RESUMEN
The human SASS6(I62T) missense mutation has been linked with the incidence of primary microcephaly in a Pakistani family, although the mechanisms by which this mutation causes disease remain unclear. The SASS6(I62T) mutation corresponds to SAS-6(L69T) in Caenorhabditis elegans. Given that SAS-6 is highly conserved, we modeled this mutation in C. elegans and examined the sas-6(L69T) effect on centrosome duplication, ciliogenesis, and dendrite morphogenesis. Our studies revealed that all the above processes are perturbed by the sas-6(L69T) mutation. Specifically, C. elegans carrying the sas-6(L69T) mutation exhibit an increased failure of centrosome duplication in a sensitized genetic background. Further, worms carrying this mutation also display shortened phasmid cilia, an abnormal phasmid cilia morphology, shorter phasmid dendrites, and chemotaxis defects. Our data show that the centrosome duplication defects caused by this mutation are only uncovered in a sensitized genetic background, indicating that these defects are mild. However, the ciliogenesis and dendritic defects caused by this mutation are evident in an otherwise wild-type background, indicating that they are stronger defects. Thus, our studies shed light on the novel mechanisms by which the sas-6(L69T) mutation could contribute to the incidence of primary microcephaly in humans.
Asunto(s)
Proteínas de Caenorhabditis elegans , Microcefalia , Animales , Humanos , Caenorhabditis elegans/genética , Centriolos/genética , Proteínas de Caenorhabditis elegans/genética , Microcefalia/genética , Proteínas de Ciclo Celular/genética , Mutación , Morfogénesis/genética , Dendritas , CentrosomaRESUMEN
Cartwheel assembly is considered the first step in the initiation of procentriole biogenesis; however, the reason for persistence of the assembled human cartwheel structure from S phase to late mitosis remains unclear. Here, we demonstrate mainly using cell synchronization, RNA interference, immunofluorescence and time-lapse-microscopy, biochemical analysis, and methods that the cartwheel persistently assembles and maintains centriole engagement and centrosome integrity during S phase to late G2 phase. Blockade of the continuous accumulation of centriolar Sas-6, a major cartwheel protein, after procentriole formation induces premature centriole disengagement and disrupts pericentriolar matrix integrity. Additionally, we determined that during mitosis, CDK1-cyclin B phosphorylates Sas-6 at T495 and S510, disrupting its binding to cartwheel component STIL and pericentriolar component Nedd1 and promoting cartwheel disassembly and centriole disengagement. Perturbation of this phosphorylation maintains the accumulation of centriolar Sas-6 and retains centriole engagement during mitotic exit, which results in the inhibition of centriole reduplication. Collectively, these data demonstrate that persistent cartwheel assembly after procentriole formation maintains centriole engagement and that this configuration is relieved through phosphorylation of Sas-6 by CDK1-cyclin B during mitosis in human cells.
Asunto(s)
Centriolos , Centrosoma , Humanos , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Centrosoma/metabolismo , Mitosis , Fosforilación , Proteínas/metabolismo , Ciclina BRESUMEN
The conserved nine-fold structural symmetry of the centriole is thought to be generated by cooperation between two mechanisms, one dependent on and the other independent of the cartwheel, a sub-centriolar structure consisting of a hub and nine spokes. However, the molecular entity of the cartwheel-independent mechanism has not been elucidated. Here, using Chlamydomonas reinhardtii mutants, we show that Bld10p/Cep135, a conserved centriolar protein that connects cartwheel spokes and triplet microtubules, plays a central role in this mechanism. Using immunoelectron microscopy, we localized hemagglutinin epitopes attached to distinct regions of Bld10p along two lines that connect adjacent triplets. Consistently, conventional and cryo-electron microscopy identified crosslinking structures at the same positions. In centrioles formed in the absence of the cartwheel, truncated Bld10p was found to significantly reduce the inter-triplet distance and frequently form eight-microtubule centrioles. These results suggest that the newly identified crosslinks are comprised of part of Bld10p/Cep135. We propose that Bld10p determines the inter-triplet distance in the centriole and thereby regulates the number of triplets in a cartwheel-independent manner.
Asunto(s)
Centriolos , Hemaglutininas , Centriolos/genética , Centriolos/metabolismo , Microscopía por Crioelectrón , Epítopos/metabolismo , Hemaglutininas/metabolismo , Microtúbulos/metabolismoRESUMEN
As a typical species of microsporidium, Nosema bombycis is the pathogen causing the pébrine disease of silkworm. Rapid proliferation of N. bombycis in host cells requires replication of genetic material. As eukaryotic origin recognition protein, origin recognition complex (ORC) plays an important role in regulating DNA replication, and Orc1 is a key subunit of the origin recognition complex. In this study, we identified the Orc1 in the microsporidian N. bombycis (NbOrc1) for the first time. The NbOrc1 gene contains a complete ORF of 987 bp in length that encodes a 328 amino acid polypeptide. Indirect immunofluorescence results showed that NbOrc1 were colocalized with Nbactin and NbSAS-6 in the nuclei of N. bombycis. Subsequently, we further identified the interaction between the NbOrc1 and Nbactin by CO-IP and Western blot. These results imply that Orc1 may be involved in the proliferation of the microsporidian N. bombycis through interacting with actin.
Asunto(s)
Bombyx , Nosema , Animales , Bombyx/genética , Bombyx/metabolismo , Nosema/genética , Nosema/metabolismo , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismoRESUMEN
Centrioles are eukaryotic organelles that template the formation of cilia and flagella, as well as organize the microtubule network and the mitotic spindle in animal cells. Centrioles have proximal-distal polarity and a 9-fold radial symmetry imparted by a likewise symmetrical central scaffold, the cartwheel. The spindle assembly abnormal protein 6 (SAS-6) self-assembles into 9-fold radially symmetric ring-shaped oligomers that stack via an unknown mechanism to form the cartwheel. Here, we uncover a homo-oligomerization interaction mediated by the coiled-coil domain of SAS-6. Crystallographic structures of Chlamydomonas reinhardtii SAS-6 coiled-coil complexes suggest this interaction is asymmetric, thereby imparting polarity to the cartwheel. Using a cryoelectron microscopy (cryo-EM) reconstitution assay, we demonstrate that amino acid substitutions disrupting this asymmetric association also impair SAS-6 ring stacking. Our work raises the possibility that the asymmetric interaction inherent to SAS-6 coiled-coil provides a polar element for cartwheel assembly, which may assist the establishment of the centriolar proximal-distal axis.
Asunto(s)
Proteínas de Ciclo Celular , Centriolos , Animales , Proteínas de Ciclo Celular/metabolismo , Centriolos/química , Centriolos/metabolismo , Microscopía por Crioelectrón , Orgánulos/metabolismo , Huso Acromático/metabolismoRESUMEN
The centrosome plays an essential role in maintaining genetic stability, ciliogenesis and cell polarisation. The core of the centrosome is made up of two centrioles that duplicate precisely once during every cell cycle to generate two centrosomes that are required for bipolar spindle assembly and chromosome segregation. Abundance of centriole proteins at optimal levels and their recruitment to the centrosome are tightly regulated in time and space in order to restrict aberrant duplication of centrioles, a phenomenon that is observed in many cancers. Recent advances have conclusively shown that dedicated ubiquitin ligase-dependent protein degradation machineries are involved in governing centriole duplication. These studies revealed intricate mechanistic insights into how the ubiquitin ligases target different centriole proteins. In certain cases, a specific ubiquitin ligase targets a number of substrate proteins that co-regulate centriole assembly, prompting the possibility that substrate-targeting occurs during formation of the sub-centriolar structures. There are also instances where a specific centriole duplication protein is targeted by several ubiquitin ligases at different stages of the cell cycle, suggesting synchronised actions. Recent evidence also indicated a direct association of E3 ubiquitin ligase with the centrioles, supporting the notion that substrate-targeting occurs in the organelle itself. In this review, we highlight these advances by underlining the mechanisms of how different ubiquitin ligase machineries control centriole duplication and discuss our views on their coordination.
Asunto(s)
Centriolos , Ubiquitina , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Centriolos/genética , Centriolos/metabolismo , Centrosoma/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Centrioles are structurally conserved organelles, composing both centrosomes and cilia. In animal cycling cells, centrioles often form through a highly characterized process termed canonical duplication. However, a large diversity of eukaryotes assemble centrioles de novo through uncharacterized pathways. This unexplored diversity is key to understanding centriole assembly mechanisms and how they evolved to assist specific cellular functions. Here, we show that, during spermatogenesis of the bryophyte Physcomitrium patens, centrioles are born as a co-axially oriented centriole pair united by a cartwheel. Interestingly, we observe that these centrioles are twisted in opposite orientations. Microtubules emanate from the bicentrioles, which localize to the spindle poles during cell division. After their separation, the two resulting sister centrioles mature asymmetrically, elongating specific microtubule triplets and a naked cartwheel. Subsequently, two motile cilia are assembled that appear to alternate between different motility patterns. We further show that centriolar components SAS6, Bld10, and POC1, which are conserved across eukaryotes, are expressed during spermatogenesis and required for this de novo biogenesis pathway. Our work supports a scenario where centriole biogenesis, while driven by conserved molecular modules, is more diverse than previously thought.
Asunto(s)
Centriolos , Centrosoma , Animales , Ciclo Celular , Centriolos/metabolismo , Centrosoma/metabolismo , Cilios/metabolismo , Eucariontes , Masculino , Microtúbulos/metabolismoRESUMEN
The duplication and ninefold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. Here, we use hydrogen-deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its subcellular localization. We identify a core stabilization sequence within Gorab's C-terminal coiled-coil domain that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an antiparallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable heterotrimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a sixteenfold reduced binding affinity for Gorab that cannot support centriole duplication. Thus, Gorab dimers at the Golgi exist in equilibrium with Sas6-associated monomers at the centriole to balance Gorab's dual role.
Asunto(s)
Centriolos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de la Matriz de Golgi/genética , Animales , Centriolos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Proteínas de la Matriz de Golgi/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , MutaciónRESUMEN
Spindle assembly abnormal protein 6 (SAS-6), a highly conserved centriolar protein, constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. Abnormalities in cartwheel assembly lead to chromosomal dysfunctions. The molecular structure of human SAS-6 (HsSAS-6) and cartwheel hub and how they direct centriole symmetry is unknown. No crystal structure of wildtype HsSAS-6 has been reported to date, since soluble recombinant partial/full-length HsSAS-6 expression and purification posed grand challenges. In the present study we have explored optimization of ten different N terminal SAS-6 fusion proteins expression in a variety of E. coli hosts. During optimization we have included some of the most commonly used purification tags: Histidine tag, maltose-binding protein (MBP), small ubiquitin-related modifier (SUMO) tag and modified MBP tag with surface entropy reduction mutations. We demonstrate several levels of tag assisted solubility and stable expression strategies. We find that the MBP tag accompanied by Surface Entropy Reduction mutations (MBP/SER) in a fixed arm approach rescues the folded SAS-6N protein with significantly improved solubility. This expression of HsSAS-6N in E. coli Rosetta DE3 pLysS expression strain gave rise to high protein expression yielding around 6.0-11.5 mg of soluble protein per liter of growth culture.
Asunto(s)
Proteínas de Ciclo Celular , Escherichia coli , Expresión Génica , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Dominios Proteicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , SolubilidadRESUMEN
The centriole is a ninefold symmetrical structure found at the core of centrosomes and, as a basal body, at the base of cilia, whose conserved duplication is regulated by Plk4 kinase. Plk4 phosphorylates a single serine residue at the N-terminus of Ana2 to promote Ana2's loading to the site of procentriole formation. Four conserved serines in Ana2's STAN motif are then phosphorylated by Plk4, enabling Sas6 recruitment. Crystallographic data indicate that the coiled-coil domain of Ana2 forms a tetramer but the structure of full-length Ana2 has not been solved. Here, we have employed hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) to uncover the conformational dynamics of Ana2, revealing the high flexibility of this protein with one rigid region. To determine the elusive nature of the interaction surfaces between Ana2 and Sas6, we have confirmed complex formation between the phosphomimetic form of Ana2 (Ana2-4D) and Sas6 in vitro and in vivo. Analysis of this complex by HDX-MS identifies short critical regions required for this interaction, which lie in the C-terminal parts of both proteins. Mutational studies confirmed the relevance of these regions for the Ana2-Sas6 interaction. The Sas6 site required for Ana2 binding is distinct from the site required for Sas6 to bind Gorab and Sas6 is able to bind both these protein partners simultaneously.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Dominios y Motivos de Interacción de Proteínas , Secuencia de Aminoácidos , Espectrometría de Masas , Unión Proteica , Mapeo de Interacción de Proteínas , Mapas de Interacción de ProteínasRESUMEN
Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS-6 proteins. A cryo-tomography map of centrioles from the termite flagellate Trichonympha spp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub-tomogram averaging (STA) in T. spp. and Trichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead, and A-C linker. Moreover, we report ~25 Å resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore, STA of centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel architecture and a distinct filamentous CID. Fitting the CrSAS-6 crystal structure into the flagellate maps and analyzing cartwheels generated in vitro indicate that SAS-6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improved STA maps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms.
Asunto(s)
Centriolos/ultraestructura , Microscopía por Crioelectrón/métodos , Tomografía/métodos , Adhesión Celular , Cilios/ultraestructura , Microtúbulos/ultraestructura , Parabasalidea/citologíaRESUMEN
Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Chlamydomonas/fisiología , Cilios/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Algáceas/química , Proteínas Algáceas/metabolismo , Línea Celular , Chlamydomonas/clasificación , Cristalografía por Rayos X , Células HEK293 , Humanos , Microtúbulos/metabolismo , Modelos Moleculares , Filogenia , Conformación Proteica , Dominios Proteicos , Multimerización de ProteínaRESUMEN
Centriole assembly requires a small number of conserved proteins. The precise pathway of centriole assembly has been difficult to study, as the lack of any one of the core assembly proteins [Plk4, Ana2 (the homologue of mammalian STIL), Sas-6, Sas-4 (mammalian CPAP) or Asl (mammalian Cep152)] leads to the absence of centrioles. Here, we use Sas-6 and Ana2 particles (SAPs) as a new model to probe the pathway of centriole and centrosome assembly. SAPs form in Drosophila eggs or embryos when Sas-6 and Ana2 are overexpressed. SAP assembly requires Sas-4, but not Plk4, whereas Asl helps to initiate SAP assembly but is not required for SAP growth. Although not centrioles, SAPs recruit and organise many centriole and centrosome components, nucleate microtubules, organise actin structures and compete with endogenous centrosomes to form mitotic spindle poles. SAPs require Asl to efficiently recruit pericentriolar material (PCM), but Spd-2 (the homologue of mammalian Cep192) can promote some PCM assembly independently of Asl. These observations provide new insights into the pathways of centriole and centrosome assembly.
Asunto(s)
Centriolos , Proteínas de Drosophila , Animales , Proteínas de Ciclo Celular/genética , Centrosoma , Drosophila , Proteínas de Drosophila/genética , Drosophila melanogaster/genéticaRESUMEN
Formation of a single new centriole from a pre-existing centriole is strictly controlled to maintain correct centrosome number and spindle polarity in cells. However, the mechanisms that govern this process are incompletely understood. Here, using several human cell lines, immunofluorescence and structured illumination microscopy methods, and ubiquitination assays, we show that the E3 ubiquitin ligase F-box and WD repeat domain-containing 7 (FBXW7), a subunit of the SCF ubiquitin ligase, down-regulates spindle assembly 6 homolog (HsSAS-6), a key protein required for procentriole cartwheel assembly, and thereby regulates centriole duplication. We found that FBXW7 abrogation stabilizes HsSAS-6 and increases its recruitment to the mother centriole at multiple sites, leading to supernumerary centrioles. Ultrastructural analyses revealed that FBXW7 is broadly localized on the mother centriole and that its presence is reduced at the site where the HsSAS-6-containing procentriole is formed. This observation suggested that FBXW7 restricts procentriole assembly to a specific site to generate a single new centriole. In contrast, during HsSAS-6 overexpression, FBXW7 strongly associated with HsSAS-6 at the centriole. We also found that SCFFBXW7 interacts with HsSAS-6 and targets it for ubiquitin-mediated degradation. Further, we identified putative phosphodegron sites in HsSAS-6, whose substitutions rendered it insensitive to FBXW7-mediated degradation and control of centriole number. In summary, SCFFBXW7 targets HsSAS-6 for degradation and thereby controls centriole biogenesis by restraining HsSAS-6 recruitment to the mother centriole, a molecular mechanism that controls supernumerary centrioles/centrosomes and the maintenance of bipolar spindles.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Centriolos/ultraestructura , Centrosoma/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/antagonistas & inhibidores , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Fase G1 , Duplicación de Gen , Humanos , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Fase S , Especificidad por Sustrato , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Drosophila spermatocytes have giant centrioles that display unique properties. Both the parent centrioles maintain a distinct cartwheel and nucleate a cilium-like region that persists during the meiotic divisions and organizes a structured sperm axoneme. Moreover, the parent centrioles are morphologically undistinguishable, unlike vertebrate cells in which mother and daughter centrioles have distinct structural features. However, our immunofluorescence analysis of the parent centrioles in mature primary spermatocytes revealed an asymmetric accumulation of the typical Sas4 and Sas6 proteins. Notably, the fluorescence intensity of Sas4 and Sas6 at the daughter centrioles is greater than the intensity found at the mother ones. In contrast, the centrioles of wing imaginal disc cells display an opposite condition in which the loading of Sas4 and Sas6 at the mother centrioles is greater. These data underlie a subtle asymmetry among the parent centrioles and point to a cell type diversity of the localization of the Sas4 and Sas6 proteins.
Asunto(s)
Centriolos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Espermatocitos/citología , Espermatocitos/metabolismo , Animales , Centriolos/ultraestructura , Drosophila melanogaster/ultraestructura , Larva/citología , Larva/ultraestructura , Masculino , Meiosis , Proteínas Asociadas a Microtúbulos , Microtúbulos/metabolismo , Espermatocitos/ultraestructuraRESUMEN
The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.
Asunto(s)
Cuerpos Basales/metabolismo , Microtúbulos/metabolismo , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genética , Animales , Axonema/química , Axonema/genética , Axonema/metabolismo , Cuerpos Basales/química , Cuerpos Basales/parasitología , Centriolos/química , Centriolos/genética , Centriolos/parasitología , Flagelos/química , Flagelos/genética , Flagelos/parasitología , Humanos , Microtúbulos/química , Microtúbulos/parasitología , Estabilidad Proteica , Proteínas Protozoarias/química , Interferencia de ARN , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/patogenicidadRESUMEN
Insects and mammals have atypical centrioles in their sperm. However, it is unclear how these atypical centrioles form. Drosophila melanogaster sperm has one typical centriole called the giant centriole (GC) and one atypical centriole called the proximal centriole-like structure (PCL). During early sperm development, centriole duplication factors such as Ana2 and Sas-6 are recruited to the GC base to initiate PCL formation. The centriolar protein, Poc1B, is also recruited at this initiation stage, but its precise role during PCL formation is unclear. Here, we show that Poc1B recruitment was dependent on Sas-6, that Poc1B had effects on cellular and PCL Sas-6, and that Poc1B and Sas-6 were colocalized in the PCL/centriole core. These findings suggest that Sas-6 and Poc1B interact during PCL formation. Co-overexpression of Ana2 and Sas-6 induced the formation of ectopic particles that contained endogenous Poc1 proteins and were composed of PCL-like structures. These structures were disrupted in Poc1 mutant flies, suggesting that Poc1 proteins stabilize the PCL-like structures. Lastly, Poc1B and Sas-6 co-overexpression also induced the formation of PCL-like structures, suggesting that they can function together during the formation of the PCL. Overall, our findings suggest that Poc1B and Sas-6 function together during PCL formation.
Asunto(s)
Centriolos/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Animales , Proteínas del Citoesqueleto/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Unión ProteicaRESUMEN
The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar material (PCM). Although centrioles are known to regulate PCM assembly, it is less known whether and how the PCM contributes to centriole assembly. Here we investigate the interaction between centriole components and the PCM by taking advantage of fission yeast, which has a centriole-free, PCM-containing centrosome, the SPB. Surprisingly, we observed that several ectopically-expressed animal centriole components such as SAS-6 are recruited to the SPB. We revealed that a conserved PCM component, Pcp1/pericentrin, interacts with and recruits SAS-6. This interaction is conserved and important for centriole assembly, particularly its elongation. We further explored how yeasts kept this interaction even after centriole loss and showed that the conserved calmodulin-binding region of Pcp1/pericentrin is critical for SAS-6 interaction. Our work suggests that the PCM not only recruits and concentrates microtubule-nucleators, but also the centriole assembly machinery, promoting biogenesis close by.
Asunto(s)
Antígenos/metabolismo , Centriolos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Animales , Animales Modificados Genéticamente , Antígenos/genética , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Microscopía Confocal , Microtúbulos/metabolismo , Unión Proteica , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Espermatozoides/citología , Espermatozoides/metabolismo , Imagen de Lapso de Tiempo/métodosRESUMEN
At the onset of procentriole formation, a structure called the cartwheel is formed adjacent to the pre-existing centriole. SAS-6 proteins are thought to constitute the hub of the cartwheel structure. However, the exact function of the cartwheel in the process of centriole formation has not been well characterized. In this study, we focused on the functions of human SAS-6 (HsSAS-6, also known as SASS6). By using an in vitro reconstitution system with recombinant HsSAS-6, we first observed its conserved molecular property of forming the central part of the cartwheel structure. Furthermore, we uncovered critical functions of HsSAS-6 by using a combination of an auxin-inducible HsSAS-6-degron (AID) system and super-resolution microscopy in human cells. Our results demonstrate that the HsSAS-6 is required not only for the initiation of centriole formation, but also for the stabilization of centriole intermediates. Moreover, after procentriole formation, HsSAS-6 is necessary for limiting Plk4 accumulation at the centrioles and thereby suppressing the formation of initiation sites that would otherwise promote the development of extra procentrioles. Overall, these findings illustrate the conserved and fundamental functions of the cartwheel in centriole duplication.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Técnicas de Cultivo de Célula , Ciclo Celular/fisiología , Humanos , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The centriole organelle consists of microtubules (MTs) that exhibit a striking 9-fold radial symmetry. Centrioles play fundamental roles across eukaryotes, notably in cell signaling, motility and division. In this Cell Science at a Glance article and accompanying poster, we cover the cellular life cycle of this organelle - from assembly to disappearance - focusing on human centrioles. The journey begins at the end of mitosis when centriole pairs disengage and the newly formed centrioles mature to begin a new duplication cycle. Selection of a single site of procentriole emergence through focusing of polo-like kinase 4 (PLK4) and the resulting assembly of spindle assembly abnormal protein 6 (SAS-6) into a cartwheel element are evoked next. Subsequently, we cover the recruitment of peripheral components that include the pinhead structure, MTs and the MT-connecting A-C linker. The function of centrioles in recruiting pericentriolar material (PCM) and in forming the template of the axoneme are then introduced, followed by a mention of circumstances in which centrioles form de novo or are eliminated.