Permanent deconstruction of intracellular primary cilia in differentiating granule cell neurons.

Primary cilia on granule cell neuron progenitors in the developing cerebellum detect sonic hedgehog to facilitate proliferation. Following differentiation, cerebellar granule cells become the most abundant neuronal cell type in the brain. While essential during early developmental stages, the fate of granule cell cilia is unknown. Here, we provide nanoscopic resolution of ciliary dynamics in situ by studying developmental changes in granule cell cilia using large-scale electron microscopy volumes and immunostaining of mouse cerebella. We found that many granule cell primary cilia were intracellular and concealed from the external environment. Cilia were disassembed in differentiating granule cell neurons in a process we call cilia deconstruction that was distinct from pre-mitotic cilia resorption in proliferating progenitors. In differentiating granule cells, ciliary loss involved unique disassembly intermediates, and, as maturation progressed, mother centriolar docking at the plasma membrane. Cilia did not reform from the docked centrioles, rather, in adult mice granule cell neurons remained unciliated. Many neurons in other brain regions require cilia to regulate function and connectivity. In contrast, our results show that granule cell progenitors had concealed cilia that underwent deconstruction potentially to prevent mitogenic hedgehog responsiveness. The ciliary deconstruction mechanism we describe could be paradigmatic of cilia removal during differentiation in other tissues.

Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis.

The endocytic protein EHD1 controls primary ciliogenesis by facilitating fusion of the ciliary vesicle and by removal of CP110 from the mother centriole. EHD3, the closest EHD1 paralog, has a similar regulatory role, but initial evidence suggested that the other two more distal paralogs, EHD2 and EHD4 may be dispensable for ciliogenesis. Herein, we define a novel role for EHD4, but not EHD2, in regulating primary ciliogenesis. To better understand the mechanisms and differential functions of the EHD proteins in ciliogenesis, we first demonstrated a requirement for EHD1 ATP-binding to promote ciliogenesis. We then identified two sequence motifs that are entirely conserved between EH domains of EHD1, EHD3 and EHD4, but display key amino acid differences within the EHD2 EH domain. Substitution of either P446 or E470 in EHD1 with the aligning S451 or W475 residues from EHD2 was sufficient to prevent rescue of ciliogenesis in EHD1-depleted cells upon reintroduction of EHD1. Overall, our data enhance the current understanding of the EHD paralogs in ciliogenesis, demonstrate a need for ATP-binding and identify conserved sequences in the EH domains of EHD1, EHD3 and EHD4 that regulate EHD1 binding to proteins and its ability to rescue ciliogenesis in EHD1-depleted cells.

C3G localizes to the mother centriole in a cenexin-dependent manner and regulates centrosome duplication and primary cilium length.

C3G (also known as RAPGEF1) plays a role in cell differentiation and is essential for early embryonic development in mice. In this study, we identify C3G as a centrosomal protein that colocalizes with cenexin (also known as ODF2) at the mother centriole in interphase cells. C3G interacts with cenexin through its catalytic domain, and the two proteins show interdependence for localization to the centrosome. C3G depletion causes a decrease in cellular cenexin levels. Centrosomal localization of C3G is lost as myocytes differentiate to form myotubes. Depletion of C3G by CRISPR/Cas9 results in the formation of supernumerary centrioles, whereas overexpression of C3G, or expression of a catalytically active C3G deletion construct, inhibits centrosome duplication. Cilium length is increased in C3G knockout cells, and this phenotype is reverted upon reintroduction of C3G or its catalytic domain alone. Association of C3G with the basal body is dynamic, decreasing upon serum starvation and increasing upon re-entry into the cell cycle. C3G inhibits cilium formation and length, and this inhibition is dependent on C3G catalytic activity. We conclude that C3G regulates centrosome duplication and maintains ciliary homeostasis, properties that could be important for its role in embryonic development.

NANOG/NANOGP8 Localizes at the Centrosome and is Spatiotemporally Associated with Centriole Maturation.

NANOG is a transcription factor involved in the regulation of pluripotency and stemness. The functional paralog of NANOG, NANOGP8, differs from NANOG in only three amino acids and exhibits similar reprogramming activity. Given the transcriptional regulatory role played by NANOG, the nuclear localization of NANOG/NANOGP8 has primarily been considered to date. In this study, we investigated the intriguing extranuclear localization of NANOG and demonstrated that a substantial pool of NANOG/NANOGP8 is localized at the centrosome. Using double immunofluorescence, the colocalization of NANOG protein with pericentrin was identified by two independent anti-NANOG antibodies among 11 tumor and non-tumor cell lines. The validity of these observations was confirmed by transient expression of GFP-tagged NANOG, which also colocalized with pericentrin. Mass spectrometry of the anti-NANOG immunoprecipitated samples verified the antibody specificity and revealed the expression of both NANOG and NANOGP8, which was further confirmed by real-time PCR. Using cell fractionation, we show that a considerable amount of NANOG protein is present in the cytoplasm of RD and NTERA-2 cells. Importantly, cytoplasmic NANOG was unevenly distributed at the centrosome pair during the cell cycle and colocalized with the distal region of the mother centriole, and its presence was markedly associated with centriole maturation. Along with the finding that the centrosomal localization of NANOG/NANOGP8 was detected in various tumor and non-tumor cell types, these results provide the first evidence suggesting a common centrosome-specific role of NANOG.

The C7orf43/TRAPPC14 component links the TRAPPII complex to Rabin8 for preciliary vesicle tethering at the mother centriole during ciliogenesis.

The primary cilium is a cellular sensor that detects light, chemicals, and movement and is important for morphogen and growth factor signaling. The small GTPase Rab11-Rab8 cascade is required for ciliogenesis. Rab11 traffics the guanine nucleotide exchange factor (GEF) Rabin8 to the centrosome to activate Rab8, needed for ciliary growth. Rabin8 also requires the transport particle protein complex (TRAPPC) proteins for centrosome recruitment during ciliogenesis. Here, using an MS-based approach for identifying Rabin8-interacting proteins, we identified C7orf43 (also known as microtubule-associated protein 11 (MAP11)) as being required for ciliation both in human cells and zebrafish embryos. We find that C7orf43 directly binds to Rabin8 and that C7orf43 knockdown diminishes Rabin8 preciliary centrosome accumulation. Interestingly, we found that C7orf43 co-sediments with TRAPPII complex subunits and directly interacts with TRAPPC proteins. Our findings establish that C7orf43 is a TRAPPII-specific complex component, referred to here as TRAPPC14. Additionally, we show that TRAPPC14 is dispensable for TRAPPII complex integrity but mediates Rabin8 association with the TRAPPII complex. Finally, we demonstrate that TRAPPC14 interacts with the distal appendage proteins Fas-binding factor 1 (FBF1) and centrosomal protein 83 (CEP83), which we show here are required for GFP-Rabin8 centrosomal accumulation, supporting a role for the TRAPPII complex in tethering preciliary vesicles to the mother centriole during ciliogenesis. In summary, our findings have revealed an uncharacterized TRAPPII-specific component, C7orf43/TRAPPC14, that regulates preciliary trafficking of Rabin8 and ciliogenesis and support previous findings that the TRAPPII complex functions as a membrane tether.

Akt Regulates a Rab11-Effector Switch Required for Ciliogenesis.

Serum starvation stimulates cilia growth in cultured cells, yet serum factors associated with ciliogenesis are unknown. Previously, we showed that starvation induces rapid Rab11-dependent vesicular trafficking of Rabin8, a Rab8 guanine-nucleotide exchange factor (GEF), to the mother centriole, leading to Rab8 activation and cilium growth. Here, we demonstrate that through the LPA receptor 1 (LPAR1), serum lysophosphatidic acid (LPA) inhibits Rab11a-Rabin8 interaction and ciliogenesis. LPA/LPAR1 regulates ciliogenesis initiation via downstream PI3K/Akt activation, independent of effects on cell cycle. Akt stabilizes Rab11a binding to its effector, WDR44, and a WDR44-pAkt-phosphomimetic mutant blocks ciliogenesis. WDR44 depletion promotes Rabin8 preciliary trafficking and ciliogenesis-initiating events at the mother centriole. Our work suggests disruption of Akt signaling causes a switch from Rab11-WDR44 to the ciliogenic Rab11-FIP3-Rabin8 complex. Finally, we demonstrate that Akt regulates downstream ciliogenesis processes associated with Rab8-dependent cilia growth. Together, this study uncovers a mechanism whereby serum mitogen signaling regulates Rabin8 preciliary trafficking and ciliogenesis initiation.

LRRC45 contributes to early steps of axoneme extension.

Cilia perform essential signalling functions during development and tissue homeostasis. A key event in ciliogenesis occurs when the distal appendages of the mother centriole form a platform that docks ciliary vesicles and removes CP110-Cep97 inhibitory complexes. Here, we analysed the role of LRRC45 in appendage formation and ciliogenesis. We show that the core appendage proteins Cep83 and SCLT1 recruit LRRC45 to the mother centriole. Once there, LRRC45 recruits the keratin-binding protein FBF1. The association of LRRC45 with the basal body of primary and motile cilia in both differentiated and stem cells reveals a broad function in ciliogenesis. In contrast to the appendage components Cep164 and Cep123, LRRC45 was not essential for either docking of early ciliary vesicles or for removal of CP110. Rather, LRRC45 promotes cilia biogenesis in CP110-uncapped centrioles by organising centriolar satellites, establishing the transition zone and promoting the docking of Rab8 GTPase-positive vesicles. We propose that, instead of acting solely as a platform to recruit early vesicles, centriole appendages form discrete scaffolds of cooperating proteins that execute specific functions that promote the initial steps of ciliogenesis.

Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex.

Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development.

Methods to analyze novel liaisons between endosomes and centrosomes.

For some time, it has been known that recycling endosomes (REs) are organized in a nebulous "pericentrosomal" region in interphase cells. However, the collective use of previously developed methods, including centrosome isolation, live cell imaging, and electron microscopy, suggested that there is much more going on between the centrosome and the RE than previously imagined. By exploiting these approaches, we uncovered novel roles of the centrosome in RE function and, conversely, novel roles for REs in centrosome function. We first found that REs dynamically localized to the centrosome throughout the cell cycle. More specifically, we found that REs interacted with appendages of the older centriole in interphase cells to control endosome recycling, and this interaction was governed by RE-machinery including the small GTPase Rab11. We next determined that REs carry centrosome proteins to spindle poles as part of the "centrosome maturation" process. Here we discuss the methods used and materials needed to complete these types of studies.

Cell Cycle-Dependent Localization of Dynactin Subunit p150 glued at Centrosome.

p150(glued) is the largest subunit of dynactin protein complex, through which cargo vesicles link to the microtubule minus-end directed motor protein dynein. In addition, p150(glued) also locates in the mother centriole where it organizes the subdistal appendage. The components of appendage are dynamically regulated throughout the cell cycle stages, but it is still unclear whether the centrosomal residency of p150(glued) correlated with cell cycle progression. Here we found that p150(glued) was located in the mother centriole during G1/S stage and its centrosomal residency was independent of microtubule transportation. However, the centrosomal p150(glued) became blurred at G2/M phase and this event was not regulated by its phosphorylation. Entering into mitosis, p150(glued) was robustly enriched in the mitotic spindle nearby the spindle poles but not in the centrosome. During serum starvation (G0 stage), p150(glued) appeared at the base of primary cilium and its depletion attenuated starvation-induced primary cilium formation. We also checked its role in the maintenance of centrosome homeostasis and configuration, and found depletion of p150(glued) did not induce centrosome amplification or splitting but inhibited U2OS cell growth. G1 arrest and reduced EdU incorporation were observed in p150(glued) deficient U2OS cells. In addition, cyclin E was downregulated following p150(glued) depletion. The p53/p21 signaling was activated indicating that CDKs were inactivated. The reduced cell growth was ameliorated in the p150(glued) depleted cells when treated with p53 inhibitor. Thus, we have identified the centrosomal targeting of p150(glued) in distinct cell cycle stage and uncovered its role in controlling G1/S transition.