T75% for isolated T cells, IS regions with no antigen, non-IS regions, and IS regions of multiple individual T cells. Error bar, SD. Data are pooled from four separate experiments. The significance test was an unpaired t test.

  • Fig. 3 Altered microvillar regional dwell time in response to ligand detection.

    (A) The scanning method for measuring regional dwell time over a contact surface. (B) Variation in occupancy, measured for a 25-pixel–sized area, illustrating a cutoff at 50% occupancy. Short-lived protrusions are highlighted with the black arrows, and a longer-lived scanning event is shown in gray. (C) A comparison of the average regional dwell time, defined as in (B), for isolated T cells, IS with no antigen, non-IS regions, and IS regions in different T cells. Teal line connects individual cells. Shaded regions denote generalized half-life for weak-agonist pMHCs. Error bar, SD. Data are pooled from four separate experiments. The significance test was an unpaired t test. (D) Membrane topology of the synaptic region of a T cell, labeled with antibody to CD45-Alexa488, interacting with a peptide-loaded APC at various time points. Stable protrusions are highlighted in red; an example transient protrusion is indicated with a black arrowhead.

  • Fig. 4 TCR-occupied projections are stabilized.

    (A) Schematic representation of bilayer-bound Qdots for SCM-based imaging. Qdots larger than the ~15-nm TCR-pMHC length are excluded when membranes closely appose. See also figs. S6 and S7. (B) Images of TCR, bilayer-bound QD605 streptavidin conjugates, and TCR/streptavidin overlays from cells fixed during synapse formation. Scale bars, 5 μm. Dashed box region is shown at the bottom. Arrow points to a contact with no apparent TCR microcluster. (C) Normalized intensities of Qdot605 and TCR line scan for the light blue line in (B). (D) Number of contacts and total IS area during IS formation. (E) Average contact density over time during IS formation. Error bars, SD. Plot represents 28 cells pooled from seven independent experiments. (F) Frequency histogram of the average intensity of all the contacts throughout the observation period. The blue Gaussian curve represents the intensity distribution expected from background sources for this region of interest. The green dashed line represents the cutoff from TCR+ to TCR. (G) Dwell times in the TCR+ and TCR populations within an 18.9-s observation window. Error bar, SD. The significance test was an unpaired t test. (H) (Top) Overlay of TCR+ (yellow) with the TCR contact populations (red) at two different time points. (Bottom) Time projection of TCR+ (left) and TCR (right). Colors represent the percentage of time of each pixel occupied by contacts over the length of the movie. (I) (Top) Image of a nascent H57-labeled TCR microcluster and corresponding contact image. Dashed line indicates the subsequent microcluster path. (Bottom) Kymograph generated from the microcluster path in each channel and overlay. (J) As in (I) but for a ZAP70-GFP cluster.

  • Fig. 5 Signaling independence of TCR-mediated protrusion stabilization.

    (A) Fura-2 calcium ratios measured in ZAP70(AS)/OT-I T cells on activating bilayers after treatment with vehicle or 10 μM 3-MB-PP1. Plots represent the average Fura-2 ratio measured in more than 100 cells pooled from two separate experiments. Error bar, 95% confidence interval. (B) QD605-streptavidin and TCR TIRF time lapse images of ZAP70(AS)/OT-I synapses after treatment with 10 μM 3-MB-PP1. Scale bars, 5 μm. (C) TCR intensity in contacts in ZAP70(AS)/OT-I T cell synapses. 3-MB-PP1, N = 12; DMSO vehicle, N = 9 cells. Data are pooled from three experiments. Error bars, SEM. (D) SCM images of bilayer-bound QD605-SA during encounter of OT-I T cell, without bilayer-bound pMHC but with ICAM-1. Scale bar, 5 μm. The boxed region measured 5 × 2 μm. (E) (Left) Boxed region shown in (D). Light blue dashed line indicates direction of cell motility. (Right) Normalized intensity line scans for the light blue dashed line shown at left. The vertical gray dashed lines correspond to the starting point along the line for two contacts. The contacts moved against the direction of cell motility. (F) Displacement vectors for contacts shown in (D). For clarity, only tracks longer than 1 μm are shown. (G) Mean lifetimes for contacts based on the bilayer-bound pMHC and TCR occupancy. For the N4, the contacts are categorized based on whether they acquired TCR microclusters. For the G4 and null conditions, TCR+ microclusters were not observed. Data are pooled from at least three separate experiments for each condition.

  • Fig. 6 Cytoskeletal independence of TCR-mediated protrusion stabilization.

    (A) Bilayer-bound QD605-streptavidin, TCR, and IRM time-lapse images of an OT-I IS during LatB challenge. LatB (1 μM) was added after acquisition of the t = 0 s image. Scale bar, 5 μm. At right, a zoomed view of a region of the synapse 3 min after LatB addition. The white arrow points to a remaining patch of contacts with TCR microclusters and low IRM intensity. (B) Number of contacts (orange) and TCR microclusters (green) before and after LatB challenge for the IS shown in (A). (C) The fraction of contacts occupied by TCRs before and after LatB challenge for the IS shown in (A). (D) Average fraction of contacts occupied before and after LatB treatment. Each plot point represents the average fraction of contacts occupied in a cell measured over the 60 s before (pre) and after (post) LatB treatment. N = 7 cells pooled from three independent experiments. The significance test was a paired t test. (E and G) (Top) QD605-streptavidin SCM and phalloidin TIRF images of OT-I IS on activating bilayers. IS in (E) was fixed 3 min after addition of the cell to the bilayer. IS in (G) was fixed after 6 min and represents a mature synapse. (Bottom) Images of inset regions from images at top. Inset regions measure 5 × 2 μm. Images are representative of at least 25 cells imaged in three independent experiments. (F and H) Normalized line scan intensities for the dashed white line at bottom in (E) and (G), respectively.

  • Movie 1 Microvillar dynamics in T cells.

    Surface projection of an OT-I T cell labeled with antibody to CD45-Alexa488 and imaged by LLS microscopy. Time resolution is 2.25 s. For this movie, the cell’s position in space was stabilized using the center of mass (see also fig. S1A).

  • Movie 2 The immunological synapse formed between a T cell and an APC.

    This movie shows an IS formed between a T cell and an APC captured by time-lapse LLS microscopy. For synapse side view, the T cell, labeled with antibody to CD45-Alexa488, is shown in red; the APC (BMDC), labeled with a lipophilic membrane dye DiD, is shown in green. The top right panel shows the en face view of this synapse. The lower left panel shows a 3D surface of the synapse rendered in Imaris. The lower right panel shows the synapse with the z scale color coded.

  • Movie 3 Comparison of dynamic behaviors of TCR+ and TCR contacts.

    Overlay of the TCR+ contact population (shown in yellow) with the TCR contact population (shown in red), as defined in Fig. 4F. Movie demonstrates the stability of the TCR+ compared to TCR contacts.

  • Movie 4 SCM time-lapse imaging of IS contacts and TCRs of a ZAP70(AS)/OT-I T cell during 3-MB-PP1 treatment.

    Time-lapse movie of QD605-streptavidin SCM and TCR TIRF images of a ZAP70(AS)/OT-I synapse formed on an activating bilayer after treatment with 10 μM 3-MB-PP1 to inhibit ZAP70(AS). Top to bottom: TCR TIRF, QD605-streptavidin SCM, and IRM images. TCRs were stained with Alexa Fluor 488–conjugated H57-597 antibody to TCRβ. Movie region measures 14 × 14 μm. Total time elapsed is 257 s. Original acquisition rate was 1 frame/sec. Movie is played at 12 frames/sec (12× real time).

  • Movie 5 SCM time-lapse imaging of IS contacts and TCRs during latrunculin B challenge.

    Time-lapse movie created from QD605-streptavidin SCM and TCR TIRF images of an OT-I synapse formed on an activating bilayer. Top to bottom: QD605-streptavidin, IRM, and TCR images. TCRs were stained with Alexa Fluor 488–conjugated H57-597 antibody to TCRβ. Movie region measures 17.5 × 17.5 μm. Total time elapsed is 4 min. Latrunculin was added after ~45 s. Original acquisition rate was 1 frame/sec. Movie is played at 12 frames/sec (12× real time).

  • Visualizing dynamic microvillar search and stabilization during ligand detection by T cells

    En Cai, Kyle Marchuk, Peter Beemiller, Casey Beppler, Matthew G. Rubashkin, Valerie M. Weaver, Audrey Gérard, Tsung-Li Liu, Bi-Chang Chen, Eric Betzig, Frederic Bartumeus, Matthew F. Krumme

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Figs. S1 to S9
    • Legends for Movies S1 to S9

    Images, Video, and Other Media

    Movie S1
    Microvillar dynamics in T cells
    Movie S2
    Saturating coverage by ongoing microvillar scanning
    Movie S3
    Stable and labile protrusions in an IS revealed by LLS imaging
    Movie S4
    T cells forming IS on an activating lipid bilayer imaged by LLS microscope make multiple microvillar-like contacts
    Movie S5
    SCM time-lapse imaging of IS contacts and TCRs
    Movie S6
    SCM time-lapse imaging of IS TCR/contact merging dynamics for two contacts that are both TCR+
    Movie S7
    SCM time-lapse imaging of TCR+ and TCR- contacts merging
    Moviei S8
    SCM time-lapse imaging of contact splitting and merging dynamics
    Movie S9
    SCM time-lapse imaging of surface contacts generated by multiple cell types demonstrates a diversity of search patterns

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