Fig. 3: LysoIP proteomics reveals a fingerprint of lysosomal dysfunction in VPS35 KO cells.

a–j A range of functional networks and protein families were relatively depleted or enriched in lysosomes derived from VPS35 KO H4 cells relative to wild-type and VPS35-GFP-expressing rescue cells. Dot-plots representing log₂ fold change and p-value in quantified abundances of (a) BORC; (b) mTORC1 complexes; (c) lysosomal lumen proteins; (d) Rab GTPases; (e) APP processing proteins; (f) vesicle SNAREs; (g) PIP metabolism; (h) cholesterol transport; (i) autophagy; and (j) lysosomal solute channels, two-tailed paired t tests. Log₂ fold change, relative change and p-value score are depicted by dot colour, size and outline respectively. k Proteolytic processing of APP is enhanced in VPS35 KO H4 cells. Cell lysates from the indicated cell lines were immuno-blotted using anti-APP (full length and CTF), VPS35 and β-actin antibodies. APP-CTF levels in VPS35 KO clone were rescued upon re-introduction of VPS35-GFP. kDa = kilodaltons. l Quantification of APP-CTF signal intensity (n = 5 independent experiments), means ± SEM, one-sample t-tests with Holm–Šídák correction, adjusted p = 0.025 (VPS35 KO Cl.15), 0.1655 (Cl.15 + VPS35-GFP). Red—VPS35 KO clone 15, blue—VPS35 Clone 15 + VPS35-GFP. m APP accumulates in LAMP1-positive compartments in VPS35 KO cells. Wild-type and VPS35 KO H4 were fixed and immuno-stained for LAMP1 and APP and linescan analysis was used to demonstrate colocalisation (arbitrary units). Scale bar = 20 µm and 1 µm in zoomed panels. Data representative of 3 independent repeats. n Proteins enriched in the VPS35 KO LysoIP dataset are associated with neurodegenerative disease. Enrichment of selected DisGeneNET disease categories represented by significantly enriched proteins in the VPS35 KO LysoIP dataset (log₂ fold change ± 0.26, p < 0.05) relative to both wild-type and VPS35-GFP-expressing control conditions, hypergeometric test.