Kinetochores form the fundamental link between chromosomal domains termed centromeres and spindle microtubules in all eukaryotes. This connection, provided by a large, multiprotein complex, is ...essential for precise chromosome segregation and thus ensures genetic stability. Here, we review recent insights into the composition and function of centromeric chromatin. Multiple approaches have converged to identify centromere-associated proteins from yeast to humans. Among them are newly characterized histone-fold family members that operate at the DNA–kinetochore interface and provide critical connections between chromosomes and microtubules. Together, these findings contribute to a unified view of how centromeric chromatin functions as a regulated scaffold for kinetochore assembly.
Dominance, hostility and autonomy are interpersonal phenomena that emerge from the complex dyadic interplay of two individuals reciprocally influencing each other. Assessing the complexity of ...interpersonal interactions usually involves its reduction, for example with self-report and observer-rated measures informed by the structural analysis of social behavior (SASB). In contrast, letting individuals generate a complex stream of interpersonal experience and behavior from moment to moment is an empirical approach not yet usual in interpersonal theory. In the present study, we developed and evaluated an interpersonal, generative paradigm that allows participants to interact nonverbally and spontaneously with a computer-controlled other player in real-time without the need for introspection or the capacity to verbalize potentially implicit interpersonal processes. In the game-like paradigm, participants use the keyboard to take over objects such as a handcar to move autonomously around and encounter interfering or freedom granting artificial other players. We expected that participants (1) experience the nonverbal so-called game mechanics of the paradigm as interpersonal in nature, (2) behave towards the other players in an interpersonally complementary way, and (3) are influenced by their own trait interpersonal expectations. During the paradigm, 40 participants appraised the majority of the game mechanics and computer-controlled other players as intended. Also, interpersonal traits affected the spontaneous behavior towards artificial characters. These findings corroborate the feasibility and validity of a generative assessment of interpersonal dynamics beyond self-reports and observer ratings. The paradigm paves the way for the empirical testing of formal, computational models of dyadic interaction.
•In a game-like, nonverbal paradigm, interpersonal experience and behavior emerged.•Interpersonal positions such as autonomy and dominance resulted from game dynamics.•Interpersonal traits affected the spontaneous behavior towards artificial characters.•The MOVES paradigm allows empirical testing of formal models of dyadic interaction.
The αβ-tubulin heterodimer, the building block of microtubules, is subject to a large number of post-translational modifications, comparable in diversity to the intensively studied histone ...modifications. Although these unusual modifications are conserved throughout evolution, their functions have remained almost completely elusive. Recently, however, important advances in the understanding of how tubulin modifications regulate function and organization have been made.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Kinesin-14s are conserved molecular motors required for high-fidelity chromosome segregation, but their specific contributions to spindle function have not been fully defined. Here, we show that key ...functions of budding yeast Kinesin-14 Cik1-Kar3 are accomplished in a complex with Bim1 (yeast EB1). Genetic complementation of mitotic phenotypes identifies a novel KLTF peptide motif in the Cik1 N-terminus. We show that this motif is one element of a tripartite binding interface required to form a high-affinity Bim1-Cik1-Kar3 complex. Lack of Bim1-binding by Cik1-Kar3 delays cells in mitosis and impairs microtubule bundle organization and dynamics. Conversely, constitutive targeting of Cik1-Kar3 to microtubule plus ends induces the formation of nuclear microtubule bundles. Cells lacking the Bim1-Cik1-Kar3 complex rely on the conserved microtubule bundler Ase1/PRC1 for metaphase spindle organization, and simultaneous loss of plus-end targeted Kar3 and Ase1 is lethal. Our results reveal the contributions of an EB1-Kinesin-14 complex for spindle formation as a prerequisite for efficient kinetochore clustering and bi-orientation.
Kinetochores must remain associated with microtubule ends, as they undergo rapid transitions between growth and shrinkage. The molecular basis for this essential activity that ensures correct ...chromosome segregation is unclear. In this study, we have used reconstitution of dynamic microtubules and total internal reflection fluorescence microscopy to define the functional relationship between two important budding yeast kinetochore complexes. We find that the Dam1 complex is an autonomous plus end-tracking complex. The Ndc80 complex, despite being structurally related to the general tip tracker EB1, fails to recognize growing ends efficiently. Dam1 oligomers are necessary and sufficient to recruit Ndc80 to dynamic microtubule ends, where both complexes remain continuously associated. The interaction occurs specifically in the presence of microtubules and is subject to regulation by Ipl1 phosphorylation. These findings can explain how the force harvested by Dam1 is transmitted to the rest of the kinetochore via the Ndc80 complex.
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 yeast kinetochore receptors Cnn1 and Dsn1 employ a related peptide motif to recruit the microtubule‐binding Ndc80 complex in a competitive, phosphorylation‐regulated manner.
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.
EB1 (end binding 1) proteins have emerged as central regulators of microtubule (MT) plus ends in all eukaryotes, but molecular mechanisms controlling the activity of these proteins are poorly ...understood. In this study, we show that the budding yeast EB1 protein Bim1p is regulated by Aurora B/Ipl1p-mediated multisite phosphorylation. Bim1p forms a stable complex with Ipl1p and is phosphorylated on a cluster of six Ser residues in the flexible linker connecting the calponin homology (CH) and EB1 domains. Using reconstitution of plus end tracking in vitro and total internal reflection fluorescence microscopy, we show that dimerization of Bim1p and the presence of the linker domain are both required for efficient tip tracking and that linker phosphorylation removes Bim1p from static and dynamic MTs. Bim1 phosphorylation occurs during anaphase in vivo, and it is required for normal spindle elongation kinetics and an efficient disassembly of the spindle midzone. Our results define a mechanism for the use and regulation of CH domains in an EB1 protein.
Abstract
The majority of individuals with problematic and pathological gambling remain untreated, and treatment barriers are high. Internet-based interventions can help to address existing barriers, ...and first studies suggest their potential for this target group. Within a randomized controlled trial (
N
= 150) with two assessment times (baseline and post-intervention), we aimed to investigate the feasibility, acceptance, and effectiveness of a self-guided Internet-based intervention targeted at gambling problems. We expected a significant reduction in gambling symptoms (primary outcome) and depressive symptoms as well gambling-specific dysfunctional thoughts (secondary outcomes) in the intervention group (IG) compared to a wait-list control group with access to treatment-as-usual (control group, CG) after the intervention period of 8 weeks. Results of the complete cases, per protocol, intention-to-treat (ITT), and frequent user analyses showed significant improvements in both groups for primary and secondary outcomes but no significant between-group differences (ITT primary outcome,
F
(1,147) = .11,
p
= .739, ηp2 < .001). Moderation analyses indicated that individuals in the IG with higher gambling and depressive symptoms, older age, and comorbid anxiety symptoms showed significant improvement relative to the CG. The intervention was positively evaluated (e.g., 96.5% rated the program as useful). Possible reasons for the nonsignificant between-group differences are discussed. Future studies should include follow-up assessments and larger samples to address limitations of the present study.
Trial Registration
ClinicalTrials.gov (NCT03372226),
http://clinicaltrials.gov/ct2/show/NCT03372226
, date of registration (13/12/2017).
Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro ...from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end-tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrations used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. Our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.
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