CRL4DCAF1 ubiquitin ligase regulates PLK4 protein levels to prevent premature centriole duplication.

Centrioles play important roles in the assembly of centrosomes and cilia. Centriole duplication occurs once per cell cycle and is dependent on polo-like kinase 4 (PLK4). To prevent centriole amplification, which is a hallmark of cancer, PLK4 protein levels need to be tightly regulated. Here, we show that the Cullin4A/B-DDB1-DCAF1, CRL4DCAF1, E3 ligase targets PLK4 for degradation in human cells. DCAF1 binds and ubiquitylates PLK4 in the G2 phase to prevent premature centriole duplication in mitosis. In contrast to the regulation of PLK4 by SCFß-TrCP, the interaction between PLK4 and DCAF1 is independent of PLK4 kinase activity and mediated by polo-boxes 1 and 2 of PLK4, suggesting that DCAF1 promotes PLK4 ubiquitylation independently of ß-TrCP. Thus, the SCFSlimb/ß-TrCP pathway, targeting PLK4 for ubiquitylation based on its phosphorylation state and CRL4DCAF1, which ubiquitylates PLK4 by binding to the conserved PB1-PB2 domain, appear to be complementary ways to control PLK4 abundance to prevent centriole overduplication.

Comparison of the Declared Nutrient Content of Plant-Based Meat Substitutes and Corresponding Meat Products and Sausages in Germany.

Plant-based meat substitutes (PBMS) are becoming increasingly popular due to growing concerns about health, animal welfare, and environmental issues associated with animal-based foods. The aim of this study was to compare the declared energy and nutrient contents of PBMS with corresponding meat products and sausages available on the German market. Mandatory nutrition labelling data of 424 PBMS and 1026 meat products and sausages, surveyed in 2021 and 2020, respectively, as part of the German national monitoring of packaged food were used to test for differences in energy and nutrient contents. Principal component analysis (PCA) was used to describe characteristics in the energy and nutrient contents. The comparison showed that most of the PBMS subcategories had significantly lower contents of fat and saturated fat but higher contents of carbohydrate and sugar than corresponding meat subcategories. For salt, the only striking difference was that PBMS salamis had lower salt content than meat salamis. Overall, the PCA revealed protein as a main characteristic for most PBMS categories, with the protein content being equivalent to or, in most protein-based PBMS, even higher than in the corresponding meat products. The wide nutrient content ranges within subcategories, especially for salt, reveal the need and potential for reformulation.

Impact of different factor VIII inhibitor kinetic profiles on the inhibitor titer quantification using the modified Nijmegen-Bethesda assay.

Background: Coagulation factor VIII (FVIII) inhibitor titer quantification is vital for optimizing care in people with hemophilia A. Objectives: This study analyzed the impact of the different kinetic profiles of four FVIII monoclonal antibodies on inhibitor titer quantification using the modified Nijmegen-Bethesda assay. Methods: Concentration-related and time-related profiles of FVIII antibodies (4A4, BO2C11, 2-54, ESH-8) were evaluated in vitro. FVIII residual activity was measured using a one-stage clotting assay and chromogenic substrate assay. Profiles of the FVIII antibodies were compared with the theoretical kinetic model: the ideal log (residual activity)-linear (inhibitor concentration) relationship. Different theoretical kinetic model-dependent and -independent criteria to calculate FVIII inhibitor titer were compared. Results: Factor VIII monoclonal antibodies had different concentration-related and time-related profiles, ideal for comparative analysis using the modified Nijmegen-Bethesda assay. The kinetic profile of 4A4 was similar to the theoretical kinetic model, while BO2C11 showed a steeper curve, and 2-54 and ESH-8 a flatter curve, than the model. In the modified Nijmegen-Bethesda assay, conversion of measured FVIII residual activities for different inhibitor dilutions into FVIII inhibitor titer is based on the theoretical kinetic model. Therefore, titer calculations for FVIII inhibitors that deviate from the model are prone to underestimation or overestimation. Calculating a theoretical dilution at 50% FVIII residual activity by sigmoidal regression reflecting different kinetic inhibition profiles can provide a more accurate titer result. Conclusion: Kinetic profiles of FVIII antibodies can deviate from the theoretical kinetic model in the modified Nijmegen-Bethesda assay, leading to differences in FVIII inhibitor titer quantification.

The SCF-FBXW7 E3 ubiquitin ligase triggers degradation of histone 3 lysine 4 methyltransferase complex component WDR5 to prevent mitotic slippage.

During prolonged mitotic arrest induced by antimicrotubule drugs, cell fate decision is determined by two alternative pathways, one leading to cell death and the other inducing premature escape from mitosis by mitotic slippage. FBWX7, a member of the F-box family of proteins and substrate-targeting subunit of the SKP1-CUL1-F-Box E3 ubiquitin ligase complex, promotes mitotic cell death and prevents mitotic slippage, but molecular details underlying these roles for FBWX7 are unclear. In this study, we report that WDR5 (WD-repeat containing protein 5), a component of the mixed lineage leukemia complex of histone 3 lysine 4 methyltransferases, is a substrate of FBXW7. We determined by coimmunoprecipitation experiments and in vitro binding assays that WDR5 interacts with FBXW7 in vivo and in vitro. SKP1-CUL1-F-Box-FBXW7 mediates ubiquitination of WDR5 and targets it for proteasomal degradation. Furthermore, we find that WDR5 depletion counteracts FBXW7 loss of function by reducing mitotic slippage and polyploidization. In conclusion, our data elucidate a new mechanism in mitotic cell fate regulation, which might contribute to prevent chemotherapy resistance in patients after antimicrotubule drug treatment.

Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization.

Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. A 16-subunit complex, the constitutive centromere associated network (CCAN), connects between the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, and the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has implications for the mechanism of specific recognition of the CENP-A nucleosome. A model consistent with our structure depicts the CENP-C-bound nucleosome as connected to the CCAN through extended, flexible regions of CENP-C. An alternative model identifies both CENP-C and CENP-N as specificity determinants but requires CENP-N to bind CENP-A in a mode distinct from the classical nucleosome octamer.

Role of Polo-like Kinases Plk1 and Plk4 in the Initiation of Centriole Duplication-Impact on Cancer.

Centrosomes nucleate and anchor microtubules and therefore play major roles in spindle formation and chromosome segregation during mitosis. Duplication of the centrosome occurs, similar to DNA, only once during the cell cycle. Aberration of the centrosome number is common in human tumors. At the core of centriole duplication is the conserved polo-like kinase 4, Plk4, and two structural proteins, STIL and Sas-6. In this review, I summarize and discuss developments in our understanding of the first steps of centriole duplication and their regulation.

The Epstein-Barr virus noncoding RNA EBER2 transactivates the UCHL1 deubiquitinase to accelerate cell growth.

The Epstein-Barr virus (EBV) transforms resting B cells and is involved in the development of B cell lymphomas. We report here that the viral noncoding RNA EBER2 accelerates B cell growth by potentiating expression of the UCHL1 deubiquitinase that itself increased expression of the Aurora kinases and of cyclin B1. Importantly, this effect was also visible in Burkitt's lymphoma cells that express none of the virus's known oncogenes. Mechanistically, EBER2 bound the UCHL1 messenger RNA (mRNA), thereby bringing a protein complex that includes PU.1, a UCHL1 transactivator, to the vicinity of its promoter. Although the EBV oncogene LMP1 has been suggested to induce UCHL1, we show here that EBER2 plays a much more important role to reach significant levels of the deubiquitinase in infected cells. However, some viruses that carried a polymorphic LMP1 had an increased ability to achieve full UCHL1 expression. This work identifies a direct cellular target of a viral noncoding RNA that is likely to be central to EBV's oncogenic properties.

Monitoring of different factor VIII replacement products using a factor VIII one-stage clotting assay on cobas t 511/711 analysers.

INTRODUCTION: Recombinant coagulation factor VIII (FVIII) products are the standard of care for patients with haemophilia A. The development of modified FVIII products has provided benefit for patients but presented challenges for monitoring FVIII activity. AIM: This single-centre study evaluated the Roche FVIII one-stage clotting assay (OSA) in measuring FVIII activity in plasma samples spiked with seven FVIII products at clinically relevant concentrations. METHODS: FVIII-deficient plasma samples were spiked with two batches of recombinant FVIII products (octocog alfa, moroctocog alfa, simoctocog alfa, efmoroctocog alfa, damoctocog alfa pegol, rurioctocog alfa pegol, lonoctocog alfa) at 1-120 IU/dL FVIII activity, according to their labelled potency. Measurement was conducted on the cobas t 511/711 analysers using the Roche FVIII OSA and the Technoclone TECHNOCHROM FVIII:C chromogenic substrate assay (CSA). RESULTS: Using the OSA, FVIII activity was close to labelled potency for most analysed FVIII products including a recombinant FVIII Fc fusion protein. PEGylated FVIII product, damoctocog alfa pegol, was marginally above and single-chain product, lonoctocog alfa, below the predefined acceptance criteria: for FVIII activity < 25 IU/dL: ± 5 IU/dL; for FVIII activity ≥ 25 IU/dL: ± 20% (relative). The different principles of OSA and CSA led to discrepancies in the estimation of all analysed FVIII products. Additionally, in vitro recovery was increased at lower levels of FVIII activity using the OSA, whereas recovery was more consistent using the CSA. CONCLUSION: These data allow the interpretation of FVIII activity results for different FVIII products using the Roche FVIII OSA on the cobas t 511/711 analysers.

Association between body mass index and macronutrients differs along the body mass index range of German adults: results from the German National Nutrition Survey II.

The aim of the present study was to determine whether the association between body mass index (BMI) and the intake of macronutrients varies along the BMI distribution of German adults. Based on a sample of 9214 men and women aged 18-80 years from the representative cross-sectional German National Nutrition Survey (NVS) II, quantile regression was used to investigate the association between BMI and the intake of macronutrients independent of energy intake and other predictors. In both sexes, BMI was positively associated with the intake of total protein and animal protein over its entire range and negatively associated with vegetable protein. A negative association between BMI and the intake of polysaccharides was found along the entire range of BMI in men. There was a weak negative association between BMI and the intake of total fat and saturated fatty acids observed in normal-weight-range women only. In conclusion, the association between BMI and the intake of macronutrients varies along the BMI range. Animal protein intake is positively associated with BMI independent of energy intake in both sexes whereas only in men an inverse association of polysaccharide intake with BMI was shown.

Types of Nutrition Knowledge, Their Socio-Demographic Determinants and Their Association With Food Consumption: Results of the NEMONIT Study.

Objectives: To investigate nutrition knowledge in the German population, its determinants and its association with food consumption. Methods: Data were obtained from the NEMONIT study (2014/15, n = 1,505, participants' age: 22-80 years). Nutrition knowledge was measured using the consumer nutrition knowledge scale (CoNKS) in a computer-assisted telephone interview. Two 24-h recalls were conducted to assess food consumption, which was evaluated using the Healthy Eating Index-NVS II. Results: Areas for knowledge enhancement were the understanding of health benefits of fruit and vegetable consumption, the concept of a balanced diet and saturated fatty acids. Nutrition knowledge was higher among females, younger and high socio-economic status participants. Correlations between nutrition knowledge and a favorable diet were significant but low. Analyses of types of nutrition knowledge yielded similar results for procedural knowledge and knowledge on nutrients but not for knowledge on calories. Conclusions: Areas for knowledge enhancement were identified, but an increase in nutrition knowledge alone seems unlikely to result in large improvements of dietary behavior.

Centrosomes in mitotic spindle assembly and orientation.

The centrosome is present in most animal cells and functions as the major microtubule-organizing center to ensure faithful chromosome segregation during cell division. As cells transition from interphase to mitosis, the duplicated centrosomes separate and move to opposite sides of the cell where the spindle assembles. Centrosomes not only nucleate but also organize microtubules of the mitotic spindle. The mitotic spindle is anchored to the cell cortex by the astral microtubules emanating from the centrosomes. Proper orientation of the mitotic spindle is essential for correct cell division. Centrosome-localized polo-like kinase Plk1 has been linked to regulation of proper spindle orientation. A number of proteins including MISP and NuMA have been implicated in the Plk1-mediated spindle orientation pathway.

Glyphosate and AMPA levels in human urine samples and their correlation with food consumption: results of the cross-sectional KarMeN study in Germany.

Glyphosate (N-[phosphonomethyl]-glycine) is the most widely used herbicide worldwide. Due to health concerns about glyphosate exposure, its continued use is controversially discussed. Biomonitoring is an important tool in safety evaluation and this study aimed to determine exposure to glyphosate and its metabolite AMPA, in association with food consumption data, in participants of the cross-sectional KarMeN study (Germany). Glyphosate and AMPA levels were measured in 24-h urine samples from study participants (n = 301). For safety evaluation, the intake of glyphosate and AMPA was calculated based on urinary concentrations and checked against the EU acceptable daily intake (ADI) value for glyphosate. Urinary excretion of glyphosate and/or AMPA was correlated with food consumption data. 8.3% of the participants (n = 25) exhibited quantifiable concentrations (> 0.2 µg/L) of glyphosate and/or AMPA in their urine. In 66.5% of the samples, neither glyphosate (< 0.05 µg/L) nor AMPA (< 0.09 µg/L) was detected. The remaining subjects (n = 76) showed traces of glyphosate and/or AMPA. The calculated glyphosate and/or AMPA intake was far below the ADI of glyphosate. Significant, positive associations between urinary glyphosate excretion and consumption of pulses, or urinary AMPA excretion and mushroom intake were observed. Despite the widespread use of glyphosate, the exposure of the KarMeN population to glyphosate and AMPA was found to be very low. Based on the current risk assessment of glyphosate by EFSA, such exposure levels are not expected to pose any risk to human health. The detected associations with consuming certain foods are in line with reports on glyphosate and AMPA residues in food.

FBXO45-MYCBP2 regulates mitotic cell fate by targeting FBXW7 for degradation.

Cell fate decision upon prolonged mitotic arrest induced by microtubule-targeting agents depends on the activity of the tumor suppressor and F-box protein FBXW7. FBXW7 promotes mitotic cell death and prevents premature escape from mitosis through mitotic slippage. Mitotic slippage is a process that can cause chemoresistance and tumor relapse. Therefore, understanding the mechanisms that regulate the balance between mitotic cell death and mitotic slippage is an important task. Here we report that FBXW7 protein levels markedly decline during extended mitotic arrest. FBXO45 binds to a conserved acidic N-terminal motif of FBXW7 specifically under a prolonged delay in mitosis, leading to ubiquitylation and subsequent proteasomal degradation of FBXW7 by the FBXO45-MYCBP2 E3 ubiquitin ligase. Moreover, we find that FBXO45-MYCBP2 counteracts FBXW7 in that it promotes mitotic slippage and prevents cell death in mitosis. Targeting this interaction represents a promising strategy to prevent chemotherapy resistance.

Robust Markers of Coffee Consumption Identified Among the Volatile Organic Compounds in Human Urine.

SCOPE: The human volatilome has gained high interest for the discovery of potential biomarkers of diseases. However, knowledge about the diet as a crucial factor affecting the volatilome is scarce. Therefore, the search for disease biomarkers, as well as the potential use of volatiles as dietary markers is so far limited. The aim of this study is to investigate the association of the diet with the urinary volatilome with the special task to find potential markers of coffee consumption in 24 h urine samples from the Karlsruhe Metabolomics and Nutrition (KarMeN) study. METHODS AND RESULTS: Acidified urine samples are analyzed using an approach combining headspace solid phase microextraction (HS-SPME) sampling with untargeted GC×GC-MS. Overall, 138 reliably occurring volatiles are detected. To account for the unequally concentrated urine samples, results of six different commonly used normalization methods are compared. Statistical analysis evidences six potential markers of coffee consumption, the most promising being 3,4-dimethyl-2,5-furandione. A correlation analysis between the 24 h dietary recall data and the urinary volatilome reveals further promising associations. CONCLUSION: The human urinary volatilome is highly affected by the diet, enabling access to a high level of information about potential diet-related biomarkers. Therefore, it is a very promising source for further investigations on dietary markers.

Comparison of food consumption and nutrient intake assessed with three dietary assessment methods: results of the German National Nutrition Survey II.

PURPOSE: Comparison of food consumption, nutrient intake and underreporting of diet history interviews, 24-h recalls and weighed food records to gain further insight into specific strength and limitations of each method and to support the choice of the adequate dietary assessment method. METHODS: For 677 participants (14-80 years) of the German National Nutrition Survey II confidence intervals for food consumption and nutrient intake were calculated on basis of bootstrapping samples, Cohen's d for the relevance of differences, and intraclass correlation coefficients for the degree of agreement of dietary assessment methods. Low energy reporters were identified with Goldberg cut-offs. RESULTS: In 7 of 18 food groups diet history interviews showed higher consumption means than 24-h recalls and weighed food records. Especially mean values of food groups perceived as socially desirable, such as fruit and vegetables, were highest for diet history interviews. For "raw" and "cooked vegetables", the diet history interviews showed a mean consumption of 144 and 109 g/day in comparison with 68 and 70 g/day in 24-h recalls and 76 and 75 g/day in weighed food records, respectively. For "fruit", diet history interviews showed a mean consumption of 256 g/day in comparison with 164 g/day in 24-h recalls and 147 g/day in weighed food records. No major differences regarding underreporting of energy intake were found between dietary assessment methods. CONCLUSIONS: With regard to estimating food consumption and nutrient intake, 24-h recalls and weighed food records showed smaller differences and better agreement than pairwise comparisons with diet history interviews.

Prácticas Preventivas dentro de la Ventana de Infectividad dental por parte de madres lactantes, pacientes del Hospital Universitario Angel Larralde, en Carabobo, Venezuela / Práticas Preventivas dentro da Janela da Infectividade Dentária por parte das mães lactentes, pacientes do Hospital Universitário Angel Larralde, em Carabobo, Venezuela / Preventive Practices within the Window of Dental Infectivity by breastfeeding mothers, patients of Angel Larralde University Hospital, in Carabobo, Venezuela

Objetivo: El objetivo de investigación es identificar mediante la educación para la salud el nivel de conocimiento de las madres de las madres referente a la ventana de infectividad bucal en el binomio madre-hijo y a las prácticas preventivas. Materiales y Métodos: La muestra fue tomada durante la consulta de obstetricia del Hospital Universitario Ángel Larralde, estado Carabobo en el período Septiembre-Diciembre del año 2017. Previamente, las madres habían recibido instrucción referente a técnicas de higiene personal y bucal por parte de los residentes del Postgrado de Odontopediatría de la Universidad de Carabobo. El enfoque de la investigación es cualitativo, modalidad de campo de tipo descriptiva, diseño no experimental transversal. La muestra fue de treinta (30) madres. Los datos se Originalrecolectaron por medio de la técnica de la encuesta usando como un cuestionario contentivo de veinticinco (25) preguntas, sobre las dimensiones contacto directo, lactancia materna e higiene personal. Los datos obtenidos fueron organizados y presentados en tablas. Resultados: Se obtuvo un nivel de conocimiento de la transmisibilidad bacteriana por contacto directo regular. El nivel de conocimiento de las prácticas preventivas durante la lactancia materna fue deficiente, a diferencia del conocimiento referente a las medidas de higiene personal que fue aceptable. Conclusión: Se concluyó que los indicadores contacto directo y lactancia materna deben ser reforzadas.

Objetivo: O objetivo da pesquisa é identificar, por meio da educação em saúde, o nível de conhecimento das mães em relação à janela de infectividade bucal no binômio mãe-filho e práticas preventivas. Materiais e Métodos: A amostra foi retirada durante a consulta de obstetrícia do Hospital Universitário Ángel Larralde, no estado de Carabobo, no período de setembro a dezembro de 2017. Anteriormente, as mães recebiam instruções sobre técnicas de higiene pessoal e bucal por parte das crianças. Residentes do Curso de Pós-Graduação em Odontopediatria da Universidade de Carabobo. O foco da pesquisa foi qualitativo, modalidade de campo do tipo descritiva, delineamento transversal não experimental. A amostra foi de trinta (30) mães. Os dados foram coletados por meio da técnica de pesquisa, utilizando-se um questionário contendo vinte e cinco (25) questões, sobre as dimensões contato direto, amamentação e higiene pessoal. Os dados obtidos foram organizados e apresentados em tabelas. Um nível de conhecimento da transmissibilidade bacteriana foi obtido por contato direto regular. O nível de conhecimento das práticas preventivas durante a amamentação foi deficiente, diferentemente do conhecimento sobre medidas de higiene pessoal aceitável. Concluiu-se que os indicadores contato direto e amamentação devem ser reforçados.

Purpose: The objective of the research is to identify, through health education, the level of knowledge of mothers regarding the window of oral infectivity in the mother-child binomial and preventive practices. Materials and Methods: The sample was taken during the obstetrics consultation of the Ángel Larralde University Hospital, Carabobo state, in the period September-December of the year 2017. Previously, the mothers had received instruction regarding techniques of personal and oral hygiene on the part of the children. Residents of the Postgraduate Course in Pediatric Dentistry of the University of Carabobo. The focus of the research is qualitative, field modality of descriptive type, transversal non-experimental design. The sample was thirty (30) mothers. The data was collected by means of the survey technique using as a questionnaire containing twenty-five (25) questions, about the dimensions direct contact, breastfeeding and personal hygiene. The data obtained were organized and presented in tables. A level of knowledge of bacterial transmissibility was obtained by regular direct contact. The level of knowledge of preventive practices during breastfeeding was deficient, unlike the knowledge regarding personal hygiene measures that was acceptable. It was concluded that the indicators direct contact and breastfeeding should be reinforced.

The complex human urinary sugar profile: determinants revealed in the cross-sectional KarMeN study.

Background: Although sugars and sugar derivatives are an important class of metabolites involved in many physiologic processes, there is limited knowledge on their occurrence and pattern in biofluids. Objective: Our aim was to obtain a comprehensive urinary sugar profile of healthy participants and to demonstrate the wide applicability and usefulness of this sugar profiling approach for nutritional as well as clinical studies. Design: In the cross-sectional KarMeN study, the 24-h urine samples of 301 healthy participants on an unrestricted diet, assessed via a 24-h recall, were analyzed by a newly developed semitargeted gas chromatography-mass spectrometry (GC-MS) profiling method that enables the detection of known and unknown sugar compounds. Statistical analyses were performed with respect to associations of sex and diet with the urinary sugar profile. Results: In total, 40 known and 15 unknown sugar compounds were detected in human urine, ranging from mono- and disaccharides, polyols, and sugar acids to currently unknown sugar-like compounds. A number of rarely analyzed sugars were found in urine samples. Maltose was found in statistically higher concentrations in the urine of women compared with men and was also associated with menopausal status. Further, a number of individual sugar compounds associated with the consumption of specific foods, such as avocado, or food groups, such as alcoholic beverages and dairy products, were identified. Conclusions: We here provide data on the complex nature of the sugar profile in human urine, of which some compounds may have the potential to serve as dietary markers or early disease biomarkers. Thus, comprehensive urinary sugar profiling not only has the potential to increase our knowledge of host sugar metabolism, but can also reveal new dietary markers after consumption of individual food items, and may lead to the identification of early disease biomarkers in the future. The KarMeN study was registered at drks.de as DRKS00004890.

Ccdc61 controls centrosomal localization of Cep170 and is required for spindle assembly and symmetry.

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.

MISP regulates the IQGAP1/Cdc42 complex to collectively orchestrate spindle orientation and mitotic progression.

Precise mitotic spindle orientation is essential for both cell fate and tissue organization while defects in this process are associated with tumorigenesis and other diseases. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. The actin-binding protein MISP controls spindle orientation and mitotic progression in human cells. However, the exact underlying mechanism remains to be elucidated. Here we report that MISP interacts with the multidomain scaffolding protein IQGAP1. We further show that MISP binds to the active form of Cdc42 through IQGAP1. Depletion of MISP promotes increased accumulation of IQGAP1 at the cell cortex and a decrease in its Cdc42-binding capacity leading to reduced active Cdc42 levels. Interestingly, overexpression of IQGAP1 can rescue mitotic defects caused by MISP downregulation including spindle misorientation, loss of astral microtubules and prolonged mitosis and also restores active Cdc42 levels. Importantly, we find that IQGAP1 acts downsteam of MISP in regulating astral microtubule dynamics and the localization of the dynactin subunit p150glued that is crucial for proper spindle positioning. We propose that MISP regulates IQGAP1 and Cdc42 to ensure proper mitotic progression and correct spindle orientation.

Activated ezrin controls MISP levels to ensure correct NuMA polarization and spindle orientation.

Correct spindle orientation is achieved through signaling pathways that provide a molecular link between the cell cortex and spindle microtubules in an F-actin-dependent manner. A conserved cortical protein complex, composed of LGN (also known as GPSM2), NuMA (also known as NUMA1) and dynein-dynactin, plays a key role in establishing proper spindle orientation. It has also been shown that the actin-binding protein MISP and the ERM family, which are activated by lymphocyte-oriented kinase (LOK, also known as STK10) and Ste20-like kinase (SLK) (hereafter, SLK/LOK) in mitosis, regulate spindle orientation. Here, we report that MISP functions downstream of the ERM family member ezrin and upstream of NuMA to allow optimal spindle positioning. We show that MISP directly interacts with ezrin and that SLK/LOK-activated ezrin ensures appropriate cortical MISP levels in mitosis by competing with MISP for actin-binding sites at the cell cortex. Furthermore, we found that regulation of the correct cortical MISP levels, by preventing its excessive accumulation, is essential for crescent-like polarized NuMA localization at the cortex and, as a consequence, leads to highly dynamic astral microtubules. Our results uncover how appropriate MISP levels at the cortex are required for proper NuMA polarization and, therefore, an optimal placement of the mitotic spindle within the cell.This article has an associated First Person interview with the first author of the paper.