A Force-Induced Directional Switch of a Molecular Motor Enables Parallel Microtubule Bundle Formation.

Microtubule-organizing centers (MTOCs) nucleate microtubules that can grow autonomously in any direction. To generate bundles of parallel microtubules originating from a single MTOC, the growth of multiple microtubules needs to coordinated, but the underlying mechanism is unknown. Here, we show that a conserved two-component system consisting of the plus-end tracker EB1 and the minus-end-directed molecular motor Kinesin-14 is sufficient to promote parallel microtubule growth. The underlying mechanism relies on the ability of Kinesin-14 to guide growing plus ends along existing microtubules. The generality of this finding is supported by yeast, Drosophila, and human EB1/Kinesin-14 pairs. We demonstrate that plus-end guiding involves a directional switch of the motor due to a force applied via a growing microtubule end. The described mechanism can account for the generation of parallel microtubule networks required for a broad range of cellular functions such as spindle assembly or cell polarization.

Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore.

Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer kinetochore, assembling into 3 MDa-sized microtubule-embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi-orientation. We show that Dam1c and the general microtubule plus end-associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c-Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c-Bim1 binding by relieving an intramolecular inhibition of the Dam1 C-terminus. In addition, Bim1 recruits Bik1/CLIP-170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end-on attachments are formed during the process of attachment error correction.

Auto-inhibition of Mif2/CENP-C ensures centromere-dependent kinetochore assembly in budding yeast.

Kinetochores are chromatin-bound multi-protein complexes that allow high-fidelity chromosome segregation during mitosis and meiosis. Kinetochore assembly is exclusively initiated at chromatin containing Cse4/CENP-A nucleosomes. The molecular mechanisms ensuring that subcomplexes assemble efficiently into kinetochores only at centromeres, but not anywhere else, are incompletely understood. Here, we combine biochemical and genetic experiments to demonstrate that auto-inhibition of the conserved kinetochore subunit Mif2/CENP-C contributes to preventing unscheduled kinetochore assembly in budding yeast cells. We show that wild-type Mif2 is attenuated in its ability to bind a key downstream component in the assembly pathway, the Mtw1 complex, and that addition of Cse4 nucleosomes overcomes this inhibition. By exchanging the N-terminus of Mif2 with its functional counterpart from Ame1/CENP-U, we have created a Mif2 mutant which bypasses the Cse4 requirement for Mtw1 binding in vitro, thereby shortcutting kinetochore assembly. Expression of this Mif2 mutant in cells leads to mis-localization of the Mtw1 complex and causes pronounced chromosome segregation defects. We propose that auto-inhibition of Mif2/CENP-C constitutes a key concept underlying the molecular logic of kinetochore assembly.

Multivalent Molecular Tweezers Disrupt the Essential NDC80 Interaction with Microtubules.

Binding of microtubule filaments by the conserved Ndc80 protein is required for kinetochore-microtubule attachments in cells and the successful distribution of the genetic material during cell division. The reversible inhibition of microtubule binding is an important aspect of the physiological error correction process. Small molecule inhibitors of protein-protein interactions involving Ndc80 are therefore highly desirable, both for mechanistic studies of chromosome segregation and also for their potential therapeutic value. Here, we report on a novel strategy to develop rationally designed inhibitors of the Ndc80 Calponin-homology domain using Supramolecular Chemistry. With a multiple-click approach, lysine-specific molecular tweezers were assembled to form covalently fused dimers to pentamers with a different overall size and preorganization/stiffness. We identified two dimers and a trimer as efficient Ndc80 CH-domain binders and have shown that they disrupt the interaction between Ndc80 and microtubules at low micromolar concentrations without affecting microtubule dynamics. NMR spectroscopy allowed us to identify the biologically important lysine residues 160 and 204 as preferred tweezer interaction sites. Enhanced sampling molecular dynamics simulations provided a rationale for the binding mode of multivalent tweezers and the role of pre-organization and secondary interactions in targeting multiple lysine residues across a protein surface.

A blueprint for kinetochores - new insights into the molecular mechanics of cell division.

Kinetochores are large proteinaceous complexes that physically link centromeric DNA to the plus ends of spindle microtubules. Stable kinetochore-microtubule attachments are a prerequisite for the accurate and efficient distribution of genetic material over multiple generations. In the past decade, concerted research has resulted in the identification of the individual kinetochore building blocks, the characterization of critical microtubule-interacting components, such as the NDC80 complex, and the development of an approximate model of the architecture of this sophisticated biological machine.

Molecular basis for inner kinetochore configuration through RWD domain-peptide interactions.

Kinetochores are dynamic cellular structures that connect chromosomes to microtubules. They form from multi-protein assemblies that are evolutionarily conserved between yeasts and humans. One of these assemblies-COMA-consists of subunits Ame1CENP-U, Ctf19CENP-P, Mcm21CENP-O and Okp1CENP-Q A description of COMA molecular organization has so far been missing. We defined the subunit topology of COMA, bound with inner kinetochore proteins Nkp1 and Nkp2, from the yeast Kluyveromyces lactis, with nanoflow electrospray ionization mass spectrometry, and mapped intermolecular contacts with hydrogen-deuterium exchange coupled to mass spectrometry. Our data suggest that the essential Okp1 subunit is a multi-segmented nexus with distinct binding sites for Ame1, Nkp1-Nkp2 and Ctf19-Mcm21. Our crystal structure of the Ctf19-Mcm21 RWD domains bound with Okp1 shows the molecular contacts of this important inner kinetochore joint. The Ctf19-Mcm21 binding motif in Okp1 configures a branch of mitotic inner kinetochores, by tethering Ctf19-Mcm21 and Chl4CENP-N-Iml3CENP-L Absence of this motif results in dependence on the mitotic checkpoint for viability.

Sleep problems and worrying precede psychotic symptoms during an online intervention for psychosis.

OBJECTIVE: Experience sampling assessments (multiple assessments per day for approximately one week) indicate that positive symptoms fluctuate over time in psychosis. Precursors, such as sleep problems or worrying, predict these fluctuations. To date, it remains unclear whether the same precursors predict symptom variability also during treatment in an online intervention for psychosis, using assessments lying temporally further apart. METHODS: Participants completed brief intermediate online self-report assessments on their computers (up to every 7 days during a 2-month waiting period and up to twice every 6 days during a 2-month intervention period) within a randomized controlled trial. We monitored the course of paranoia, auditory verbal hallucinations, and their theory-driven precursors worrying, negative affect, self-esteem, self-reported cognitive biases, and quality of sleep in n = 124 participants (M = 10.32 assessments per participant; SD = 6.07). We tested group differences regarding the course of the composite of precursors, group differences regarding the effect of the composite on subsequent momentary psychotic symptoms, and the effect of each individual precursor on subsequent psychotic symptoms, using (lagged) linear mixed models. RESULTS: The course composite precursors over time and their lagged effect on subsequent momentary psychotic symptoms did not differ between groups. During the intervention, increased worrying and decreased quality of sleep preceded heightened momentary psychotic symptoms. CONCLUSION: The regression-based design does not allow drawing causal conclusions. However, worrying and sleep problems likely represent underlying mechanisms of psychotic symptom variability during online psychosis treatment, indicating that experience sampling findings from everyday life generalize to interventions with assessments lying several days apart. PRACTITIONER POINTS: Worrying and sleep problems represent important mechanisms of symptom fluctuations during an online intervention for people with psychosis. Our findings further support the notion that worrying and sleep problems are important treatment targets in psychological interventions for people with psychosis. Momentary levels of worrying and quality of sleep can signal subsequent fluctuations of psychotic symptom severity so practitioners should monitor these variables during treatment. Worrying seems to predict subsequent paranoia specifically during treatment whereas quality of sleep predicts both paranoia and auditory verbal hallucinations.

Unguided Internet-based cognitive-behavioral therapy for obsessive-compulsive disorder: A randomized controlled trial.

BACKGROUND: Many individuals with obsessive-compulsive disorder (OCD) do not receive professional treatment due to various idiosyncratic barriers. Internet-based cognitive-behavioral therapy (iCBT) is increasingly used to narrow treatment gaps, but the efficacy of such interventions without guidance of therapists has not been well studied. This study evaluated the efficacy of an unguided iCBT that includes third-wave approaches for the treatment of OCD symptoms. METHODS: A total of 128 individuals with self-reported OCD symptoms were randomly allocated to either an intervention group (unguided iCBT) or to a care-as-usual (CAU) control group following an anonymous baseline assessment via an online survey. Eight weeks after inclusion, a reassessment was carried out online. The Yale-Brown Obsessive-Compulsive Scale served as the primary outcome parameter for detecting symptom changes in the per-protocol sample with at least 60 minutes utilization. RESULTS: The iCBT group showed a significantly stronger reduction of OCD symptoms with a medium effect size (η²p = 0.06) compared with the control condition. This effect was moderated by the general frequency of Internet usage (η²p = 0.08); the more time per day users spent online, the less they benefited from the intervention. Secondary outcomes revealed (1) a medium effect size on self-esteem (η²p = 0.06); (2) no statistically significant effects on quality of life, depression symptoms, impulsivity, or social insecurity; and (3) good acceptability of the intervention. CONCLUSIONS: The current study provides evidence that unguided iCBT for OCD may be a viable option for individuals who experience treatment barriers. As non-compliance remains a challenge, this topic needs further research.

Brief Web-Based Intervention for Depression: Randomized Controlled Trial on Behavioral Activation.

BACKGROUND: Web-based interventions have been shown to be effective for the treatment of depression. However, interventions are often complex and include a variety of elements, making it difficult to identify the most effective component(s). OBJECTIVE: The aim of this pilot study was to shed light on mechanisms in the online treatment of depression by comparing a single-module, fully automated intervention for depression (internet-based behavioral activation [iBA]) to a nonoverlapping active control intervention and a nonactive control group. METHODS: We assessed 104 people with at least mild depressive symptoms (Patient Health Questionnaire-9, >4) via the internet at baseline (t0) and 2 weeks (t1) and 4 weeks (t2) later. After the t0 assessment, participants were randomly allocated to one of three groups: (1) iBA (n=37), (2) active control using a brief internet-based mindfulness intervention (iMBI, n=32), or (3) care as usual (CAU, n=35). The primary outcome was improvement in depressive symptoms, as measured using the Patient Health Questionnaire-9. Secondary parameters included changes in activity, dysfunctional attitudes, and quality of life. RESULTS: While groups did not differ regarding the change in depression from t0 to t1 (ηp2=.007, P=.746) or t0 to t2 (ηp2=.008, P=.735), iBA was associated with a larger decrease in dysfunctional attitudes from t0 to t2 in comparison to CAU (ηp2=.053, P=.04) and a larger increase in activity from t0 to t1 than the pooled control groups (ηp2=.060, P=.02). A change in depression from t0 to t2 was mediated by a change in activity from t0 to t1. At t1, 22% (6/27) of the participants in the iBA group and 12% (3/25) of the participants in the iMBI group indicated that they did not use the intervention. CONCLUSIONS: Although we did not find support for the short-term efficacy of the single-module iBA regarding depression, long-term effects are still conceivable, potentially initiated by changes in secondary outcomes. Future studies should use a longer intervention and follow-up interval. TRIAL REGISTRATION: DKRS (#DRKS00011562).

biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes.

Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular "mix and match" approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes.

Diminishing Effects After Recurrent Use of Self-Guided Internet-Based Interventions in Depression: Randomized Controlled Trial.

BACKGROUND: Self-guided internet-based interventions have several advantages over guided interventions and are generally effective in treating psychiatric symptoms. OBJECTIVE: We aimed to investigate whether the use of a new self-guided internet-based intervention (MOOD) would lead to a significant reduction in depressive symptoms compared with a care-as-usual (CAU) control group in a sample of individuals with depressive symptoms, most of whom had already used a different self-guided internet-based intervention in a previous trial. METHODS: A total of 125 individuals were randomized to the intervention condition (MOOD) and received access to the intervention for a period of six weeks or a CAU group. After six weeks, all participants were invited to take part in the post assessment. The Beck Depression Inventory-II served as the primary outcome. RESULTS: Both intention-to-treat as well as per-protocol analyses indicated that the depressive symptomatology decreased in both conditions but showed no advantage for those who had used MOOD. Subsequent moderation analyses suggested that those individuals who had less experience with psychotherapy benefitted to a greater extent compared with those with more experience. CONCLUSIONS: Self-guided internet-based interventions are deemed a suitable first-step approach to the treatment of depression. However, our results indicate that they are more efficacious in those with less psychotherapy experience. TRIAL REGISTRATION: ClinicalTrials.gov NCT03795480; http://clinicaltrials.gov/ct2/show/NCT03795480.

An internet-based intervention for people with psychosis (EviBaS): study protocol for a randomized controlled trial.

BACKGROUND: Evidence shows that internet-based self-help interventions are effective in reducing symptoms for a wide range of mental disorders. To date, online interventions treating psychotic disorders have been scarce, even though psychosis is among the most burdensome disorders worldwide. Furthermore, the implementation of cognitive-behavioral therapy (CBT) for psychosis in routine health care is challenging. Internet-based interventions could narrow this treatment gap. Thus, a comprehensive CBT-based online self-help intervention for people with psychosis has been developed. The aim of this study is the evaluation of the feasibility and efficacy of the intervention compared with a waiting list control group. METHODS: The intervention includes modules on delusion, voice hearing, social competence, mindfulness, and seven other domains. Participants are guided through the program by a personal moderator. Usage can be amended by an optional smartphone app. In this randomized controlled trial, participants are allocated to a waiting list or an intervention of eight weeks. Change in positive psychotic symptoms of both groups will be compared (primary outcome) and predictors of treatment effects will be assessed. DISCUSSION: To our knowledge, this project is one of the first large-scale investigations of an internet-based intervention for people with psychosis. It may thus be a further step to broaden treatment options for people suffering from this disorder. TRIAL REGISTRATION: NCT02974400 (clinicaltrials.gov), date of registration: November 28th 2016.

A structural basis for kinetochore recruitment of the Ndc80 complex via two distinct centromere receptors.

The Ndc80 complex is the key microtubule-binding element of the kinetochore. In contrast to the well-characterized interaction of Ndc80-Nuf2 heads with microtubules, little is known about how the Spc24-25 heterodimer connects to centromeric chromatin. Here, we present molecular details of Spc24-25 in complex with the histone-fold protein Cnn1/CENP-T illustrating how this connection ultimately links microtubules to chromosomes. The conserved Ndc80 receptor motif of Cnn1 is bound as an α helix in a hydrophobic cleft at the interface between Spc24 and Spc25. Point mutations that disrupt the Ndc80-Cnn1 interaction also abrogate binding to the Mtw1 complex and are lethal in yeast. We identify a Cnn1-related motif in the Dsn1 subunit of the Mtw1 complex, necessary for Ndc80 binding and essential for yeast growth. Replacing this region with the Cnn1 peptide restores viability demonstrating functionality of the Ndc80-binding module in different molecular contexts. Finally, phosphorylation of the Cnn1 N-terminus coordinates the binding of the two competing Ndc80 interaction partners. Together, our data provide structural insights into the modular binding mechanism of the Ndc80 complex to its centromere recruiters.

The customer is always right? Subjective target symptoms and treatment preferences in patients with psychosis.

Clinicians and patients differ concerning the goals of treatment. Eighty individuals with schizophrenia were assessed online about which symptoms they consider the most important for treatment, as well as their experience with different interventions. Treatment of affective and neuropsychological problems was judged as more important than treatment of positive symptoms (p < 0.005). While most individuals had experience with Occupational and Sports Therapy, only a minority had received Cognitive-Behavioral Therapy, Family Therapy, and Psychoeducation with family members before. Patients appraised Talk, Psychoanalytic, and Art Therapy as well as Metacognitive Training as the most helpful treatments. Clinicians should carefully take into consideration patients' preferences, as neglect of consumers' views may compromise outcome and adherence to treatment.

Expressive Suppression of Emotions and Overeating in Individuals with Overweight and Obesity.

Emotions have a considerable impact on eating behaviour; however, research addressing emotion regulation in obesity is rare. The present study is the first to investigate the association between emotional suppression and overeating in individuals with overweight. In total, 314 participants including 190 individuals with obesity filled in a cross-sectional online survey, which assessed emotional suppression, eating behaviour and psychopathology. A hierarchical linear regression analysis was conducted to identify factors associated with overeating. Individuals with obesity reported more frequent overeating compared with individuals without obesity. The habitual use of emotional suppression was associated with more overeating; however, this link was moderated by increased body mass index (BMI). The results suggest that suppression of emotional expression contributes to overeating and is maladaptive especially in individuals with obesity. Further research should longitudinally investigate the predictive value of emotional suppression on overweight, as the training of emotion regulation could contribute to treating obesity. Copyright © 2016 John Wiley & Sons, Ltd and Eating Disorders Association.

"Uno, nessuno e centomila": the different faces of the budding yeast kinetochore.

"One, no one and one hundred thousand" is a masterpiece of Italian literature, written by Luigi Pirandello. The central theme is that in each individual there are multiple personalities, since one's perception of one's self differs from the view of others. As a consequence, a unique identity does not exist, but rather one hundred thousand. This concept can be very well applied to the kinetochore, one of the largest macromolecular complexes conserved in eukaryotes. The kinetochore is essential during cell division and fulfills different sophisticated functions, including linking chromosomes to spindle microtubules and delaying anaphase onset in case of incorrect bi-orientation. In order to perform these tasks, the kinetochore shapes its structure by recruiting different subunits, such as the components of the spindle assembly checkpoint (SAC) or the monopolin complex during meiosis. It also modifies its internal organization by rearranging intramolecular connections and acquiring a distinct identity at different time points of cell division. In this review, we describe recent insights into the changes in composition and configuration of the kinetochore in mitosis and meiosis, focusing on the kinetochore of Saccharomyces cerevisiae.

Cognitive deficits are a matter of emotional context: inflexible strategy use mediates context-specific learning impairments in OCD.

The present study examines the interplay between cognitive deficits and emotional context in obsessive-compulsive disorder (OCD) and social phobia (SP). Specifically, this study examines whether the inflexible use of efficient learning strategies in an emotional context underlies impairments in probabilistic classification learning (PCL) in OCD, and whether PCL impairments are specific to OCD. Twenty-three participants with OCD, 30 participants with SP and 30 healthy controls completed a neutral and an OCD-specific PCL task. OCD participants failed to adopt efficient learning strategies and showed fewer beneficial strategy switches than controls only in an OCD-specific context, but not in a neutral context. Additionally, OCD participants did not show any explicit memory impairments. Number of beneficial strategy switches in the OCD-specific task mediated the difference in PCL performance between OCD and control participants. Individuals with SP were impaired in both PCL tasks. In contrast to neuropsychological models postulating general cognitive impairments in OCD, the present findings suggest that it is the interaction between cognition and emotion that is impaired in OCD. Specifically, activated disorder-specific fears may impair the flexible adoption of efficient learning strategies and compromise otherwise unimpaired PCL. Impairments in PCL are not specific to OCD.

Microtubule end-on attachment maturation regulates Mps1 association with its kinetochore receptor.

Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear. The conserved kinase Mps1 initiates SAC signaling by localizing transiently to kinetochores in prometaphase and is released upon bi-orientation. Using biochemistry, structure predictions, and cellular assays, we shed light on this dynamic behavior in Saccharomyces cerevisiae. A conserved N-terminal segment of Mps1 binds the neck region of Ndc80:Nuf2, the main microtubule receptor of kinetochores. Mutational disruption of this interface, located at the backside of the paired CH domains and opposite the microtubule-binding site, prevents Mps1 localization, eliminates SAC signaling, and impairs growth. The same interface of Ndc80:Nuf2 binds the microtubule-associated Dam1 complex. We demonstrate that the error correction kinase Ipl1/Aurora B controls the competition between Dam1 and Mps1 for the same binding site. Thus, binding of the Dam1 complex to Ndc80:Nuf2 may release Mps1 from the kinetochore to promote anaphase onset.

Advancing the neuroscience of social emotions with social immersion.

Second-person neuroscience offers a framework for the study of social emotions, such as embarrassment and pride. However, we propose that an enduring mental representation of oneself in relation to others without a continuous direct social interaction is possible. We call this state "social immersion" and will explain its impact on the neuroscience of social emotions.

Elaborative encoding during REM dreaming as prospective emotion regulation.

Rapid eye movement (REM) dreaming results in "emotionally intelligent encoding," according to the target article. Building on this, we argue that elaborative encoding alters emotional processing of upcoming events and thereby functions as prospective emotion regulation. After elaborative encoding, future events are appraised differently and result in a redirected emotional response. Disturbed elaborative encoding might be relevant for emotional dysregulation in psychopathology.