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Elucidating a Role for CEP290 in Bardet-Biedl Syndrome and other Cilia-related DisordersZhang, Yan 01 July 2013 (has links)
Ciliopathies are a group of heterogeneous diseases associated with ciliary dysfunction. Diseases in this group display considerable phenotypic variation within individual diseases as well as overlapping phenotypes among clinically distinct diseases. In particular, mutations in CEP290 cause phenotypically diverse ciliopathies ranging from isolated retinal degeneration, nephronophthisis (NPHP), and Bardet-Biedl Syndrome (BBS) to the neonatal lethal Meckel-Gruber syndrome (MKS). However, the underlying mechanisms of the variable expressivity in ciliopathies are not well understood. This thesis focuses on evaluating the molecular and biological processes behind the retinal degeneration and obesity observed in cilia disorders with respect to CEP290 and other ciliopathy genes using the zebrafish and mouse model systems.
CEP290 is the most frequently mutated gene underlying the non-syndromic blinding disorder, Leber's congenital amaurosis (LCA). We first aimed to characterize the function of various CEP290 domains and to characterize a zebrafish model aimed at progressing towards future therapy for patients with CEP290 LCA. To this end, an antisense oligonucleotide [Morpholino(MO)] was used for gene knockdown. We showed that cep290 MO-injected embryos have reduced Kupffer's vesicle size and delays in melanosome transport, two phenotypes that are observed upon knockdown of BBS genes in zebrafish. More importantly, the embryos had a statistically significant reduction in visual function, and this vision impairment caused by the disruption of cep290 can be rescued by expressing only the N-terminal region of the human CEP290 protein. These data indicate a specific region of the CEP290 protein, which is necessary for visual function.
We examined the contribution of BBS genes to the clinical variability of CEP290-associated ciliopathies. We demonstrated that the BBSome binds to the N-terminal region of CEP290 and co-localizes with CEP290 to the centriolar satellite in ciliated cells and to the connecting cilium of photoreceptor cells. We further showed that the BBSome is required for proper localization of CEP290 in these structures. Genetic interactions were tested using Cep290rd16, a Cep290 hypomorphic allele with an in-frame deletion of 299 residues, and Bbs4 null mutant mouse lines. Additional loss of Bbs4 alleles in Cep290rd/rd mutants results in increased body weight and accelerated photoreceptor degeneration compared to mice without Bbs4 mutations. Furthermore, double heterozygous mice (Cep290+/rd16; Bbs4+/-) have increased body weight compared to single heterozygous animals. Our data indicated that genetic interactions between the BBSome components and CEP290 underlie the variable expression and overlapping phenotypes of ciliopathies caused by CEP290 mutations.
Finally, this work was extended to other cilia disorders through the characterization of genetic interactions between CEP290 and other ciliopathy genes. We found that different NPHP and MKS proteins interact with CEP290 via its different regions, suggesting the central role of CEP290 in CEP290 biological/cellular functions. To characterize the functional interaction between these proteins, we used in vitro systems to double knockdown CEP290 with other NPHP and MKS genes and showed that depletion of a certain combination set of these proteins disrupted the localization of proteins into the cilia. The data indicated that the phenotypic variability of human ciliopathies is associated with different degree of compromise of cilia function.
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Sensational Propellers: Novel Protein Functions in Cilia Assembly and MotilityAustin, Christina Anne January 2013 (has links)
Cilia and flagella are hair-like projections found on the surface of virtually every vertebrate cell. These microtubule-based organelles are historically known for their striking motility, a valuable tool for the manipulation of fluid environments. In addition, immotile (or 'primary') cilia play critical roles in cell signaling. More than ten human diseases have been linked to cilia function, with pleiotropic phenotypes including obesity, kidney and liver disease, skeletal abnormalities, situs defects, mental retardation, and sterility. In this dissertation, I first examine the function of Cep290, a putative master regulator of cilia biology, which is mutated in five human ciliopathies. I found that the zebrafish Cep290 protein was localized in a cell-type specific fashion to two distinct ciliary compartments: transition zones and centriolar satellites. Through morpholino knockdown, I demonstrated that Cep290 regulates the length of photoreceptor, Kupffer’s vesicle, and spinal canal cilia, while it was dispensable for normal cilia length in other tissues. Rescue of Cep290 associated cilia length defects by overexpression of cilia membrane proteins implicated Cep290 in cilia vesicle trafficking. Unexpectedly, I found that Cep290 deficiency in Kupffer’s vesicle and spinal canal resulted in cilia paralysis, accounting for left right asymmetry and hydrocephalus phenotypes, and identifying a novel function for Cep290 in dynein arm assembly. In the second chapter I identify and characterize three novel ciliopathy genes. We performed a small-scale morpholino screen to test the function of predicted cilia proteins. Three genes essential to cilia motility were identified: c21orf59, ccdc65, and c15orf26. Parallel studies in other systems revealed that C21orf59 was a component of the flagellar matrix required for the assembly of outer dynein arms, while Ccdc65 was part of the dynein regulatory complex, which regulates ciliary beat patterns. Importantly, we discovered that both C21ORF59 and CCDC65 were mutated in patients diagnosed with the human motile cilia disorder primary ciliary dyskinesia, identifying two novel human disease genes. Taken together, this work analyses multiple requirements for the assembly of motile and primary cilia and highlights the utility of the zebrafish system in investigations of cilia biology, particularly in the discovery and characterization of human disease genes.
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Genetic Modifiers of <i>CEP290</i>-Dependent Retinal PathologyLessieur Contreras, Emma Mercedes 01 June 2018 (has links)
No description available.
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Études fonctionnelles de deux nouvelles protéines centrosomales, NPHP5 et Cep76, et leurs implications dans les maladies humainesBarbelanne, Marine 08 1900 (has links)
Les centrosomes sont de petits organites qui régulent divers processus cellulaires
comme la polarité ou la mitose dans les cellules de mammifères. Ils sont composés de deux
centrioles entourés par une matrice péricentriolaire. Ces centrosomes sont les principaux
centres organisateurs de microtubules. De plus, ils favorisent la formation de cils, des
protubérances sur la surface des cellules quiescentes qui sont critiques pour la transduction du
signal. Une grande variété de maladies humaines telles que les cancers ou les ciliopathies sont
liées à un mauvais fonctionnement des centrosomes et des cils. C’est pourquoi le but de mes
projets de recherche est de comprendre les mécanismes nécessaires à la biogénèse et au
fonctionnement des centrosomes et des cils.
Tout d'abord, j’ai caractérisé une nouvelle protéine centrosomale nommée
nephrocystine - 5 (NPHP5). Cette protéine est localisée dans les cellules en interphase au
niveau de la région distale des centrioles. Sa déplétion inhibe la migration des centrosomes à
la surface cellulaire lors de l’étape précoce de la formation des cils. NPHP5 interagit avec la
protéine CEP290 via sa région C-terminale qui est essentielle pour la ciliogenèse. Elle
interagit également avec la calmoduline ce qui empêche son auto-agrégation. J’ai démontré
que les domaines de liaison de NHPH5 à CEP290 et à la calmoduline, ainsi que son domaine
de localisation centrosomale sont séparables. De plus, j’ai démontré que les protéines NPHP5
présentant des mutations pathogènes ne peuvent plus interagir avec CEP290 et ne sont plus
localisées aux centrosomes, rendant ainsi ces protéines non fonctionnelles. Enfin, en utilisant
une approche pharmacologique pour moduler les événements en aval dans la voie
ciliogénique, j’ai montré que la formation des cils peut être restaurée même en absence de
NPHP5.
D’autre part, j’ai étudié le rôle de NPHP5 dans l'assemblage et le trafic du complexe
BBSome dans le cil. Le BBSome est composé de huit sous-unités différentes qui s’assemblent
en un complexe fonctionnel dont on sait peu de chose sur la régulation spatiotemporelle de son
processus d'assemblage. J’ai précédemment montré que NPHP5 favorisait la formation des cils
et que son dysfonctionnement contribuait au développement de néphronophtise (NPHP).
Bien que la NPHP et le syndrome de Bardet-Biedl (BBS) soient des ciliopathies qui partagent
des caractéristiques cliniques communes, la base moléculaire de ces ressemblances
phénotypiques n’est pas comprise. J’ai constaté que NPHP5, localisé à la base du cil, contient
deux sites de liaison distincts pour le BBSome. De plus, j’ai démontré que NPHP5 et son
partenaire CEP290 interagissent de façon dynamique avec le BBSome pendant la transition de
la prolifération à la quiescence. La déplétion de NPHP5 ou CEP290 conduit à la dissociation
d’au moins deux sous-unités du BBSome formant alors un sous-complexe dont la capacité de
migration dans le cil n’est pas compromise. J’ai montré que le transport des cargos vers le
compartiment ciliaire par ce sous-complexe n’est que partiellement altéré.
Enfin, j’ai également concentré mes recherches sur une autre protéine centrosomale
peu caractérisée. La protéine centrosomale de 76 kDa (Cep76) a été précédemment impliquée
dans le maintien d’une duplication unique des centrioles par cycle cellulaire, et dans une
interaction avec la kinase cycline-dépendante 2 (CDK2). Cep76 est préférentiellement
phosphorylée par le complexe cycline A/CDK2 sur le site unique S83. Cet événement est
essentiel pour supprimer l'amplification des centrioles en phase S. J’ai démontré que Cep76
inhibe cette amplification en bloquant la phosphorylation de Plk1 au niveau des centrosomes.
D’autre part, Cep76 peut être acétylée au site K279 en phase G2, ce qui régule négativement
son activité et sa phosphorylation sur le site S83.
Ces études permettent d'améliorer notre compréhension de la biologie des
centrosomes et des cils et pourraient conduire au développement de nouvelles applications
diagnostiques et thérapeutiques. / Centrosomes are small organelles that regulate diverse cellular processes such as
polarity or mitosis in mammalian cells. They are composed of two centrioles surrounded by a
pericentriolar matrix. These centrosomes are the major microtubule organizing centers.
Moreover, they promote the formation of cilia, protrusions on the surface of quiescent cells
that are critical for signal transduction. A wide variety of human diseases such as cancers or
ciliopathies are linked to a malfunction of centrosomes and cilia. Therefore the aim of my
research is to understand the mechanisms necessary for the biogenesis and function of
centrosomes and cilia.
First, I have characterized a novel centrosomal protein called nephrocystin - 5
(NPHP5). This protein is localized, in interphase cells, in the distal region of centrioles. Its
depletion inhibits the migration of centrosomes to the cell surface during the early stage of
cilia formation. NPHP5 interacts with CEP290 via its C-terminal region that is essential for
ciliogenesis. It also interacts with calmodulin, which prevents its self-aggregation. I have
demonstrated that the Cep290- and CaM-binding domains as well as the centrosomal
localization domain of NPHP5 are separable. Moreover, I have shown that NPHP5 proteins
with pathogenic mutations can no longer interact with CEP290 and are not localized to
centrosomes, rendering these proteins non-functional. Finally, using a pharmacological
approach to modulate the downstream events in the ciliogenic pathway, I showed that cilia
formation can be restored even without NPHP5.
On the other hand, I studied the role of NPHP5 in the assembly and trafficking of the
BBSome into the cilium. The BBSome consists of eight different subunits that assemble into a
functional complex of which little is known about the spatiotemporal regulation of its
assembly process. I have previously shown that NPHP5 favored the formation of cilia and its
dysfunction contributes to the development of nephronophthisis (NPHP). Although the NPHP
and BBS syndrome (BBS) are ciliopathies that share common clinical features, molecular
basis of these phenotypic similarities is not understood. I found that NPHP5, located at the
base of the cilium, contains two separate binding sites for BBSome.
Furthermore, I demonstrated that NPHP5 and his partner CEP290 interact dynamically with
the BBSome during the transition from quiescence to proliferation. Depletion NPHP5 or
CEP290 leads to the dissociation of at least two subunits of BBSome forming a sub-complex
that can still traffic into the cilium. I have shown that the transport of cargo to the ciliary
compartment through this sub-complex is only partially altered.
Finally, I have also focused my research on another centrosomal protein poorly
characterized. The centrosomal protein of 76 kDa (Cep76) was previously involved in the
maintenance of a single duplication of centrioles per cell cycle, and interacts with the cyclindependent
kinase 2 (CDK2). Cep76 is preferentially phosphorylated by cyclin A/CDK2 on the
single site S83. This event is essential to suppress centrioles amplification in S phase. I have
demonstrated that Cep76 inhibits amplification by blocking the phosphorylation of Plk1 at the
centrosome. Moreover, Cep76 can be acetylated at the K279 site in G2 phase, which
negatively regulates its activity and phosphorylation on the site S83.
These studies will improve our understanding of the biology of centrosomes and cilia
and could lead to development of new diagnostic and therapeutic applications.
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