PKCθ links proximal T cell and Notch signaling through localized regulation of the actin cytoskeleton
- University of Bristol, United Kingdom
Figures
Figure 1 with 1 supplement
PKCθ enhances antigen-induced Notch activation.
(A) Tg4WT and Tg4KO mice were injected subcutaneously with 80 µg of MBPAc1-9[4Y] peptide or PBS. After 18 hr splenocytes were immunostained to assess intracellular Notch1 expression and analyzed by …
Figure 1—figure supplement 1
NICD expression is comparable across Tg4 thymocytes, naïve and primed T cells and substantially enhanced upon retroviral expression.
A is a representative NICD Western blot of protein extracts from Tg4WT T cells stimulated or not with plate-bound anti-CD3 and anti-CD28 for 1 hr. (B) Data from four experiments are presented. (C) …
Figure 2 with 1 supplement
Constitutively active Notch rescues defective proliferation, Th17 polarization and IL-17A secretion in PKCθ deficient T cells.
(A) Splenocytes from Tg4WT and Tg4KO mice were stained for the indicated molecules and absolute cell numbers or percentages are given as indicated. Each data point represents one mouse. Data are …
Figure 2—figure supplement 1
Tg4KO mice display largely unperturbed immune cell distributions.
Splenocytes from Tg4WT and Tg4KO mice were stained for the indicated molecules and absolute cell numbers or percentages are given as indicated. Each data point represents one mouse. Data are …
Figure 3 with 2 supplements
PKCθ mediates transient actin-dependent recruitment of ADAM10 to the T cell lamellum.
(A) Tg4WT CD4+ T cells, treated with 40 nM Jasplakinolide (bottom) or not (top), and Tg4KO (middle) CD4+ T cells expressing ADAM10-GFP were activated with PL8 cells presenting the Ac1-9[4Y] peptide. …
Figure 3—figure supplement 1
PKCθ enables transient recruitment of ADAM10 to the T cell lamellum.
(A) For the ADAM10-GFP experiments displayed in Figure 3A,B the proportion of T cells with accumulation in one of the six interface patterns at the time relative to couple formation is given ± SEM. …
Figure 3—figure supplement 2
PKCθ enhances interface actin dynamics.
(A) For the F-tractin-GFP experiments displayed in Figure 3D,E the proportions of T cells with accumulation in one of the six interface patterns at the time relative to couple formation are given ± …
Figure 4 with 1 supplement
PKCθ phosphorylates and localizes Coronin1A.
(A) Tg4WT and Tg4KO T cell expressing coronin1A-GFP were activated with PL8 cells presenting the Ac1-9[4Y] peptide. Representative images are given as in Figure 3A. The entire image sequences are …
Figure 4—figure supplement 1
Interface recruitment of signaling intermediates peaks within the first three minutes in Tg4KO CD4+ T cells.
(A) In the top row, for the Coronin1A-GFP experiments displayed in Figure 4A,B the proportions of T cells with accumulation in one of the six interface patterns at the time relative to couple …
Videos
Video 1
ADAM10-GFP accumulates rapidly and transiently at the interface between Tg4WT CD4+ T cells and PL8 APCs.
A representative interaction of a Tg4WT CD4+ T cell expressing ADAM10-GFP with a PL8 APC presenting the Ac1-9[4Y] peptide is shown. Top: DIC images. Bottom: Top-down maximum projections of 3D …
Video 2
ADAM10-GFP does not accumulate at the interface between Tg4KO CD4+ T cells and PL8 APCs.
A representative interaction of a Tg4KO CD4+ T cell expressing ADAM10-GFP with a PL8 APC presenting the Ac1-9[4Y] peptide is shown as in Video 1. Tight cell coupling occurs in frame 5 (2 s indicated …
Video 3
F-tractin-GFP accumulates rapidly at the interface between Tg4WT CD4+ T cells and PL8 APCs.
A representative interaction of a Tg4WT CD4+ T cell expressing F-tractin-GFP with a PL8 APC presenting the Ac1-9[4Y] peptide is show as in Video 1. Tight cell coupling occurs in frame 6 (4 s …
Video 4
F-tractin-GFP accumulates rapidly at the interface between Tg4KO CD4+ T cells and PL8 APCs.
A representative interaction of a Tg4KO CD4+ T cell expressing F-tractin-GFP with a PL8 APC presenting the Ac1-9[4Y] peptide is shown as in Video 1. Tight cell coupling occurs in frame 4 (2 s …
Video 5
Coronin1A-GFP accumulates rapidly at the interface between Tg4WT CD4+ T cells and PL8 APCs.
A representative interaction of a Tg4WT CD4+ T cell expressing Coronin1A-GFP with a PL8 APC presenting the Ac1-9[4Y] peptide is shown as in Video 1. Tight cell coupling occurs in frame 4 (2 s …
Video 6
Coronin1A-GFP accumulates rapidly at the interface between Tg4KO CD4+ T cells and PL8 APCs.
A representative interaction of a Tg4KO CD4+ T cell expressing Coronin1A-GFP with a PL8 APC presenting the Ac1-9[4Y] peptide is shown as in Video 1. Tight cell coupling occurs in frame 3 (1 s …
Video 7
PKCθ-GFP accumulates at the center of the interface between Tg4WT CD4+ T cells and PL8 APCs.
A representative interaction of a Tg4WT CD4+ T cell expressing PKCθ-GFP with a PL8 APC presenting the Ac1-9[4Y] peptide is shown as in Video 1. Tight cell coupling occurs in frame 4 (2 s indicated …
