Citron kinase-dependent F-actin maintenance at midbody secondary ingression sites mediates abscission.

Abscission is the final step of cytokinesis whereby the intercellular bridge (ICB) linking the two daughter cells is cut. The ICB contains a structure called the midbody, required for the recruitment and organization of the abscission machinery. Final midbody severing is mediated by formation of secondary midbody ingression sites, where the ESCRT III component CHMP4B is recruited to mediate membrane fusion. It is presently unknown how cytoskeletal elements cooperate with CHMP4B to mediate abscission. Here, we show that F-actin is associated with midbody secondary sites and is necessary for abscission. F-actin localization at secondary sites depends on the activity of RhoA and on the abscission regulator citron kinase (CITK). CITK depletion accelerates loss of F-actin proteins at the midbody and subsequent cytokinesis defects are reversed by restoring actin polymerization. Conversely, midbody hyperstabilization produced by overexpression of CITK and ANLN is reversed by actin depolymerization. CITK is required for localization of F-actin and ANLN at the abscission sites, as well as for CHMP4B recruitment. These results indicate that control of actin dynamics downstream of CITK prepares the abscission site for the final cut.

ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules.

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT-stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral-MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins.

Assessing vertical jump developmental levels in childhood using a low-cost motion capture approach.

Understanding the developmental levels of fundamental movement skills has a critical role in the improvement of motor competence in childhood. In this respect, the use of Microsoft Kinect to assess vertical jumping skill and to predict developmental levels in 9- to 12-yr.-old children was evaluated. 41 boys and girls repeated the countermovement jump test three times. Vertical jumping skill levels were categorized using observational records, while kinematic and temporal parameters were estimated using a biomechanical model based on data acquired by the Kinect. Multivariate analysis of variance (MANOVA) and discriminant analysis verified that the height of the jump and the flight height predict the primary differences in jumping skill developmental levels, and the Kinect-based assessment discriminates these levels.

A Multidimensional Approach to Talent Identification in Youth Volleyball through Declarative Tactical Knowledge and Functional Fitness.

This study aimed to assess which multidimensional performance indexes were the best predictors of talent identification in volleyball. Fifty-five female players (age: 13.8 ± 1.81 years; mass: 55.12 ± 8.12 kg; height: 158.23 ± 7.62 cm) were clustered into two groups according to some physical characteristics (i.e., the first group included players with more favorable performance predictors). Musculoskeletal Fitness (MSF), Functional Motor Competence (FMC), and Declarative Tactical Knowledge (DTK) were measured as multidimensional indexes of performance. Moderate-to-large differences between groups were found for each index in favor of the first group. Regression analyses were performed to examine the variance explained by MSF, FMC, and DTK in the two groups. A model with FMC components explained slightly more variance in the group predictor variables (R2 = 0.53) than a model using only MSF components (R2 = 0.45). Among FMC components, the score of the Throw-and-Catch test resulted in the best predictor (Odds Ratio = 1.58) for determining group selection, followed by the score of the Supine-to-Stand-and-Go test (Odds Ratio = 0.02). An additional model composed by MSF and FMC significant predictors (i.e., functional fitness index) and DTK explained 63% of the variance (R2 = 0.63), and these were significant predictors of group membership (Odds Ratio = 6.32 and Odds Ratio = 1.51, respectively). A more comprehensive multidimensional analysis of youth performances is warranted to identify and monitor the best players in a youth volleyball context.

Effects of Different Teaching Approaches on Proxy Measures of Physical Fitness of Italian Kindergarten Children.

Developing physical fitness (PF) behaviors early in life enhances physical development and facilitates sustained participation in physical activity and sports across childhood. This study addressed the effect of different teaching approaches on precursors of PF in kindergarten children. A total of 178 children (5.45 ± 0.40 years, female = 92) from 11 classes were organized into three groups. Group 1 (structured activity + free play) and Group 2 (only free play) attended the same playground (PrimoSport0246) for one hour/week for 10 weeks. Group 3 (structured activity + free play in kindergarten) followed their standard physical education curriculum at school. PF tests (standing long jump, medicine ball throw, and 20 m running speed) were administered pre- and post-intervention. Factorial ANOVA was implemented using a percentage change in PF performance (PFC) as the dependent variable, and teaching approaches, gender, and age as factors. Group 1 demonstrated significant improvements in fitness performance compared with Groups 2 and 3. Moderate to large effect sizes (Cohen's d range: 0.68-1.40) were noted in both males and females. Six-year-old demonstrated the greatest improvement in composite PFC compared to Groups 2 and 3. A structured teaching plan, even with a limited dose of once per week, supported the development of PF attributes in kindergarteners.

Structural and functional differences between KRIT1A and KRIT1B isoforms: a framework for understanding CCM pathogenesis.

KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed. Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms. We found that the 15th exon encodes for the distal beta-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner. As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.

Inactivation of Citron Kinase Inhibits Medulloblastoma Progression by Inducing Apoptosis and Cell Senescence.

Medulloblastoma is the most common malignant brain tumor in children. Current treatment for medulloblastoma consists of surgery followed by irradiation of the whole neuraxis and high-dose multiagent chemotherapy, a partially effective strategy associated with highly invalidating side effects. Therefore, identification and validation of novel target molecules capable of contrasting medulloblastoma growth without disturbing brain development is needed. Citron kinase protein (CITK), encoded by primary microcephaly gene MCPH17, is required for normal proliferation and survival of neural progenitors. Constitutive loss of CITK leads to cytokinesis failure, chromosome instability, and apoptosis in the developing brain, but has limited effects on other tissues. On this basis, we hypothesized that CITK could be an effective target for medulloblastoma treatment. In medulloblastoma cell lines DAOY and ONS-76, CITK knockdown increased both cytokinesis failure and DNA damage, impairing proliferation and inducing cell senescence and apoptosis via TP53 or TP73. Similar effects were obtained in the NeuroD-SmoA1 transgenic mouse model, in which CITK deletion increased apoptotic cells and senescence markers such as P21CIP1, P27KIP1, and P16INK4A Most importantly, CITK deletion decreased tumor growth and increased overall survival in these mice, with no apparent side effects. These results suggest that CITK can be a useful molecular target for medulloblastoma treatment.Significance:In vitro and in vivo proof of concept identifies citron kinase protein as a suitable target for medulloblastoma treatment.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4599/F1.large.jpg Cancer Res; 78(16); 4599-612. ©2018 AACR.

Correction to: ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly.

The authors wish to point out that the name of the first author is appearing incorrectly on Pubmed: it should be El Ghouzzi V (and not Ghouzzi VE). In addition, the words "and p53" appear at the end of the title in the original publication ( https://www.nature.com/articles/cddis2016266 ) and in the previous erratum version ( https://www.nature.com/articles/cddis2016446 ). This is not correct.

Assessing Standing Long Jump Developmental Levels Using an Inertial Measurement Unit.

This study tested the feasibility of using an inertial measurement unit to discriminate different developmental levels of children performing the standing long jump (SLJ). Sixty-four children (54.6% boys, 46.4% girls) aged 8 to 11 years old ( M = 9.17, SD = 0.97) performed the SLJ three times and were classified by rater observations into three developmental levels. Concurrently, they wore an inertial measurement unit from which a set of temporal and kinematic parameters for the quantitative assessment of their jumps. A multivariate analysis of variance revealed that variability among the developmental groups was identified by the overall set of parameters, while discriminant function analysis verified that these parameters discriminated among developmental levels. Primary predictors for developmental group discrimination were maximum peak acceleration in the vertical and anteroposterior directions, respectively, and normalized preparation time. These outcomes represent significant steps toward improving the assessment of SLJ rate of development in childhood and supporting physical education.

RNAs competing for microRNAs mutually influence their fluctuations in a highly non-linear microRNA-dependent manner in single cells.

BACKGROUND: Distinct RNA species may compete for binding to microRNAs (miRNAs). This competition creates an indirect interaction between miRNA targets, which behave as miRNA sponges and eventually influence each other's expression levels. Theoretical predictions suggest that not only the mean expression levels of targets but also the fluctuations around the means are coupled through miRNAs. This may result in striking effects on a broad range of cellular processes, such as cell differentiation and proliferation. Although several studies have reported the functional relevance of this mechanism of interaction, detailed experiments are lacking that study this phenomenon in controlled conditions by mimicking a physiological range. RESULTS: We used an experimental design based on two bidirectional plasmids and flow cytometry measurements of cotransfected mammalian cells. We validated a stochastic gene interaction model that describes how mRNAs can influence each other's fluctuations in a miRNA-dependent manner in single cells. We show that miRNA-target correlations eventually lead to either bimodal cell population distributions with high and low target expression states, or correlated fluctuations across targets when the pool of unbound targets and miRNAs are in near-equimolar concentration. We found that there is an optimal range of conditions for the onset of cross-regulation, which is compatible with 10-1000 copies of targets per cell. CONCLUSIONS: Our results are summarized in a phase diagram for miRNA-mediated cross-regulation that links experimentally measured quantities and effective model parameters. This phase diagram can be applied to in vivo studies of RNAs that are in competition for miRNA binding.

Citron Kinase Deficiency Leads to Chromosomal Instability and TP53-Sensitive Microcephaly.

Mutations in citron (CIT), leading to loss or inactivation of the citron kinase protein (CITK), cause primary microcephaly in humans and rodents, associated with cytokinesis failure and apoptosis in neural progenitors. We show that CITK loss induces DNA damage accumulation and chromosomal instability in both mammals and Drosophila. CITK-deficient cells display "spontaneous" DNA damage, increased sensitivity to ionizing radiation, and defective recovery from radiation-induced DNA lesions. In CITK-deficient cells, DNA double-strand breaks increase independently of cytokinesis failure. Recruitment of RAD51 to DNA damage foci is compromised by CITK loss, and CITK physically interacts with RAD51, suggesting an involvement of CITK in homologous recombination. Consistent with this scenario, in doubly CitK and Trp53 mutant mice, neural progenitor cell death is dramatically reduced; moreover, clinical and neuroanatomical phenotypes are remarkably improved. Our results underscore a crucial role of CIT in the maintenance of genomic integrity during brain development.

Offensive strategies in the European Football Championship 2012.

The aim of this study was to identify the independent and interactive effects of possession strategy, pitch location, and game period on the offensive actions performed by the winning teams in the 2012 European Football Championship. The non-clinical magnitude-based inferences method was used to interpret the true effect of the performance indicators on the response variable. The offensive team possessions were grouped into winning (n = 2035) and losing (n = 2071). The winning teams performed offensive processes mainly using the possession play strategy (OR: 0.75, very likely negative effect of the direct play). When the analysis included the pitch location, negative interaction effect was found for the direct play, which ended up in the central path (OR: 0.70, very likely negative effect). On the contrary, the direct play in the second half of the match seemed to produce an effect on the probability of the winning teams performing offensive processes (OR: 1.59, most likely positive effect). The results of multivariate analyses showed that the offensive team possession profiles required a careful investigation because the possession strategy changed under the conjoint effect of pitch location and game period.

ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly.

Epidemiological evidence from the current outbreak of Zika virus (ZIKV) and recent studies in animal models indicate a strong causal link between ZIKV and microcephaly. ZIKV infection induces cell-cycle arrest and apoptosis in proliferating neural progenitors. However, the mechanisms leading to these phenotypes are still largely obscure. In this report, we explored the possible similarities between transcriptional responses induced by ZIKV in human neural progenitors and those elicited by three different genetic mutations leading to severe forms of microcephaly in mice. We found that the strongest similarity between all these conditions is the activation of common P53 downstream genes. In agreement with these observations, we report that ZIKV infection increases total P53 levels and nuclear accumulation, as well as P53 Ser15 phosphorylation, correlated with genotoxic stress and apoptosis induction. Interestingly, increased P53 activation and apoptosis are induced not only in cells expressing high levels of viral antigens but also in cells showing low or undetectable levels of the same proteins. These results indicate that P53 activation is an early and specific event in ZIKV-infected cells, which could result from cell-autonomous and/or non-cell-autonomous mechanisms. Moreover, we highlight a small group of P53 effector proteins that could act as critical mediators, not only in ZIKV-induced microcephaly but also in many genetic microcephaly syndromes.

The DCR protein TTC3 affects differentiation and Golgi compactness in neurons through specific actin-regulating pathways.

In neuronal cells, actin remodeling plays a well known role in neurite extension but is also deeply involved in the organization of intracellular structures, such as the Golgi apparatus. However, it is still not very clear which mechanisms may regulate actin dynamics at the different sites. In this report we show that high levels of the TTC3 protein, encoded by one of the genes of the Down Syndrome Critical Region (DCR), prevent neurite extension and disrupt Golgi compactness in differentiating primary neurons. These effects largely depend on the capability of TTC3 to promote actin polymerization through signaling pathways involving RhoA, ROCK, CIT-N and PIIa. However, the functional relationships between these molecules differ significantly if considering the TTC3 activity on neurite extension or on Golgi organization. Finally, our results reveal an unexpected stage-dependent requirement for F-actin in Golgi organization at different stages of neuronal differentiation.