Fig 2: Average protein levels of BAG2 and cathepsin D are decreased in the plasma of PD patients. (a) Preparation of the albumin/IgG‐depleted plasma samples from control and PD patients (20 subjects each) was verified by SDS‐PAGE. The plasma samples from the 20 control and patients with PD were each pooled for the quantitative analysis of the average levels of autophagy‐related proteins. The pooled samples were immunoblotted with antibodies specific to BAG2 (b), BAG3 (c), cathepsin B (d), cathepsin D (e), LAMP1 (f), LAMP2 (g), and WDFY3 (h). Each analysis was repeated as necessary. Arrows and asterisks indicate specific and cross‐reacting bands, respectively. (i) Intensities of specific bands of b–h were quantitatively analyzed, and the level of changes in the PD group was compared to that in the control group. Each blue dot indicates the result from each blot, and the grey bars represent mean values. (j) The fold change in BAG2 levels of the PD group. Values represent mean ± SEM of three independent experiments. (k) The fold change in cathepsin D levels of the PD group. Values represent mean ± SEM of two independent experiments. Molecular weight standards (in kDa) are shown to the left. **P < 0.01 (Student t‐test). BAG, BCL2‐associated athanogene; CBB, Coomassie Brilliant Blue; PD, Parkinson’s disease; SDS‐PAGE, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.

Fig 3: ALFY localizes to early endosomes in a PtdIns(3)P-dependent manner. (A) ALFYKO1-1 EGFP–ALFY rescue cells were fixed and immunostained against EEA1 and analyzed by confocal microscopy. Yellow (white in inset) arrowheads highlight EGFP–ALFY structures positive for EEA1. The colocalization histogram is from the two vesicles in the line marked. (B) ALFYKO1-1 EGFP–ALFY cells stably expressing mScarlet-I–RAB5 were treated with tetracycline overnight and analyzed with live-cell imaging. mScarlet-I–RAB5- and EGFP–ALFY-positive puncta are indicated by arrowheads, and colocalization is shown in the histogram for the line marked. (C) HeLa ALFYKO1-1 cells rescued with EGFP–ALFY, EGFP–ALFY LIRmut or EGFP–ALFY FYVEmut were transiently transfected with Myc–RAB5Q79L, fixed at 18 h post transfection, and immunostained against Myc for visualization of the RAB5Q79L structures before confocal microscopy analysis. Colocalization histograms from the indicated Myc–RAB5Q79L structures are shown. (D) ALFYKO1-1 EGFP–ALFY cells with stable expression of mScarlet-I-tagged LC3B were treated with tetracycline and imaged live. Colocalization was measured from the indicated structures. (E) ALFYKO1-1 EGFP–ALFY cells with stable expression of mScarlet-I-tagged GABARAP were treated with tetracycline and imaged live. Colocalization was measured from the indicated structures. Images are representative of three experiments. Scale bars: 10 µm.

Fig 4: ALFY is required for directional cell migration. (A) Wound healing analysis of HeLaWT and ALFYKO1-1 cells using the Incucyte® live-cell imaging system. The mean relative wound density was quantified from three replicates from three independent experiments. The timepoints marked by a dotted square in the left panel are shown as a magnified graph to the right (mean±s.e.m., n=3). *P<0.05 by multiple unpaired two-tailed t-tests. (B) Representative images from experiments as in A showing the wound densities of HeLaWT and ALFYKO1-1 at 0 and 28 h. Images show 1460×1970 µm. (C) Graphs representing movements of HeLaWT and ALFYKO1-1 cells obtained by tracking individual cells in the images from experiments as in A using the Chemotaxis and migration tool (Ibidi). (D) Manual tracking of images from wound healing assays of HeLaWT, ALFYKO1-1, ALFYKO2-6, ALFYKO2-9 and ALFYKO2-11, showing velocity, directionality and forward migration index along the y-axis (mean±s.e.m., n=3). *P<0.05, **P<0.01; ***P<0.001; ****P<0.0001 (unpaired two-tailed Student's t-test). (E) Analysis of leading edge formation in HeLaWT cells, ALFYKO1-1 cells and ALFYKO1-1 rescue cells with doxycycline-induced expression of EGFP–ALFY, immunostained against cortactin for marking the leading edge. The white lines indicate the introduced wound. (F) Quantification of the percentage (%) of cells from E with cortactin at the leading edge. (mean±s.e.m., n=3). **P<0.01; ***P<0.001; ns, not significant (unpaired two-tailed Student's t-test).

Fig 5: ALFY regulates cell attachment and integrin trafficking. (A) Quantification of proportion of attached HeLaWT and ALFYKO1-1, ALFYKO2-6, ALFYKO2-9 and ALFYKO2-11 cells relative to wild type (stained with CellMask) (mean±s.e.m., n=4). *P<0.05; **P<0.01; ***P<0.001 (unpaired two-tailed Student's t-test). (B) Analysis of integrin protein levels from HeLaWT and ALFYKO1-1, ALFYKO2-6, ALFYKO2-9 and ALFYKO2-11 cells as well as ALFYKO2-9 EGFP–ALFY rescue cells. The blots are representative of three independent experiments. (C) HeLaWT and ALFYKO2-9 were treated or not with PNGaseF or CIP for 1 h at 37°C, followed by western blot analysis using the indicated antibodies. (D) Representative live-cell image of ALFYKO2-9 EGFP–ALFY rescue cells, co-expressed with integrin-a5–mScarlet-I. Arrowheads indicate structures positive for both. Scale bar: 10 µm. Images in C,D are representative of three experiments. (E) Model for role of ALFY in cell migration. ALFY localizes to cell protrusions in the leading and trailing edge of migrating cells, and to early endosomes. By being present on these structures, ALFY regulates the proper sorting and bi-directional trafficking of adhesion proteins such as integrins, thereby controlling directional cell migration.