bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.10.18.562882

Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

Maib, H.·Adarska, P.·Hunton, R.·Vines, J.·Strutt, D.·Bottanelli, F.·Murray, D. H.
View original source ↗Export metadata ↓

Abstract

Phosphoinositides are a small family of phospholipids, acting as signalling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organisation and is most commonly done by over-expression of biosensors. However, this leads to perturbations of phosphoinositide signalling and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of all 8 phosphoinositides using a unifying staining approach for fixed cells and tissue, based on recombinant biosensors with self-labelling SNAP tags. These recombinant biosensors are highly specific, and reliably visualise the subcellular distributions of phosphoinositides across scales, ranging from 2D or 3D cell culture to Drosophila tissue. Using stimulated emission depletion (STED) microscopy, we reveal the nanoscale organisation of PI(3)P on endosomes and PI(4)P on the Golgi and confirm the preservation of subcellular membranes. Multiplex staining enables the investigation of phosphoinositide conversions and reveals an unexpected presence of residual PI(3,5)P2 positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualisation of multiple phosphoinositide species in an unprecedented manner.

Source connections

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Maib, H., Adarska, P., Hunton, R., Vines, J., Strutt, D., Bottanelli, F., Murray, D. H.. 2023-10-19. Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides. https://doi.org/10.1101/2023.10.18.562882

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

KEEP EXPLORING

Related preprints

bioRxiv2026

Functional characterization of Rho GTPase activating proteins SYDE1 and SYDE2

The human genome encodes more than 60 proteins containing Rho GTPase activating protein (RhoGAP) domains, many of which remain understudied with respect to their target specificity and biological roles. SYDE1 and SYDE2 are two such orphan RhoGAPs, for which there are few studies characterizing their biochemical and cellular functions and conflicting reports identifying their cognate GTPases. We previously identified SYDE1 and SYDE2 in a screen for substrates of the c-Jun N-terminal kinases. Here, we show that SYDE1 and SYDE2 are preferentially phosphorylated by JNK1 relative to other mitogen-activated protein kinases (MAPKs) at sites proximal to a kinase docking region. Purified SYDE1 and SYDE2 are shown to have significant catalytic GAP activity toward RhoA, Rac1, and Cdc42. However, neither up- nor down-regulation of SYDE1/2 expression leads to detectable changes in bulk GTP loading of any of these GTPases. Nevertheless, we demonstrate that SYDE1 and SYDE2, in a partially GAP-dependent manner, increase cell spreading and number of focal adhesions, and promote more directionally persistent migration in HEK293 cells. Together, these findings establish SYDE1 and SYDE2 as robust JNK substrates with catalytic activity toward a set of Rho GTPases and reveal basic functions of SYDE1 and SYDE2 in regulating cell morphology, adhesion, and migration.

cell biology↗
bioRxiv2026

The filopodial scaffold polyphosphate dictates cell adhesion-versus-invasion decisions

Inorganic polyphosphate (polyP) is an ancient polymer conserved across all life, serving cell type and location specific functions in every major compartment. Yet its role at the plasma membrane, where it accumulates to peak levels in many primary cells, is largely unknown. Here we identify polyP as a stabilizing component of filopodia, actin based membrane protrusions that govern cell adhesion, contact inhibition, and chemotaxis. Elevating cellular polyP increases filopodial stability and enhances cell adhesion, whereas reducing polyP accelerates filopodial disassembly and promotes cell migration. Mechanistically, we find that polyP acts as a structural filopodial scaffold, recruiting and organizing IRSp53, a membrane curvature inducing protein. We show that metastatic fibroblasts and breast cancer organoids carry markedly reduced and intracellularly reorganized polyP levels relative to their non transformed counterparts. Restoring endogenous polyP via lipid nanoparticle delivery suppresses their invasive phenotypes and reverses prometastatic gene expression signatures, implicating polyP as a primordial tumor suppressor.

cell biology↗
bioRxiv2026

Mitochondrial transfer mediates metabolic communication between beta cells and islet macrophages

Pancreatic islet macrophages support islet homeostasis and adapt their metabolic program in response to environmental cues, including beta cell released factors. Intercellular mitochondrial transfer is a biological process that modulates cellular responses. To test whether beta cells, which are strongly secretory, transfer mitochondria to islet macrophages, we generated mice with beta cell-specific expression of mitochondrial GFP (PhAMfloxIns1Cre). We demonstrate that beta cells transfer mitochondria to islet macrophages in vivo and in vitro. Diabetogenic stressors did not alter the frequency of mitochondrial transfer and macrophages containing beta cell-derived GFP exhibit increased protein synthesis rates. RNA-seq identified upregulation of activity-regulated cytoskeleton associated protein (Arc) in macrophages receiving beta cell-derived mitochondria, while disruption of actin cytoskeleton dynamics prevented mitochondrial transfer. Together, these findings identify mitochondrial transfer as a previously unrecognized mechanism of beta cell-macrophage communication that may contribute to islet homeostasis and immune regulation.

cell biology↗