bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.04.07.717016

Systematic analysis of RhoGAP expression and function in border cell morphology and migration

Mishra, A. K.·Gemmil, E.·Campanale, J. P.·Mondo, J.·Lisi, V.·Kosik, K. S.·Montell, D. J.
View original source ↗Export metadata ↓

Abstract

Rho family GTPases are central hubs in the signaling and cytoskeletal networks that govern cell morphology and behavior. GTPase-activating proteins (GAPs) inactivate them by accelerating GTP hydrolysis. However, a systematic analysis of GAPs in cell migration is lacking. Here, we report screens for RhoGAP expression and function in migratory Drosophila border cells. Constitutively active Cdc42, Rac, or Rho causes defects, demonstrating that negative regulation is critical. Integrating single-cell RNAseq with published datasets reveals that most of the 22 RhoGAPs are expressed in border cells. RNAi knockdown shows most RhoGAPs are functionally required. We developed automated image analysis tools to sensitively and objectively classify border cell morphologies, defining a normal morphological phase space. RhoGAP perturbations push clusters outside this range. In-depth analysis of RhoGAPp190 reveals that loss-of-function resembles Rho hyperactivation and gain-of-function resembles myosin II inhibition. Thus, complex spatiotemporal sculpting of RhoGTPase activities requires diverse RhoGAPs within a single cell type to control morphology and motility in vivo.

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Mishra, A. K., Gemmil, E., Campanale, J. P., Mondo, J., Lisi, V., Kosik, K. S., Montell, D. J.. 2026-04-08. Systematic analysis of RhoGAP expression and function in border cell morphology and migration. https://doi.org/10.64898/2026.04.07.717016

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Deep generative embeddings of gene expression and splicing reposition the interpretation of single-cell transcriptomic signatures

Single-cell transcriptomic analysis predominantly derives cell identity from gene expression analysis, while alternative splicing is processed separately despite its fundamental role for cell homeostasis. To overcome the limits of separate investigations, we developed a probabilistic deep learning framework, Crecerelle, enabling resolution of the contributions of gene expression and alternative splicing in each cell. Crecerelle learns cell embeddings from gene expressions and alternative splicing isoforms, to decipher their mutually dependent impact on the functional characterisation of cells in a data-driven manner, exemplified for the Tabula Muris dataset. This is enabled through a zero-and-N-inflated Dirichlet-Multinomial for a variational autoencoder that learns cell embeddings solely from splicing profiles, as well as a bi-modal variational autoencoder with a relevance-weighted mixture-of-experts variational posterior to consolidate the modality-specific contribution at single-cell level. Crecerelle reveals cell-type-specific isoform markers as well as subpopulations with unique isoforms and uncovers regulatory and disease-associated pathways not detected by gene expression analyses alone. This scalable and interpretable framework thus allows a more holistic study of transcriptomic regulation and will open a route to modality-relevance-weighted investigations across single-cell multiomics datasets and their influence on cellular homeostasis, tissue development and disease phenotypes.

cell biology↗
bioRxiv2026

MHC Molecules on B Cell Microvilli Are Spatially Associated with IL-15Rα

Interleukin-15 (IL-15) trans-presentation (TP) by B cells is an important mechanism of T-cell activation; however, the spatial organisation of interleukin-15 receptor (IL-15R) relative to major histocompatibility complex (MHC) molecules on B-cell microvilli remains poorly understood. As microvilli protrude from the B-cell surface and may serve as sites of initial B cell-T-cell contact, the distribution of IL-15R and MHC molecules within these structures may be important during the earliest stages of T-cell recognition and activation. Here, we investigated the spatial association and molecular proximity of IL-15R with MHC class I and class II molecules on B-cell microvilli before immunological synapse formation, using confocal microscopy, stimulated emission depletion (STED) microscopy, stochastic optical reconstruction microscopy (STORM), and fluorescence lifetime imaging microscopy-based Forster resonance energy transfer (FLIM-FRET). Both MHC class I and class II molecules showed significant spatial association with IL-15R; however, the extent of colocalisation decreased as spatial resolution increased. STED microscopy revealed significant colocalisation between IL-15R and MHC class I, whereas STORM did not detect this association. In contrast, IL-15R and MHC class II remained significantly colocalised at both resolutions. FLIM-FRET further demonstrated molecular proximity between IL-15R and both MHC class I and class II molecules, with higher FRET efficiency observed for MHC class II. Collectively, these findings indicate that IL-15R is spatially organised in proximity to both MHC class I and class II molecules on B-cell microvilli before immunological synapse formation. This arrangement at potential sites of initial B-cell-T-cell contact may facilitate the coordination of IL-15 trans-presentation and antigen presentation during the earliest stages of B-cell-T-cell interactions.

cell biology↗
bioRxiv2026

Pulsed-SILAC in single mouse embryos reveals early embryonic protein synthesis dynamics and phosphosite regulation

Early embryogenesis relies extensively on maternally deposited products until zygotic genome activation, yet the dynamics for the synthesis of new proteins in mammalian embryos remains poorly characterized. To address this, we applied pulsed stable isotope labelling by amino acids in cell culture (pSILAC) combined with narrow-window data-independent acquisition mass spectrometry to single mouse oocytes and embryos to resolve de novo protein synthesis during early embryogenesis. This revealed that the maternal proteome is not a static reservoir, with components of the subcortical maternal complex and amino acid transporters SLC7A1/2 being actively synthesized during the earliest developmental stages. Furthermore, phosphoproteomic analysis identified hundreds of previously unreported phosphosites and extensive regulation during the oocyte-to-embryo transition. Notably, phosphorylation of the PRC2-interacting KLP motif of EZHIP emerged as a potential regulatory mechanism, with modification of this region reducing EZHIP-PRC2 interaction and coinciding with H3K27me3 remodelling. Together, single embryo pSILAC revealed a maternal proteome that is continuously synthesized, recycled, and post-translationally regulated during early embryogenesis.

cell biology↗