Fig 2: DYNC1LI2-dependent rescue of ER stress requires functional RAB7 and RAB11 but not RAB5. (A) Immunofluorescence analysis of the endogenous ER stress marker, HSPA5 (Red), expression in wild-type, ctns-/-, ctns-/–DYNC1LI2 and dominant negative (DN)-expressing RAB5DN, RAB7DN, RAB11DN, ctns-/–DYNC1LI2 cells. GFP-DYNC1LI2 is shown in green. DN-RAB GTPases are pseudo-colored orange. Scale bar: 10 µm. (B) Quantification of ER stress marker expression. A total of 19–41 fields and 30–50 cells were quantified in each category, from three independent experiments. Mean ± SEM, *, p < 0.05, **, p < 0.01, ***, p < 0.001. (C) Analysis of HSPA5 expression by western blot in WT, ctns-/-, ctns-/–DYNC1LI2 or cells expressing DN-RABs. DsRed-RAB expression was verified by fluorescence microscopy and it is shown here by immunoblot. For detection of GFP-DYNC1LI1, GFP-DYNC1LI2 and exogenous expressed RABs the same sample mix was run in parallel and probed on separate membranes. (D) Quantification of ER stress expression levels by immunoblot from 3 independent experiments. Mean ± SEM, **, p < 0.01, ***, p < 0.001.

Fig 3: The C2 helical insertion (C2HH) of VPS34 is a critical element for the interaction of complex II with Rab5a.a, b GTP-locked Rab5a (Q79L) and all four components of complex II, containing either C-terminally EGFP-tagged WT (a) or C2HH mutant REIE > AAAA VPS34 (b), were coexpressed in HEK293T cells. Confocal images show cellular localisation of mCherry–Rab5a-Q79L (magenta) and VPS34-EGFP (cyan). To the right of each panel, traces for the fluorescence along the transect indicated by the line in the micrograph are shown. The C2HH REIE mutant markedly increased colocalization of complex II with Rab5a (b, e) compared to VPS34 WT (a, e). c, d GTP-locked Rab5a (Q79L) and all four components of complex I, containing either C-terminally EGFP-tagged WT (c) or mutant REIE > AAAA VPS34 (d), were coexpressed in HEK293T cells. The mutant had no impact on the colocalization of complex I with Rab5a (c–e), quantitated as described in methods. Error bars: standard deviation. ***: p < 0.0001; n.s.: p > 0.05. Scale bars: 10 µm. f GUV assay of activity of complex II shows that the VPS34 C2HH REIE > AAAA mutation greatly potentiates the activation of complex II by Rab5a–GTP, without affecting the basal activity of complex II. Micrographs: AF647-PX signals at the end of reactions. Scale bars: 5 µm. g The initial rates in the GUV assay in f (AF647-PX fluorescence change/min in arbitrary units, AU) are depicted. ***: p < 0.001; n.s.: p > 0.05. Source data are provided as a Source Data file.

Fig 4: KIF13A knockdown impairs endocytic recycling of VE-cadherin in ECs. (A) Confocal images of VE-cadherin and Rab5 double immunostained HUVECs showing increased VE-cadherin/Rab5 double positive early endosomes in KIF13A knockdown HUVECs. Scale bar: 20 μm (upper panel) and 8 μm (lower panel). (B) Quantification showing increased size of Rab5-positive early endosomes in KIF13A knockdown HUVECs (266 endosomes from control HUVECs, 289 endosomes from siHgs-1-treated HUVECs, and 275 endosomes from siHgs-2-treated HUVECs were analysed). (C) Western blot analyses of VE-cadherin and KIF13A expression in control and KIF13A knockdown HUVECs. (D–G) VE-cadherin internalization assay (D and E) and the corresponding quantifications (F and G) showing increased colocalization of VE-cadherin with Rab5-positive early endosomes and decreased colocalization of VE-cadherin with Rab11-positive recycling endosomes in KIF13A knockdown HUVECs (n = 5). Scale bars: 15 µm. (H) Immunostaining for VE-cadherin, Rab11, and F-actin showing impaired recruitment of VE-cadherin-containing recycling endosomes to the AMIS of KIF13A knockdown HUVECs. (Right) Quantification of VE-cadherin/Rab11-positive endosomes enriched at the AMIS of control and KIF13A knockdown HUVECs (n = 5). Scale bar: 15 µm. Values are presented as means ± standard deviation. **P < 0.01 by Mann–Whitney test.

Fig 5: Restoration of endosomal recycling rescues VE-cadherin polarity in Hgs knockdown ECs. (A–D) HUVECs were treated with control or Hgs-siRNA for 24 h and then transfected with control or KIF13A plasmids for 24–48 h before fixation in 4% PFA. (A) Confocal images showing that KIF13A overexpression reduced the intracellular accumulation of VE-cadherin and VE-cadherin-containing Rab5-positive early endosomes (upper panel) and normalized the size of Rab5-positive early endosomes (lower panel) in Hgs knockdown HUVECs. Scale bar: 10 µm. (B) Quantification of the size of Rab5-positive endosomes (333 endosomes from control HUVECs, 291 endosomes from siHgs-1-treated HUVECs, and 263 endosomes from siHgs-2-treated HUVECs transfected with KIF13A plasmids were analysed). (C) Quantification of junctionally distributed VE-cadherin (n = 5). (D) KIF13A overexpression restored the recruitment of VE-cadherin to the AMIS in Hgs knockdown HUVECs (n = 5). Scale bar: 10 µm. Values are presented as means ± standard deviation. **P < 0.01 by Mann–Whitney test and one-way analysis of variance.