Different degrees of lever arm rotation control myosin step size

Myosins are actin-based motors that are generally believed to move by amplifying small structural changes in the core motor domain via a lever arm rotation of the light chain binding domain. However, the lack of a quantitative agreement between observed step sizes and the length of the proposed lever arms from different myosins challenges this view. We analyzed the step size of rat myosin 1d (Myo1d) and surprisingly found that this myosin takes unexpectedly large steps in comparison to other myosins. Engineering the length of the light chain binding domain of rat Myo1d resulted in a linear increase of step size in relation to the putative lever arm length, indicative of a lever arm rotation of the light chain binding domain. The extrapolated pivoting point resided in the same region of the rat Myo1d head domain as in conventional myosins. Therefore, rat Myo1d achieves its larger working stroke by a large calculated ∼90° rotation of the light chain binding domain. These results demonstrate that differences in myosin step sizes are not only controlled by lever arm length, but also by substantial differences in the degree of lever arm rotation

Predicting the Fission Yeast Protein Interaction Network

A systems-level understanding of biological processes and information flow requires the mapping of cellular component interactions, among which protein–protein interactions are particularly important. Fission yeast (Schizosaccharomyces pombe) is a valuable model organism for which no systematic protein-interaction data are available. We exploited gene and protein properties, global genome regulation datasets, and conservation of interactions between budding and fission yeast to predict fission yeast protein interactions in silico. We have extensively tested our method in three ways: first, by predicting with 70–80% accuracy a selected high-confidence test set; second, by recapitulating interactions between members of the well-characterized SAGA co-activator complex; and third, by verifying predicted interactions of the Cbf11 transcription factor using mass spectrometry of TAP-purified protein complexes. Given the importance of the pathway in cell physiology and human disease, we explore the predicted sub-networks centered on the Tor1/2 kinases. Moreover, we predict the histidine kinases Mak1/2/3 to be vital hubs in the fission yeast stress response network, and we suggest interactors of argonaute 1, the principal component of the siRNA-mediated gene silencing pathway, lost in budding yeast but preserved in S. pombe. Of the new high-quality interactions that were discovered after we started this work, 73% were found in our predictions. Even though any predicted interactome is imperfect, the protein network presented here can provide a valuable basis to explore biological processes and to guide wet-lab experiments in fission yeast and beyond. Our predicted protein interactions are freely available through PInt, an online resource on our website (www.bahlerlab.info/PInt)

Critical importance of DNA binding for CSL protein functions in fission yeast

CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) proteins are conserved transcription factors found in animals and fungi. In fission yeast, they regulate various cellular processes, including cell cycle progression, lipid metabolism, and cell adhesion. CSL proteins bind to DNA through their N-terminal Rel-like domain and central beta-trefoil domain. Here, we investigated the importance of DNA binding for CSL functions in the fission yeast Schizosaccharomyces pombe. We created CSL mutants with disrupted DNA binding and found that the vast majority of CSL functions depend on intact DNA binding. Specifically, DNA binding is crucial for the regulation of cell adhesion, lipid metabolism, cell cycle progression, long non-coding RNA expression, and genome integrity maintenance. Interestingly, perturbed lipid metabolism leads to chromatin structure changes, potentially linking lipid metabolism to the diverse CSL-associated phenotypes. Our study highlights the critical role of DNA binding for CSL protein functions in fission yeast

Eingriffe und Behandlungen von Nutz­tieren durch Nicht-Tierärzte: quo vadis?

English Interventions and treatments of farm animals by non-veterinarians: quo vadis? The increasing professionalization and the cost pressure on farms combined with a decreasing availability of veterinary expertise procures the demand for treatments and interventions performed by non-veterinarians. A current survey resulting in 56 responses of food animal practitioners showed that a majority delivered pour-on anthelmintics, drugs for oral application and ointments after respective instruction to farmers. Udder injectors, antimicrobials and vaccines were handled more restrictively, but even hormones were committed by more than half of the veterinarians to livestock owners. In respect to interventions, most practitioners supported that farmers perform functional foot trimming, administration of boli, inseminations, dehorning and castrations of calves. By contrast, many veterinarians refused that farmers apply intravenous injections, insert a trocar or treat sole ulcers. In respect to the future development, implications of non-veterinarian interventions on animal welfare, consumer protection and drug safety have to be considered. Indispensable are qualification and skills of the person performing interventions which can be acquired by certificated training courses. Moreover, a basic conception and taking responsibility in particular for application of veterinary drugs is mandatory – in this respect, at present marked deficits exist among many farmers. Based on the current legal situation, several interventions can be delegated to non-veterinarians, but the technical responsibility for the proper implementation is due to the veterinarian. This includes an evaluation of interventions and treatments on a regular basis together with the farmer. On this basis, the assessment of clinical symptoms as well as the initial treatment can be useful and appropriate to ensure a rapid therapy especially in animals suffering from factorial diseases. Having in mind profound differences in respect to the competence of non-veterinarians, general procedures to deliver drugs and to delegate interventions have to be rejected. Advanced diagnostic procedures and the prescription of veterinary drugs remain an exclusive mission of the veterinarian. Keywords: Animal welfare, consumer protection, drug safety, interventions by non-veterinarians, livestock production Deutsch Eingriffe und Behandlungen von Nutz­tieren durch Nicht-Tierärzte: quo vadis? Die fortschreitende Professionalisierung und der Kostendruck auf landwirtschaftlichen Betrieben führen in Verbindung mit einer zumindest regional abnehmenden Verfügbarkeit tierärztlicher Expertise zu der Forderung, dass Behandlungen und Eingriffe bei Nutztieren vermehrt auch durch Nicht-Tierärzte* vorgenommen werden dürfen. Eine aktuelle Umfrage mit Rückmeldungen von 56 Nutztierpraxen ergab, dass die Mehrheit Pour-on-Anthelmintika, oral zu verabreichende Präparate und Wundsalben an Tierhalter abgibt. Mit Euterinjektoren, Antibiotika und Impfstoffen wird restriktiver umgegangen, doch selbst Hormone werden nach Einweisung von mehr als der Hälfte der Tierärzte an Landwirte abgegeben. Hinsichtlich von Eingriffen befürwortete die Mehrzahl der Praktiker die Durchführung der funktionellen Klauenpflege, die Eingabe von Boli, das Enthornen von Kälbern, das Kastrieren sowie die Besamung durch Nicht-Tierärzte. Hingegen sprachen sich die meisten Praktiker dafür aus, dass intravenöse Injektionen, das Einsetzen eines Trokars sowie das Ausschneiden eines Klauengeschwürs ausschliesslich durch Tierärzte durchgeführt werden. Im Hinblick auf die zukünftige Entwicklung gilt es, die Implikationen für Tierschutz, Konsumentenschutz und Arzneimittelsicherheit zu berücksichtigen. Unabdingbar sind stets die Fähigkeit und Fertigkeit des Durchführenden, die an den Nachweis der Sachkunde gebunden sind. Zudem sind ein fachliches Grundverständnis und die Übernahme von Verantwortung insbesondere im Hinblick auf den Einsatz von Tierarzneimitteln notwendig. Zwar kann der Bestandestierarzt aufgrund der aktuellen Gesetzeslage durchaus Massnahmen delegieren, übernimmt dann jedoch die fachliche Verantwortung für die Qualität der jeweiligen Interventionen. Zudem erfordert das Delegieren zwingend die regelmässige retrospektive Auswertung der Art und Anzahl von Behandlungen, sowie gemäss Tierarzneimittelverordnung die Überwachung des korrekten Tierarzneimitteleinsatzes. Unter dieser Prämisse kann die Befunderhebung in einem klar definierten Rahmen unter Einhaltung der gesetzlichen Vorgaben mit anschliessender Erstbehandlung durch den Landwirt in der Praxis insbesondere bei Faktorenkrankheiten sinnvoll sein, um die schnellstmögliche Versorgung erkrankter Tiere sicherzustellen. Angesichts der erheblichen Unterschiede bzgl. der fachlichen Kompetenz unter Nicht-Tierärzten sind pauschale Regelungen abzulehnen. Die weiterführende Diagnostik sowie die Verschreibung von Tierarzneimitteln müssen grundsätzlich eine exklusive Aufgabe des Bestandestierarztes bleiben. Schlüsselwörter: Arzneimittelsicherheit, Behandlung durch Laien, Konsumentenschutz, Nutztierhaltung, Tierschutz Français Interventions et traitements des animaux de rente par les non-vétérinaires: quo vadis? La professionnalisation progressive et la pression des coûts sur les exploitations agricoles, combinées à une disponibilité d’expertise vétérinaire décroissante au moins au niveau régional, font que de plus en plus de traitements et d’interventions sur le bétail peuvent être effectués par des non-vétérinaires. Une enquête récente portant sur les réponses de 56 cabinets pour animaux de rente a révélé que la plupart d’entre eux remettent des anthelminthiques pour-on, des médicaments à administrer par voie orale et des onguents aux propriétaires d’animaux. On est plus restrictif avec les injecteurs intra-mammaires, les antibiotiques et les vaccins, mais même des hormones sont remises aux agriculteurs après instruction par plus de la moitié des vétérinaires. En termes d’interventions, la majorité des praticiens soutiennent la réalisation de soins fonctionnels des onglons, l’administration de boli, l’écornage des veaux, la castration et l’insémination par des non-vétérinaires. En revanche, la plupart des praticiens considèrent que les injections intraveineuses, la mise en place d’un trocart et le traitement d’un ulcère de la sole doivent être effectués par des vétérinaires. En ce qui concerne l’évolution future, il est important de prendre en compte les implications pour le bien-être des animaux, la protection des consommateurs et la sécurité en matière de médicaments. Il est essentiel que l’exécutant ait la compétence nécessaire ainsi que la capacité de prouver qu’il possède une expertise. En outre, une compréhension de base du sujet et la prise de responsabilité, notamment en ce qui concerne l’utilisation de médicaments vétérinaires, sont nécessaires. Bien que le vétérinaire puisse déléguer des actes en raison de la situation juridique actuelle, il assume ensuite la responsabilité professionnelle de la qualité des interventions réalisées. En outre la délégation nécessite impérativement une évaluation rétrospective régulière du type et du nombre des traitements ainsi qu’un contrôle du bon usage des médicaments vétérinaires conformément à l’Ordonnance sur les médicaments vétérinaires. Sous cette prémisse, le recueil des commémoratifs dans un cadre clairement défini et conformément aux exigences légales avec un traitement initial par l’agriculteur peut être, dans la pratique, judicieux, en particulier dans les maladies factorielles pour assurer le traitement le plus rapide possible des animaux malades. Compte tenu des différences considérables en termes de compétence professionnelle parmi les non-vétérinaires, des réglementations forfaitaires devraient être rejetées. Les diagnostics plus approfondis et la prescription de médicaments vétérinaires doivent en principe rester une tâche exclusive du vétérinaire. Mots-clés: sécurité des médicaments, traitement par des profanes, protection des consommateurs, élevage, protection des animaux. Italiano Interventi e trattamenti eseguiti da non-veterinari sugli animali da reddito: quo vadis? La crescente professionalizzazione e la pressione sui costi nelle aziende agricole abbinate a una diminuzione, per ora regionale, di competenze veterinarie sfociano in una domanda di trattamenti e interventi veterinari sugli animali da reddito prodigati da non-veterinari. Un recente sondaggio con risposte ricevute da 56 prassi veterinarie per animali da reddito ha rilevato che la maggior parte degli intervistati procuravano ai detentori degli animali formulazioni pour-on di prodotti antielmintici, di preparati per via orale e di unguenti per le ferite. Un uso più restrittivo si trova negli iniettori della mammella, negli antibiotici e nei vaccini e anche gli ormoni vengono procurati agli allevatori, dietro istruzioni, da più della metà dei veterinari. Per quel che concerne gli interventi come la cura degli zoccoli, somministrazione di boli, inseminazioni, decornazioni e castrazioni di vitelli, la maggior parte dei professionisti si schiera dalla parte dei non-veterinari. Per contro molti veterinari hanno rifiutato che gli allevatori effettuino iniezioni endovenose, inserimenti di trocar e rimozioni di ulcere dello zoccolo. In previsione degli sviluppi futuri bisogna tener presente le implicazioni degli interventi prodigati da non-veterinari sulla protezione degli animali, sulla protezione dei consumatori e sulla sicurezza dei medicamenti. Indispensabili sono delle qualifiche e delle competenze che la persona che esegue gli interventi deve dimostrare. Inoltre sono indispensabili conoscenze professionali e l’assunzione delle responsabilità in particolare per quel che riguarda l’uso di medicamenti veterinari. Anche se il veterinario è autorizzato legalmente a delegare certi interventi a non-veterinari, egli deve sempre assumerne la responsabilità professionale per la qualità di tutti i trattamenti. Questa delega richiede inoltre obbligatoriamente una valutazione periodica retrospettiva del tipo e della quantità di trattamenti e, in conformità all’Ordinanza sui medicamenti veterinari, il controllo di un uso corretto dei medicamenti veterinari. In tale ottica, può avere senso di effettuare una valutazione dei sintomi in un quadro chiaramente definito, e nel rispetto dei requisiti di legge, con un trattamento iniziale da parte dell’allevatore, in particolare nel caso di malattie multifattoriali, al fine di garantire una rapida cura degli animali. Considerando le differenze in competenze tecniche tra i non-veterinari è necessario respingere le regolamentazioni generali. In ogni caso la diagnosi definitiva e la prescrizione dei medicamenti veterinari devono essere di competenza esclusiva dei veterinari. Parole chiavi: sicurezza dei medicamenti, trattamenti da parte di non-veterinari, protezione dei consumatori, allevamento di animali da reddito, protezione degli animal

Multiple roles of filopodial dynamics in particle capture and phagocytosis and phenotypes of Cdc42 and Myo10 deletion

Macrophage filopodia, finger-like membrane protrusions, were first implicated in phagocytosis more than 100 years ago, but little is still known about the involvement of these actin-dependent structures in particle clearance. Using spinning disk confocal microscopy to image filopodial dynamics in mouse resident Lifeact-EGFP macrophages, we show that filopodia, or filopodia-like structures, support pathogen clearance by multiple means. Filopodia supported the phagocytic uptake of bacterial (Escherichia coli) particles by (i) capturing along the filopodial shaft and surfing toward the cell body, the most common mode of capture; (ii) capturing via the tip followed by retraction; (iii) combinations of surfing and retraction; or (iv) sweeping actions. In addition, filopodia supported the uptake of zymosan (Saccharomyces cerevisiae) particles by (i) providing fixation, (ii) capturing at the tip and filopodia-guided actin anterograde flow with phagocytic cup formation, and (iii) the rapid growth of new protrusions. To explore the role of filopodia-inducing Cdc42, we generated myeloid-restricted Cdc42 knock-out mice. Cdc42-deficient macrophages exhibited rapid phagocytic cup kinetics, but reduced particle clearance, which could be explained by the marked rounded-up morphology of these cells. Macrophages lacking Myo10, thought to act downstream of Cdc42, had normal morphology, motility, and phagocytic cup formation, but displayed markedly reduced filopodia formation. In conclusion, live-cell imaging revealed multiple mechanisms involving macrophage filopodia in particle capture and engulfment. Cdc42 is not critical for filopodia or phagocytic cup formation, but plays a key role in driving macrophage lamellipodial spreading

PomBase: a comprehensive online resource for fission yeast.

PomBase (www.pombase.org) is a new model organism database established to provide access to comprehensive, accurate, and up-to-date molecular data and biological information for the fission yeast Schizosaccharomyces pombe to effectively support both exploratory and hypothesis-driven research. PomBase encompasses annotation of genomic sequence and features, comprehensive manual literature curation and genome-wide data sets, and supports sophisticated user-defined queries. The implementation of PomBase integrates a Chado relational database that houses manually curated data with Ensembl software that supports sequence-based annotation and web access. PomBase will provide user-friendly tools to promote curation by experts within the fission yeast community. This will make a key contribution to shaping its content and ensuring its comprehensiveness and long-term relevance

Phenotypic analysis of Myo10 knockout (Myo10tm2/tm2) mice lacking full-length (motorized) but not brain-specific headless myosin X

We investigated the physiological functions of Myo10 (myosin X) using Myo10 reporter knockout (Myo10tm2) mice. Full-length (motorized) Myo10 protein was deleted, but the brain-specific headless (Hdl) isoform (Hdl-Myo10) was still expressed in homozygous mutants. In vitro, we confirmed that Hdl-Myo10 does not induce filopodia, but it strongly localized to the plasma membrane independent of the MyTH4-FERM domain. Filopodia-inducing Myo10 is implicated in axon guidance and mice lacking the Myo10 cargo protein DCC (deleted in colorectal cancer) have severe commissural defects, whereas MRI (magnetic resonance imaging) of isolated brains revealed intact commissures in Myo10tm2/tm2 mice. However, reminiscent of Waardenburg syndrome, a neural crest disorder, Myo10tm2/tm2 mice exhibited pigmentation defects (white belly spots) and simple syndactyly with high penetrance (>95%), and 24% of mutant embryos developed exencephalus, a neural tube closure defect. Furthermore, Myo10tm2/tm2 mice consistently displayed bilateral persistence of the hyaloid vasculature, revealed by MRI and retinal whole-mount preparations. In principle, impaired tissue clearance could contribute to persistence of hyaloid vasculature and syndactyly. However, Myo10-deficient macrophages exhibited no defects in the phagocytosis of apoptotic or IgG-opsonized cells. RNA sequence analysis showed that Myo10 was the most strongly expressed unconventional myosin in retinal vascular endothelial cells and expression levels increased 4-fold between P6 and P15, when vertical sprouting angiogenesis gives rise to deeper layers. Nevertheless, imaging of isolated adult mutant retinas did not reveal vascularization defects. In summary, Myo10 is important for both prenatal (neural tube closure and digit formation) and postnatal development (hyaloid regression, but not retinal vascularization)

Revised fission yeast gene and allele nomenclature guidelines for machine readability

Standardized nomenclature for genes, gene products, and isoforms is crucial to prevent ambiguity and enable clear communication of scientific data, facilitating efficient biocuration and data sharing. Standardized genotype nomenclature, which describes alleles present in a specific strain that differ from those in the wild-type reference strain, is equally essential to maximize research impact and ensure that results linking genotypes to phenotypes are Findable, Accessible, Interoperable, and Reusable (FAIR). In this publication, we extend the fission yeast clade gene nomenclature guidelines to support the curation efforts at PomBase (www.pombase.org), the Schizosaccharomyces pombe Model Organism Database. This update introduces nomenclature guidelines for noncoding RNA genes, following those set forth by the Human Genome Organisation Gene Nomenclature Committee. Additionally, we provide a significant update to the allele and genotype nomenclature guidelines originally published in 1987, to standardize the diverse range of genetic modifications enabled by the fission yeast genetic toolbox. These updated guidelines reflect a community consensus between numerous fission yeast researchers. Adoption of these rules will improve consistency in gene and genotype nomenclature, and facilitate machine-readability and automated entity recognition of fission yeast genes and alleles in publications or datasets. In conclusion, our updated guidelines provide a valuable resource for the fission yeast research community, promoting consistency, clarity, and FAIRness in genetic data sharing and interpretation

Model of For3p-Mediated Actin Cable Assembly in Fission Yeast

Formin For3p nucleates actin cables at the tips of fission yeast cells for polarized cell growth. The results of prior experiments have suggested a possible mechanism for actin cable assembly that involves association of For3p near cell tips, For3p-mediated actin polymerization, retrograde flow of actin cables toward the cell center, For3p dissociation from cell tips, and cable disassembly. We used analytical and computational modeling to test the validity and implications of the proposed coupled For3p/actin mechanism. We compared the model to prior experiments quantitatively and generated predictions for the expected behavior of the actin cable system upon changes of parameter values. We found that the model generates stable steady states with realistic values of rate constants and actin and For3p concentrations. Comparison of our results to previous experiments monitoring the FRAP of For3p-3GFP and the response of actin cables to treatments with actin depolymerizing drugs provided further support for the model. We identified the set of parameter values that produces results in agreement with experimental observations. We discuss future experiments that will help test the model's predictions and eliminate other possible mechanisms. The results of the model suggest that flow of actin cables may establish actin and For3p concentration gradients in the cytoplasm that could be important in global cell patterning

TOR complex 2 localises to the cytokinetic actomyosin ring and controls the fidelity of cytokinesis.

The timing of cell division is controlled by the coupled regulation of growth and division. The TOR signalling network synchronises these processes with the environmental setting. Here we describe a novel interaction of the fission yeast TOR Complex 2 (TORC2) with the Cytokinetic Actomyosin Ring (CAR), and a novel role for TORC2 in regulating the timing and fidelity of cytokinesis. Disruption of TORC2 or its localisation results in defects in CAR morphology and constriction. We provide evidence that a myosin II, Myp2, and myosin V, Myo51, play roles in recruiting TORC2 to the CAR. We show that Myp2 and TORC2 are co-dependent upon each other for their normal localisation to the cytokinetic machinery. We go on to show that TORC2 dependent phosphorylation of Acp1 (Actin Capping Protein, a known regulator of cytokinesis) controls CAR stability and the modulation of CAPZA/BAcp1/2 heterodimer formation and is essential for survival upon stress. Thus TORC2 localisation to the CAR and TORC2 dependent CAPZAAcp1 phosphorylation contributes to timely control and fidelity of cytokinesis and cell division