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 pre-ciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle (CV) formation from smaller distal appendage vesicles (DAV). 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 CV assembly. Interestingly, only after CV 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 DAV prior to coordinated ciliary membrane and axoneme growth.

SUMMARY The response to DNA damage, which regulates nuclear processes such as DNA repair, transcription, and cell cycle, has been studied thoroughly. However, the cytoplasmic response to DNA damage is poorly understood. Here, we demonstrate that DNA damage triggers dramatic reorganization of the Golgi, resulting in its dispersal throughout the cytoplasm. We further show that DNA-damage-induced Golgi dispersal requires GOLPH3/MYO18A/F-actin and the DNA damage protein kinase, DNA-PK. In response to DNA damage, DNA-PK phosphorylates GOLPH3, resulting in increased interaction with MYO18A, which applies a tensile force to the Golgi. Interference with the Golgi DNA damage response by depletion of DNA-PK, GOLPH3, or MYO18A reduces survival after DNA damage, whereas over-expression of GOLPH3, as is observed frequently in human cancers, confers resistance to killing by DNA-damaging agents. Identification of the DNA-damage-induced Golgi response reveals an unexpected pathway through DNA-PK, GOLPH3, and MYO18A that regulates cell survival following DNA damage.

Eps15 homology domain (EHD) 1 enables membrane recycling by controlling the exit of internalized molecules from the endocytic recycling compartment (ERC) en route to the plasma membrane, similar to the role described for Rab11. However, no physical or functional connection between Rab11 and EHD-family proteins has been demonstrated yet, and the mode by which they coordinate their regulatory activity remains unknown. Here, we demonstrate that EHD1 and EHD3 (the closest EHD1 paralog), bind to the Rab11-effector Rab11-FIP2 via EH-NPF interactions. The EHD/Rab11-FIP2 associations are affected by the ability of the EHD proteins to bind nucleotides, and Rab11-FIP2 is recruited to EHD-containing membranes. These results are consistent with a coordinated role for EHD1 and Rab11-FIP2 in regulating exit from the ERC. However, because no function has been attributed to EHD3, the significance of its interaction with Rab11-FIP2 remained unclear. Surprisingly, loss of EHD3 expression prevented the delivery of internalized transferrin and early endosomal proteins to the ERC, an effect differing from that described upon EHD1 knockdown. Moreover, the subcellular localization of Rab11-FIP2 and endogenous Rab11 were altered upon EHD3 knockdown, with both proteins absent from the ERC and retained in the cell periphery. The results presented herein promote a coordinated role for EHD proteins and Rab11-FIP2 in mediating endocytic recycling and provide evidence for the function of EHD3 in early endosome to ERC transport. INTRODUCTIONInternalization of plasma membrane proteins is a critical event required for multiple physiological cellular processes and its regulation is mediated by a complex web of molecular components (Mellman, 1996;Conner and Schmid, 2003;Benmerah, 2004).Proteins at the plasma membrane are either internalized into clathrin-coated vesicles (Conner and Schmid, 2003;Benmerah, 2004), or they can be internalized independently of clathrin (Nichols and Lippincott-Schwartz, 2001;Naslavsky et al., 2004b). In either case, the internalized vesicles deliver their cargo to the endocytic pathway by fusing with early endosomes (Naslavsky et al., 2004b). Although many proteins are transported along the endosomal pathway to late endosomes and lysosomes where they ultimately undergo degradation, some endocytic receptors are returned to the plasma membrane where they continue to exert their physiological effects (Maxfield and McGraw, 2004).Recycling to the plasma membrane can occur either directly from the early endosome in a process that is not well understood (Sheff et al., 1999;Hao and Maxfield, 2000;Sheff et al., 2002;van Dam et al., 2002) or indirectly through a pericentriolar-localized organelle known as the endocytic recycling compartment (ERC) (Gruenberg and Maxfield, 1995;Maxfield and McGraw, 2004). This compartment is a condensed cellular region containing tubular membrane structures that emanate from the microtubule organizing center (Hopkins and Trowbridge, 1983;Yamashiro et al., 1984).Evidence suggests that endocytic rec...

Endocytic recycling of receptors and lipids occurs via a complex network of tubular and vesicular membranes. EHD1 is a key regulator of endocytosis and associates with tubular membranes to facilitate recycling. Although EHD proteins tubulate membranes in vitro, EHD1 primarily associates with preexisting tubules in vivo. How EHD1 is recruited to these tubular endosomes remains unclear. We have determined that the Rab8-interacting protein, MICAL-L1, associates with EHD1, with both proteins colocalizing to long tubular membranes, in vitro and in live cells. MICAL-L1 is a largely uncharacterized member of the MICAL-family of proteins that uniquely contains two asparagine-proline-phenylalanine motifs, sequences that typically interact with EH-domains. Our data show that the MICAL-L1 C-terminal coiled-coil region is necessary and sufficient for its localization to tubular membranes. Moreover, we provide unexpected evidence that endogenous MICAL-L1 can link both EHD1 and Rab8a to these structures, as its depletion leads to loss of the EHD1-Rab8a interaction and the absence of both of these proteins from the membrane tubules. Finally, we demonstrate that MICAL-L1 is essential for efficient endocytic recycling. These data implicate MICAL-L1 as an unusual type of Rab effector that regulates endocytic recycling by recruiting and linking EHD1 and Rab8a on membrane tubules.

Endocytosis is a conserved process across species in which cell surface receptors and lipids are internalized from the plasma membrane. Once internalized, receptors can either be degraded or recycled back to the plasma membrane. A variety of small GTP-binding proteins regulate receptor recycling. Despite our familiarity with many of the key regulatory proteins involved in this process, our understanding of the mode by which these proteins cooperate and the sequential manner in which they function remains limited. In this study, we identify two GTP-binding proteins as interaction partners of the endocytic regulatory protein MICAL-L1. First, we demonstrate that Rab35 is a MICAL-L1 binding partner in vivo. Over-expression of active Rab35 impairs the recruitment of MICAL-L1 to tubular recycling endosomes, whereas Rab35 depletion promotes enhanced MICAL-L1 localization to these structures. Moreover, we demonstrate that Arf6 forms a complex with MICAL-L1 and plays a role in its recruitment to tubular endosomes. Overall, our data suggest a model in which Rab35 is a critical upstream regulator of MICAL-L1 and Arf6, while both MICAL-L1 and Arf6 regulate Rab8a function.