Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11–Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11–Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby EHD1 and EHD3 reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.

Exosomes are extracellular vesicles of endosomal origin that are released by practically all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs, and proteins between different cells, tissues, or organs. Here, we describe a mechanism whereby proteins containing a KFERQ motif pentapeptide are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, we demonstrate that this mechanism is independent of the ESCRT machinery but dependent on HSC70, CD63, Alix, Syntenin-1, Rab31, and ceramides. We show that the master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. In addition, by tagging fluorescent proteins with KFERQ-like sequences, we were able to follow the interorgan transfer of exosomes. Our findings open new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs.

The Arf-like protein Arl13b has been implicated in ciliogenesis and Sonic hedgehog signaling. Furthermore, we have previously shown that it regulates endocytic recycling traffic and interacts with actin. Herein, we report that the non-muscle myosin heavy chain IIA, also known as Myh9, is an Arl13b effector. Moreover, we found that both proteins localize to circular dorsal ruffles (CDRs) induced by platelet-derived growth factor stimulation and are required for their formation. CDRs are ring-shaped actin-dependent structures formed on the dorsal cell surface and have been involved in diverse processes such as macropinocytosis, integrin recycling, internalization of receptor tyrosine kinases and cell migration. We found that Arl13b or Myh9 silencing impairs cell migration, suggesting that Arl13b is required for this function through the interaction with Myh9. Moreover, Arl13b silencing impairs neural crest cell migration in zebrafish embryos. Furthermore, we show that Arl13b is required for the formation of CDRs in migrating cells. Thus, our results indicate a novel role for Arl13b in actin cytoskeleton remodeling through the interaction with Myh9, by driving the formation of CDRs necessary for cell migration.