Browsing by Author "Camelo, Carolina"
Now showing 1 - 9 of 9
Results Per Page
Sort Options
- Centrosome positioning and development of ciliopathies: role of the human centrosomal protein TBCCD1Publication . Carmona, Bruno; Camelo, Carolina; Mehraz, Manon; Lemullois, Michel; Ferreira, David C.; Nolasco, Sofia; Lince-Faria, Mariana; Marinho, H. Susana; Bettencourt-Dias, Mónica; Tassin, Anne-Marie; Soares, HelenaAims/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.
- From centrosomal microtubule anchoring and organization to basal body positioning: TBCCD1 an elusive proteinPublication . Carmona, Bruno; Camelo, Carolina; Mehraz, Manon; Lemullois, Michel; Ferreira, David C.; Nolasco, Sofia; Lince-Faria, Mariana; Marino, H. Susana; Bettencourt-Dias, Mónica; Tassin, Anne-Marie; Koll, France; Soares, HelenaCilia 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.
- Opi1p translocation to the nucleus is regulated by hydrogen peroxide in Saccharomyces cerevisiaePublication . Camelo, Carolina; Vilas-Boas, Filipe; Cepeda, Andreia Pereira; Real, Carla; Barros-Martins, Joana; Pinto, Francisco; Soares, Helena; Marinho, H. Susana; Cyrne, LuisaDuring exposure of yeast cells to low levels of hydrogen peroxide (H2 O2 ), the expression of several genes is regulated for cells to adapt to the surrounding oxidative environment. Such adaptation involves modification of plasma membrane lipid composition, reorganization of ergosterol-rich microdomains and altered gene expression of proteins involved in lipid and vesicle traffic, to decrease permeability to exogenous H2 O2. Opi1p is a transcriptional repressor that is inactive when present at the nuclear membrane/endoplasmic reticulum, but represseses transcription of inositol upstream activating sequence (UASINO )-containing genes, many of which are involved in the synthesis of phospholipids and fatty acids, when it is translocated to the nucleus. We investigated whether H2 O2 in concentrations inducing adaptation regulates Opi1p function. We found that, in the presence of H2 O2, GFP-Opi1p fusion protein translocates to the nucleus and, concomitantly, the expression of UASINO -containing genes is affected. We also investigated whether cysteine residues of Opi1p were implicated in the H2 O2 -mediated translocation of this protein to the nucleus and identified cysteine residue 159 as essential for this process. Our work shows that Opi1p is redox-regulated and establishes a new mechanism of gene regulation involving Opi1p, which is important for adaptation to H2 O2 in yeast cells.
- OPI1P translocation to the nucleus is regulated through oxidation by hydrogen peroxide in saccharomyces cerevisiaePublication . Camelo, Carolina; Vilas-Boas, F.; Cepeda, A.; Real, C.; Pinto, F.; Costa, G. da; Soares, Helena; Marinho, H. Susana; Cyrne, L.Adaptation of yeast cells to hydrogen peroxide (H2O2) leads to a rapid change in membrane permeability accompanied by a decrease of membrane fluidity and the alteration of its lipid composition, allowing cells to survive to higher doses of H2O2. During adaptation to H2O2 several genes that contain the regulatory element UASINO, and which codify for enzymes involved in phospholipid and fatty acid metabolism, are repressed. This repression is due to the translocation into the nucleus of the endoplasmic reticulum-bound transcriptional repressor Opi1p. However, the mechanisms of regulation of this translocation regulated by H2O2 are still unknown. Oxidation of particular cysteine residues in proteins by H2O2 is involved in signaling cascades that culminate in the regulation of transcription. Opi1p has in its structure four cysteine residues that may be targets of oxidation. Such oxidation might be responsible for the H2O2-dependent translocation of Opi1p to the nucleus and subsequent transcriptional repression of target genes. Opi1p oxidation when cells were exposed to adaptive doses of H2O2 was confirmed by a protein electrophoresis (SDS-PAGE), after tagging oxidized protein cysteine sulphydryl groups with methoxy-polyethylene glycolmaleimide (MAL-PEG). To determine whether Opi1p cysteine residues are responsible for H2O2-mediated translocation of Opi1p to the nucleus, yeast strains with individual mutations in cysteine residues were prepared (cysteine to alanine substitutions). These mutations did not compromise the function of Opi1p as a transcriptional repressor since all cells presented similar levels of expression of a reporter gene containing UASINO. As expected, the wild-type strain displayed Opi1p mainly in the endoplasmic reticulum, which translocated to the nucleus in the presence of H2O2. In clear contrast, Opi1p translocation to the nucleus in cells treated with H2O2 was impaired in the C159A mutant, with Opi1p being equally distributed in the periphery and inside the nucleus. These results identify cysteine residue 159 of Opi1p as responsible for H2O2-mediated translocation of this protein to the nucleus.
- TBCCD1 and OFD1, a novel centrosomal protein partnership with a role in human ciliopathies?Publication . Veiga, Joaquim; Carmona, Bruno; Camelo, Carolina; Pereira, Dulce; Marinho, H. Susana; Soares, HelenaCilia are hair-like appendages, consisting of a microtubule (MT)-based ciliary axoneme, that 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 (primary cilia). 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 in mitosis they assist spindle pole formation.
- TBCCD1 is a key regulator of centrosomal microtubule anchor and basal body positioning/attachmentPublication . Carmona, Bruno; Camelo, Carolina; Mehraz, Manon; Lemullois, Michel; Nolasco, Sofia; Marinho, H. Susana; Tassin, Anne-Marie; Koll, France; Soares, HelenaSuccessful 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.
- TBCCD1: a new player in the development of ciliopathies?Publication . Carmona, Bruno; Camelo, Carolina; Mehraz, Manon; Lemullois, Michel; Ferreira, David C.; Nolasco, Sofia; Lince-Faria, Mariana; Marinho, H. Susana; Bettencourt-Dias, Mónica; Tassin, Anne-Marie; Koll, France; Soares, HelenaCilia 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.
- The maintenance of centriole appendages and motile cilia basal body anchoring relies on TBCCD1Publication . Carmona, Bruno; Camelo, Carolina; Mehraz, Manon; Lemullois, Michel; Faria, Mariana Lince; Coyaud, Étienne; Marinho, H. Susana; Gonçalves, João; Nolasco, Sofia; Pinto, Francisco; Raught, Brian; Tassin, Anne-Marie; Koll, France; Soares, HelenaCentrosomes 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.
- Unraveling the role of TBCCD1 protein on cell size control: the regulation of cytoskeleton dynamics and cell junctionsPublication . Camelo, Carolina; Peneda, Catarina; Coyaud, Étienne; Raught, Brian; Câmara, Ana I.; Carmona, Bruno; Pinto, Francisco; Narinho, H. Susana; Soares, HelenaDuring their lifetime most cells maintain their size. There is increasing evidence showing that this process may be dynamic and that cells can adapt their size in response to external signals and changes in the environment [1], which strongly suggests that cell size is regulated. Both Hippo and IGF/PI3K/AKT/mTORC1 pathways have been described as being involved in cell size/growth control [1]. Interestingly, these pathways are in a cross-talk with others involved and/or dependent on cellular polarity [2]. Our group characterized a centrosomal protein, TBCCD1 (TBCC domain – containing human protein 1) which, when depleted in human retinal epithelial (RPE–1) cells, leads to an abnormal localization of the centrosome at the cell periphery accompanied by the fragmentation of the Golgi apparatus, resulting in the disruption of the intrinsic cell polarity axis “Nucleus-Centrosome-Golgi Apparatus”. Moreover, TBCCD1 – depleted cells are larger, slower and have a lower efficiency in primary cilia assembly than control cells [3]. We identified the TBCCD1 interactome that showed that most of its partners are involved in cell polarity. Furthermore, most of them participate in the formation/maintenance of cell junctions, which are main regulators of cell polarity in epithelia and are upstream of pathways, like Hippo pathway. We also observed that TBCCD1 overexpression affects tubulin acetylation, which supports our results showing that some of the partners are involved in the regulation of the cytoskeleton dynamics, which may affect cell size. Therefore, it is tempting to hypothesize that the mechanisms involved in the establishment of intrinsic cell polarity may also directly/indirectly participate in the regulation of cell size.