Fig 2: Rab4 and Rab10 facilitate the transition of the LCV from an sER to an rER identity(A) HeLa Fcγ cells were subjected to subcellular fractionation, and immunoblot analysis was done against the indicated proteins.(B) Sucrose gradient fractionation was done to obtain the sER and rER fractions and immunoblotted against indicated antibodies.(C) HeLa Fcγ cells expressing mRFP-Rab4 or mRFP-Rab10 were infected with L.p. for the indicated times, stained with L.p. for immunofluorescence analysis, and the vacuoles positive for mRFP-Rab4 or -Rab10 were quantified. Bar, 5 μm. The purple box represents the magnified image. Results represent three independent experiments (100 vacuoles were scored in each). Error bars represent mean ± SD.(D) HEK293 Fcγ cells were transfected with either mock, Rab4, or Rab10 siRNA for 72 h, and cell extracts were immunoblotted against the indicated antibodies.(E and F) HeLa Fcγ cells were silenced for Rab4 or Rab10 for 72 h, infected with L.p. for 4 and 8 h, and the vacuoles positive for Rtn4 or Sec61β were counted. Results represent three independent experiments, and statistical significance was measured using one-way ANOVA (100 vacuoles were scored in each experiment). Bars represent mean ± SD.(G) HeLa Fcγ cells were silenced with Rab4 and Rab10 siRNA, and the ER morphology was observed by immunofluorescence analysis. Bars, 5 μm. Arrows indicate disruption of the sER tubular network. Arrowheads indicate diffusion of rER to the cell periphery.(H) Triplicate experiments (30 cells for each condition) from 3G were quantitated and are shown as a histogram. Bars represent mean ± SD.

Fig 3: Increased colocalization of Rab4-positive and Rab11-positive vesicles in SynDIG4 KO neurons(A) Representative images showing the colocalization of SynDIG4 and Rab4 or Rab11 and quantification of Manders’ colocalization coefficient for SynDIG4 and Rab4 or Rab11 in WT neurons. (B) Representative images showing the colocalization of Rab4 and Rab11 and quantification of Manders’ colocalization coefficient for Rab4 and Rab11 in WT and SynDIG4 KO neurons. (C) Representative images showing the colocalization of Rab4 and EEA1 and quantification of Manders’ colocalization coefficient for Rab4 and EEA1 in WT and SynDIG4 KO neurons. Data are presented as mean ± SEM, with statistical significance determined using unpaired t-test; n = ~40 dendrite stretches, 2–3 dendritic stretches were cropped from individual neuron images; ****p < 0.0001. Scale bar, 5 μm.

Fig 4: BAP31 binds to Rab4 and Rab10, while BAP29 interacts with Rab4(A) The membranes of sER and rER fractions were collected by centrifugation and subjected to immunoblotting against the indicated antibodies.(B) HeLa Fcγ cells expressing GFP vector and either 3x-FLAG-Rab4, -Rab5, -Rab10, or -Rab35 were fixed, proximity ligation assay (PLA) was performed using the indicated antibodies, and the number of PLA dots was quantified. Bar, 5 μm. Green asterisks indicate the cells expressing GFP, implicating the expression of 3x-FLAG constructs.(C) Results from 3B are representative of three independent experiments, and statistical significance was achieved using one-way ANOVA (PLA dots in 25 cells expressing GFP were scored in each experiment). Error bars represent mean ± SD. ****p < 0.0001.(D and E) HEK293 Fcγ cells were transfected with the indicated plasmids, and cell extracts were subjected to immunoprecipitation assay using the anti-FLAG antibody and immunoblotted against the indicated antibodies.(F) Results from 3D and 3E are quantitated and shown as a histogram.

Fig 5: Model for the role of SynDIG4 in AMPAR distribution via endosomal recycling mechanismIn WT neurons, SynDIG4 (SD4) facilitates the trafficking of AMPARs between Rab4-positive and Rab11-positive endosomes under baseline conditions. In SynDIG4 KO neurons, the recycling of GluA1-containing AMPARs is reduced, and Rab11-positive endosomes fail to undergo fission from Rab4-positive endosomes. Consequently, the levels of intracellular AMPARs are increased, impairing the Rab4-Rab11-dependent recycling process. This SD4-mediated mechanism is crucial for the synaptic distribution of surface AMPARs. Created in BioRender. He, C. (2025) https://BioRender.com/k52o982.