Fig 2: Protrudin overexpression enhances ER presence at growth cones and this interaction is required for successful axon regeneration.a Schematic representation of Protrudin endoplasmic reticulum domain mutants. b Representative images of RTN4 immunofluorescence (green) in the distal axon of neurons expressing the indicated m-Cherry Protrudin constructs (magenta). Scale bar is 10 µm. c, d Quantification of RTN4 fluorescence intensity at the axon tip and shaft (p < 0.0001, Kruskal–Wallis with Dunn’s multiple comparisons test). Error bars represent mean ± SEM. e Percentage of regenerating axons in neurons expressing mCherry-Protrudin domain mutants—FFAT (n = 4 independent experiments, 60 neurons) and TM1-3 (n = 5 independent experiments, 45 neurons) compared to phosphomimetic Protrudin as a positive control (n = 2 independent experiments, 21 neurons), and mCherry as a negative control (n = 3 independent experiments, 41 neurons). Error bars represent mean ± SEM.

Fig 3: Knockdown of Protrudin promotes perinuclear accumulation of mTOR-positive LE/Lys and diminished mTORC1 activity. a Deconvolved confocal micrographs showing distribution of mTOR-positive LE/Lys. HUVECs were seeded on glass coverslips along with lentivirus carrying shProtrudin, shProtrudin#2 or shNT sequence and cells were grown in ECGM2 complete media. Post-transduction, the cells were fixed in 4% PFA and immunostained with anti-Lamp1 and anti-mTOR antibodies. Experiment was done thrice, and the data are representative of at least 15 image captures for each condition per experiment. The high-magnification inset on the right illustrates dispersed mTOR-Lamp1-positive endosomes. b Quantification of perinuclear distributed mTOR-positive lysosomes. Graph represents percentage intensities of perinuclearly distributed mTOR relative to total cellular mTOR-positive vesicles. 150 cells per condition were analyzed from 3 independent experiments. Data represent mean ± SD for 3 independent experiments. *p < 0.05 as compared to control samples. c Immunoblots showing phosphorylation level of S6K in Protrudin knockdown HUVECs grown in complete media. d Immunoblots of Protrudin knockdown or control cells showing the phosphorylation level of S6K. Cells were serum starved in 0.1% BSA for 5 h followed by stimulation with ( +) or without ( -) VEGF (50 ng/ml) for 20 min. Cells were lysed and immunoblotted with total or phospho-p70S6K (Thr 389) antibodies. Data represent mean ± SD for 5 independent experiments.*p < 0.05 as compared to shNT, - VEGF samples #p < 0.05 compared to shNT, + VEGF samples. d Immunoblots showing the phosphorylation level of Akt(Ser473) in Protrudin knockdown HUVECs as described in (c). Graphs represent relative densitometric values of pAkt(Ser473) normalized to Akt. Data represent mean ± SD for 3 independent experiments. n.s. indicates statistically not significant compared to shNT, - VEGF samples or shNT, + VEGF samples

Fig 4: PDZD8 and Protrudin associate independently with late endosomes.a PDZD8-GFP co-localizes with Protrudin-mCherry on BFP-Rab7-labeled late endosomes. Representative image of a U2OS cell co-expressing PDZD8-GFP, BFP-Rab7, and Protrudin-mCherry. Mitochondria were labeled with MitoTracker DeepRed. Scale bar: 10 µm. b The PDZD8 TM domain is required for its interaction with Protrudin. Extracts from HEK293T cells expressing various GFP-tagged truncated versions of PDZD8 (as illustrated in Fig. 3a) and Protrudin-mCherry were immunoprecipitated with GFP-Trap beads and western blot analysis was performed using anti-GFP, anti-Actin anti-Protrudin antibodies for detection of PDZD8-GFP truncated versions, actin and Protrudin, respectively. Source data are provided as a Source Data file. c PDZD8-GFP and Protrudin-mCherry are independently recruited to BFP-Rab7-labeled endosomes. Representative images of PDZD8 KO HeLa cells expressing either wildtype Protrudin-GFP or Protrudin FYVE4A-GFP and co-expressing BFP-Rab7 with either PDZD8-mCherry or PDZD8?CC-mCherry. Scale bar: 10 µm.

Fig 5: Protrudin gene deletion retards physiological postnatal angiogenesis. a Isolectin B4 staining of postnatal day 7 retinas from Zfyve27+/+ (wild-type) and Protrudin KO (Zfyve27-/-) mice, showing vascular progression. Scale bar = 500 µm; n = 5. b Bar graph representing quantification of retinal vascular progression; n = 5. Data represent mean ± SD, *p < 0.05, two-tailed Student’s t-test. c Body weight of Zfyve27+/+ and Zfyve27–/– mice at 7 to 9 days of age, n = 9, two-tailed Student’s t-test. Data represent mean ± SD, **p < 0.01. d FACS-based cell counts for CD31+CD45— cell populations isolated for mice lungs (postnatal day 7, n = 3) and cerebrum (postnatal day 13–16, n = 3). n.s., statistically not significant