Setting out zebrafish (Danio rerio) as a model to study nervous necrosis virus-host interaction.

Viral nervous necrosis is responsible for important economic losses in aquaculture facilities. The causative agent is the nervous necrosis virus (NNV). Four NNV species have been described, although only RGNNV and SJNNV have been detected in the Mediterranean area. RGNNV- SJNNV reassortants have also been isolated from several species. In order to design strategies to improve fish resistance to NNV, in vivo studies in commercial and model species are required to study the mechanisms underlying fish susceptibility to viral isolates. The aim of this work was to set up zebrafish as model of NNV infection. To fulfil this aim, zebrafish susceptibility to three NNV isolates was determined, and viral replication and innate immune response were characterized. Three days post-fertilisation zebrafish larvae were infected by intracerebral injection with 107 TCID50/mL of SJ93Nag (SJNNV), Dl956 (RGNNV from seabass), and RG/SJ (from seabream). Larvae were daily monitored for 4 days to record clinical signs and mortality. At 1 and 4 days post-infection (dpi), 3 pools of 6 larvae were sampled for viral genome quantification. Innate immune response was also assessed. Transcriptional analyses were completed by in vivo 3D imaging approaches on a zebrafish transgenic line expressing GFP in neutrophils (Tg (mpx:GFP) to monitor neutrophils recruitment in brain. RGNNV was the most virulent isolate compared to SJNNV and RG/SJ. These observations were consistent with viral genome replication, as the highest number of viral genome copies was in RGNNV-infected larvae. The induction of immune-related genes and the recruitment of neutrophils in brain, was also higher in RGNNV-infected larvae. Therefore, further experiments can be designed in this successfully model to better understand the mechanisms underlying NNV virulence in its hosts. Acknowledgments: Projects PID2020-115954RB-100/AEI/10.13039/501100011033 (Spanish Government) and EU H2020 VBN_22_73 (VetBioNet project).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The maintenance of centriole appendages and motile cilia basal body anchoring relies on TBCCD1

Centrosomes are organelles consisting of two structurally and functionally distinct centrioles, with the mother centriole having complex distal (DA) and subdistal appendages (SDA). Despite their importance, how appendages are assembled and maintained remains unclear. This study investigated human TBCCD1, a centrosomal protein essential for centrosome positioning, to uncover its localization and role at centrioles. We found that TBCCD1 localizes at both proximal and distal regions of the two centrioles, forming a complex structure spanning from SDA to DA and extending inside and outside the centriole lumen. TBCCD1 depletion caused centrosome mispositioning, which was partially rescued by taxol, and the loss of microtubules (MTs) anchored to centrosomes. TBCCD1 depletion also reduced levels of SDA proteins involved in MT anchoring such as Centriolin/CEP110, Ninein, and CEP170. Additionally, TBCCD1 was essential for the correct positioning of motile cilia basal bodies and associated structures in Paramecium. This study reveals that TBCCD1 is an evolutionarily conserved protein essential for centriole and basal body localization and appendage assembly and maintenance. A BioID screening also linked TBCCD1 to ciliopathy-associated protein networks.info:eu-repo/semantics/publishedVersio

The mesoSPIM initiative Paris-SaclayAn open-source light-sheet microscope for 3D imaging of large cleared tissues and organisms

International audienc

TBCCD1 is a key regulator of centrosomal microtubule anchor and basal body positioning/attachment

Successful cilia assembly requires correct positioning and anchoring of the centrosome's mother centriole/basal body (BB) to the cell membrane. A clear picture of the different signals and players involved in centrosome positioning/anchoring is still not available. Published work from our group identified a new TBCC domain-containing human protein (TBCCD1). Depletion of TBCCD1 in human RPE-1 cells severely affects the relative position of the centrosome to the nucleus and the efficiency of cells to assemble primary cilia. Aim of the study: To dissect the mechanisms involving TBCCD1 in human RPE-1 positioning and anchoring during ciliogenesis.info:eu-repo/semantics/publishedVersio

Centrosome positioning and development of ciliopathies: role of the human centrosomal protein TBCCD1

Project IPL/2019/MOONOFCILI/ESTeSLAims/Context: Primary cilia are specialized microtubule-based signaling organelles that convey extracellular signaling and cellular polarity into a cellular response. Defects in primary cilia assembly/function cause severe diseases known as ciliopathies, typified by clinical manifestations, like infertility, obesity, brain problems, blindness, and kidney cysts. Primary cilia assembly entails centrosome migration to the plasma membrane where a centriole docks, maturates into a basal body (BB), and assembles the cilia axoneme. The human centrosomal TBCCD1 is a critical factor in centrosome positioning previously identified by us. Our aim is to discover the mechanisms/signals required for the correct positioning of the centrosome during cilia assembly, and how these mechanisms, when compromised, are related to ciliopathies. Methods: The proximity-dependent identification (BioID) assay was used to screen for TBCCD1 interactors. Immunofluorescent and super-resolution microscopy, as well as Western blot, were used to study the levels and cellular localization of the identified TBCCD1 interactors in human RPE1 cells overexpressing or depleted of TBCCD1. To study the impact of TBCCD1 knockdown in motile cilia the ciliate Paramecium, containing ∼3,000 motile cilia, was used. Results: Our BioID screen for TBCCD1 interactors identified several well-known proteins encoded by ciliopathy genes, e.g. the centrosomal protein OFD1 involved in the Orofacial-Digital Syndrome. We show that TBCCD1 knockdown and overexpression in RPE1 cells affects OFD1 distribution. Super-resolution microscopy shows TBCCD1 is localized at the distal region of the centrosome and that its depletion dramatically affects the centrosome subdistal protein CEP170, a component of cilia basal feet. In Paramecium, the TBCCD1 knockdown causes abnormal BB-associated structures organization and anomalous BB positioning/anchoring defects. Conclusions: Our data support a role for TBCCD1 in the maintenance of centrosome structure and in BB anchoring at the cell membrane during ciliogenesis. TBCCD1 is emerging as a novel protein with a role in human ciliopathies.info:eu-repo/semantics/publishedVersio

From centrosomal microtubule anchoring and organization to basal body positioning: TBCCD1 an elusive protein

Cilia are microtubule-based organelles that protrude from the cell surface and fulfill critical motility and sensory functions being required for normal embryonic development and for homeostasis of human adult tissues. Cilia loss or dysfunction is associated with human ciliopathies. At their base cilia have a centriole/basal body (BB), which can be derived from the centrosome and assembles the ciliary axoneme. This process requires the correct positioning/anchoring of the centrosome’s mother centriole/BB to the cell membrane. A clear picture of the different signals and players involved in centrosome positioning/anchoring is still not available. Published work from our group identified a new centrosomal TBCC domain-containing human protein (TBCCD1) that is involved in centrosome correct positioning and primary cilia assembly. In mammalian cells, TBCCD1 is observed at pericentriolar satellites, in basal bodies of primary and motile cilia and at primary cilia ciliopathy hot domain, the transition zone. Super-resolution microscopy shows that TBCCD1 is localized at the distal region of the centrosome and its depletion dramatically affects the centrosome subdistal protein CEP170, a component of primary and motile cilia basal feet. By doing a proximity-dependent biotin identification (BioID-MS) screen for TBCCD1 interactors several well-known proteins encoded by ciliopathy genes were identified, e.g. the centrosomal proteins OFD1 and Moonraker/KIAA0753 associated with Digital Syndrome 1 and Joubert syndrome, respectively. OFD1 and Moonraker are required for the maintenance of centrosome structure and both proteins localization is dramatically disturbed by TBCCD1 depletion. To clarify the role of human TBCCD1 in cilia biogenesis we used the ciliate Paramecium. Noteworthy, in Paramecium TBCCD1 knockdown causes abnormal basal body associated rootlets organization, anomalous BB positioning/anchoring defects. Our data using human cells and the ciliate Paramecium support a role of TBCCD1 in centrosome structure maintenance and BB anchoring at the cell membrane. The Paramecium phenotypes confirm that TBCCD1 is a new candidate to a ciliopathic gene probably by founding the TBCCD1/Moonraker/OFD1 functional conserved module required for cilia assembly.info:eu-repo/semantics/publishedVersio

TBCCD1: a new player in the development of ciliopathies?

Project IPL/2016/TBCCentro_ESTeSL.Project IPL/2017/CILIOPAT/ESTeSLCilia are hair-like appendages, consisting of a microtubule (MT)-based ciliary axoneme, which fulfill critical motility and sensory functions required for normal embryonic development and also for homeostasis of adult tissues. At their base, cilia have a centriole/basal body, which can be derived from the centrosome, and that nucleates the ciliary axoneme. Centrosomes consist of a pair of centrioles surrounded by the pericentriolar matrix that nucleate/organize the cytoskeleton and are implicated in cell migration, adhesion, and polarity, while during mitosis they assist spindle pole formation. Centriolar satellites are cytoplasmic granules that are located and move around the centrosome. These particles are involved in centrosome assembly and primary cilium formation by delivering cytoplasmatic centriolar/centrosomal components to the centrosome. Mutations in genes encoding centrosome and/or centriolar satellite components and regulators lead to various human disorders such as ciliopathies. Ciliopathies are typified by often overlapping clinical manifestations, e.g. infertility, obesity, brain and skeletal developmental problems, blindness and kidney cysts.info:eu-repo/semantics/publishedVersio