RESUMEN
With over 450 genes, solute carriers (SLCs) constitute the largest transporter superfamily responsible for the uptake and efflux of nutrients, metabolites, and xenobiotics in human cells. SLCs are associated with a wide variety of human diseases, including cancer, diabetes, and metabolic and neurological disorders. They represent an important therapeutic target class that remains only partly exploited as therapeutics that target SLCs are scarce. Additionally, many small molecules reported in the literature to target SLCs are poorly characterized. Both features may be due to the difficulty of developing SLC transport assays that fulfill the quality criteria for high-throughput screening. Here, we report one of the main limitations hampering assay development within the RESOLUTE consortium: the lack of a resource providing high-quality information on SLC tool compounds. To address this, we provide a systematic annotation of tool compounds targeting SLCs. We first provide an overview on RESOLUTE assays. Next, we present a list of SLC-targeting compounds collected from the literature and public databases; we found that most data sources lacked specificity data. Finally, we report on experimental tests of 19 selected compounds against a panel of 13 SLCs from seven different families. Except for a few inhibitors, which were active on unrelated SLCs, the tested inhibitors demonstrated high selectivity for their reported targets. To make this knowledge easily accessible to the scientific community, we created an interactive dashboard displaying the collected data in the RESOLUTE web portal (https://re-solute.eu). We anticipate that our open-access resources on assays and compounds will support the development of future drug discovery campaigns for SLCs.
RESUMEN
The Cellular Thermal Shift Assay (CETSA) enables the study of protein-ligand interactions in a cellular context. It provides valuable information on the binding affinity and specificity of both small and large molecule ligands in a relevant physiological context, hence forming a unique tool in drug discovery. Though high-throughput lab protocols exist for scaling up CETSA, subsequent data analysis and quality control remain laborious and limit experimental throughput. Here, we introduce a scalable and robust data analysis workflow which allows integration of CETSA into routine high throughput screening (HT-CETSA). This new workflow automates data analysis and incorporates quality control (QC), including outlier detection, sample and plate QC, and result triage. We describe the workflow and show its robustness against typical experimental artifacts, show scaling effects, and discuss the impact of data analysis automation by eliminating manual data processing steps.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Flujo de Trabajo , Ensayos Analíticos de Alto Rendimiento/métodos , Control de Calidad , Análisis de Datos , Automatización/métodos , Humanos , Ligandos , Descubrimiento de Drogas/métodos , Unión ProteicaRESUMEN
This article presents detailed descriptions of procedures and troubleshooting tips for solid-supported membrane (SSM)-based electrophysiology assays (SURFE²R) to measure electrogenic solute carrier transporter proteins (SLCs) and assess the effects of compounds that modulate their activity. SURFE²R allows the use of the standard 96-well format, making it an ideal platform for tertiary assays in a drug-discovery campaign. The assays are performed with cell-line-derived membrane fractions or proteoliposomes containing the transporter of interest. Three main protocols are described for the isolation of membrane fractions from cell culture and the generation of proteoliposomes containing the transporter of interest. Additionally, detailed protocols for SURFE²R single concentration and dose-response experiments are included to measure the potencies of test compounds in stimulating or inhibiting transporter function (EC50 or IC50 values, respectively) and kinetic functional assays to calculate apparent affinity (kM ) and maximal velocity (Vmax ) of substrate uptake. © 2023 Sanofi. Current Protocols published by Wiley Periodicals LLC. PROTOCOL GROUP 1: Sample preparation for SSM-based electrophysiology assays Support Protocol 1: Production of cell batches Support Protocol 2: Simple isolation of cell membranes Alternate Protocol 1: Isolation of cell membranes with sucrose gradient pre-purification Support Protocol 3: Production and isolation of liposomes Support Protocol 4: Preparation of sensor with isolated cell membranes Alternate Protocol 2: Preparation of sensor with isolated proteoliposomes PROTOCOL GROUP 2: Determination of assay parameters for SSM-based electrophysiology assay Support Protocol 5: Assay with stable buffer Alternate Protocol 3: Assay with ion gradient Support Protocol 6: Determination of membrane/liposome concentration Support Protocol 7: Determination of substrate dependency kM PROTOCOL GROUP 3: Determination of advanced assay parameters for SSM-based electrophysiology assays Support Protocol 8: Assessment of ion concentration dependency Support Protocol 9: Assessment of pH dependency Support Protocol 10: Assessment of DMSO dependency Support Protocol 11: Assessment of signal stability with multiple activations PROTOCOL GROUP 4: Compound testing through SSM-based electrophysiology assays using SURFE²R apparatus Support Protocol 12: Assessment of signal specificity of a published inhibitor or unknown compound(s) Support Protocol 13: Compound wash-out Support Protocol 14: Statistical analysis.
Asunto(s)
Descubrimiento de Drogas , Proteínas de Transporte de Membrana , Electrofisiología/métodos , Proteínas de Transporte de Membrana/metabolismo , Membranas/metabolismo , Membrana Celular/metabolismo , LiposomasRESUMEN
Acoustic droplet ejection mass spectrometry (ADE-MS) has recently emerged as a promising label-free, MS-based readout method for high throughput screening (HTS) campaigns in early pharmaceutical drug discovery, since it enables high-speed analysis directly from 384- or 1536-well plates. In this manuscript we describe our characterization of an ADE-MS based high sample content enzymatic assay for mutant isocitrate dehydrogenase 1 (IDH1) R132H with a strong focus on assay development. IDH1 R132H has become a very attractive therapeutic target in the field of antitumor drug discovery, and several pharmaceutical companies have attempted to develop novel small molecule inhibitors against mutant IDH1. With the development of an mIDH1 ADE-MS based HTS assay and a detailed comparison of this new readout technique to the commonly used fluorescence intensity mIDH1 assay, we demonstrated good correlation of both methods and were able to identify new potent inhibitors of mIDH1.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Isocitrato Deshidrogenasa , Acústica , Ensayos Analíticos de Alto Rendimiento/métodos , Isocitrato Deshidrogenasa/genética , Espectrometría de Masas , Preparaciones FarmacéuticasRESUMEN
The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.
RESUMEN
ERAP1 is a key aminopeptidase involved in peptide trimming before major histocompatibility complex (MHC) presentation. A single nucleotide polymorphism (SNP) in the ERAP1 gene can lead to impaired trimming activity and affect ERAP1 function. ERAP1 genetic variations have been linked to an increased susceptibility to cancer and autoimmune disease. Here, we report the discovery of novel ERAP1 inhibitors using a high throughput screening approach. Due to ERAP1 broad substrate specificity, the hit finding strategy included testing inhibitors with a range of biochemical assays. Based on the hit potency, selectivity, and in vitro absorption, distribution, metabolism, excretion, and toxicity, the benzofuran series was selected. Fifteen derivatives were designed and synthesized, the compound potency was improved to the nanomolar range, and the structure-activity relationship supported by modeling studies.
RESUMEN
BACKGROUND: Epidemiological studies have shown that increased circulating branched-chain amino acids (BCAAs) are associated with insulin resistance and type 2 diabetes (T2D). This may result from altered energy metabolism or dietary habits. OBJECTIVE: We hypothesized that a lower intake of BCAAs improves tissue-specific insulin sensitivity. METHODS: This randomized, placebo-controlled, double-blinded, crossover trial examined well-controlled T2D patients receiving isocaloric diets (protein: 1 g/kg body weight) for 4 wk. Protein requirements were covered by commercially available food supplemented ≤60% by an AA mixture either containing all AAs or lacking BCAAs. The dietary intervention ensured sufficient BCAA supply above the recommended minimum daily intake. The patients underwent the mixed meal tolerance test (MMT), hyperinsulinemic-euglycemic clamps (HECs), and skeletal muscle and white adipose tissue biopsies to assess insulin signaling. RESULTS: After the BCAA- diet, BCAAs were reduced by 17% during fasting (P < 0.001), by 13% during HEC (P < 0.01), and by 62% during the MMT (P < 0.001). Under clamp conditions, whole-body and hepatic insulin sensitivity did not differ between diets. After the BCAA- diet, however, the oral glucose sensitivity index was 24% (P < 0.01) and circulating fibroblast-growth factor 21 was 21% higher (P < 0.05), whereas meal-derived insulin secretion was 28% lower (P < 0.05). Adipose tissue expression of the mechanistic target of rapamycin was 13% lower, whereas the mitochondrial respiratory control ratio was 1.7-fold higher (both P < 0.05). The fecal microbiome was enriched in Bacteroidetes but depleted of Firmicutes. CONCLUSIONS: Short-term dietary reduction of BCAAs decreases postprandial insulin secretion and improves white adipose tissue metabolism and gut microbiome composition. Longer-term studies will be needed to evaluate the safety and metabolic efficacy in diabetes patients.This trial was registered at clinicaltrials.gov as NCT03261362.
Asunto(s)
Aminoácidos de Cadena Ramificada/administración & dosificación , Diabetes Mellitus Tipo 2/microbiología , Microbioma Gastrointestinal , Secreción de Insulina , Tejido Adiposo Blanco/metabolismo , Estudios Cruzados , Método Doble Ciego , Femenino , Humanos , Masculino , Comidas , Persona de Mediana Edad , Mitocondrias/fisiología , Músculo Esquelético/metabolismoRESUMEN
Antisense oligonucleotide knockdown (ASO-KD) of nicotinamide N-methyltransferase (NNMT) in high-fat diet (HFD)-fed mice has been reported to reduce weight gain and plasma insulin levels and to improve glucose tolerance. Using NNMT-ASO-KD or NNMT knockout mice (NNMT-/-), we tested the hypothesis that Nnmt deletion protects against diet-induced obesity and its metabolic consequences in males and females on obesity-inducing diets. We also examined samples from a human weight reduction (WR) study for adipose NNMT (aNNMT) expression and plasma 1-methylnicotinamide (MNAM) levels. In Western diet (WD)-fed female mice, NNMT-ASO-KD reduced body weight, fat mass, and insulin level and improved glucose tolerance. Although NNMT-/- mice fed a standard diet had no obvious phenotype, NNMT-/- males fed an HFD showed strongly improved insulin sensitivity (IS). Furthermore, NNMT-/- females fed a WD showed reduced weight gain, less fat, and lower insulin levels. However, no improved glucose tolerance was observed in NNMT-/- mice. Although NNMT expression in human fat biopsy samples increased during WR, corresponding plasma MNAM levels significantly declined, suggesting that other mechanisms besides aNNMT expression modulate circulating MNAM levels during WR. In summary, upon NNMT deletion or knockdown in males and females fed different obesity-inducing diets, we observed sex- and diet-specific differences in body composition, weight, and glucose tolerance and estimates of IS.
Asunto(s)
Intolerancia a la Glucosa/metabolismo , Resistencia a la Insulina/fisiología , Nicotinamida N-Metiltransferasa/metabolismo , Obesidad/metabolismo , Animales , Composición Corporal/genética , Composición Corporal/fisiología , Peso Corporal/genética , Peso Corporal/fisiología , Dieta Alta en Grasa/efectos adversos , Intolerancia a la Glucosa/genética , Resistencia a la Insulina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nicotinamida N-Metiltransferasa/genética , Obesidad/genéticaRESUMEN
Microtubule nucleation was uncovered as a key principle of spindle assembly. However, the mechanistic details about microtubule nucleation and the organization of spindle formation and symmetry are currently being revealed. Here we describe the function of coiled-coil domain containing 61 (Ccdc61), a so far uncharacterized centrosomal protein, in spindle assembly and symmetry. Our data describe that Ccdc61 is required for spindle assembly and precise chromosome alignments in mitosis. Microtubule tip-tracking experiments in the absence of Ccdc61 reveal a clear loss of the intrinsic symmetry of microtubule tracks within the spindle. Furthermore, we show that Ccdc61 controls the centrosomal localization of centrosomal protein of 170 kDa (Cep170), a protein that was shown previously to localize to centrosomes as well as spindle microtubules and promotes microtubule organization and microtubule assembly. Interestingly, selective disruption of Ccdc61 impairs the binding between Cep170 and TANK binding kinase 1, an interaction that is required for microtubule stability. In summary, we have discovered Ccdc61 as a centrosomal protein with an important function in mitotic microtubule organization.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Mitosis , Proteínas Serina-Treonina Quinasas/genética , Huso Acromático/metabolismo , Factores de Transcripción/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Centrosoma , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Expresión Génica , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Osteoblastos/metabolismo , Osteoblastos/ultraestructura , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/ultraestructura , Factores de Transcripción/metabolismoRESUMEN
Fatty acid esters of hydroxylated fatty acids (FAHFAs) were discovered as a novel class of endogenous mammalian lipids whose profound effects on metabolism have been shown. In the current study, in vitro and in vivo the metabolic effects of two of these FAHFAs, namely palmitic acid-5- (or -9) -hydroxy-stearic acid (5- or 9-PAHSA, respectively) were profiled. In DIO mice fed with differentially composed low- or high-fat diets, acute and subchronic treatment with 5-PAHSA and 9-PAHSA alone, or in combination, did not significantly improve the deranged metabolic status. Neither racemic 5- or 9-PAHSA, nor the enantiomers were able to: (1) increase basal or insulin-stimulated glucose uptake in vitro, (2) stimulate GLP-1 release from GLUTag cells, or (3) induce GSIS in rat, mouse, or human islets or in a human pancreatic ß cell line. Therefore, our data do not support the further development of PAHSAs or their derivatives for the control of insulin resistance and hyperglycemia.
Asunto(s)
Hiperglucemia/tratamiento farmacológico , Resistencia a la Insulina , Islotes Pancreáticos , Obesidad , Ácido Palmítico , Ácidos Esteáricos , Animales , Dieta con Restricción de Grasas , Dieta Alta en Grasa , Péptido 1 Similar al Glucagón/metabolismo , Glucosa/metabolismo , Células HEK293 , Humanos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Ácido Palmítico/administración & dosificación , Ácido Palmítico/farmacología , Ratas , Ratas Sprague-Dawley , Ácidos Esteáricos/administración & dosificación , Ácidos Esteáricos/farmacologíaRESUMEN
The assembly of the first centrosome occurs upon fertilisation when male centrioles recruit pericentriolar material (PCM) from the egg cytoplasm. The mechanisms underlying the proper assembly of centrosomes during early embryogenesis remain obscure. We identify Wdr8 as a novel maternally essential protein that is required for centrosome assembly during embryonic mitoses of medaka (Oryzias latipes). By CRISPR-Cas9-mediated knockout, maternal/zygotic Wdr8-null (m/zWdr8-/-) blastomeres exhibit severe defects in centrosome structure that lead to asymmetric division, multipolar mitotic spindles and chromosome alignment errors. Via its WD40 domains, Wdr8 interacts with the centriolar satellite protein SSX2IP. Combining targeted gene knockout and in vivo reconstitution of the maternally essential Wdr8-SSX2IP complex reveals an essential link between maternal centrosome proteins and the stability of the zygotic genome for accurate vertebrate embryogenesis. Our approach provides a way of distinguishing maternal from paternal effects in early embryos and should contribute to understanding molecular defects in human infertility.
Asunto(s)
Centrosoma/metabolismo , Proteínas de Peces/metabolismo , Mitosis , Oryzias/embriología , Oryzias/genética , Animales , Blastómeros/metabolismo , Centriolos/metabolismo , Femenino , Proteínas de Peces/genética , Masculino , Herencia Materna , Oryzias/metabolismo , Cigoto/citología , Cigoto/metabolismoRESUMEN
Centrioles are core components of centrosomes, the major microtubule-organizing centers of animal cells, and act as basal bodies for cilia formation. Control of centriole number is therefore crucial for genome stability and embryogenesis. Centriole duplication requires the serine/threonine protein kinase Plk4. Here, we identify Cep78 as a human centrosomal protein and a new interaction partner of Plk4. Cep78 is mainly a centriolar protein that localizes to the centriolar wall. Furthermore, we find that Plk4 binds to Cep78 through its N-terminal domain but that Cep78 is not an in vitro Plk4 substrate. Cep78 colocalizes with Plk4 at centrioles and is required for Plk4-induced centriole overduplication. Interestingly, upon depletion of Cep78, newly synthesized Plk4 is not localized to centrosomes. Our results suggest that the interaction between Cep78 and the N-terminal catalytic domain of Plk4 is a new and important element in the centrosome overduplication process.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células HeLa , Humanos , Interfase , Unión Proteica , Transporte de ProteínasRESUMEN
Duplication of centrioles, namely the formation of a procentriole next to the parental centriole, is regulated by the polo-like kinase Plk4. Only a few other proteins, including STIL (SCL/TAL1 interrupting locus, SIL) and Sas-6, are required for the early step of centriole biogenesis. Following Plk4 activation, STIL and Sas-6 accumulate at the cartwheel structure at the initial stage of the centriole assembly process. Here, we show that STIL interacts with Plk4 in vivo. A STIL fragment harboring both the coiled-coil domain and the STAN motif shows the strongest binding affinity to Plk4. Furthermore, we find that STIL is phosphorylated by Plk4. We identified Plk4-specific phosphorylation sites within the C-terminal domain of STIL and show that phosphorylation of STIL by Plk4 is required to trigger centriole duplication.
RESUMEN
In differentiated human cells, primary cilia fulfill essential functions in converting mechanical or chemical stimuli into intracellular signals. Formation and maintenance of cilia require multiple functions associated with the centriole-derived basal body, from which axonemal microtubules grow and which assembles a gate to maintain the specific ciliary proteome. Here we characterize the function of a novel centriolar satellite protein, synovial sarcoma X breakpoint-interacting protein 2 (SSX2IP), in the assembly of primary cilia. We show that SSX2IP localizes to the basal body of primary cilia in human and murine ciliated cells. Using small interfering RNA knockdown in human cells, we demonstrate the importance of SSX2IP for efficient recruitment of the ciliopathy-associated satellite protein Cep290 to both satellites and the basal body. Cep290 takes a central role in gating proteins to the ciliary compartment. Consistent with that, loss of SSX2IP drastically reduces entry of the BBSome, which functions to target membrane proteins to primary cilia, and interferes with efficient accumulation of the key regulator of ciliary membrane protein targeting, Rab8. Finally, we show that SSX2IP knockdown limits targeting of the ciliary membrane protein and BBSome cargo, somatostatin receptor 3, and significantly reduces axoneme length. Our data establish SSX2IP as a novel targeting factor for ciliary membrane proteins cooperating with Cep290, the BBSome, and Rab8.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Proteínas Portadoras/metabolismo , Centriolos/metabolismo , Cilios/metabolismo , Células Epiteliales/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Antígenos de Neoplasias/genética , Axonema/metabolismo , Axonema/ultraestructura , Cuerpos Basales/metabolismo , Cuerpos Basales/ultraestructura , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Línea Celular , Centriolos/ultraestructura , Cilios/ultraestructura , Proteínas del Citoesqueleto , Células Epiteliales/ultraestructura , Regulación de la Expresión Génica , Humanos , Ratones , Proteínas Asociadas a Microtúbulos , Células 3T3 NIH , Proteínas de Neoplasias/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Transducción de Señal , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Meiotic maturation in vertebrate oocytes is an excellent model system for microtubule reorganization during M-phase spindle assembly. Here, we surveyed changes in the pattern of microtubule-interacting proteins upon Xenopus laevis oocyte maturation by quantitative proteomics. We identified the synovial sarcoma X breakpoint protein (SSX2IP) as a novel spindle protein. Using X. laevis egg extracts, we show that SSX2IP accumulated at spindle poles in a Dynein-dependent manner and interacted with the γ-tubulin ring complex (γ-TuRC) and the centriolar satellite protein PCM-1. Immunodepletion of SSX2IP impeded γ-TuRC loading onto centrosomes. This led to reduced microtubule nucleation and spindle assembly failure. In rapidly dividing blastomeres of medaka (Oryzias latipes) and in somatic cells, SSX2IP knockdown caused fragmentation of pericentriolar material and chromosome segregation errors. We characterize SSX2IP as a novel centrosome maturation and maintenance factor that is expressed at the onset of vertebrate development. It preserves centrosome integrity and faithful mitosis during the rapid cleavage division of blastomeres and in somatic cells.
Asunto(s)
Centriolos/metabolismo , Centrosoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Blastómeros/metabolismo , Blastómeros/patología , Centriolos/genética , Segregación Cromosómica , Cromosomas/genética , Cromosomas/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Técnicas de Silenciamiento del Gen , Mitosis , Proteínas de Neoplasias/genética , Oocitos/metabolismo , Oryzias/embriología , Oryzias/genética , Oryzias/metabolismo , Proteómica , Proteínas Represoras/genética , Huso Acromático/genética , Huso Acromático/metabolismo , Imagen de Lapso de Tiempo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Xenopus laevis/genéticaRESUMEN
Accurate control of centrosome number is critical for the maintenance of genomic integrity. A recent study reveals that the deubiquitinating enzyme USP33 regulates centrosome biogenesis by stabilizing the centriolar protein CP110.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centrosoma/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación , Animales , HumanosRESUMEN
Since its first description by Theodor Boveri in 1888, the centrosome has been studied intensely, and it revealed detailed information about its structure, molecular composition and its various functions. The centrosome consists of two centrioles, which generally appear in electron microscopy as barrel-shaped structures usually composed of nine microtubule triplets. An amorphous mass of pericentriolar material surrounds the centrioles and accumulates many proteins important for the integrity and function of centrosomes, such as the γ-tubulin ring complex (γ-TuRC) that mediates microtubule nucleation and capping. In animal somatic cells, the centrosome generally accounts for the major microtubule organizing center, and the duplicated pair of centrosomes determines the poles of the microtubule-based mitotic spindle. Despite detailed insights into the centrosome's structure and function, it has been a complete mystery until a few years ago how centrosomes duplicate and assemble. Moreover, it is still largely unclear if and how centrosomal proteins or protein complexes are exchanged, replaced or qualitatively altered. Previously identified cytoplasmic granules, named "pericentriolar" or "centriolar satellites", might fulfil such functions in protein targeting and exchange, and communication between the centrosomes and the cytoplasm. In this review, we summarize current knowledge about the structure, molecular composition and possible roles of the satellites that seem to surround the core of the centrosome in most animal cells.