Biology subjects

Cabernard, C.

Publications and source records attributed to Cabernard, C..

9 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

Asymmetric centromere and gene locus positioning in Drosophila neural stem cells

Chromatin organization is important for cell division, epigenetic memory, and gene regulation. It is often reflected in the non-random positioning of centromeres, but the underlying mechanisms and functions remain largely unknown. Here, we demonstrate that asymmetrically dividing Drosophila neural stem cells cluster centromeres near the nuclear envelope, adjacent to the apical centrosome. This asymmetric centromere positioning is regulated through microtubules, originating from the apical centrosome that connect to apical nuclear pore complexes. The minus-end directed motor protein Dynein, its binding partner Mushroom body defect, and Lamin are also required. Asymmetric centromere positioning persists throughout interphase in neural stem cells but is lost in more differentiated progeny. We also reveal that the genes hunchback (hb) and pendulin (pen) occupy specific nuclear regions, correlating with polarized centromere localization. We propose that fly neural stem cells translate their inherent polarity into stereotypical chromatin organization, potentially influencing cell fate decisions and stem cell behavior.

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↗

bioRxiv2021

In vivo regulation of fluorescent fusion proteins by engineered kinases

Reversible protein phosphorylation by kinases in extensively used to control a plethora of processes essential for proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases for direct, robust and tissue-specific phosphorylation of target fluorescent protein fusions in vivo. We show that synthetic Rok kinases, based on the Drosophila ortholog of Rho-associated protein kinase (ROCK), are functional enzymes and can activate myosin II through phosphorylation of Sqh::GFP or Sqh::mCherry in different morphogenetic processes in a developing fly embryo. We next use the system to study the impact of actomyosin activation specifically in the developing tracheal branches and showed that ectopic activation of actomyosin with engineered Rok kinase did not prevent cell intercalation nor the formation of autocellular junctions. We assume that this approach can be adapted to other kinases and targets in various eukaryotic genetic systems.

developmental biology↗

bioRxiv2020

Asymmetric chromatin capture and nuclear envelopes separate endogenous and ectopic chromosomes after induced cell fusion

Hybrid cells derived through fertilization or somatic cell fusion recognize and separate chromosomes of different origin. The underlying mechanisms are unknown but could prevent aneuploidy and tumor formation. Here, we acutely induce fusion between Drosophila neural stem cells (neuroblasts; Nbs) and differentiating ganglion mother cells (GMCs) in vivo to define how epigenetically distinct chromatin is recognized and segregated. We find that Nb-GMC hybrid cells align both endogenous (neuroblast-origin) and ectopic (GMC-origin) chromosomes at the metaphase plate through centrosome derived dual-spindles. Mixing of endogenous and ectopic chromatin is prevented through an asymmetric, microtubule-dependent chromatin capture mechanism during interphase and physical boundaries imposed by nuclear envelopes. Although hybrid cells fail to accurately segregate ectopic chromatin, hybrid cells neither reduce the lifespan nor form visible tumors in host flies. We propose that Nb-GMC derived hybrid cells utilize asymmetric centrosome activity in interphase and nuclear envelopes to physically separate epigenetically distinct chromatin.

cell biology↗

bioRxiv2018

Drosophila scribble mutant tumors undergo a transition from a growth arrest state to a proliferative state over time

The Drosophila neoplastic tumor suppressor gene (nTSG) mutant tumors have successfully modeled many aspects of human tumor progression. However, the fly nTSG mutant tumors progress rapidly over days. This is in contrast with most human tumors which develop slowly, harbor heterogeneous cell populations for selection and undergo an evolution-like process. Whether the fast-growing fly nTSG mutant tumors have capacity for evolution remains unclear. Through quantitative analysis of the scrib mutant tumor growth, we found that the scrib mutant tumors evolve to display different growth rates and cell cycle profiles over time. Multiple growth-regulatory signaling pathways show quantitative differences in early versus late scrib mutant tumors. These data suggest that the scrib mutant tumors undergo a transition from a growth arrest state to a proliferative state. Through longitudinal single cell RNA (scRNA) data analysis we found that the scrib mutant tumors harbor heterogeneous cell populations likely of distinct proliferative states, which are available for potential selection. This study raises the possibility of studying tumor evolution in a genetically accessible and fast-growing invertebrate tumor model.

developmental biology↗

bioRxiv2018

Spatiotemporally controlled Myosin relocalization and internal pressure cause biased cortical extension to generate sibling cell size asymmetry

Metazoan cells can generate unequal sized sibling cells during cell division. This form of asymmetric cell division depends on spindle geometry and Myosin distribution but the underlying mechanics are unclear. Here, we use atomic force microscopy and live cell imaging to elucidate the biophysical forces involved in the establishment of physical asymmetry in Drosophila neural stem cells. We show that the force driving initial apical membrane expansion is provided by hydrostatic pressure, peaking shortly after anaphase onset, and enabled by a relieve of actomyosin contractile tension on the apical cell cortex. The subsequent increase in contractile forces at the cleavage furrow, combined with the relocalization of basally located Myosin results in basal membrane extension and sustained apical expansion. We propose that spatiotemporally controlled actomyosin contractile tension and hydrostatic pressure enables stereotypic biased membrane expansion to generate sibling cell size asymmetry.

cell biology↗

bioRxiv2018

A centrosome asymmetry switch in fly neural stem cells

Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older mother and a younger daughter centriole. Stem cells either inherit the mother or daughter centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the dynamics and mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-Structured Illumination Microscopy (3D-SIM) and live cell imaging we show that in fly neural stem cells (neuroblasts) the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) dynamically relocalize from the mother to the daughter centriole during mitosis. This mechanism generates a centrosome, containing two molecularly distinct centrioles by telophase. Cnbs timely relocalization is regulated by Polo-mediated phosphorylation whereas Polos daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments we propose that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.

cell biology↗