The present work characterizes one of the earliest sequences in the establishment of pre-AC and suggests that subsets of EE-derived tubular membranes may serve as the earliest biomarkers in pre-AC biogenesis. Our study also indicates that the pre-AC biogenesis is complex and likely involves multiple parallel processes, of which Rab10-PD expansion is one. Our experiments, particularly our silencing experiments, show that Rab10 and EHBP-1 do not play a significant role in the later stages of inner pre-AC biogenesis or in the expansion of downstream tubular domains. A more comprehensive understanding of the tubular domain expansion remains to be established.

We performed long-term live imaging of EGFP-Rab10 with epifluorescence imaging-enhanced digital holotomographic microscopy (DHTM), confocal imaging of known Rab10 interactors and identification of important Rab10 interactors with the proximity-dependent biotin identification assay (BioID). The accumulation of Rab10-PD was analyzed after knock-down of EHBP1 and Rabin8, two proteins that facilitate Rab10 recruitment to membranes, and after blocking of PI(4,5)P2 by PI(4,5)P2-binding protein domains.

Our study shows the gradual expansion of Rab10-PD in the inner pre-AC, the association of Rab10 with EHBP1 and MICAL-L1, and the dependence of Rab10-PD expansion on EHBP1 and PI(4,5)P2 but not Rabin8, indicating the expansion of EE-derived tubular recycling endosome-like membranes in the pre-AC. Silencing of Rab10 and EHBP1 suggests that Rab10-PD expansion is not required for the establishment of the inner pre-AC nor for the expansion of downstream tubular domains.