Vesicular trafficking plays a role in centriole disengagement and duplication

Abstract

Centrosomes are the major microtubule-nucleating and microtubule-organizing centers of cells and play crucial roles in microtubule anchoring, organelle positioning, and ciliogenesis. At the centrosome core lies a tightly associated or "engaged" mother-daughter centriole pair. During mitotic exit, removal of centrosomal proteins pericentrin and Cep215 promotes "disengagement" by the dissolution of intercentriolar linkers, ensuring a single centriole duplication event per cell cycle. Herein, we explore a new mechanism involving vesicular trafficking for the removal of centrosomal Cep215. Using small interfering RNA and CRISPR/Cas9 gene-edited cells, we show that the endocytic protein EHD1 regulates Cep215 transport from centrosomes to the spindle midbody, thus facilitating disengagement and duplication. We demonstrate that EHD1 and Cep215 interact and show that Cep215 displays increased localization to vesicles containing EHD1 during mitosis. Moreover, Cep215-containing vesicles are positive for internalized transferrin, demonstrating their endocytic origin. Thus, we describe a novel relationship between endocytic trafficking and the centrosome cycle, whereby vesicles of endocytic origin are used to remove key regulatory proteins from centrosomes to control centriole duplication.

Cellular functions and intrinsic attributes of the ATP‐binding Eps15 homology domain‐containing proteins

“…The latter function links EHD to the recruitment of the SNARE protein SNAP29 for fusion of DAVs to form the CV. In addition, a recent report indicates that MICAL L1 is required for EHD1 recruitment during these early steps, similar to the reported functions of MICAL L1 at the endocytic recycling compartment 62 . Other membrane remodeling proteins such as the F‐BAR domain‐containing Pacsin1 and 2 are also required during these early steps of ciliogenesis 63 .…”

MICAL-L1 coordinates ciliogenesis by recruiting EHD1 to the primary cilium

“…1H,K), we hypothesized that MICAL-L1 is involved in EHD1 recruitment to the cilium. Accordingly, we serum-starved CRISPR/ CAS9 gene-edited NIH3T3 cells expressing endogenous levels of EHD1 as a GFP fusion protein (Xie et al, 2018;Yeow et al, 2017), and immunostained them for EHD1, MICAL-L1 and acetylated tubulin. Under these serum-starved conditions, both EHD1 and MICAL-L1 could be detected along acetylated tubulin-stained cilia ( Fig.…”

Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission

“…As we have recently demonstrated that EHD1-depletion impairs fission and induces enlarged SE [27], we next asked whether simultaneous depletion of EHD1 and EHD4 further impedes endosomal fission and leads to increased SE size. To this aim, we used CRISPR/Cas9 NIH3T3 cells that were gene-edited and chronically lack EHD1 (EHD1 KO), EHD4 (EHD4 KO) or both EHD1 and EHD4 (EHD1/EHD4 DKO) [34,35] (Fig 3; protein expression validated in 3I). As demonstrated, in both the EHD1 and EHD4 single KO cell lines, SE size was modestly but significantly larger than in the wild-type parental NIH3T3 cell line (compare B and the inset in F, and C and the inset in G, to A and the inset E, and quantified in J).…”

“…The HeLa cervical cancer cell line was acquired from the American Type Culture Collection (ATCC, CRM-CCL-2). NIH3T3 (ATCC, CRL-1658) parental cells were subjected to CRISPR/ Cas9 to generate the NIH3T3 cell line expressing endogenous levels of EHD1 with GFP attached to the C-terminus, as well as the EHD1 knock-out, EHD4 knock-out and EHD1/ EHD4 double knock-out cells as described [34,35]. Both HeLa and NIH3T3 cells were cultured at 37˚C in 5% CO2 in DMEM/High Glucose (HyClone, SH30243.01) containing 10% heat-inactivated Fetal Bovine Serum (Atlanta Biologicals, S1150), 1x Penicillin Streptomycin (Gibco, 15140-122), 50 mg of Normocin (InvivoGen, NOL-40-09), and 2 mM L-Glutamine (Gibco, 25030-081).…”