SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling
- EMBL Australia Node in Single Molecule Science, School of Medical Sciences and the ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Australia
- Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Switzerland
- Department of Biology, University of Konstanz, Germany
- Electron Microscopy Unit, Mark Wainwright Analytical Centre, University of New South Wales, Australia
- Institute for Molecular Bioscience (IMB), University of Queensland, Australia
Figures
Figure 1 with 2 supplements
Recruitment of SNX9 to the immunological synapse upon T cell activation.
(A) Representative images of maximum intensity projections from Z-stacks (0.19 µm per slice) of WT Jurkat T cells conjugated to SEE pulsed (+ SEE) or non-pulsed (-SEE) antigen presenting cells …
Figure 1—figure supplement 1
SNX9 is recruited to the immunological synapse of primary T cells where it co-localises with CD28.
(A) Representative images of maximum intensity projections of 5 Z-stacks of OT-II T cells expressing GFP-SNX9 and conjugated for 15 min to bone marrow-derived dendritic cells pulsed with OT-II …
Figure 1—figure supplement 2
Worflow for identification of intracellular structures.
1. A threshold is applied to the original data to identify endosomal structures in the z-plane with maximal identifiable structures. 2. The threshold is applied to the entire Z-stack to generate a …
Figure 2 with 5 supplements
Recruitment of SNX9 to CD28 clusters upon T cell activation.
(A) Representative TIRF images of fixed WT Jurkat T cells expressing CD28WT-EGFP or CD28YF-EGFP (cyan) and activated for 10 min. (B) Fold fluorescent intensities of CD28WT/YF-EGFP (cyan) and …
Figure 2—figure supplement 1
Representative confocal images from three independent experiments of WT Jurkat T cells expressing SNX9-GFP (cyan) and mCherry-Rab5 (magenta) and activated for 10 min.
Scale bar = 5 µm.
Figure 2—figure supplement 2
SNX9 is recruited into CD28 microclusters upon T cell activation.
(A) Representative images of Jurkat T cells expressing SNX9-EGFP (cyan) and CD28WT (top)- or CD28YF (bottom) fused to PA-mCherry (magenta), plated onto activating cover glass (anti-CD3ε and …
Figure 2—figure supplement 3
SNX9 is recruited into CD28 microclusters upon T cell activation by antigen-presenting cells.
(A) Upper row: representative images of maximum intensity projections from Z-stacks of Jurkat T cell expressing mCherry-SNX9 and CD28-EGFP conjugated to Raji B cells in absence of SEE for 15 min, …
Figure 2—figure supplement 4
SNX9 expression in Jurkat WT and SNX9KO T cells.
Western blot probed with antibodies against SNX9 and α-tubulin (loading control) in WT Jurkat (first lane) and CRISPR/Cas9 gene edited Jurkat T cells with SNX9 knocked out (second SNX9KO#3 and third …
Figure 2—video 1
Representative live cell movie showing SNX9 recruitment to CD28 clusters during T cell activation.
WT Jurkat T cells expressing SNX9-mCherry (magenta) and CD28-EGFP (cyan), activated live on anti-CD3ε and anti-CD28 antibody-coated glass surfaces and imaged live in TIRF for 700 s with 100 ms …
Figure 3 with 3 supplements
Dynamics and persistence of SNX9 within CD28 clusters.
(A) Schematic of photoactivation of fluorescent proteins at the plasma membrane of cover glass activated cells with the photoactivated protein diffusing either though the cell membrane or through …
Figure 3—figure supplement 1
Example of custom-made FIJI quantification of cluster versus cytosol.
The GFP channel is thresholded to define GFP positive CD28 clusters and the cytosol (no GFP positive clusters) creating a cluster mask and cytosol mask, respectively. These masks are then divided by …
Figure 3—figure supplement 2
Quantification approach used to count the number of PAmCherrry-positive vesicles after photoactivation.
(A) Representative images of SNX9 and TCRζ-PAmCherry and the endosome masks generated from thresholded PAmCherry signal to identify endosomal structures. (B) Quantification of endosomal structures …
Figure 3—video 1
Representative movie showing the dynamic localisation of SNX9 in CD28 clusters.
Confocal time series of SNX9 KO#4 cells expressing CD28-EGFP and SNX9-PA-mCherry and repetitively photoactivated by 405 nm laser at the membrane region of interest (dashed line).
Figure 4 with 3 supplements
SNX9 defines tubules that are connected to CD28 clusters.
(A) Correlation of SNX9-mCherry transfected cell with the same cell re-registered by transmission electron microscopy. White box: region of interest positive for CD28-GFP and SNX9-mCherry. Scale 5 …
Figure 4—figure supplement 1
Identification and quantification of tubular structure in correlative electron tomography.
(A) Top-down rendering from electron tomography of cell plasma membrane (white contours), SNX9-positive tubules (purple rendering), SNX9-negative tubules or tubules with a density consistent with …
Figure 4—video 1
Confocal slices of the WT Jurkat cell expressing CD28-EGFP and SNX9-mCherry used to overlaid on the EM pictures.
Figure 4—video 2
Optical slices from a reconstructed tomogram of different z-depths demonstrating abundant tubules emerging from the plasma membrane.
Figure 5 with 4 supplements
SNX9 promotes CD28 cluster stability.
(A) Representative live images of activated WT Jurkat (left) and SNX9 KO#4 (right) T cells expressing CD28WT-EGFP or CD28YF-EGFP with or without SNX9WT-mCherry. (B) Number of CD28WT/YF-EGFP positive …
Figure 5—figure supplement 1
Fitting of the Fluorescence recovery after photobleaching (FRAP) curves for CD28 in WT, SNX9KO and SNX9 overexpressing Jurkat T cells.
(A) Normalised FRAP curves fitted with a bi-exponential decay model. (B) Goodness-of-fit of the mono-, bi-, or triple-exponential decay model evaluated by the sum of the weighted summed square of …
Figure 5—figure supplement 2
SNX9 has no impact on the recovery of Lck fluorescence intensity after FRAP.
(A) Confocal images of WT and SNX9KO Jurkat T cells expressing Lck10WT-EGFP and SNX9-mCherry, or only Lck10-EGFP, activated on anti-CD3ε and anti-CD28-coated glass surfaces. Photobleaching of the …
Figure 5—video 1
Representative movie showing the diffusion of CD28 through clusters (identified by SNX9-mCherry).
Top: WT Jurkat T cells expressing CD28-PA-mCherry (magenta) and SNX9-EGFP (cyan). Bottom: TCRζ-PA-mCherry (magenta) and SNX9-EGFP (cyan). Cells were activated on antiCD3 and anti-CD28 coated …
Figure 5—video 2
Representative movie showing two-photon FRAP of CD28-EGFP clusters.
WT Jurkat T cells expressing CD28WT co-expressing SNX9-mCherryor not and SNX9KO#4T cells expressing CD28WT-EGFP. Cells were activated on antiCD3 and anti-CD28-coated surfaces and the region of …
Figure 6 with 2 supplements
Surface levels and endocytosis of CD28 upon SNX9 knock-out.
(A) Mean fluorescence intensity of resting and activated (20 min) WT Jurkat and SNX9KO#3 and SNX9KO#4T cells incubated with an antibody against CD28 (CD28-FITC). (B) Mean fluorescence intensity of …
Figure 6—figure supplement 1
Flow cytometry gating strategy.
(A) FSC and SSC dot plots were used to identify live T Lymphocytes, doublets were eliminated by using a pulse geometry gate with FSC-H and FSC-A. Jurkat T cell viability was further assessed by …
Figure 6—figure supplement 2
CD28 receptor internalisation very low compared to TCR.
Remaining CD28 and TCR at the surface of WT Jurkat T cells upon activation with soluble anti-CD3ε and anti-CD28. Surface TCR (CD3 ε) and CD28 were labelled at t = 0 min with biotinylated antibodies …
Figure 7
SNX9 specifically contributes to CD28-mediated signalling.
(A) Phosphorylation of CD28 and (C) TCRζ in activated WT Jurkat and SNX9KO T cells detected by phospho-specific antibodies in flow cytometry. (B) Normalised (WT = 100%) percentage of pCD28 and (D) …
Author response image 1
Co-immunoprecipitation (Co-IP) analysis of the interaction between SNX9 and CD28.
SNX9knock-out Jurkat T cells were retrovirally reconstituted to stably express GFP-SNX9. The Jurkat T cells were activated or not for 20-30 min with anti-CD3ε (OKT3) and anti-CD28 (CD28.2) …
