Biology subjects

von Barnau Sythoff, A.

Publications and source records attributed to von Barnau Sythoff, A..

3 recordsLinked to original sources

bioRxiv2025

Altering cell size asymmetry in Drosophila neural stem cells creates supernumerary stem cells with limited lineage expansion potential

Asymmetrically dividing invertebrate and vertebrate stem cells can generate unequal sized sibling cells. However, the functional implications of cell size asymmetry (CSA) are underexplored. Here, we use Drosophila neural stem cells (NSCs) to investigate how changes in CSA impact NSC proliferation and cell fate decisions. Using live cell imaging, NSC lineage analysis, and gene expression profiling, we find that altering CSA increases the NSC pool but decreases lineage size and the number of differentiating progeny cells. Modeling CSA in silico with a volume-sensitivity and NSC self-inhibition model can recapitulate these findings. Gene expression profiling further revealed that the NSC growth regulator Imp and the G1-S cell cycle regulator CycE are upregulated in NSCs with altered CSA, providing a potential molecular link to the volume-sensitivity model. We propose that cell size and position regulate NSC proliferation and differential potential, impacting lineage progression and progeny cell differentiation in the developing Drosophila brain.

cell biology↗

bioRxiv2025

CrisprBuildr: an open-source application for CRISPR-mediated genome engineering in Drosophila melanogaster

CRISPR/Cas9 is a powerful tool for targeted genome engineering experiments. With CRISPR/Cas9, genes can be deleted or modified by inserting small peptides, fluorescent proteins or other tags for protein labelling experiments. Such experiments are important for detailed protein characterization in vivo. However, designing and cloning the corresponding constructs can be repetitive, time consuming and laborious. To aid users in CRISPR/Cas9-based genome engineering experiments, we built CrisprBuildr, a web-based application that allows users to delete genes or insert fluorescent proteins at the N- or C-terminus of their gene of choice. The application is built on the Drosophila melanogaster genome but can be used as a template for other available genomes. We have also generated new tagging vectors, using EGFP and mCherry combined with the small peptide SspB-Q73R for use in iLID-based optogenetic experiments. CrisprBuildr guides users through the process of designing guide RNAs and repair template vectors. CrisprBuildr is an open-source application and future releases could incorporate additional tagging or deletion vectors, genomes or CRISPR applications.

genomics↗

bioRxiv2025

Protein phosphatase 4 is required for centrosome asymmetry in fly neural stem cells

Asymmetric cell division is used by stem cells to create diverse cell types while self-renewing the stem cell population. Biased segregation of molecularly distinct centrosomes could provide a mechanism to maintain stem cell fate, induce cell differentiation or both. However, the molecular mechanisms generating molecular and functional asymmetric centrosomes remain incompletely understood. Here, we show that in asymmetrically dividing fly neural stem cells, Protein phosphatase 4 (Pp4) is necessary for correct centrosome asymmetry establishment during mitosis, and microtubule organizing center (MTOC) maintenance in interphase. Using in-vivo live cell imaging we show that while wild type neural stem cells always maintain one active MTOC, Pp4 mutant neuroblasts contain two inactive centrioles in interphase. Furthermore, centrosomes of Pp4 mutant neural stem cells mature in mitosis but fail to correctly transfer the centriolar protein Centrobin (Cnb) from the mother to the daughter centriole. Using superresolution imaging, we find that phosphomimetic Centrobin fails to accurately relocalize in mitosis. We propose that Pp4 regulates the timely relocalization of Cnb in mitosis to establish two molecularly distinct centrosomes. In addition, Pp4 is also necessary to maintain MTOC activity in interphase, ensuring biased centrosome segregation. Mechanistically, Pp4 could regulate centrosome asymmetry by dephosphorylating both Cnb and gamma-Tubulin. SIGNIFICANCE STATEMENTO_LIAsymmetric centrosome segregation occurs in stem cells and has been linked with cell fate decisions. C_LIO_LIProtein phosphatase 4 (Pp4), a conserved Serine/Threonine phosphatase, regulates centrosome asymmetry in Drosophila neural stem cells by acting upon gamma tubulin and Centrobin. C_LIO_LIPp4 regulates centrosome asymmetry establishment in mitosis and interphase, necessary for biased centrosome segregation. C_LI

cell biology↗