MID1 and MID2 are closely related members of the TRIM family of E3 ubiquitin ligases, involved in regulating cytoskeletal organization, intracellular signaling, and spatial protein dynamics. Mutations in MID1 are causative of X-linked Opitz GBBB syndrome (OS), a congenital disorder affecting developmental structures of the midline, whereas MID2 mutations are linked to intellectual disability. Although most evidence links these E3 ligases to cell architecture, the broader cellular functions of both MID1 and MID2 remain poorly understood. In this study, using the BioE3 approach in combination with proteomic analysis, we identified endosome-associated substrates potentially regulated by these E3 ligases. We investigated whether depletion of MID1 and MID2 using siRNA-mediated knockdown in human cells could affect endosomal organization. Phenotypes were analyzed by immunofluorescence and quantitative image analysis, including changes in the expression and subcellular distribution of well-established markers of early endosomes. Our findings show that the silencing of MID1 and/or MID2 leads to altered endosomal properties, including changes in EEA1 signal intensity and dispersion. These findings support the hypothesis that MID1 and MID2, through their regulatory roles in microtubule regulation and endosome distribution and/or maturation, contribute to the cellular vesicular trafficking pathways.
