Fig 2: Altered subcellular location of VAMP7 in ep Paneth cells. (A) Representative immunofluorescence images of ep/+ (Ctrl) and ep ileum section stained with anti-VAMP7 (green) and anti-GM130 (purple). UEA1 (red) indicates LDCVs. The arrow heads indicate the co-localization of VAMP7 and GM130, the open triangles indicate the co-localization of VAMP7 and UEA-1 and the white arrows indicate the co-localization of all those three makers. (B) Boxed areas are shown at higher magnification. Bar = 5 µm.

Fig 3: Protrudin colocalises with Calnexin in the ER and forms contact sites with VAMP7 and FYCO1-positive LELys. A RPE1 cells were transiently transfected with GFP-Protrudin, fixed and stained with antibodies against GFP and the ER marker Calnexin, showing that Protrudin resides in the ER. Representative of 12 micrographs. B RPE1 cells were transiently transfected with GFP-Protrudin, fixed and stained with anti-VAMP7 and anti-FYCO1 antibodies. The insets highlight two VAMP7 and FYCO1 co-positive dots in close apposition to GFP-Protrudin in the ER. The fluorescence intensity line plots give two examples of how VAMP7 and FYCO1-positive dots overlap with Protrudin in the ER, indicative of ER-endosome contact sites

Fig 4: Model illustrating the role of the Protrudin pathway in phagocytosis. A Left: inactive Protrudin pathway leads to incomplete phagocytic cup formation. Non-successful uptake of PS-rich silica beads or apoptotic bodies (green sphere). Right: active Protrudin pathway results in efficient phagocytic cup formation and particle internalisation. The Protrudin pathway promotes Kinesin-1 dependent delivery of vesicles to the forming phagocytic cup. FYCO1 acts as an adapter protein between the motor protein Kinesin-1 and the RAB7 and PtdIns3P-positive vesicle. The endosomes fuse in a Ca2+-dependent manner with the PM mediated by SYT7 and VAMP7 to aid internalisation of particles. Internalised beads cannot be stained by Streptavidin-Alexa-488 and are visible as colourless spheres by brightfield microscopy (grey sphere, Fig. 7). Overexpression of Protrudin increases the cell’s capacity to internalise PS-rich particles. Created with BioRender.com. B Detailed illustration of Kinesin-1 handover from Protrudin to LELys at ER-endosome contact sites. (1) Kinesin-1 binds to Protrudin in the ER [67]. (2) Protrudin binds to RAB7 and PdIns3P at the LELy, forming contact sites. The endosomal adaptor protein FYCO1 receives Kinesin-1 [24]. (3) Kinesin-1 detaches from Protrudin and binds to a microtubule, resulting in plus end directed transport of the SYT7 and VAMP7-positive LELy. Created with BioRender.com

Fig 5: Recruitment of FYCO1 and VAMP7-positive endosomes to the phagocytic cup requires Protrudin. A RPE1 cells were treated with siRNA Protrudin #1 and seeded on coverslips. 48 h after transfection the cells were fixed and stained with anti-VAMP7 antibody and Phalloidin Alexa Fluor 647. In the Protrudin depleted cells, VAMP7-positive endosomes cluster more perinuclearly, implicating a role for Protrudin in VAMP7-positive endosome positioning. Representative images of a minimum of 10 images per experiment, n = 4. The graph represents the relative sum fluorescence intensity of VAMP7-positive puncta in the perinuclear region as % of the complete cellular population of VAMP7 dots from at least 190 cells per condition quantified using the NisElements software. Shown is mean ± s.d., n = 4 experiments ***P < 0.001, unpaired two-tailed t test. B RPE1 cells were treated as in A and stained with anti-VAMP7 and anti-FYCO1 antibodies. Numbers denote Pearson correlation coefficient calculated from 5 images per condition. Insets highlight the co-occurrence of VAMP7 and FYCO1 on the same endosomal structures. The graph represents the quantification of the degree of perinuclear positioning of FYCO1 and VAMP7 co-positive dots detected automatically using NisElements. Shown is mean ± s.d., n = 4 experiments ***P < 0.001, unpaired two-tailed t test. > 190 cells were quantified per condition, same dataset as in A. C Maximum projection, orthographic view and 3D rendering of confocal z-stacks showing VAMP7, FYCO1 and F-actin in RPE1 cells transfected with control siRNA or Protrudin siRNA. Note that FYCO1 and VAMP7 co-positive dots accumulate at the base of an actin-rich cup in the control treated cells, whereas the Protrudin depleted cell does not form a cup and shows a perinuclear localization of FYCO1 and VAMP7.