The V3 linker dictates the kinetics of PKCε and PKCθ recruitment.

<p>5C.C7 T cells expressing the indicated GFP-labeled nPKC chimeras together with PKCθ-RFP were imaged by TIRF microscopy and UV irradiated on surfaces containing photoactivatable pMHC. (A) Representative time-lapse montages (time shown in seconds at lower right corner), with the time and region of UV irradiation indicated by white circles. Dotted lines in the first image denote the extent of the T cell membrane on the glass. Scale bar = 10 µm. (B) Offset times separating the recruitment of nPKC chimeras from the recruitment of PKCθ, calculated by cross-correlation analysis of at least 10 paired responses. Error bars = s.e.m. Data are representative of at least two independent experiments.</p

The Variable Hinge Region of Novel PKCs Determines Localization to Distinct Regions of the Immunological Synapse

<div><p>The immunological synapse (IS) formed between a T cell and its cognate antigen-presenting cell (APC) enables the directional secretion of cytolytic and inflammatory molecules. Synaptic architecture is established in part by a two-step cascade of novel protein kinase C (nPKC) isozymes. PKCε and PKCη arrive at the IS first, and occupy the entire synaptic membrane. Then, PKCθ accumulates in a smaller zone at the center of the contact. We investigated the molecular basis for this differential recruitment behavior using chimeric nPKC constructs and total internal reflection fluorescence microscopy. Our studies revealed that the V3 linker just N-terminal to the kinase domain plays a crucial role in specifying nPKC localization. Substitution of this linker switched the scope and the kinetics of PKCθ accumulation to that of PKCε and PKCη, and vice versa. Although the V3 was necessary for synaptic compartmentalization, it was not sufficient, as the tandem C1 domains were also required to mediate membrane association. Together, these results suggest a model whereby the V3 linker controls nPKC sub-compartmentalization after initial C1 domain-mediated accumulation at the IS.</p></div

The V3 region is insufficient for recruitment to the IS.

<p>(A–B) 5C.C7 T cells expressing the indicated GFP-labeled nPKC constructs and Lifeact-RFP were stimulated on bilayers containing pMHC, ICAM-1, and B7.1 as indicated, and then imaged by TIRF microscopy. (A) Representative images are shown to the left (scale bar = 10 µm), with linescans (derived from the white lines shown in the rightmost images) depicting the radial fluorescence intensity of nPKCs and Lifeact to the right. (B) Quantification of the width ratio for each PKC construct (n≥20 cells), with black lines and red error bars denoting mean and s.e.m., respectively. **** indicates p<0.0001 (C) 5C.C7 T cells expressing the indicated GFP-labeled nPKC constructs were mixed with CH12 APCs pre-loaded with MCC peptide, fixed, and stained with an anti-CD4 antibody (to label T cells). Images of T cell-APC conjugates (representative of >20 conjugates) are shown, with T cells and APCs indicated in the transmission images. Yellow dotted lines in the images on the right denote the edge of the APC. (D) 5C.C7 T cells expressing the indicated GFP-labeled nPKC constructs were imaged by TIRF microscopy and UV irradiated on surfaces containing photoactivatable pMHC. Left, representative time-lapse montages (time indicated in seconds at lower right corner) with the time and region of UV irradiation indicated by white circles. Right, background corrected accumulation (ΔF/F) in the irradiated region (n = 6 per graph), with red lines indicating UV irradiation and error bars denoting s.e.m. Data are representative of at least two independent experiments.</p