Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy.

Autophagy is a lysosome-dependent degradation pathway essential to maintain cellular homeostasis. Therefore, either defective or excessive autophagy may be detrimental for cells and tissues. The past decade was characterized by significant advances in molecular dissection of stimulatory autophagy inputs; however, our understanding of the mechanisms that restrain autophagy is far from complete. Here, we describe a negative feedback mechanism that limits autophagosome biogenesis based on the selective autophagy-mediated degradation of ATG13, a component of the ULK1 autophagy initiation complex. We demonstrate that the centrosomal protein OFD1 acts as bona fide autophagy receptor for ATG13 via direct interaction with the Atg8/LC3/GABARAP family of proteins. We also show that patients with Oral-Facial-Digital type I syndrome, caused by mutations in the OFD1 gene, display excessive autophagy and that genetic inhibition of autophagy in a mouse model of the disease, significantly ameliorates polycystic kidney, a clinical manifestation of the disorder. Collectively, our data report the discovery of an autophagy self-regulated mechanism and implicate dysregulated autophagy in the pathogenesis of renal cystic disease in mammals.

The HOPS complex subunit VPS39 controls ciliogenesis through autophagy.

Primary cilia are microtubule-based organelles that assemble and protrude from the surface of most mammalian cells during quiescence. The biomedical relevance of cilia is indicated by disorders ascribed to cilia dysfunction, known as ciliopathies, that display distinctive features including renal cystic disease. In this report, we demonstrate that vacuolar protein sorting 39 (VPS39), a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, acts as a negative regulator of ciliogenesis in human renal cells, by controlling the localization of the intraflagellar transport 20 protein at the base of cilia through autophagy. Moreover, we show that VPS39 controls ciliogenesis through autophagy also in vivo in renal tubules of medaka fish. These observations suggest a direct involvement of the HOPS complex in the regulation of autophagy-mediated ciliogenesis and eventually in target selection. Interestingly, we show that the impact of autophagy modulation on ciliogenesis is cell-type dependent and strictly related to environmental stimuli. This report adds a further tile to the cilia-autophagy connection and suggests that VPS39 could represent a new biological target for the recovery of the cilia-related phenotypes observed in the kidneys of patients affected by ciliopathies.

Development of a novel SNP assay to detect lactase persistence associated genetic variants.

BACKGROUND: In adulthood the activity of the lactase enzyme is inherited as autosomal dominant form associated to Single nucleotide polymorphisms (SNPs). The present research was aimed to develop a novel genetic method to test lactase non persistence more powerfully. METHODS AND RESULTS: In our study, we selected eight different SNPs that are associated with lactase persistence from Caucasian, Arabian Bedouins, sub-Saharian Africans and Asian populations to set up an approach to detect all the eight different SNPs at the same time in the same sample. This technique is centred on the identification of SNPs with a single nucleotide primer extension method using Sanger sequencing and capillary electrophoresis. CONCLUSIONS: Our method allowed us to check the genotype asset of eight SNPs related to lactase persistence simultaneously and in a very efficient manner. It could be applied to a higher number of SNPs in a single reaction.

Oral-facial-digital syndrome type I cells exhibit impaired DNA repair; unanticipated consequences of defective OFD1 outside of the cilia network.

Defects in OFD1 underlie the clinically complex ciliopathy, Oral-Facial-Digital syndrome Type I (OFD Type I). Our understanding of the molecular, cellular and clinical consequences of impaired OFD1 originates from its characterised roles at the centrosome/basal body/cilia network. Nonetheless, the first described OFD1 interactors were components of the TIP60 histone acetyltransferase complex. We find that OFD1 can also localise to chromatin and its reduced expression is associated with mis-localization of TIP60 in patient-derived cell lines. TIP60 plays important roles in controlling DNA repair. OFD Type I cells exhibit reduced histone acetylation and altered chromatin dynamics in response to DNA double strand breaks (DSBs). Furthermore, reduced OFD1 impaired DSB repair via homologous recombination repair (HRR). OFD1 loss also adversely impacted upon the DSB-induced G2-M checkpoint, inducing a hypersensitive and prolonged arrest. Our findings show that OFD Type I patient cells have pronounced defects in the DSB-induced histone modification, chromatin remodelling and DSB-repair via HRR; effectively phenocopying loss of TIP60. These data extend our knowledge of the molecular and cellular consequences of impaired OFD1, demonstrating that loss of OFD1 can negatively impact upon important nuclear events; chromatin plasticity and DNA repair.

Mutations in COX7B cause microphthalmia with linear skin lesions, an unconventional mitochondrial disease.

Microphthalmia with linear skin lesions (MLS) is an X-linked dominant male-lethal disorder associated with mutations in holocytochrome c-type synthase (HCCS), which encodes a crucial player of the mitochondrial respiratory chain (MRC). Unlike other mitochondrial diseases, MLS is characterized by a well-recognizable neurodevelopmental phenotype. Interestingly, not all clinically diagnosed MLS cases have mutations in HCCS, thus suggesting genetic heterogeneity for this disorder. Among the possible candidates, we analyzed the X-linked COX7B and found deleterious de novo mutations in two simplex cases and a nonsense mutation, which segregates with the disease, in a familial case. COX7B encodes a poorly characterized structural subunit of cytochrome c oxidase (COX), the MRC complex IV. We demonstrated that COX7B is indispensable for COX assembly, COX activity, and mitochondrial respiration. Downregulation of the COX7B ortholog (cox7B) in medaka (Oryzias latipes) resulted in microcephaly and microphthalmia that recapitulated the MLS phenotype and demonstrated an essential function of complex IV activity in vertebrate CNS development. Our results indicate an evolutionary conserved role of the MRC complexes III and IV for the proper development of the CNS in vertebrates and uncover a group of mitochondrial diseases hallmarked by a developmental phenotype.

Drug repurposing screen to identify inhibitors of the RNA polymerase (nsp12) and helicase (nsp13) from SARS-CoV-2 replication and transcription complex.

Coronaviruses contain one of the largest genomes among the RNA viruses, coding for 14-16 non-structural proteins (nsp) that are involved in proteolytic processing, genome replication and transcription, and four structural proteins that build the core of the mature virion. Due to conservation across coronaviruses, nsps form a group of promising drug targets as their inhibition directly affects viral replication and, therefore, progression of infection. A minimal but fully functional replication and transcription complex was shown to be formed by one RNA-dependent RNA polymerase (nsp12), one nsp7, two nsp8 accessory subunits, and two helicase (nsp13) enzymes. Our approach involved, targeting nsp12 and nsp13 to allow multiple starting point to interfere with virus infection progression. Here we report a combined in-vitro repurposing screening approach, identifying new and confirming reported SARS-CoV-2 nsp12 and nsp13 inhibitors.

ProfhEX: AI-based platform for small molecules liability profiling.

Off-target drug interactions are a major reason for candidate failure in the drug discovery process. Anticipating potential drug's adverse effects in the early stages is necessary to minimize health risks to patients, animal testing, and economical costs. With the constantly increasing size of virtual screening libraries, AI-driven methods can be exploited as first-tier screening tools to provide liability estimation for drug candidates. In this work we present ProfhEX, an AI-driven suite of 46 OECD-compliant machine learning models that can profile small molecules on 7 relevant liability groups: cardiovascular, central nervous system, gastrointestinal, endocrine, renal, pulmonary and immune system toxicities. Experimental affinity data was collected from public and commercial data sources. The entire chemical space comprised 289'202 activity data for a total of 210'116 unique compounds, spanning over 46 targets with dataset sizes ranging from 819 to 18896. Gradient boosting and random forest algorithms were initially employed and ensembled for the selection of a champion model. Models were validated according to the OECD principles, including robust internal (cross validation, bootstrap, y-scrambling) and external validation. Champion models achieved an average Pearson correlation coefficient of 0.84 (SD of 0.05), an R2 determination coefficient of 0.68 (SD = 0.1) and a root mean squared error of 0.69 (SD of 0.08). All liability groups showed good hit-detection power with an average enrichment factor at 5% of 13.1 (SD of 4.5) and AUC of 0.92 (SD of 0.05). Benchmarking against already existing tools demonstrated the predictive power of ProfhEX models for large-scale liability profiling. This platform will be further expanded with the inclusion of new targets and through complementary modelling approaches, such as structure and pharmacophore-based models. ProfhEX is freely accessible at the following address: https://profhex.exscalate.eu/ .

Diketo acid inhibitors of nsp13 of SARS-CoV-2 block viral replication.

For RNA viruses, RNA helicases have long been recognized to play critical roles during virus replication cycles, facilitating proper folding and replication of viral RNAs, therefore representing an ideal target for drug discovery. SARS-CoV-2 helicase, the non-structural protein 13 (nsp13) is a highly conserved protein among all known coronaviruses, and, at the moment, is one of the most explored viral targets to identify new possible antiviral agents. In the present study, we present six diketo acids (DKAs) as nsp13 inhibitors able to block both SARS-CoV-2 nsp13 enzymatic functions. Among them four compounds were able to inhibit viral replication in the low micromolar range, being active also on other human coronaviruses such as HCoV229E and MERS CoV. The experimental investigation of the binding mode revealed ATP-non-competitive kinetics of inhibition, not affected by substrate-displacement effect, suggesting an allosteric binding mode that was further supported by molecular modelling calculations predicting the binding into an allosteric conserved site located in the RecA2 domain.

DHFR Inhibitors Display a Pleiotropic Anti-Viral Activity against SARS-CoV-2: Insights into the Mechanisms of Action.

During the COVID-19 pandemic, drug repurposing represented an effective strategy to obtain quick answers to medical emergencies. Based on previous data on methotrexate (MTX), we evaluated the anti-viral activity of several DHFR inhibitors in two cell lines. We observed that this class of compounds showed a significant influence on the virus-induced cytopathic effect (CPE) partly attributed to the intrinsic anti-metabolic activity of these drugs, but also to a specific anti-viral function. To elucidate the molecular mechanisms, we took advantage of our EXSCALATE platform for in-silico molecular modelling and further validated the influence of these inhibitors on nsp13 and viral entry. Interestingly, pralatrexate and trimetrexate showed superior effects in counteracting the viral infection compared to other DHFR inhibitors. Our results indicate that their higher activity is due to their polypharmacological and pleiotropic profile. These compounds can thus potentially give a clinical advantage in the management of SARS-CoV-2 infection in patients already treated with this class of drugs.

Kidney-specific inactivation of Ofd1 leads to renal cystic disease associated with upregulation of the mTOR pathway.

The oral-facial-digital type I syndrome (OFDI; MIM 311200) is a rare syndromic form of inherited renal cystic disease. It is transmitted as an X-linked dominant, male lethal disorder and is caused by mutations in the OFD1 gene. Previous studies demonstrated that OFDI belongs to the growing number of disorders ascribed to dysfunction of primary cilia. We generated a conditional inactivation of the mouse Ofd1 gene using the Ksp-Cre transgenic line, which resulted in a viable model characterized by renal cystic disease and progressive impairment of renal function. The study of this model allowed us to demonstrate that primary cilia initially form and then disappear after the development of cysts, suggesting that the absence of primary cilia is a consequence rather than the primary cause of renal cystic disease. Immunofluorescence and western blotting analysis revealed upregulation of the mTOR pathway in both dilated and non-dilated renal structures. Treatment with rapamycin, a specific inhibitor of the mTOR pathway, resulted in a significant reduction in the number and size of renal cysts and a decrease in the cystic index compared with untreated mutant animals, suggesting that dysregulation of this pathway in our model is mTOR-dependent. The animal model we have generated could thus represent a valuable tool to understand the molecular link between mTOR and cyst development, and eventually to the identification of novel drug targets for renal cystic disease.

Natural Compounds Inhibit SARS-CoV-2 nsp13 Unwinding and ATPase Enzyme Activities.

SARS-CoV-2 infection is still spreading worldwide, and new antiviral therapies are an urgent need to complement the approved vaccine preparations. SARS-CoV-2 nps13 helicase is a validated drug target participating in the viral replication complex and possessing two associated activities: RNA unwinding and 5'-triphosphatase. In the search of SARS-CoV-2 direct antiviral agents, we established biochemical assays for both SARS-CoV-2 nps13-associated enzyme activities and screened both in silico and in vitro a small in-house library of natural compounds. Myricetin, quercetin, kaempferol, and flavanone were found to inhibit the SARS-CoV-2 nps13 unwinding activity at nanomolar concentrations, while licoflavone C was shown to block both SARS-CoV-2 nps13 activities at micromolar concentrations. Mode of action studies showed that all compounds are nsp13 noncompetitive inhibitors versus ATP, while computational studies suggested that they can bind both nucleotide and 5'-RNA nsp13 binding sites, with licoflavone C showing a unique pattern of interaction with nsp13 amino acid residues. Overall, we report for the first time natural flavonoids as selective inhibitors of SARS-CoV-2 nps13 helicase with low micromolar activity.

Cytopathic SARS-CoV-2 screening on VERO-E6 cells in a large-scale repurposing effort.

Worldwide, there are intensive efforts to identify repurposed drugs as potential therapies against SARS-CoV-2 infection and the associated COVID-19 disease. To date, the anti-inflammatory drug dexamethasone and (to a lesser extent) the RNA-polymerase inhibitor remdesivir have been shown to be effective in reducing mortality and patient time to recovery, respectively, in patients. Here, we report the results of a phenotypic screening campaign within an EU-funded project (H2020-EXSCALATE4COV) aimed at extending the repertoire of anti-COVID therapeutics through repurposing of available compounds and highlighting compounds with new mechanisms of action against viral infection. We screened 8702 molecules from different repurposing libraries, to reveal 110 compounds with an anti-cytopathic IC50 < 20 µM. From this group, 18 with a safety index greater than 2 are also marketed drugs, making them suitable for further study as potential therapies against COVID-19. Our result supports the idea that a systematic approach to repurposing is a valid strategy to accelerate the necessary drug discovery process.

Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions.

Chemotherapy-induced peripheral neuropathy (CIPN) and hypersensitivity reactions (HSRs) are among the most frequent and impairing side effects of the antineoplastic agent paclitaxel. Here, we demonstrated that paclitaxel can bind and activate complement component 5a receptor 1 (C5aR1) and that this binding is crucial in the etiology of paclitaxel-induced CIPN and anaphylaxis. Starting from our previous data demonstrating the role of interleukin (IL)-8 in paclitaxel-induced neuronal toxicity, we searched for proteins that activate IL-8 expression and, by using the Exscalate platform for molecular docking simulations, we predicted the high affinity of C5aR1 with paclitaxel. By in vitro studies, we confirmed the specific and competitive nature of the C5aR1-paclitaxel binding and found that it triggers intracellularly the NFkB/P38 pathway and c-Fos. In F11 neuronal cells and rat dorsal root ganglia, C5aR1 inhibition protected from paclitaxel-induced neuropathological effects, while in paclitaxel-treated mice, the absence (knock-out mice) or the inhibition of C5aR1 significantly ameliorated CIPN symptoms-in terms of cold and mechanical allodynia-and reduced the chronic pathological state in the paw. Finally, we found that C5aR1 inhibition can counteract paclitaxel-induced anaphylactic cytokine release in macrophages in vitro, as well as the onset of HSRs in mice. Altogether these data identified C5aR1 as a key mediator and a new potential pharmacological target for the prevention and treatment of CIPN and HSRs induced by paclitaxel.

Characterization of raloxifene as a potential pharmacological agent against SARS-CoV-2 and its variants.

The new coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic, which so far has caused over 6 million deaths in 2 years, despite new vaccines and antiviral medications. Drug repurposing, an approach for the potential application of existing pharmaceutical products to new therapeutic indications, could be an effective strategy to obtain quick answers to medical emergencies. Following a virtual screening campaign on the most relevant viral proteins, we identified the drug raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a new potential agent to treat mild-to-moderate COVID-19 patients. In this paper we report a comprehensive pharmacological characterization of raloxifene in relevant in vitro models of COVID-19, specifically in Vero E6 and Calu-3 cell lines infected with SARS-CoV-2. A large panel of the most common SARS-CoV-2 variants isolated in Europe, United Kingdom, Brazil, South Africa and India was tested to demonstrate the drug's ability in contrasting the viral cytopathic effect (CPE). Literature data support a beneficial effect by raloxifene against the viral infection due to its ability to interact with viral proteins and activate protective estrogen receptor-mediated mechanisms in the host cells. Mechanistic studies here reported confirm the significant affinity of raloxifene for the Spike protein, as predicted by in silico studies, and show that the drug treatment does not directly affect Spike/ACE2 interaction or viral internalization in infected cell lines. Interestingly, raloxifene can counteract Spike-mediated ADAM17 activation in human pulmonary cells, thus providing new insights on its mechanism of action. A clinical study in mild to moderate COVID-19 patients (NCT05172050) has been recently completed. Our contribution to evaluate raloxifene results on SARS-CoV-2 variants, and the interpretation of the mechanisms of action will be key elements to better understand the trial results, and to design new clinical studies aiming to evaluate the potential development of raloxifene in this indication.

The SARS-CoV-2 spike protein binds and modulates estrogen receptors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 as its primary infection mechanism. Interactions between S and endogenous proteins occur after infection but are not well understood. We profiled binding of S against >9000 human proteins and found an interaction between S and human estrogen receptor α (ERα). Using bioinformatics, supercomputing, and experimental assays, we identified a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects. Non-invasive imaging in SARS-CoV-2-infected hamsters localized lung pathology with increased ERα lung levels. Postmortem lung experiments from infected hamsters and humans confirmed an increase in cytoplasmic ERα and its colocalization with S in alveolar macrophages. These findings describe the discovery of a S-ERα interaction, imply a role for S as an NRC, and advance knowledge of SARS-CoV-2 biology and coronavirus disease 2019 pathology.

The SARS-CoV-2 spike protein binds and modulates estrogen receptors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) at the cell surface, which constitutes the primary mechanism driving SARS-CoV-2 infection. Molecular interactions between the transduced S and endogenous proteins likely occur post-infection, but such interactions are not well understood. We used an unbiased primary screen to profile the binding of full-length S against >9,000 human proteins and found significant S-host protein interactions, including one between S and human estrogen receptor alpha (ERα). After confirming this interaction in a secondary assay, we used bioinformatics, supercomputing, and experimental assays to identify a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit and an S-ERα binding mode. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects and ACE2 expression. Noninvasive multimodal PET/CT imaging in SARS-CoV-2-infected hamsters using [ 18 F]fluoroestradiol (FES) localized lung pathology with increased ERα lung levels. Postmortem experiments in lung tissues from SARS-CoV-2-infected hamsters and humans confirmed an increase in cytoplasmic ERα expression and its colocalization with S protein in alveolar macrophages. These findings describe the discovery and characterization of a novel S-ERα interaction, imply a role for S as an NRC, and are poised to advance knowledge of SARS-CoV-2 biology, COVID-19 pathology, and mechanisms of sex differences in the pathology of infectious disease.

S-Carboxymethyl Cysteine Protects against Oxidative Stress and Mitochondrial Impairment in a Parkinson's Disease In Vitro Model.

The mucolytic agent S-carboxymethylcysteine is widely used as an expectorant for the treatment of numerous respiratory disorders. The metabolic fate of S-carboxymethyl-L-cysteine is complex. Several clinical studies have demonstrated that the metabolism of this agent differs within the same individual, with sulfur oxygenated metabolites generated upon night-time administration. It has been indicated that this drug behaves like a free radical scavenger and that, in this regard, the sulfide is the active species with sulphoxide metabolites (already oxidized) being inactive. Consequently, a night-time consumption of the drug should be more effective upon daytime administration. Still, this diurnal variation in biotransformation (deactivation) is dependent on the genetic polymorphism on which relies the patient population capacities of S-carboxymethyl-L-cysteine sulphoxidation. It has been reported that those cohorts who are efficient sulfur oxidizers will generate inactive oxygenated metabolites. In contrast, those who have a relative deficiency in this mechanism will be subjected to the active sulfide for a more extended period. In this regard, it is noteworthy that 38-39% of Parkinson's disease patients belong to the poor sulphoxide cohort, being exposed to higher levels of active sulfide, the active antioxidant metabolite of S-carboxymethyl-L-cysteine. Parkinson's disease is a neurodegenerative disorder that affects predominately dopaminergic neurons. It has been demonstrated that oxidative stress and mitochondrial dysfunction play a crucial role in the degeneration of dopaminergic neurons. Based on this evidence, in this study, we evaluated the effects of S-carboxymethyl cysteine in an in vitro model of Parkinson's disease in protecting against oxidative stress injury. The data obtained suggested that an S-carboxymethylcysteine-enriched diet could be beneficial during aging to protect neurons from oxidative imbalance and mitochondrial dysfunction, thus preventing the progression of neurodegenerative processes.

L-Methionine Protects against Oxidative Stress and Mitochondrial Dysfunction in an In Vitro Model of Parkinson's Disease.

Methionine is an aliphatic, sulfur-containing, essential amino acid that has been demonstrated to have crucial roles in metabolism, innate immunity, and activation of endogenous antioxidant enzymes, including methionine sulfoxide reductase A/B and the biosynthesis of glutathione to counteract oxidative stress. Still, methionine restriction avoids altered methionine/transmethylation metabolism, thus reducing DNA damage and possibly avoiding neurodegenerative processes. In this study, we wanted to study the preventive effects of methionine in counteracting 6-hydroxydopamine (6-OHDA)-induced injury. In particular, we analyzed the protective effects of the amino acid L-methionine in an in vitro model of Parkinson's disease and dissected the underlying mechanisms compared to the known antioxidant taurine to gain insights into the potential of methionine treatment in slowing the progression of the disease by maintaining mitochondrial functionality. In addition, to ascribe the effects of methionine on mitochondria and oxidative stress, methionine sulfoxide was used in place of methionine. The data obtained suggested that an L-methionine-enriched diet could be beneficial during aging to protect neurons from oxidative imbalance and mitochondrial dysfunction, thus preventing the progression of neurodegenerative processes.

Identification of Inhibitors of SARS-CoV-2 3CL-Pro Enzymatic Activity Using a Small Molecule in Vitro Repurposing Screen.

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro and have identified 62 additional compounds with IC50 values below 1 µM and profiled their selectivity toward chymotrypsin and 3CL-Pro from the Middle East respiratory syndrome virus. A subset of eight inhibitors showed anticytopathic effect in a Vero-E6 cell line, and the compounds thioguanosine and MG-132 were analyzed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Å, showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.

The centrosomal/basal body protein OFD1 is required for microtubule organization and cell cycle progression.

Oral-Facial-Digital type I (OFD1) is a rare inherited form of renal cystic disease associated with ciliary dysfunction. This disorder is due to mutations in the OFD1 gene that encodes a protein localized to centrosomes and basal bodies in different cell types. Immunofluorescence analysis demonstrated that OFD1 displays a dynamic distribution during cell cycle. High-content microscopy analysis of Ofd1-depleted fibroblasts revealed impaired cell cycle progression. Immunofluorescence analysis and cell proliferation assays also indicated the presence of a variety of defects such as centrosome accumulation, nuclear abnormalities and aneuploidy. In addition, Ofd1-depleted cells displayed an abnormal microtubule network that may underlie these defects. All together our results suggest that OFD1 contributes to the function of the microtubule organizing center (MTOC) in the cell, controlling cell cycle progression both in vitro and in vivo.