Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C.

The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a kinetochore. CENP-C and CENP-N are CENP-A binding proteins. We previously demonstrated that vertebrate CENP-C binding to the CENP-A nucleosome is regulated by CDK1-mediated CENP-C phosphorylation. However, it is still unknown how the phosphorylation of CENP-C regulates its binding to CENP-A. It is also not completely understood how and whether CENP-C and CENP-N act together on the CENP-A nucleosome. Here, using cryo-electron microscopy (cryo-EM) in combination with biochemical approaches, we reveal a stable CENP-A nucleosome-binding mode of CENP-C through unique regions. The chicken CENP-C structure bound to the CENP-A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP-C residue. The stable CENP-A-CENP-C complex excludes CENP-N from the CENP-A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1-mediated CENP-C phosphorylation.

Two new competing pathways establish the threshold for cyclin-B-Cdk1 activation at the meiotic G2/M transition.

Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K-Akt pathway through the Gßγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B-Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B-Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gßγ-dependent pathway enhances PI3K-Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gßγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries.

High-gamma activity in an attention network predicts individual differences in elderly adults' behavioral performance.

The current study used a magnetoencephalogram to investigate the relationship between high-gamma (52-100 Hz) activity within an attention network and individual differences in behavioral performance among healthy elderly adults. We analyzed brain activity in 41 elderly subjects performing a 3-stimulus visual oddball task. In addition to the average amplitude of event-related fields in the left intraparietal sulcus (IPS), high-gamma power in the left middle frontal gyrus (MFG), the strength of high-gamma imaginary coherence between the right MFG and the left MFG, and those between the right MFG and the left thalamus predicted individual differences in reaction time. In addition, high-gamma power in the left MFG was correlated with task accuracy, whereas high-gamma power in the left thalamus and left IPS was correlated with individual processing speed. The direction of correlations indicated that higher high-gamma power or coherence in an attention network was associated with better task performance and, presumably, higher cognitive function. Thus, high-gamma activity in different regions of this attention network differentially contributed to attentional processing, and such activity could be a fundamental process associated with individual differences in cognitive aging.

Reliability of Spectral Features of Resting-State Brain Activity: A Magnetoencephalography Study.

Background Cognition is a vital sign and its deterioration is a major concern in clinical medicine. It is usually evaluated using neuropsychological assessments, which have innate limitations such as the practice effect. To compensate for these assessments, the oscillatory power of resting-state brain activity has recently become available. The power is obtained noninvasively using magnetoencephalography and is summarized by spectral parameters such as the median frequency (MF), individual alpha frequency (IAF), spectral edge frequency 95 (SEF95), and Shannon's spectral entropy (SSE). As these parameters are less sensitive to practice effects, they are suitable for longitudinal studies. However, their reliability remains unestablished, hindering their proactive use in clinical practice. Therefore, we aimed to quantify the within-participant reliability of these parameters using repeated measurements of healthy participants to facilitate their clinical use and to evaluate the observed changes/differences in these parameters reported in previous studies. Methodology Resting-state brain activity with eyes closed was recorded using magnetoencephalography for five minutes from 15 healthy individuals (29.3 ± 4.6 years old: ranging from 23 to 28 years old). The following four spectral parameters were calculated: MF, IAF, SEF95, and SSE. To quantify reliability, the minimal detectable change (MDC) and intraclass correlation coefficient (ICC) were computed for each parameter. In addition, we used MDCs to evaluate the changes and differences in the spectral parameters reported in previous longitudinal and cross-sectional studies. Results The MDC at 95% confidence interval (MDC95) of MF, IAF, SEF95, and SSE were 0.61 Hz, 0.44 Hz, 2.91 Hz, and 0.028, respectively. The ICCs of these parameters were 0.96, 0.92, 0.94, and 0.83, respectively. The MDC95 of these parameters was smaller than the mean difference in the parameters between cognitively healthy individuals and patients with dementia, as reported in previous studies. Conclusions The spectral parameter changes/differences observed in prior studies were not attributed to measurement errors but rather reflected genuine effects. Furthermore, all spectral parameters exhibited high ICCs (>0.8), underscoring their robust within-participant reliability. Our results support the clinical use of these parameters, especially in the longitudinal monitoring and evaluation of the outcomes of interventions.

A single starfish Aurora kinase performs the combined functions of Aurora-A and Aurora-B in human cells.

Aurora, an essential mitotic kinase, is highly conserved during evolution. Most vertebrates have at least two Aurora kinases, Aurora-A and Aurora-B, which have distinct functions in the centrosome-spindle and inner centromere-midbody, respectively. However, some non-vertebrate deuterostomes have only a single Aurora. It remains to be verified whether the single Aurora performs the same functions as vertebrate Auroras A and B combined. We have isolated a cDNA of a single Aurora (ApAurora) from the echinoderm starfish, Asterina pectinifera, and show that ApAurora displays most features of both Aurora-A and Aurora-B in starfish oocytes and early embryos. Furthermore, ApAurora that is stably expressed in HeLa cells can substitute for both human Aurora-A and Aurora-B when either is reduced by RNAi. A single ApAurora thus has properties of both Aurora-A and Aurora-B in starfish eggs and HeLa cells. Together with phylogenetic analysis indicating that ApAurora forms a clade with all types of vertebrate Auroras and single Auroras of non-vertebrate deuterostomes, our observations support the idea that the single Aurora found in non-vertebrate deuterostomes represents the ancestor that gave rise to various types of vertebrate Auroras. This study thus provides functional evidence for phylogenetic considerations.

Akt inhibits Myt1 in the signalling pathway that leads to meiotic G2/M-phase transition.

In eukaryotes, entry into M-phase of the cell cycle is induced by activation of cyclin B-Cdc2 kinase. At G2-phase, the activity of its inactivator, a member of the Wee1 family of protein kinases, exceeds that of its activator, Cdc25C phosphatase. However, at M-phase entry the situation is reversed, such that the activity of Cdc25C exceeds that of the Wee1 family. The mechanism of this reversal is unclear. Here we show that in oocytes from the starfish Asterina pectinifera, the kinase Akt (or protein kinase B (PKB)) phosphorylates and downregulates Myt1, a member of the Wee1 family. This switches the balance of regulator activities and causes the initial activation of cyclin B-Cdc2 at the meiotic G2/M-phase transition. These findings identify Myt1 as a new target of Akt, and demonstrate that Akt functions as an M-phase initiator.

The Starfish Asterina pectinifera: Collection and Maintenance of Adults and Rearing and Metamorphosis of Larvae.

Here we describe methods for (a) collecting starfish during their breeding period; (b) maintaining adults with fully grown gonads in laboratory aquaria; (c) rearing fertilized eggs to brachiolaria larvae, and (d) inducing larvae to metamorphose into juveniles under laboratory conditions. Such protocols should facilitate various analyses of starfish development throughout the entire life cycle of these model organisms.

CDK1-mediated CENP-C phosphorylation modulates CENP-A binding and mitotic kinetochore localization.

The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A-CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A-binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A-CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.

Multiple phosphorylations control recruitment of the KMN network onto kinetochores.

To establish a functional kinetochore, the constitutive centromere-associated network (CCAN) forms a foundation on the centromere and recruits the KMN network, which directly binds to spindle microtubules. The CENP-C and CENP-T pathways in the CCAN recruit the KMN network to kinetochores, independently. The CENP-C pathway has been considered the major scaffold for the KMN network in vertebrate CCAN. However, we demonstrate that it is mainly the CENP-T pathway that recruits the KMN network onto the kinetochores and that CENP-T-KMN interactions are essential in chicken DT40 cells. By contrast, less Ndc80 binds to the CENP-C pathway in mitosis and the Mis12-CENP-C association is decreased during mitotic progression, which is consistent with the finding that the Mis12 complex-CENP-C binding is dispensable for cell viability. Furthermore, we find that multiple phosphoregulations of CENP-T and the Mis12 complex make the CENP-T pathway dominant. These results provide key insights into kinetochore dynamics during mitotic progression.

Publisher Correction: Multiple phosphorylations control recruitment of the KMN network onto kinetochores.

In the version of this Article originally published, the 'ON' and 'OFF' labels in panel c of Fig. 6 were incorrect. For the Tet treated cells (+Tet) in both image panels, CENP-T should have been 'OFF' and CENP-T Δ90 should have been 'ON'. For the cells untreated with Tet (-Tet) in both graph panels, CENP-T Δ90 should have been 'ON'. This has now been amended.

Localization and dynamics of Cdc2-cyclin B during meiotic reinitiation in starfish oocytes.

The Cdc2-cyclin B kinase has a central role in regulating the onset of M phase. In starfish oocytes, Cdc2-cyclin B begins to be activated approximately 10 min after application of maturation hormone, followed by accumulation in the nucleus then nuclear envelope breakdown. By immunofluorescence and by expressing a green fluorescent (GFP) chimera of cyclin B, we find that cyclin B is present in aggregates in the cytoplasm of immature oocytes. The aggregates disperse at approximately 10 min, suggesting that the dispersal is closely related to the activation of the kinase. Using cyclin B-GFP, the dispersion begins from the region containing the centrosomes. Extractability of Cdc2-cyclin B changes with similar kinetics during maturation. Active Cdc25 phosphatase released Cdc2-cyclin B from the detergent-insoluble fraction independently of its phosphatase activity. Live cell imaging also showed that Cdc2-cyclin B begins to accumulate in the nucleus before changes in nuclear pore permeability, consistent with Cdc2-cyclin B-induced disassembly of the pores.

High frequency oscillations are less frequent but more specific to epileptogenicity during rapid eye movement sleep.

OBJECTIVE: We hypothesized that high frequency oscillations (HFOs) are differently suppressed during rapid eye movement sleep (REM) between epileptogenic and less epileptogenic cortices, and that the suppressive effect can serve as a specific marker of epileptogenicity. METHODS: Intracranial electroencephalography (EEG) was recorded in 13 patients with drug-resistant epilepsy. HFOs between 80 and 200Hz were semi-automatically detected from total 15-min EEG epochs each for REM and slow wave sleep (SWS). z-Score of HFO occurrence rate was calculated from the baseline rate derived from non-epileptogenic cortex. Intracranial electrodes were labeled as REM dominant HFO (RdH) if REM z-score was greater than SWS z-score or as SWS dominant HFO (SdH) if SWS z-score was greater than REM z-score. Relationship of electrode location to the area of surgical resection was compared between RdH and SdH electrodes. RESULTS: Out of 1070 electrodes, 101 were defined as RdH electrodes and 115 as SdH electrodes. RdH electrodes were associated with the area of resection in patients with postoperative seizure freedom (P<0.001), but not in patients without seizure freedom. CONCLUSIONS: HFOs near the epileptogenic zone are less suppressed during REM. SIGNIFICANCE: The less suppressive effect of REM may provide a specific marker of epileptogenicity.

MEG Frequency Analysis Depicts the Impaired Neurophysiological Condition of Ischemic Brain.

PURPOSE: Quantitative imaging of neuromagnetic fields based on automated region of interest (ROI) setting was analyzed to determine the characteristics of cerebral neural activity in ischemic areas. METHODS: Magnetoencephalography (MEG) was used to evaluate spontaneous neuromagnetic fields in the ischemic areas of 37 patients with unilateral internal carotid artery (ICA) occlusive disease. Voxel-based time-averaged intensity of slow waves was obtained in two frequency bands (0.3-4 Hz and 4-8 Hz) using standardized low-resolution brain electromagnetic tomography (sLORETA) modified for a quantifiable method (sLORETA-qm). ROIs were automatically applied to the anterior cerebral artery (ACA), anterior middle cerebral artery (MCAa), posterior middle cerebral artery (MCAp), and posterior cerebral artery (PCA) using statistical parametric mapping (SPM). Positron emission tomography with 15O-gas inhalation (15O-PET) was also performed to evaluate cerebral blood flow (CBF) and oxygen extraction fraction (OEF). Statistical analyses were performed using laterality index of MEG and 15O-PET in each ROI with respect to distribution and intensity. RESULTS: MEG revealed statistically significant laterality in affected MCA regions, including 4-8 Hz waves in MCAa, and 0.3-4 Hz and 4-8 Hz waves in MCAp (95% confidence interval: 0.020-0.190, 0.030-0.207, and 0.034-0.213), respectively. We found that 0.3-4 Hz waves in MCAp were highly correlated with CBF in MCAa and MCAp (r = 0.74, r = 0.68, respectively), whereas 4-8 Hz waves were moderately correlated with CBF in both the MCAa and MCAp (r = 0.60, r = 0.63, respectively). We also found that 4-8 Hz waves in MCAp were statistically significant for misery perfusion identified on 15O-PET (p<0.05). CONCLUSIONS: Quantitatively imaged spontaneous neuromagnetic fields using the automated ROI setting enabled clear depiction of cerebral ischemic areas. Frequency analysis may reveal unique neural activity that is distributed in the impaired vascular metabolic territory, in which the cerebral infarction has not yet been completed.

Sex Differences in Gamma Band Functional Connectivity Between the Frontal Lobe and Cortical Areas During an Auditory Oddball Task, as Revealed by Imaginary Coherence Assessment.

We studied sex-related differences in gamma oscillation during an auditory oddball task, using magnetoencephalography and electroencephalography assessment of imaginary coherence (IC). We obtained a statistical source map of event-related desynchronization (ERD) / event-related synchronization (ERS), and compared females and males regarding ERD / ERS. Based on the results, we chose respectively seed regions for IC determinations in low (30-50 Hz), mid (50-100 Hz) and high gamma (100-150 Hz) bands. In males, ERD was increased in the left posterior cingulate cortex (CGp) at 500 ms in the low gamma band, and in the right caudal anterior cingulate cortex (cACC) at 125 ms in the mid-gamma band. ERS was increased in the left rostral anterior cingulate cortex (rACC) at 375 ms in the high gamma band. We chose the CGp, cACC and rACC as seeds, and examined IC between the seed and certain target regions using the IC map. IC changes depended on the height of the gamma frequency and the time window in the gamma band. Although IC in the mid and high gamma bands did not show sex-specific differences, IC at 30-50 Hz in males was increased between the left rACC and the frontal, orbitofrontal, inferior temporal and fusiform target regions. Increased IC in males suggested that males may acomplish the task constructively, analysingly, emotionally, and by perfoming analysis, and that information processing was more complicated in the cortico-cortical circuit. On the other hand, females showed few differences in IC. Females planned the task with general attention and economical well-balanced processing, which was explained by the higher overall functional cortical connectivity. CGp, cACC and rACC were involved in sex differences in information processing and were likely related to differences in neuroanatomy, hormones and neurotransmitter systems.

High-gamma power changes after cognitive intervention: preliminary results from twenty-one senior adult subjects.

INTRODUCTION: Brain-imaging techniques have begun to be popular in evaluating the effectiveness of cognitive intervention training. Although gamma activities are rarely used as an index of training effects, they have several characteristics that suggest their potential suitability for this purpose. This pilot study examined whether cognitive training in elderly people affected the high-gamma activity associated with attentional processing and whether high-gamma power changes were related to changes in behavioral performance. METHODS: We analyzed (MEG) magnetoencephalography data obtained from 35 healthy elderly subjects (60-75 years old) who had participated in our previous intervention study in which the subjects were randomly assigned to one of the three types of intervention groups: Group V trained in a vehicle with a newly developed onboard cognitive training program, Group P trained with a similar program but on a personal computer, and Group C was trained to solve a crossword puzzle as an active control group. High-gamma (52-100 Hz) activity during a three-stimulus visual oddball task was measured before and after training. As a result of exclusion in the MEG data analysis stage, the final sample consisted of five subjects in Group V, nine subjects in Group P, and seven subjects in Group C. RESULTS: Results showed that high-gamma activities were differently altered between groups after cognitive intervention. In particular, members of Group V, who showed significant improvements in cognitive function after training, exhibited increased high-gamma power in the left middle frontal gyrus during top-down anticipatory target processing. High-gamma power changes in this region were also associated with changes in behavioral performance. CONCLUSIONS: Our preliminary results suggest the usefulness of high-gamma activities as an index of the effectiveness of cognitive training in elderly subjects.

Attentional control and interpretation of facial expression after oxytocin administration to typically developed male adults.

Deficits in attentional-inhibitory control have been reported to correlate to anger, hostility, and aggressive behavior; therefore, inhibitory control appears to play an important role in prosocial behavior. Moreover, recent studies have demonstrated that oxytocin (OT) exerts a prosocial effect (e.g., decreasing negative behaviors, such as aggression) on humans. However, it is unknown whether the positively valenced effect of OT on sociality is associated with enhanced attentional-inhibitory control. In the present study, we hypothesized that OT enhances attentional-inhibitory control and that the positively valenced effect of OT on social cognition is associated with enhanced attentional-inhibitory control. In a single-blind, placebo-controlled crossover trial, we tested this hypothesis using 20 healthy male volunteers. We considered a decrease in the hostility detection ratio, which reflects the positively valenced interpretation of other individuals' facial expressions, to be an index of the positively valenced effects of OT (we reused the results of our previously published study). As a measure of attentional-inhibitory control, we employed a modified version of the flanker task (i.e., a shorter conflict duration indicated higher inhibitory control). These results failed to demonstrate any significant behavioral effects of OT (i.e., neither a positively valenced effect on facial cognition nor an effect on attentional-inhibitory control). However, the enhancement of attentional-inhibitory control after OT administration significantly correlated to the positively valenced effects on the interpretation of uncertain facial cognition (i.e., neutral and ambiguous facial expressions).

Dependence of removal of sperm-specific proteins from Xenopus sperm nuclei on the phosphorylation state of nucleoplasmin.

In Xenopus laevis, nucleoplasmin from fully grown oocytes is not highly phosphorylated, but is more extensively phosphorylated during oocyte maturation to retain this state until mid-blastula transition. Incubation of demembranated sperm with nucleoplasmin from oocytes or mature eggs revealed that egg nucleoplasmin is twice as potent as oocyte nucleoplasmin in removing sperm-specific basic proteins from chromatin (protamine-removing activity: PRA). Dephosphorylation of egg nucleoplasmin by alkaline phosphatase induced a remarkable decline of PRA in nucleoplasmin. Treatment of oocyte nucleoplasmin with cdc2 protein kinase induced an increase of the extent of phosphorylation, but to a level lower than that exhibited by egg nucleoplasmin, suggesting the involvement of other unspecified kinase(s) in phosphorylating nucleoplasmin during oocyte maturation. Incubation of sperm with cdc2 kinase induced selective phosphorylation of sperm-specific basic proteins, accompanied by their enhanced removal from sperm chromatin upon exposure to high-salt solutions. These results suggest that removal of sperm-specific basic proteins from sperm chromatin in fertilized eggs is facilitated by phosphorylation of both nucleoplasmin and sperm-specific basic proteins.

Antibody inhibition of protein activity in starfish oocytes.

Antibodies are widely utilized in cell and molecule biology for immunoblots, immunostaining, immunoprecipitation, immunoaffinity purification, and immunoassay. Some antibodies can be used for in vivo inhibition experiments. These antibodies bind to their target molecules and neutralize their functions, providing functional information in the study of their biological role. Here, we describe our methods for obtaining inhibitory antibodies against desired proteins. We then describe in the starfish oocyte system how to inhibit a target protein, even in the nucleus, by injection of antibody into the cytoplasm, and how to evaluate antibody inhibition of cell cycle regulators in small numbers of oocytes.

Cyclin B-Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase-independent mechanism.

Entry into M phase is governed by cyclin B-Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B-Cdk1-dependent inhibition of the protein phosphatase 2A (PP2A)-B55, which antagonizes cyclin B-Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B-Cdk1 to phosphorylate and activate α-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B-Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. We show here that cyclin B-Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B-Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles.

Electro- and magneto-encephalographic spike source localization of small focal cortical dysplasia in the dorsal peri-rolandic region.

OBJECTIVE: Small focal cortical dysplasia (FCD) may be ambiguous or overlooked on magnetic resonance (MR) imaging. Source localization of EEG and magnetoencephalography (MEG) spikes was evaluated to confirm the diagnosis of small FCD. METHODS: This study included 6 epilepsy patients with a single small lesion on MR imaging suggesting FCD within a single gyrus among 181 consecutive epilepsy patients admitted to our epilepsy monitoring unit over 27 months. Stereotypical interictal spikes were detected on simultaneous EEG and MEG recordings and the onset-related source of averaged spikes was estimated. RESULTS: All 6 patients had unique clinical characteristics as follows: leg sensori-motor seizures in 5 patients and eye version in 1 patient; a small MR imaging lesion suggesting FCD in the dorsal peri-rolandic region, which had been overlooked until our evaluation; and both EEG and MEG dipoles were estimated adjacent to the MR imaging lesion. CONCLUSIONS: Source localization of EEG and MEG spikes can confirm the diagnosis of FCD based on a single small MR imaging lesion, which was overlooked by previous examination of MR images. SIGNIFICANCE: Examination of MR images should be based on spike source localization as well as seizure semiology to identify subtle MR imaging abnormalities.