Fig 1.
Critical regulators of T cell activation show transient localization to the T cell lamellum.
(A) The panel represents six categories to classify spatiotemporal signaling features that reflect underlying cell biological structures. The APC above the T cell is not shown. Central reflects a large central signaling complex, peripheral the part of the actin network stabilizing the interface edge. Diffuse reflects cortical accumulation, invagination enrichment in a transient large T cell invagination suggested to contribute to early signal resetting [11]. Asymmetric reflects individual small lamella and the lamellal pattern is characterized here. (B-D) Representative interactions of 5C.C7 T cells expressing (B) SLP-76-GFP (S1 Video) (C) PLCδPH-GFP (PIP2 sensor)[3] or (D) Vav1-GFP (S2 Video) with peptide-loaded CH27s (10μM MCC) over the indicated time relative to formation of a tight cell conjugate are given. DIC images are shown on top and top-down maximum projections of 3D fluorescence data are shown in the bottom panels in a rainbow-like, false-color intensity scale (increasing from blue to red)(scale bar = 2μm). (E, F) 5C.C7 T cells expressing GFP-tagged signaling intermediates or sensors were stimulated with peptide loaded CH27 B cell lymphoma APCs (10μM MCC). The percentage of all T cells analyzed showing accumulation in defined patterns (A)[3] are shown for (E) SLP-76-GFP (number of cell couples analyzed across multiple independent experiments, n = 56) and (F) PLCδPH-GFP (PIP2 sensor) (n = 115). Vav1-GFP data are similarly quantified in Figure 3C of [10]. (G) Amounts relative to maximum as a function of the distance from the interface measured from live cell conjugates expressing GFP-actin (n = 15), PLCδPH-GFP (PIP2) (n = 18), SLP-76-GFP (n = 19), Vav1-GFP (n = 18) and TCRζ-GFP (n = 15) measured at the 1min time point across the entire interface are given. Error bars are s.e.m.
Fig 2.
Actin displays transient lamellal accumulation.
(A, B) Phalloidin stained 5C.C7 T cell:CH27 APC conjugates imaged by STED 1-2min (A) and >2min (B) after cell conjugate formation are shown in a rainbow-like color scale (scale bars = 2μm)(see methods for precise time point definition). (C) Actin amounts relative to maximum as a function of the distance from the interface in the periphery (outer 25% of the interface diameter) and center (middle 50% of the interface) measured from fixed Phalloidin stained 5C.C7 T cell:CH27 APC conjugates at an early (1-2min, number of cell couples analyzed across multiple independent experiments, n = 26) and late (2-5min, n = 16) time point (see methods for precise time point definition) by STED and live cell conjugates expressing GFP-actin (n = 15, same cells as in Fig 1G) or F-tractin-GFP (n = 19) (both measured at 1min) are given. (D) A representative interaction of a 5C.C7 T cell expressing F-tractin-GFP (S3 Video) is given similar to Fig 1B. In addition, an en face view of the T cell is shown at the bottom. (E) The pattern classification graph is given for F-tractin-GFP similar to Fig 1E (n = 51). (F) The depth to width ratio of resolved phalloidin stained F-actin structures (>135% above cellular background) in early (n = 12) and late (n = 12) STED imaged 5C.C7 T cell:CH27 APC conjugates is given (see S1 Fig for analysis details). (G) F-actin structure depth and width from F are given with dotted lines connecting paired measurements from the same structure for early and late cell conjugates. (H) The average intensity to area ratio of F-actin structures from F is given. Error bars are s.e.m. Significance was determined by Student’s t-test (*p<0.05, **p<0.001, ***p<0.0001).
Fig 3.
An undulating and highly interdigitated T cell:APC interface is dynamically regulated.
(A) From an EM tomogram of an early (1-2min, see methods for precise time point definition) 5C.C7:CH27 APC (10μM MCC) interface (T cell in red, APC in green) a representative z-plane (top, from S4 Video) an a model of the entire reconstruction (bottom, from S5 Video) are given. (scale bar = 1μm). (B) A representative early (interface outlined in red) and (C) late electron micrograph of 5C.C7:CH27 APC interfaces (10μM MCC) are given. (D) The interface length to diameter ratio is given for early (number of cell couples analyzed across multiple independent experiments, n = 22) and late (n = 16) cell conjugates (see S1 Fig for analysis details)(see methods for precise time point definition). (E) The separate interface length and diameter measurements are given for the same cells as in D. Differences in interface length between the early and late time point are significant with p = 0.002. Differences in interface diameter are not significant. (F) Lengths of tight interface contact between at the T cell:APC (i.e. regions of opposing T cell:APC membrane <20nm apart rather than the entire interface outline denoted as ‘interface length’) are given from the same electron micrographs analyzed for D. (G) CFSE-labeled 5C.C7 T cell:CH27 APC interactions in the presence of 10 μM MCC peptide were imaged with an 100 x objective. Midplane sections of a representative T cell at 20s and 3min after tight cell coupling (as indicated) are given in a rainbow-like, false-color intensity scale (increasing from blue to red). The APC (not visible) is on the right (scale bar = 1μm). (H) The interface length to diameter ratios are given at the 20s and 3min time points for 22 cell couples. Error bars are s.e.m. Significance was determined by Student’s t-test (*p<0.05, ***p<0.001, ****p<0.0001).
Fig 4.
A large part of T cell signaling associates with the lamellum.
(A) 5C.C7 T cells expressing the indicated sensors were activated on peptide loaded CH27 APCs (10μM MCC) and percentage occurrence of each pattern of interface enrichment (Fig 1A)[3] among all cell couples analysed across multiple experiments is given in shades of red from -40 to 420 s. In addition, to address the rate of pattern change, the percentage change per 20-s interval was tabulated (C-40 to L300 in the bottom part of the figure). Red indicates an increase and green a decrease in the percentage occurrence of a pattern relative to the previous time point. The cluster tree is given in pink. The source data for this figure and the sensors used are listed in S1 Table. (B-D) Pattern classification graphs, similar to Fig 1E, are given for (B) GFP-SKAP55 (number of cell couples analyzed across multiple independent experiments, n = 59), (C) Myosin 1C-GFP (n = 56), (D) NFκB p65-GFP (n = 44, % cell couples with nuclear accumulation of NFκB are given in addition in grey). Error bars are s.e.m.
Fig 5.
The active forms of the signaling intermediates SLP-76 and LAT display lamellal and central localization, respectively.
(A) A representative STED image is given of two 5C.C7 T cells conjugated to a CH27 APC (10μM MCC) stained for pSLP-76 (Y128) at a single central z-plane as a DIC and fluorescence overlay (scale bar = 2μm). (B) F-actin amounts as a function of the distance from the interface measured from STED images are given for the early (<2min.) time point as in Fig 2C and plotted on the left y-axis (number of cell couples analyzed across multiple independent experiments, n = 26)(see methods for precise time points definition). The percentage of 5C.C7:CH27 conjugates (10μM MCC) imaged by STED with pSLP-76 (Y128) clusters is shown as a function of distance from the interface and plotted on the right y-axis (n = 21) (data from single color stains). (C) Individual pSLP-76 (Y128) clusters were identified and the fraction of these pSLP-76 clusters in each normalized interface region (interface diameter = 1, interface diameter divided into eight equal size sections) across the T cell:APC interface diameter from the same cells as in B is given. (D) To analyze pSLP-76 irrespective of clustering, the entire intensity distribution of pSLP-76 (Y128) is binned into normalized interface regions (interface diameter = 1, interface diameter divided into eight equal size sections) for the same cells as in B. (E) 5C.C7:CH27 conjugates (10μM MCC) stained for pLAT (Y191) were imaged by deconvolution microscopy and a representative image is given. (F) The intensity of pLAT (Y191) is given as in D (n = 12). Error bars are s.e.m.
Fig 6.
Activated SLP-76 localizes to lamellal F-actin structures.
(A, B) Fixed 5C.C7 T cell:CH27 APC conjugates were stained for F-actin (Phalloidin, shown in red), pSLP-76 (Y128, shown in green), and APCs with Cell Trace Violet (shown in blue). The T cell outline is given in red. Representative images of (A) APC proximal and (B) lamellal localized pSLP-76 clusters are shown on top while intensity line scans perpendicular to the interface (dotted white line) are shown below with the black dashed line denoting the T cell:APC interface. A binary mask of above background F-actin (red) is shown with the centers of mass of pSLP-76 clusters (green) next to the representative image in panel B. (C) Pearson’s correlation coefficients for colocalization of pSLP-76 (Y128) with F-actin is given (number of cell couples analyzed across multiple independent experiments, n = 15). (D) Mander’s coefficient reporting the percentage of pSLP-76 (Y128) that overlaps with F-actin and vice versa was calculated for the same cell conjugates as in C. (E) The extent to which F-actin reaches into the T cell away from the interface was measured at the point of half-maximal fluorescence (average of peripheral and central measurements) and plotted against the distance of the deepest pSLP-76 (Y128) cluster from the interface with Pearson’s correlation coefficient for the same cell conjugates as in C. Error bars are s.e.m.
Fig 7.
Actin and lamellal signaling intermediates localize and move comparably.
(A) A fluorescence recovery after photobleaching (FRAP) time series is shown for GFP-actin with an initial prebleached image and post-bleached images spaced 255ms (APC is represented by white line, images shown in 0.5s intervals, bleach spot = red circle). (B) The corresponding fitted recovery curve is given. (C) Average fitted recovery curves for GFP fusions with the lamellal localized signaling intermediates PLCδPH (number of cell couples analyzed across multiple independent experiments, n = 11) and Themis (n = 18) and centrally localized PKCθ (n = 9), LAT (n = 10), an active Rac sensor [3](n = 13) plotted with GFP-actin (n = 10) and GFP (n = 10) control are shown. (D) The average half times of recovery for the same cell conjugates analyzed for C. (the line indicates significance by 1 way ANOVA, p>0.05). Error bars are s.e.m.
Fig 8.
The lamellum is an integrated component of the spatiotemporal organization of T cell signaling.
Pattern classification graphs, similar to Fig 1E, are given for (A) Grb2-GFP (number of cell couples analyzed across multiple independent experiments, n = 54), (B) Itk-GFP (n = 23), (C) the tandem C1 domain of PKCθ-GFP (Diacylglycerol (DAG) sensor) (n = 56) (late time points are not analyzable because of close apposition of Golgi-localized DAG to the interface), (D) SHP1-GFP (n = 49). Error bars are s.e.m.