Fig 1: Characterisation of the binding of novel interactors with the APP tail.(A) A schematic of point mutations introduced in the cytosolic tail of APP (649–695). Residues are numbered according to APP isoform 695. Important consensus motifs and binding sites have been highlighted. (B) Affinity chromatography from Fig. 1 was repeated using either GST fused to the APP WT tail, a series of APP tail mutants, or GST only. Eluates were used for immunoblotting and probed using antibodies against hits previously identified by mass spectrometry. (C) Quantification of RABGAP1, as shown in (B). The protein detected in each condition is expressed as a percentage of the WT tail. (D) Quantification of SNX17, as shown in (B). (E) Quantification of NUMB, as shown in (B). N = 3 biological repeats. Bars represent the mean ± standard error of the mean (SEM) (C–E). Statistical significance (C–E) was determined using one-way ANOVA followed by Tukey’s Honest Significant Difference (HSD) multiple comparisons post-hoc test (FWER = 0.05). ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; ns not significant. Source data are available online for this figure.
Fig 2: APP processing is perturbed upon RABGAP1 KO in HeLa cells.(A) Schematic of the dual-tagged APP construct, showing a HaloTag fused to the N-terminus of APP and an mNeonGreen tag fused to the cytosolic side. Major APP cleavage sites have been highlighted. (B) Immunoblotting validation of transient CRISPR/Cas9 knockout (KO) of RABGAP1 in HeLa cells. β-tubulin has been used as a loading control. (C) N-terminal HaloTag-JF646 fluorescence levels of WT HeLa and RABGAP1 KO cells, measured by flow cytometry. Fluorescence intensities are expressed as a percentage of WT HeLa cells. N = 3 biological repeats. (D) C-terminal mNeonGreen fluorescence levels of WT HeLa and RABGAP1 KO cells, measured by flow cytometry. N = 3 biological repeats. Box plots (C, D) display the median (centre line), the interquartile range (box bounds = 25th and 75th percentiles), and whiskers extending to the smallest and largest data points. (E) Ratio of mNeonGreen:HaloTag-JF646 fluorescence as quantified by flow cytometry. N = 3 biological repeats. (F) Quantitative real-time PCR (qPCR) of APP transcription in HeLa cells upon KO of RABGAP1. N = 3 biological repeats. (G) Co-localisation of APP and RABGAP1 in HeLa cells pre-treated with 25 μM DAPT for 24 h. Live cell structured illumination microscopy of steady state HaloTag-APP-mNeonGreen and mCherry-RABGAP1. Images were taken from Movies EV1 and 2. Images were taken on a Zeiss Elyra 7 Lattice SIM microscope. N = 3 biological repeats. Scale bars, 5 μm. (H) Live cell structured illumination microscopy of steady state HaloTag-APP-mNeonGreen and RAB11A-mScarlet in HeLa cells. Scale bars, 10 μm. (I) Quantification of imaging shown in (H). Pearson’s correlation of HaloTag-APP and RAB11A-mScarlet. N = 3 biological repeats. Each point represents the average from one biological replicate. A minimum of 28 cells were analysed in total in each condition. (J) Quantification of imaging shown in (H). Pearson’s correlation of APP-mNeonGreen and RAB11A-mScarlet. (K) Live cell structured illumination microscopy of steady state HaloTag-APP-mNeonGreen and RAB5A-mScarlet. Scale bars, 10 μm. (L) Quantification of imaging shown in (K). Pearson’s correlation of HaloTag-APP and RAB5A-mScarlet. N = 3 biological repeats. Each point represents the average from one biological replicate. A minimum of 30 cells were analysed in total in each condition. (M) Quantification of imaging shown in (K). Pearson’s correlation of APP-mNeonGreen and RAB5A-mScarlet. N = 3 biological repeats. Bars represent the mean ± SEM (E–M). Statistical significance (C–M) was assessed using an unpaired t test. ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; ns not significant. Source data are available online for this figure.
Fig 3: Complementation with WT RABGAP1 can recover the loss of C99 in RABGAP1 KD i3 neurons.(A) Stable iPSC lines were generated where the RABGAP1 KD line was complemented with either WT RABGAP1, RABGAP1 GAP-deficient mutant (R612A) or a RABGAP1 mutant that is predicted to no longer interact with the APP tail (L216S, S236F, A264F). A negative control line was also made using an empty vector. The cell lines were differentiated, lysed on day 15 and subsequently used for immunoblot analysis of APP processing. The D54D2 antibody (Cell Signalling, 8243) was used to assess levels of CTF C99. RABGAP1 levels were also probed for, along with β-tubulin that was used as a loading control. N = 4 biological repeats. (B) Quantification of C99 levels shown in (A). Levels of C99 are expressed relative to the WT RABGAP1 recovery condition. (C) Stable iPSC lines were generated with overexpression of either WT RABGAP1, a RABGAP1 GAP-deficient mutant (R612A) or a RABGAP1 mutant that is predicted to no longer interact with the APP tail (L216S, S236F, A264F). The cell lines were differentiated and C99 levels were assessed, as described in (A). N = 3 biological repeats. (D) Quantification of C99 levels shown in (C). Bars represent the mean ± SEM (B–D). Statistical significance (B–D) was determined using one-way ANOVA followed by Tukey’s Honest Significant Difference (HSD) multiple comparisons post-hoc test (FWER = 0.05). **P ≤ 0.01; ns = not significant. (E) Predicted interaction interface between RABGAP1 PTB domain and the YENPTY motif of the APP tail, modelled in AlphaFold3. The binding of the PTB domain of RABGAP1 (blue) to the APP tail (orange) is predicted to be abolished upon triple mutation of A264F, L216S and S236F (purple). Source data are available online for this figure.
Fig 4: Endogenous C99 production is significantly decreased in RABGAP1 KD i3 neurons.(A) Co-localisation of APP and endosomes in day 15 i3 neurons. Live cell imaging of steady state HaloTag-APP-mNeonGreen and mScarlet-FYVE. Images were taken from Movie EV3. Images were taken on a Zeiss LSM880 Airyscan microscope. Scale bars, 10 μm. (B) Quantification of imaging shown in (A). Pearson’s correlation of mScarlet-FYVE and both HaloTag and mNeonGreen from HaloTag-APP-mNeonGreen expression. N = 3 biological repeats. Each point represents the average from one biological replicate. A minimum of 30 cells were analysed in total in each condition. (C) qPCR of APP transcription in WT and RABGAP1 KD day 15 i3 neurons. N = 3 biological repeats. (D) A schematic of the C-terminus of APP, showing secretase cleavage sites and antibody epitopes. The β-amyloid CT695 (Invitrogen, 51-2700) binds to the far C-terminus of APP and detects both C99 and C83. The D54D2 antibody (Cell Signalling Technologies, 8243) binds to the N-terminus of the amyloid-β peptide and can therefore only detect C99 and not C83. (E) Immunoblotting of endogenous APP processing in WT and RABGAP1 KD i3 neurons. Neurons were lysed at day 15 of differentiation. APP processing levels were assessed using the D54D2 antibody (Cell Signalling Technologies, 8243) which detects C99, but not C83, as well as the β-amyloid CT695 (Invitrogen, 51-2700) antibody to detect full length APP levels. β-tubulin was used as a loading control. RABGAP1 was probed to monitor RABGAP1 KD efficiency. (F) Quantification of RABGAP1 KD efficiency, as seen in (E). Protein abundance is normalised to WT levels. N = 7 biological repeats. (G) Quantification of full length APP (110 kDa), as seen in (E). Full length APP was probed using the β-amyloid CT695 (Invitrogen, 51-2700) antibody. N = 7 biological repeats. (H) Quantification of C99 abundance (16 kDa), as seen in (E). C99 was probed using the D54D2 antibody (Cell Signalling Technologies, 8243) antibody. N = 7 biological repeats. (I) Quantification of the ratio of C99 (16 kDa) over full length APP (110 kDa), as shown in (E). Bars represent the mean ± SEM (B–I). Statistical significance (C–I) was assessed using an unpaired t test. N = 7 biological repeats. ****P ≤ 0.0001; ns = not significant. Source data are available online for this figure.
Supplier Page from OriGene Technologies for Rabgap1 Rat shRNA Plasmid (Locus ID 311911)