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1.
bioRxiv ; 2024 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-39314311

RESUMO

The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying electron cryotomography and subtomogram averaging, we obtained subnanometer resolution structures of the inner junction in three distinct regions of the cilium: the proximal region of the basal body, the central core of the basal body, and the flagellar axoneme. The structures allowed us to identify several basal body and axoneme components. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to particular regions of the cilium, forming intricate local interaction networks and bolstering local structural stability. Finally, by knocking out a critical basal body inner junction component Poc1, we found the triplet MT was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to "infiltrate" into the mutant basal body. Our findings provide molecular insight into cilium assembly at its inner Junctions, underscoring its precise spatial regulation.

2.
Open Biol ; 14(9): 240036, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39255847

RESUMO

Family with sequence similarity 161 (Fam161) is an ancient family of microtubule-binding proteins located at the centriole and cilium transition zone (TZ) lumen that exhibit rapid evolution in mice. However, their adaptive role is unclear. Here, we used flies to gain insight into their cell type-specific adaptations. Fam161 is the sole orthologue of FAM161A and FAM161B found in flies. Mutating Fam161 results in reduced male reproduction and abnormal geotaxis behaviour. Fam161 localizes to sensory neuron centrioles and their specialized TZ (the connecting cilium) in a cell type-specific manner, sometimes labelling only the centrioles, sometimes labelling the centrioles and cilium TZ and sometimes labelling the TZ with varying lengths that are longer than other TZ proteins, defining a new ciliary compartment, the extra distal TZ. These findings suggest that Fam161 is an essential centriole and TZ protein with a unique cell type-specific localization in fruit flies that can produce cell type-specific adaptations.


Assuntos
Centríolos , Cílios , Proteínas de Drosophila , Animais , Centríolos/metabolismo , Cílios/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Masculino , Drosophila melanogaster/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Especificidade de Órgãos
3.
Cells ; 13(17)2024 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-39272975

RESUMO

Ciliated epithelia are widespread in animals and play crucial roles in many developmental and physiological processes. Epithelia composed of multi-ciliated cells allow for directional fluid flow in the trachea, oviduct and brain cavities. Monociliated epithelia play crucial roles in vertebrate embryos, from the establishment of left-right asymmetry to the control of axis curvature via cerebrospinal flow motility in zebrafish. Cilia also have a central role in the motility and feeding of free-swimming larvae in a variety of marine organisms. These diverse functions rely on the coordinated orientation (rotational polarity) and asymmetric localization (translational polarity) of cilia and of their centriole-derived basal bodies across the epithelium, both being forms of planar cell polarity (PCP). Here, we review our current knowledge on the mechanisms of the translational polarity of basal bodies in vertebrate monociliated epithelia from the molecule to the whole organism. We highlight the importance of live imaging for understanding the dynamics of centriole polarization. We review the roles of core PCP pathways and of apicobasal polarity proteins, such as Par3, whose central function in this process has been recently uncovered. Finally, we emphasize the importance of the coordination between polarity proteins, the cytoskeleton and the basal body itself in this highly dynamic process.


Assuntos
Polaridade Celular , Centríolos , Cílios , Animais , Cílios/metabolismo , Cílios/fisiologia , Centríolos/metabolismo , Epitélio/metabolismo , Epitélio/fisiologia , Humanos , Células Epiteliais/metabolismo , Células Epiteliais/citologia , Corpos Basais/metabolismo
4.
Curr Biol ; 2024 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-39317195

RESUMO

Centrosomes have critical roles in microtubule organization, ciliogenesis, and cell signaling.1,2,3,4,5,6,7,8 Centrosomal alterations also contribute to diseases, including microcephaly, cancer, and ciliopathies.9,10,11,12,13 To date, over 150 centrosomal proteins have been identified, including several kinases and phosphatases that control centrosome biogenesis, function, and maintenance.2,3,4,5,14,15,16,17,18,19,20,21 However, the regulatory mechanisms that govern centrosome function are not fully defined, and thus how defects in centrosomal regulation contribute to disease is incompletely understood. Using a systems genetics approach, we find here that PPP2R3C, a poorly characterized PP2A phosphatase subunit, is a distal centriole protein and functional partner of centriolar proteins CEP350 and FOP. We further show that a key function of PPP2R3C is to counteract the kinase activity of MAP3K1. In support of this model, MAP3K1 knockout suppresses growth defects caused by PPP2R3C inactivation, and MAP3K1 and PPP2R3C have opposing effects on basal and microtubule stress-induced JNK signaling. Illustrating the importance of balanced MAP3K1 and PPP2R3C activities, acute overexpression of MAP3K1 severely inhibits centrosome function and triggers rapid centriole disintegration. Additionally, inactivating PPP2R3C mutations and activating MAP3K1 mutations both cause congenital syndromes characterized by gonadal dysgenesis.22,23,24,25,26,27,28 As a syndromic PPP2R3C variant is defective in centriolar localization and binding to centriolar protein FOP, we propose that imbalanced activity of this centrosomal kinase-phosphatase pair is the shared cause of these disorders. Thus, our findings reveal a new centrosomal phospho-regulatory module, shed light on disorders of gonadal development, and illustrate the power of systems genetics to identify previously unrecognized gene functions.

5.
Dev Biol ; 517: 148-156, 2024 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-39304174

RESUMO

PLK4 plays a crucial role in centriole duplication, which is essential for maintaining cellular processes such as cell division, cytoskeletal stability, and cilia formation. However, the mechanisms of PLK4 remain incompletely understood, especially in the embryonic development of vertebrate species. In this study, we observed that Plk4 dysfunction led to abnormal embryonic development in zebrafish, characterized by symptoms such as dark and wrinkled skin, microphthalmia, and body axis curvature. In plk4 mutants, defects in centriole duplication led to abnormal cell division, apoptosis, and ciliogenesis defects. Moreover, overexpression of plk4 in zebrafish embryos caused excessive centrosome amplification, disrupting embryonic gastrulation through abnormal cell division and ultimately resulting in embryonic lethality. Furthermore, we identified the "cryptic" polo box (CPB) domain, consisting of two PBs (PB1 and PB2), as the critical centrosome localization domain of Plk4. Surprisingly, overexpression of these two PB domains alone was sufficient to induce embryonic lethality. Additionally, we discovered a truncated form of CPB that localizes to the centrosome without causing defects in embryonic development. Our results demonstrate that Plk4 tightly controls centriole duplication, which is essential for early embryonic development in zebrafish.

6.
Biol Methods Protoc ; 9(1): bpae059, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39206452

RESUMO

CRISPR/Cas9 genome editing is a pervasive research tool due to its relative ease of use. However, some systems are not amenable to generating edited clones due to genomic complexity and/or difficulty in establishing clonal lines. For example, Drosophila Schneider 2 (S2) cells possess a segmental aneuploid genome and are challenging to single-cell select. Here, we describe a streamlined CRISPR/Cas9 methodology for knock-in and knock-out experiments in S2 cells, whereby an antibiotic resistance gene is inserted in-frame with the coding region of a gene-of-interest. By using selectable markers, we have improved the ease and efficiency for the positive selection of null cells using antibiotic selection in feeder layers followed by cell expansion to generate clonal lines. Using this method, we generated the first acentrosomal S2 cell lines by knocking-out centriole genes Polo-like Kinase 4/Plk4 or Ana2 as proof of concept. These strategies for generating gene-edited clonal lines will add to the collection of CRISPR tools available for cultured Drosophila cells by making CRISPR more practical and therefore improving gene function studies.

7.
J Cell Sci ; 137(16)2024 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-39092789

RESUMO

The structure of the sperm flagellar axoneme is highly conserved across species and serves the essential function of generating motility to facilitate the meeting of spermatozoa with the egg. During spermiogenesis, the axoneme elongates from the centrosome, and subsequently the centrosome docks onto the nuclear envelope to continue tail biogenesis. Mycbpap is expressed predominantly in mouse and human testes and conserved in Chlamydomonas as FAP147. A previous cryo-electron microscopy analysis has revealed the localization of FAP147 to the central apparatus of the axoneme. Here, we generated Mycbpap-knockout mice and demonstrated the essential role of Mycbpap in male fertility. Deletion of Mycbpap led to disrupted centrosome-nuclear envelope docking and abnormal flagellar biogenesis. Furthermore, we generated transgenic mice with tagged MYCBPAP, which restored the fertility of Mycbpap-knockout males. Interactome analyses of MYCBPAP using Mycbpap transgenic mice unveiled binding partners of MYCBPAP including central apparatus proteins, such as CFAP65 and CFAP70, which constitute the C2a projection, and centrosome-associated proteins, such as CCP110. These findings provide insights into a MYCBPAP-dependent regulation of the centrosome-nuclear envelope docking and sperm tail biogenesis.


Assuntos
Centrossomo , Camundongos Knockout , Membrana Nuclear , Cauda do Espermatozoide , Animais , Masculino , Membrana Nuclear/metabolismo , Centrossomo/metabolismo , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/ultraestrutura , Camundongos , Espermatogênese/genética , Camundongos Transgênicos , Fertilidade , Axonema/metabolismo , Axonema/ultraestrutura , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura
8.
Bioessays ; 46(10): e2400048, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39128131

RESUMO

The accuracy of cell division requires precise regulation of the cellular machinery governing DNA/genome duplication, ensuring its equal distribution among the daughter cells. The control of the centrosome cycle is crucial for the formation of a bipolar spindle, ensuring error-free segregation of the genome. The cell and centrosome cycles operate in close synchrony along similar principles. Both require a single duplication round in every cell cycle, and both are controlled by the activity of key protein kinases. Nevertheless, our comprehension of the precise cellular mechanisms and critical regulators synchronizing these two cycles remains poorly defined. Here, we present our hypothesis that the spatiotemporal regulation of a dynamic equilibrium of mitotic kinases activities forms a molecular clock that governs the synchronous progression of both the cell and the centrosome cycles.


Assuntos
Proteínas de Ciclo Celular , Centrossomo , Quinase 1 Polo-Like , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Centrossomo/metabolismo , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Animais , Mitose , Ciclo Celular , Fuso Acromático/metabolismo
9.
J Cell Sci ; 137(17)2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39166297

RESUMO

Proper connection between the sperm head and tail is critical for sperm motility and fertilization. Head-tail linkage is mediated by the head-tail coupling apparatus (HTCA), which secures the axoneme (tail) to the nucleus (head). However, the molecular architecture of the HTCA is poorly understood. Here, we use Drosophila to investigate formation and remodeling of the HTCA throughout spermiogenesis by visualizing key components of this complex. Using structured illumination microscopy, we demonstrate that key HTCA proteins Spag4 and Yuri form a 'centriole cap' that surrounds the centriole (or basal body) as it invaginates into the surface of the nucleus. As development progresses, the centriole is laterally displaced to the side of the nucleus while the HTCA expands under the nucleus, forming what we term the 'nuclear shelf'. We next show that the proximal centriole-like (PCL) structure is positioned under the nuclear shelf, functioning as a crucial stabilizer of centriole-nucleus attachment. Together, our data indicate that the HTCA is a complex, multi-point attachment site that simultaneously engages the PCL, the centriole and the nucleus to ensure proper head-tail connection during late-stage spermiogenesis.


Assuntos
Núcleo Celular , Centríolos , Proteínas de Drosophila , Espermatogênese , Espermatozoides , Centríolos/metabolismo , Centríolos/ultraestrutura , Masculino , Animais , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura , Drosophila melanogaster/metabolismo , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/ultraestrutura , Cabeça do Espermatozoide/ultraestrutura , Cabeça do Espermatozoide/metabolismo , Axonema/metabolismo , Axonema/ultraestrutura
10.
bioRxiv ; 2024 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-39131325

RESUMO

Excess centrosomes cause defects in mitosis, cell-signaling, and cell migration, and therefore their assembly is tightly regulated. Plk4 controls centriole duplication at the heart of centrosome assembly, and elevation of Plk4 promotes centrosome amplification (CA), a founding event of tumorigenesis. Here, we investigate the transcriptional consequences of elevated Plk4 and find Unkempt, a gene encoding an RNA binding protein with roles in translational regulation, to be one of only two upregulated mRNAs. Unk protein localizes to centrosomes and Cep131-positive centriolar satellites and is required for Plk4-induced centriole overduplication in an RNA-binding dependent manner. Translation is enriched at centrosomes and centriolar satellites with Unk and Cep131 promoting this localized translation. A transient centrosomal downregulation of translation occurs early in Plk4-induced CA. CNOT9, an Unk interactor and component of the translational inhibitory CCR4-NOT complex, localizes to centrosomes at this time. In summary, centriolar satellites and Unk promote local translation as part of a translational program that ensures centriole duplication.

11.
Eur J Med Genet ; 70: 104955, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38857829

RESUMO

CCP110 (centriolar coiled coil protein 110, also known as CP110) is one of the essential proteins localized in the centrosome that plays critical roles in the regulation of the cell cycle and also in the initiation of ciliogenesis. So far, no human congenital disorders have been identified to be associated with pathogenic variants of CCP110. Mice with biallelic loss-of-function variants of Ccp110 (Ccp110-/-) are known to manifest multiple organ defects, including a small body size, polydactyly, omphalocele, congenital heart defects, cleft palate, short ribs, and a small thoracic cage, a pattern of abnormalities closely resembling that in "ciliopathies" in humans. Herein, we report a 7-month-old male infant who presented with growth failure and skeletal abnormalities, including a narrow thorax and severe brachydactyly. Trio exome analysis of the genomic DNA of the patient and his parents showed that the patient was a compound heterozygote for truncating variants of CCP110, including a frameshift variant NM_001323572.2:c.856_857del, p.(Val286Leufs*5) inherited from the father, and a nonsense variant NM_001323572.2:c.1129C>T, p.(Arg377*) inherited from the mother. The strikingly similar pattern of malformations between Ccp110-/- mice and the 7-month-old male infant reported herein carrying unequivocal truncating CCP110 variants strongly supports the contention that CCP110 is a novel disease-causative gene.


Assuntos
Proteínas de Ciclo Celular , Ciliopatias , Fenótipo , Humanos , Masculino , Ciliopatias/genética , Ciliopatias/patologia , Lactente , Proteínas de Ciclo Celular/genética , Mutação com Perda de Função , Proteínas Associadas aos Microtúbulos/genética , Alelos , Proteínas do Citoesqueleto
12.
EMBO Rep ; 25(8): 3373-3405, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38943004

RESUMO

Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility.


Assuntos
Centríolos , Segregação de Cromossomos , Proteínas Associadas aos Microtúbulos , Centro Organizador dos Microtúbulos , Espermatócitos , Espermatogênese , Centríolos/metabolismo , Centríolos/genética , Animais , Masculino , Camundongos , Espermatogênese/genética , Espermatócitos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Meiose/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética
13.
Int J Mol Sci ; 25(11)2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38892227

RESUMO

The primary cilium is a microtubule-based sensory organelle that plays a critical role in signaling pathways and cell cycle progression. Defects in the structure and/or function of the primary cilium result in developmental diseases collectively known as ciliopathies. However, the constituents and regulatory mechanisms of the primary cilium are not fully understood. In recent years, the activity of the epigenetic modifier SMYD3 has been shown to play a key role in the regulation of cell cycle progression. However, whether SMYD3, a histone/lysine methyltransferase, contributes to the regulation of ciliogenesis remains unknown. Here, we report that SMYD3 drives ciliogenesis via the direct and indirect regulation of cilia-associated components. We show that SMYD3 is a novel component of the distal appendage and is required for centriolar appendage assembly. The loss of SMYD3 decreased the percentage of ciliated cells and resulted in the formation of stumpy cilia. We demonstrated that SMYD3 modulated the recruitment of centrosome proteins (Cep164, Fbf1, Ninein, Ttbk2 and Cp110) and the trafficking of intraflagellar transport proteins (Ift54 and Ift140) important for cilia formation and maintenance, respectively. In addition, we showed that SMYD3 regulated the transcription of cilia genes and bound to the promoter regions of C2cd3, Cep164, Ttbk2, Dync2h1 and Cp110. This study provides insights into the role of SMYD3 in cilia biology and suggests that SMYD3-mediated cilia formation/function may be relevant for cilia-dependent signaling in ciliopathies.


Assuntos
Centrossomo , Cílios , Histona-Lisina N-Metiltransferase , Transporte Proteico , Cílios/metabolismo , Humanos , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Centrossomo/metabolismo , Animais , Flagelos/metabolismo , Camundongos , Proteínas Associadas a Centrossomos
14.
Mol Hum Reprod ; 30(7)2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38870534

RESUMO

Acephalic spermatozoa syndrome (ASS) is a severe teratospermia with decaudated, decapitated, and malformed sperm, resulting in male infertility. Nuclear envelope protein SUN5 localizes to the junction between the sperm head and tail. Mutations in the SUN5 gene have been identified most frequently (33-47%) in ASS cases, and its molecular mechanism of action is yet to be explored. In the present study, we generated Sun5 knockout mice, which presented the phenotype of ASS. Nuclear membrane protein LaminB1 and cytoskeletal GTPases Septin12 and Septin2 were identified as potential partners for interacting with SUN5 by immunoprecipitation-mass spectrometry in mouse testis. Further studies demonstrated that SUN5 connected the nucleus by interacting with LaminB1 and connected the proximal centriole by interacting with Septin12. The binding between SUN5 and Septin12 promoted their aggregation together in the sperm neck. The disruption of the LaminB1/SUN5/Septin12 complex by Sun5 deficiency caused separation of the Septin12-proximal centriole from the nucleus, leading to the breakage of the head-to-tail junction. Collectively, these data provide new insights into the pathogenesis of ASS caused by SUN5 deficiency.


Assuntos
Proteínas de Membrana , Camundongos Knockout , Membrana Nuclear , Septinas , Cabeça do Espermatozoide , Cauda do Espermatozoide , Animais , Humanos , Masculino , Camundongos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/genética , Lamina Tipo B/metabolismo , Lamina Tipo B/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Membrana Nuclear/metabolismo , Septinas/metabolismo , Septinas/genética , Cabeça do Espermatozoide/metabolismo , Cabeça do Espermatozoide/patologia , Cauda do Espermatozoide/metabolismo , Espermatozoides/metabolismo , Teratozoospermia/metabolismo , Teratozoospermia/genética
15.
FEBS J ; 2024 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-38935637

RESUMO

Centrosomes are dominant microtubule organizing centers in animal cells with a pair of centrioles at their core. They template cilia during interphase and help organize the mitotic spindle for a more efficient cell division. Here, we review the roles of centrosomes in the early developing mouse and during organ formation. Mammalian cells respond to centrosome loss-of-function by activating the mitotic surveillance pathway, a timing mechanism that, when a defined mitotic duration is exceeded, leads to p53-dependent cell death in the descendants. Mouse embryos without centrioles are highly susceptible to this pathway and undergo embryonic arrest at mid-gestation. The complete loss of the centriolar core results in earlier and more severe phenotypes than that of other centrosomal proteins. Finally, different developing tissues possess varying thresholds and mount graded responses to the loss of centrioles that go beyond the germ layer of origin.

16.
Artigo em Inglês | MEDLINE | ID: mdl-38715433

RESUMO

Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and embedding conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both preclinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.

17.
EMBO Rep ; 25(6): 2698-2721, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38744971

RESUMO

Centrioles organize centrosomes, the cell's primary microtubule-organizing centers (MTOCs). Centrioles double in number each cell cycle, and mis-regulation of this process is linked to diseases such as cancer and microcephaly. In C. elegans, centriole assembly is controlled by the Plk4 related-kinase ZYG-1, which recruits the SAS-5-SAS-6 complex. While the kinase activity of ZYG-1 is required for centriole assembly, how it functions has not been established. Here we report that ZYG-1 physically interacts with and phosphorylates SAS-5 on 17 conserved serine and threonine residues in vitro. Mutational scanning reveals that serine 10 and serines 331/338/340 are indispensable for proper centriole assembly. Embryos expressing SAS-5S10A exhibit centriole assembly failure, while those expressing SAS-5S331/338/340A possess extra centrioles. We show that in the absence of serine 10 phosphorylation, the SAS-5-SAS-6 complex is recruited to centrioles, but is not stably incorporated, possibly due to a failure to coordinately recruit the microtubule-binding protein SAS-4. Our work defines the critical role of phosphorylation during centriole assembly and reveals that ZYG-1 might play a role in preventing the formation of excess centrioles.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteínas de Ciclo Celular , Centríolos , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Centríolos/metabolismo , Fosforilação , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Serina/metabolismo , Sequência de Aminoácidos , Proteínas Quinases
18.
bioRxiv ; 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38617270

RESUMO

Centrosomes have critical roles in microtubule organization and in cell signaling.1-8 However, the mechanisms that regulate centrosome function are not fully defined, and thus how defects in centrosomal regulation contribute to disease is incompletely understood. From functional genomic analyses, we find here that PPP2R3C, a PP2A phosphatase subunit, is a distal centriole protein and functional partner of centriolar proteins CEP350 and FOP. We further show that a key function of PPP2R3C is to counteract the kinase activity of MAP3K1. In support of this model, MAP3K1 knockout suppresses growth defects caused by PPP2R3C inactivation, and MAP3K1 and PPP2R3C have opposing effects on basal and microtubule stress-induced JNK signaling. Illustrating the importance of balanced MAP3K1 and PPP2R3C activities, acute overexpression of MAP3K1 severely inhibits centrosome function and triggers rapid centriole disintegration. Additionally, inactivating PPP2R3C mutations and activating MAP3K1 mutations both cause congenital syndromes characterized by gonadal dysgenesis.9-15 As a syndromic PPP2R3C variant is defective in centriolar localization and binding to centriolar protein FOP, we propose that imbalanced activity of this centrosomal kinase-phosphatase pair is the shared cause of these disorders. Thus, our findings reveal a new centrosomal phospho-regulatory module, shed light on disorders of gonadal development, and illustrate the power of systems genetics to identify previously unrecognized gene functions.

19.
Front Oncol ; 14: 1370565, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38606093

RESUMO

Breast cancer is the most prevalent malignancy among women worldwide. Despite significant advances in treatment, it remains one of the leading causes of female mortality. The inability to effectively treat advanced and/or treatment-resistant breast cancer demonstrates the need to develop novel treatment strategies and targeted therapies. Centrosomes and their associated proteins have been shown to play key roles in the pathogenesis of breast cancer and thus represent promising targets for drug and biomarker development. Centrosomes are fundamental cellular structures in the mammalian cell that are responsible for error-free execution of cell division. Centrosome amplification and aberrant expression of its associated proteins such as Polo-like kinases (PLKs), Aurora kinases (AURKs) and Cyclin-dependent kinases (CDKs) have been observed in various cancers, including breast cancer. These aberrations in breast cancer are thought to cause improper chromosomal segregation during mitosis, leading to chromosomal instability and uncontrolled cell division, allowing cancer cells to acquire new genetic changes that result in evasion of cell death and the promotion of tumor formation. Various chemical compounds developed against PLKs and AURKs have shown meaningful antitumorigenic effects in breast cancer cells in vitro and in vivo. The mechanism of action of these inhibitors is likely related to exacerbation of numerical genomic instability, such as aneuploidy or polyploidy. Furthermore, growing evidence demonstrates enhanced antitumorigenic effects when inhibitors specific to centrosome-associated proteins are used in combination with either radiation or chemotherapy drugs in breast cancer. This review focuses on the current knowledge regarding the roles of centrosome and centrosome-associated proteins in breast cancer pathogenesis and their utility as novel targets for breast cancer treatment.

20.
Cell ; 187(9): 2158-2174.e19, 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38604175

RESUMO

Centriole biogenesis, as in most organelle assemblies, involves the sequential recruitment of sub-structural elements that will support its function. To uncover this process, we correlated the spatial location of 24 centriolar proteins with structural features using expansion microscopy. A time-series reconstruction of protein distributions throughout human procentriole assembly unveiled the molecular architecture of the centriole biogenesis steps. We found that the process initiates with the formation of a naked cartwheel devoid of microtubules. Next, the bloom phase progresses with microtubule blade assembly, concomitantly with radial separation and rapid cartwheel growth. In the subsequent elongation phase, the tubulin backbone grows linearly with the recruitment of the A-C linker, followed by proteins of the inner scaffold (IS). By following six structural modules, we modeled 4D assembly of the human centriole. Collectively, this work provides a framework to investigate the spatial and temporal assembly of large macromolecules.


Assuntos
Centríolos , Microtúbulos , Centríolos/metabolismo , Humanos , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas de Ciclo Celular/metabolismo
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