Phosphorylation and Ubiquitylation Regulate Protein Trafficking, Signaling, and the Biogenesis of Primary Cilia

The primary cilium is a solitary, microtubule-based membrane protrusion extending from the surface of quiescent cells that senses the cellular environment and triggers specific cellular responses. The functions of primary cilia require not only numerous different components but also their regulated interplay. The cilium performs highly dynamic processes, such as cell cycle-dependent assembly and disassembly as well as delivery, modification, and removal of signaling components to perceive and process external signals. On a molecular level, these processes often rely on a stringent control of key modulatory proteins, of which the activity, localization, and stability are regulated by post-translational modifications (PTMs). While an increasing number of PTMs on ciliary components are being revealed, our knowledge on the identity of the modifying enzymes and their modulation is still limited. Here, we highlight recent findings on cilia-specific phosphorylation and ubiquitylation events. Shedding new light onto the molecular mechanisms that regulate the sensitive equilibrium required to maintain and remodel primary cilia functions, we discuss their implications for cilia biogenesis, protein trafficking, and cilia signaling processes

YAP不活性化におけるFurryの役割とCEP97分解における14-3-3の関与

Tohoku University大橋一正課

CEP78 functions downstream of CEP350 to control biogenesis of primary cilia by negatively regulating CP110 levels.

CEP78 is a centrosomal protein implicated in ciliogenesis and ciliary length control, and mutations in the CEP78 gene cause retinal cone-rod dystrophy associated with hearing loss. However, the mechanism by which CEP78 affects cilia formation is unknown. Based on a recently discovered disease-causing CEP78 p.L150S mutation, we identified the disease-relevant interactome of CEP78. We confirmed that CEP78 interacts with the EDD1-DYRK2-DDB1 <sup>VPRBP</sup> E3 ubiquitin ligase complex, which is involved in CP110 ubiquitination and degradation, and identified a novel interaction between CEP78 and CEP350 that is weakened by the CEP78 <sup>L150S</sup> mutation. We show that CEP350 promotes centrosomal recruitment and stability of CEP78, which in turn leads to centrosomal recruitment of EDD1. Consistently, cells lacking CEP78 display significantly increased cellular and centrosomal levels of CP110, and depletion of CP110 in CEP78-deficient cells restored ciliation frequency to normal. We propose that CEP78 functions downstream of CEP350 to promote ciliogenesis by negatively regulating CP110 levels via an EDD1-dependent mechanism

The role of centrosome distal appendage proteins (DAPs) in nephronophthisis and ciliogenesis

The primary cilium is found in most mammalian cells and plays a functional role in tissue homeostasis and organ development by modulating key signaling pathways. Ciliopathies are a group of genetically heterogeneous disorders resulting from defects in cilia development and function. Patients with ciliopathic disorders exhibit a range of phenotypes that include nephronophthisis (NPHP), a progressive tubulointerstitial kidney disease that commonly results in end-stage renal disease (ESRD). In recent years, distal appendages (DAPs), which radially project from the distal end of the mother centriole, have been shown to play a vital role in primary ciliary vesicle docking and the initiation of ciliogenesis. Mutations in the genes encoding these proteins can result in either a complete loss of the primary cilium, abnormal ciliary formation, or defective ciliary signaling. DAPs deficiency in humans or mice commonly results in NPHP. In this review, we outline recent advances in our understanding of the molecular functions of DAPs and how they participate in nephronophthisis development

Phosphorylation of CEP83 by TTBK2 is necessary for cilia initiation

© 2019 Lo et al. Primary cilia are microtubule-based organelles that play important roles in development and tissue homeostasis. Tau-tubulin kinase-2 (TTBK2) is genetically linked to spinocerebellar ataxia type 11, and its kinase activity is crucial for ciliogenesis. Although it has been shown that TTBK2 is recruited to the centriole by distal appendage protein CEP164, little is known about TTBK2 substrates associated with its role in ciliogenesis. Here, we perform superresolution microscopy and discover that serum starvation results in TTBK2 redistribution from the periphery toward the root of distal appendages. Our biochemical analyses uncover CEP83 as a bona fide TTBK2 substrate with four phosphorylation sites characterized. We also demonstrate that CEP164-dependent TTBK2 recruitment to distal appendages is required for subsequent CEP83 phosphorylation. Specifically, TTBK2-dependent CEP83 phosphorylation is important for early ciliogenesis steps, including ciliary vesicle docking and CP110 removal. In summary, our results reveal a molecular mechanism of kinase regulation in ciliogenesis and identify CEP83 as a key substrate of TTBK2 during cilia initiation.Ministry of Science and Technology, Taiwan (MOST 105-2628-B-010-004-MY3, MOST 107-2313-B-010-001, MOST 108-2628-B-010-007, MOST 107- 2633-B-009-003, and Shackleton Program Grant); Yen Tjing Ling Medical Foundation (CI-107-17 and CI-108-12); Ministry of Education, Taiwan, Higher Education Sprout Project (107AC-D920); National Core Facility for Biopharmaceuticals, Taiwan, Clinical and Industrial Genomic Application Development Service (MOST 107-2319-B-010-002)

The role of primary cilia in Townes-Brocks Syndrome

216 p.El Síndrome de Townes-Brocks (TBS1, MIM 107480 ) está causado por mutaciones en el gen SALL1, dando lugar, en la mayoría de los casos, a una proteína truncada. TBS se caracteriza por un espectro de malformaciones en dedos, oídos, corazón y riñones. Curiosamente, estos síntomas coinciden con aquellos observados en un número creciente de enfermedades y síndromes genéticos ligados a la formación y función del cilio primario conocidos como ciliopatías. Hasta ahora, el estudio del TBS se ha centrado en los defectos que, a nivel transcripcional, las mutaciones en SALL1 podrían ocasionar en el núcleo. Sin embargo, en esta tesis postulamos que las truncaciones en SALL1 podrían interferir con la función o señalización de los cilios debido a su interacción dominante negativa con proteínas del centrosoma o del cilio primario. Hemos descubierto que la proteína SALL1 truncada causa un incremento de la ciliogenesis y de la señalización de la vía Sonic-Hedgehog (Shh). También descubrimos que la forma truncada de SALL1 interactúa con los reguladores negativos de la ciliogénesis CCP110 y CEP97, posiblemente desestabilizándolos o desplazándolos del centríolo madre que da lugar al cilio primario y permitiendo, por tanto, la ciliogenesis. Además, SALL1 interactúa con la proteína LUZP1 que contiene cremalleras de leucina y tiene un papel fundamental durante el desarrollo. De hecho, el modelo de ratón knock-out de LUZP1 fenocopia algunas características asociadas con ciliopatías humanas, como defectos del tubo neural o de la formación del corazón. Nuestros datos respaldan el descubrimiento de que LUZP1 puede participar en la integración de la ciliogénesis y la dinámica de la actina durante el desarrollo.CICbioGUNE, Excelencia Severo Ocho

MECHANISMS OF HUMAN PAPILLOMAVIRUS TYPE 16 E7 (HPV-16 E7)-INDUCED DISRUPTION OF CENTRIOLE DUPLICATION CONTROL

Infection with high-risk human papillomaviruses (HPVs) is the main cause of cervical cancer, the second most common cause of cancer-related mortality in women worldwide. High-risk HPV types, such as HPV-16, express two oncoproteins, E6 and E7, which function to subvert critical host cell cycle control mechanisms in order to promote viral genome amplification. Disruption of the pRB signaling axis and the p53-mediated stress response by the HPV E7 and E6 oncoproteins, respectively, results not only in aberrant proliferation but also in host cellular changes that can promote genomic instability. The high-risk HPV-16 E7 oncoprotein was found to induce centrosome abnormalities thereby disrupting mitotic fidelity and increasing the risk for chromosome missegregation and aneuploidy. Aneuploidy is frequently found in pre-malignant high-risk HPV-associated lesions and is a critical factor for malignant progression. This thesis was designed to determine the molecular mechanisms behind the ability of HPV-16 E7 to rapidly induce centriole overduplication. This rapid induction was found to be possible through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication). It was previously discovered that the centriole multiplication pathway relied on cyclin E, CDK2 and PLK4. However, it was not known before how these molecular players cooperate in the centriole multiplication pathway or how HPV-16 E7 expression promotes the activation of this pathway. Here, we report that cyclin E/CDK2 mediates the aberrant recruitment of PLK4 to maternal centrioles. This initial recruitment step was not sufficient to induce centriole multiplication unless PLK4 protein levels were increased. We found that PLK4 protein levels were controlled by proteolysis, specifically by CUL1-based E3 ubiquitin ligase complexes localized at maternal centrioles. SCF activity was found to control not only baseline PLK4 protein stability but its activity-dependent degradation following cyclin E/CDK2 overexpression. High-risk HPV-16 E7 is known to deregulate cyclin E/CDK2 complexes and we found that ectopic expression of HPV-16 E7 promoted the aberrant recruitment of PLK4 to maternal centrioles. Since our previous experiments have shown that aberrant recruitment of PLK4 is not sufficient to drive centriole overduplication, we determined whether HPV-16 E7 may also disrupt PLK4 expression. We found that HPV-16 E7, but not low-risk HPV proteins or mutants of HPV-16 E7 that lack the ability to induce centriole overduplication, causes a moderate but significant upregulation of PLK4 mRNA. Besides centriole duplication control, we discovered that proteolysis also regulates other aspects of centriole synthesis such as regulation of daughter centriole length. Defining the precise molecular circuitry of centriole biogenesis will aid not only in deepening the current understanding of centriole biogenesis but also aid in identification of novel targets, such as CDK2 or PLK4, for small molecules to prevent centriole abnormalities, mitotic infidelity and malignant progression in pre-invasive high-risk HPV-associated lesions

The role of primary cilia in Townes-Brocks Syndrome

216 p.El Síndrome de Townes-Brocks (TBS1, MIM 107480 ) está causado por mutaciones en el gen SALL1, dando lugar, en la mayoría de los casos, a una proteína truncada. TBS se caracteriza por un espectro de malformaciones en dedos, oídos, corazón y riñones. Curiosamente, estos síntomas coinciden con aquellos observados en un número creciente de enfermedades y síndromes genéticos ligados a la formación y función del cilio primario conocidos como ciliopatías. Hasta ahora, el estudio del TBS se ha centrado en los defectos que, a nivel transcripcional, las mutaciones en SALL1 podrían ocasionar en el núcleo. Sin embargo, en esta tesis postulamos que las truncaciones en SALL1 podrían interferir con la función o señalización de los cilios debido a su interacción dominante negativa con proteínas del centrosoma o del cilio primario. Hemos descubierto que la proteína SALL1 truncada causa un incremento de la ciliogenesis y de la señalización de la vía Sonic-Hedgehog (Shh). También descubrimos que la forma truncada de SALL1 interactúa con los reguladores negativos de la ciliogénesis CCP110 y CEP97, posiblemente desestabilizándolos o desplazándolos del centríolo madre que da lugar al cilio primario y permitiendo, por tanto, la ciliogenesis. Además, SALL1 interactúa con la proteína LUZP1 que contiene cremalleras de leucina y tiene un papel fundamental durante el desarrollo. De hecho, el modelo de ratón knock-out de LUZP1 fenocopia algunas características asociadas con ciliopatías humanas, como defectos del tubo neural o de la formación del corazón. Nuestros datos respaldan el descubrimiento de que LUZP1 puede participar en la integración de la ciliogénesis y la dinámica de la actina durante el desarrollo.CICbioGUNE, Excelencia Severo Ocho

Correlative light and electron microscopy to study ciliogenesis

The primary cilium – a “microtubule-based organelle” (Mirvis et al. 2018) originating from a basal body – fulfills a variety of sensory functions in “most mammalian cell types” (Satir et al. 2010) (Anderson et al. 2008; Breslow & Holland 2019; Pedersen et al. 2012; Witzgall 2018a). Its formation is a complex, “multistep process” (Pitaval et al. 2017). The small GTPase RAB8 is an important molecular player for ciliogenesis in cell culture as it contributes to the extension of the ciliary vesicle at the distal end of the basal body, a process known as prerequisite for axoneme outgrowth (Lu et al. 2015; Nachury et al. 2007). However, studies investigating centrosomal RAB8 recruitment in combination with the three-dimensional visualization of membrane remodeling processes at this period of ciliogenesis are rare to date. Correlative light and electron microscopy (CLEM) techniques provide options to achieve this. We adapted two CLEM techniques to assign “fluorescently tagged proteins […] to their ultrastructural compartments” (Buerger et al. 2021). First, we extended the Kukulski protocol (Kukulski et al. 2011, 2012) and combined it with 200 kV bright-field scanning transmission electron (STEM) tomography with an electron beam with low semi-convergence angle (Rachel et al. 2020). Thereby it was possible to identify cellular structures such as mature primary cilia but also multivesicular bodies and to three-dimensionally visualize almost their entire ultrastructure in single tomograms. However, resin embedding prior to fluorescence microscopy resulted in loss of fluorescence intensity which made it impossible to preserve weak fluorescence signals of fluorescent proteins at the onset of cilia formation. For investigating centrosomal RAB8 recruitment, fluorescence microscopy was performed before sample preparation for electron microscopy. Retinal pigment epithelial (RPE1) cells showing the earliest moment detectable by fluorescence microscopy with RAB8A localizing to the centriole of a developing cilium were selected. Subsequent STEM tomography revealed a donut-like membrane structure formation during ciliogenesis which, according to our knowledge, has not been described so far: vesicles docked to the distal appendages of the basal body primarily fuse with incoming vesicles via a laterally directed process to form a donut-like structure. The central hole of this structure is closed afterwards to form the large so-called ciliary vesicle. Vesicles within the lumen of the microtubule barrel of the basal body are not enriched during these ciliogenetic processes. In contrast, fusion/budding events were observed at the distal part of docked membrane structures. RAB8A-positive membrane structures docked to the distal appendages of the basal body were present starting with the donut-like stadium of ciliogenesis. This suggests a role of RAB8A during the extension of the ciliary vesicle. By overexpressing dominant-negative mutants of RAB proteins in RPE1 cells we confirmed a role of the recycling endosome during ciliogenesis but could not unambiguously reproduce its contribution to centrosomal RAB8A recruitment. Early and late endosomes are dispensable for ciliogenesis in general. The formation of primary cilia requires incoming membrane material and a variety of proteins (Witzgall 2018b). According to the prevailing opinion, a diffusion barrier has to be passed to enter the “ciliary compartment” (Hu & Nelson 2011) (Garcia-Gonzalo & Reiter 2012; Nachury et al. 2010; Pedersen et al. 2012; Rohatgi & Snell 2010). Nature and localization of this barrier is largely obscure to date. By visualizing the ciliary membrane on an ultrastructural level, a more detailed knowledge regarding the dimension of the “ciliary compartment” (Hu & Nelson 2011) should be generated. A fusion protein of the M2 (Incardona et al. 2002; Xie et al. 1998) mutant of murine Smoothened (SmoM2) with horseradish peroxidase (HRP) and EGFP was stably expressed in Lilly Laboratories cell porcine kidney 1 (LLC-PK1) cells (Miyamoto et al. 2019) as marker for the ciliary membrane (Corbit et al. 2005); reviewed in (Hoffmeister et al. 2011). Via an HRP induced histochemical reaction (Connolly et al. 1994; Graham & Karnovsky 1966; reviewed in Witzgall 2018b) the HRP-SmoM2-EGFP fusion protein was visualized by transmission electron microscopy. Electron micrographs revealed a localization of HRP-SmoM2-EGFP not only at the membrane enclosing the ciliary axoneme but also at the membrane surrounding the base of primary cilia which is referred to as periciliary membrane in literature (Garcia et al. 2018; Garcia-Gonzalo & Reiter 2012; Long & Huang 2019; Pedersen et al. 2016). Therefore results of our work provide a hint that the composition of the membrane compartment at the ciliary base is more related to the ciliary membrane than to the plasma membrane