In briefCentrioles play a critical role in mitotic centrosome assembly. Here, Garbrecht et al. show that pericentriolar material not only persists but continues to assemble in C. elegans ciliated sensory neurons in the absence of centrioles and known mitotic regulators, suggesting different mechanisms for mitotic and non-mitotic centrosome assembly.

Proximity-dependent labeling approaches such as BioID have been a great boon to studies of protein-protein interactions in the context of cytoskeletal structures such as centrosomes which are poorly amenable to traditional biochemical approaches like immunoprecipitation and tandem affinity purification. Yet, these methods have so far not been applied extensively to invertebrate experimental models such as C. elegans given the long labeling times required for the original promiscuous biotin ligase variant BirA*. Here, we show that the recently developed variant TurboID successfully probes the interactomes of both stably associated (SPD-5) and dynamically localized (PLK-1) centrosomal components. We further develop an indirect proximity labeling method employing a GFP nanobody-TurboID fusion, which allows the identification of protein interactors in a tissue-specific manner in the context of the whole animal. Critically, this approach utilizes available endogenous GFP fusions, avoiding the need to generate multiple additional strains for each target protein and the potential complications associated with overexpressing the protein from transgenes. Using this method, we identify homologs of two highly conserved centriolar components, Cep97 and BLD10/Cep135, which are present in various somatic tissues of the worm. Surprisingly, neither protein is expressed in early embryos, likely explaining why these proteins have escaped attention until now. Our work expands the experimental repertoire for C. elegans and opens the door for further studies of tissue-specific variation in centrosome architecture.

SummaryDuring sexual reproduction in the ciliate, Tetrahymena thermophila, cells of complementary mating type pair (“conjugate”) undergo simultaneous meiosis and fertilize each other. In both mating partners only one of the four meiotic products is “selected” to escape autophagy, and this nucleus divides mitotically to produce two pronuclei. The migrating pronucleus of one cell translocates to the mating partner and fuses with its stationary pronucleus and vice versa. Selection of the designated gametic nucleus was thought to depend on its position within the cell because it always attaches to the junction with the partner cell. Here we show that a transmembrane protein, Semi1, is crucial for attachment. Loss of Semi1 causes failure to attach and consequent infertility. However, a nucleus is selected and gives rise to pronuclei regardless of Semi1 expression, indicating that attachment of a nucleus to the junction is not a precondition for selection but follows the selection process.

Proximity-dependent labeling approaches such as BioID have been a great boon to studies of protein-protein interactions in the context of cytoskeletal structures such as centrosomes which are poorly amenable to traditional biochemical approaches like immunoprecipitation and tandem affinity purification. Yet, these methods have so far not been applied extensively to invertebrate experimental models such as C. elegans given the long labeling times required for the original promiscuous biotin ligase variant BirA*. Here, we show that the recently developed variant TurboID successfully probes the interactomes of both stably associated (SPD-5) and dynamically localized (PLK-1) centrosomal components. We further develop an indirect proximity labeling method employing a GFP nanobody- TurboID fusion, which allows the identification of protein interactors in a tissue-specific manner in the context of the whole animal. Critically, this approach utilizes available endogenous GFP fusions, avoiding the need to generate multiple additional strains for each target protein and the potential complications associated with overexpressing the protein from transgenes. Using this method, we identify homologs of two highly conserved centriolar components, Cep97 and Bld10/Cep135, which are present in various somatic tissues of the worm. Surprisingly, neither protein is expressed in early embryos, likely explaining why these proteins have escaped attention until now. Our work expands the experimental repertoire for C. elegans and opens the door for further studies of tissue-specific variation in centrosome architecture.

In animal cells the functions of the cytoskeleton are coordinated by centriole-based centrosomes via microtubule-nucleating g-tubulin complexes embedded in the pericentriolar material or PCM [1]. PCM assembly has been best studied in the context of mitosis, where centriolar SPD-2 recruits PLK-1, which in turn phosphorylates key scaffolding components such as SPD-5 and CNN to promote expansion of the PCM polymer [2-4]. To what extent these mechanisms apply to centrosomes in interphase or in differentiated cells remains unclear [5]. Here, we examine a novel type of centrosome found at the ciliary base of C. elegans sensory neurons, which we show plays important roles in neuronal morphogenesis, cellular trafficking and ciliogenesis. These centrosomes display similar dynamic behavior to canonical, mitotic centrosomes, with a stable PCM scaffold and dynamically localized client proteins. Unusually, however, they are not organized by centrioles, which degenerate early in terminal differentiation [6]. Yet, PCM not only persists but continues to grow with key scaffolding proteins including SPD-5 expressed under control of the RFX transcription factor DAF-19. This assembly occurs in the absence of the mitotic regulators SPD-2, AIR-1 and PLK-1, but requires tethering by PCMD-1, a protein which also plays a role in the initial, interphase recruitment of PCM in early embryos [7]. These results argue for distinct mechanisms for mitotic and non-mitotic PCM assembly, with only the former requiring PLK- 1 phosphorylation to drive rapid expansion of the scaffold polymer.

ZusammenfassungDie vorliegende Interviewstudie untersucht ethische Herausforderungen des Pflegealltags in Einrichtungen der Langzeitpflege aus Sicht der Pflegepersonen während der Covid-19-Pandemie. Durch das explorative, wie auch deskriptive methodische Vorgehen liegen Interviewdaten vor, die vier Themenbereichen zugeordnet werden können, die eine komplexe und teilweise konfliktreiche Arbeits- und Lebenswirklichkeit der Langzeitpflege in der Pandemie aufzeigen. Zum einen werden von den Pflegepersonen die staatlich und institutionell getroffenen Schutzmaßnahmen sowie die daraus resultierenden Einschränkungen der persönlichen Freiheit der Bewohner:innen kritisch reflektiert und damit verbunden der Grad der Selbstbestimmtheit der Bewohner:innen von Alten- und Pflegeheimen in der Pandemie hinterfragt. Zum anderen wird – gegeben der pandemiebedingten Maßnahmen – das pflegerische Handeln im Arbeits- und Lebensort Heim als herausfordernd und die Möglichkeiten pflegerischer Fürsorge als stark eingeschränkt beschrieben. Genannt werden hier als konkrete Herausforderungen veränderte pflegerische Prozesse und Pflegequalität, wie auch eine veränderte Beziehungsqualität zu den Bewohner:innen.Diese vier Themenbereiche können grundsätzlich den ethischen Prinzipien der Autonomie und Fürsorge zugeordnet werden und präsentieren eine inhaltlich relevante Konkretisierung der prinzipienorientierten ethischen Herausforderungen. In der Zusammenschau und Interpretation der Ergebnisse wird ersichtlich, dass die interviewten Pflegepersonen die Beachtung und Umsetzung des Autonomie-Prinzips im pflegerischen Alltag eng mit dem Fürsorgeprinzip und dem daraus resultierenden Wohltun für die Bewohner:innen verknüpfen.Unsere Ergebnisse verdeutlichen, dass der in den letzten Jahrzehnten erarbeitete Paradigmenwechsel in der Langzeitpflege durch die Covid-19-Pandemie (zeitweise massiv) geschwächt wurde. Zudem machen die berichteten moralischen Unsicherheiten und Konflikte klar, dass Pflegepersonen Unterstützung hinsichtlich der ethischen Reflexion ihres pflegerischen Handelns benötigen und eine nachhaltige Integration von Ethikberatung in Langzeitpflegeeinrichtungen einen möglichen Lösungsansatz darstellen kann.

The reliability analysis shows that the items are less reliable. Currently, BISAD does not make a reliable contribution to clinical decision making in the tested setting.

No abstract