Exosomes, important players in cell-cell communication, are small extracellular vesicles of endocytic origin. Although single cells are known to release various kinds of exosomes (referred to as exosomal heterogeneity), very little is known about the mechanisms by which they are produced and released. Here, we established methods of studying exosomal heterogeneity by using polarized epithelial cells and showed that distinct types of small extracellular vesicles (more specifically CD9-and CD63-positive, Annexin I-negative small extracellular vesicles, which we refer to as exosomes herein) are differentially secreted from the apical and basolateral sides of polarized epithelial cells. We also identify GPRC5C (G protein-coupled receptor class C group 5 member C) as an apical exosome-specific protein. We further demonstrate that basolateral exosome release depends on ceramide, whereas ALIX, an ESCRT (endosomal sorting complexes required for transport)-related protein, not the ESCRT machinery itself, is required for apical exosome release. Thus, two independent machineries, the ALIX-Syntenin1-Syndecan1 machinery (apical side) and the sphingomyelinase-dependent ceramide production machinery (basolateral side), are likely to be responsible for the polarized exosome release from epithelial cells.

The small GTPase Rab11 plays pivotal roles in diverse physiological phenomena, including the recycling of membrane proteins, cytokinesis, neurite outgrowth, and epithelial morphogenesis. One effective method of analyzing the function of endogenous Rab11 is to overexpress a Rab11-binding domain of one of its effectors, e.g., the C-terminal domain of Rab11-FIP2 (Rab11-FIP2-C), as a dominant-negative construct. However, the drawback of this method is the broader Rab binding specificity of the effector domain, because Rab11-FIP2-C binds to Rabs other than Rab11, e.g., to Rab14 and Rab25. In this study, we bioengineered an artificial Rab11-specific binding domain, named RBD11. Expression of RBD11 visualized endogenous Rab11 without affecting its localization or function, whereas expression of a tandem RBD11, named 2×RBD11, inhibited epithelial morphogenesis and induced a multi-lumen phenotype characteristic of Rab11-deficient cysts. We also developed two tools for temporally and reversibly analyzing Rab11-dependent membrane trafficking: tetracycline-inducible 2×RBD11 and an artificially oligomerized domain (FM)-tagged RBD11.

Exosomes, new players in cell-cell communication, are extracellular vesicles of endocytic origin. Although single cells are known to release various kinds of exosomes (referred to as exosomal heterogeneity), very little is known about the mechanisms by which they are produced and released. Here, we established methods for studying exosomal heterogeneity by using polarized epithelial cells and showed that distinct types of exosomes are differentially secreted from the apical and basolateral sides. We also identified GPRC5C (G protein-coupled receptor class C group 5 member C) as an apical-exosome-specific protein. We further demonstrated that basolateral exosome release depends on ceramide, whereas ALIX, an ESCRT (endosomal sorting complexes required for transport)-related protein, not the ESCRT machinery itself, is required for apical exosome secretion. Thus, two independent machineries, the ALIX–Syntenin1– Syndecan1 machinery (apical side) and the sphingomyelinase-dependent ceramide production machinery (basolateral side), are likely to be responsible for the polarized exosome release from epithelial cells.

No abstract

The small GTPase Rab11 plays pivotal roles in diverse physiological phenomena, including the recycling of membrane proteins, cytokinesis, neurite outgrowth, and epithelial morphogenesis. One effective method of analyzing the function of endogenous Rab11 is to overexpress a Rab11-binding domain of one of its effectors, e.g., the C-terminal domain of Rab11-FIP2 (Rab11-FIP2-C), as a dominant-negative construct. However, the drawback of this method is the broader Rab binding specificity of the effector domain, because Rab11-FIP2-C binds to Rabs other than Rab11, e.g., to Rab14 and Rab25. In this study, we bioengineered an artificial Rab11-specific binding domain, named RBD11. Expression of RBD11 visualized endogenous Rab11 without affecting its localization or function, whereas expression of a tandem RBD11, named 2×RBD11, inhibited epithelial morphogenesis and induced a multi-lumen phenotype characteristic of Rab11-deficient cysts. We also developed two tools for temporally and reversibly analyzing Rab11-dependent membrane trafficking: tetracycline-inducible 2×RBD11 and an artificially oligomerized domain (FM)-tagged RBD11.