Biology subjects

Murray, D. H.

Publications and source records attributed to Murray, D. H..

6 recordsLinked to original sources

bioRxiv2024

Structural extension of the human exocyst is enabled by a minimal interface

In multicellular organisms, the machinery responsible for polarized trafficking directs constitutive cargo secretion at distinct sites of the plasma membrane, cilia, and junctional structures. Central to this machinery is the exocyst complex, which tethers cargo vesicles to their destination membrane, alongside other intracellular membrane tethering roles. Precisely how the exocyst spatially integrates membranes and membrane resident binding partners is unclear. Here, we address the structural morphology and formation of the human exocyst complex. Through structural approaches coupled to predictive models, we determined that the exocyst and its subcomplexes have extended arm-like structures that help maximize its reach. Moreover, we demonstrate minimal intersubunit interaction, in contrast to prior models. Nucleation of the holocomplex occurs through a single site, explaining its spatial extension. Our results provide the biochemical basis for exocyst complex assembly, suggesting an ornate extended architecture. SignificanceCargo transport to the eukaryotic cell plasma membrane predominantly relies on the exocyst as a central, signal-integrating polarized trafficking complex. How this single cargo vesicle tethering complex can target diverse vesicles to destination membrane is poorly understood, partly due to inconsistent structural models. Here, we show an extended architecture of the human exocyst complex, revealing a minimal nucleation interface between its two subcomplexes. The distinct morphology of human exocyst, compared to yeast models, suggests a novel mechanism that supports its versatile role in membrane trafficking.

biochemistry↗

bioRxiv2023

Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

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.

cell biology↗

bioRxiv2023

Asymmetric cell division-specific phosphorylation of PAR-3 regulates neuroblasts polarisation and sensory organ formation in Drosophila

The generation of distinct cell fates during development depends on asymmetric cell division of progenitor cells. In the central and peripheral nervous system of Drosophila, progenitor cells respectively called neuroblasts or sensory organ precursors use PAR polarity during mitosis to control cell fate determination in their daughter cells. How polarity and the cell cycle are coupled, and how the cell cycle machinery regulates PAR protein function and cell fate determination is poorly understood. Here, we generate an analog sensitive allele of CDK1 and reveal that its partial inhibition weakens but does not abolish apical polarity in embryonic and larval neuroblasts, and leads to defects in polarisation of fate determinants. We describe a novel in vivo phosphorylation of Bazooka, the Drosophila homolog of PAR-3, on Serine180, a consensus CDK phosphorylation site. Remarkably, phosphorylation of Serine180 occurs in asymmetrically dividing neuroblasts and sensory organ precursors, and not in their symmetrically dividing neighbours. We further show that Serine180 phosphomutants disrupt the timing of basal polarisation in neuroblasts and sensory organ formation in sensory organ precursors. Finally, we show that CDK1 can phosphorylate human PARD3 in vitro, suggestive of a conserved kinase-substrate relationship between CDK1 and PAR-3.

developmental biology↗

bioRxiv2023

Rme-6 integrates EGFR trafficking and signalling to regulate ERK1/2 signalosome dynamics

Epidermal growth factor receptor (EGFR) signalling results in a variety of cell behaviours, including cell proliferation, migration and apoptosis, which depend on cell context. Here we have explored how the Rab5GEF, Rme-6, regulates EGFR signalling by modulating endocytic flux. We demonstrate that Rme-6, which acts early in the endocytic pathway, regulates EGFR trafficking through an endocytic compartment that is competent for ERK1/2 signalling. While overexpression of Rme-6 results in enhanced ERK1/2 nuclear localisation and c-Fos activation, loss of Rme-6 results in aberrant ERK1/2 signalling with increased cytoplasmic ERK1/2 phosphorylation (Thr202/Tyr204) but decreased ERK1/2 nuclear translocation and c-Fos activation, the latter leading to decreased cell proliferation. Phosphorylation of ERK1/2 by protein kinase 2 (CK2) is required for its nuclear translocation and our data support a model whereby Rme-6 provides a scaffold for a population of CK2 which is required for efficient nuclear translocation of ERK1/2. Rme-6 is itself a substrate for CK2 on Thr642 and Ser996 and phosphorylation on these sites can activate its Rab5GEF activity and endocytic trafficking of EGFR. Together our results indicate that Rme-6 co-ordinates EGFR trafficking and signalling to regulate the assembly and disassembly of an ERK1/2 signalosome. Summary statementHere we demonstrate how Rme-6, a Rab5GEF, co-ordinates trafficking and signalling of EGFR on the early endocytic pathway to ensure appropriate regulation of downstream ERK1/2 signalling.

cell biology↗

bioRxiv2022

A PI(3,5)P2 probe reveals PIKfyve is required for Rab7 acquisition and the delivery and fusion of early macropinosomes to phagosomes

Phagosome maturation is tightly regulated to ensure efficient delivery of the complex arsenal of antimicrobial activities that kill and digest captured microbes. Like other endocytic pathways, phagosome maturation is regulated by a combination of Rab GTPases and phosphoinositide signalling lipids (PIPs) which define membrane identity and recruit specific effectors. PIKfyve is a PI-5 kinase, which converts PI(3)P to PI(3,5)P2 on endosomes. Disruption of PIKfyve results in severe defects in phagosomal maturation but the underlying mechanism remains unclear. Here, we use the model professional phagocyte, Dictyostelium discoideum to dissect the role of PIKfyve in the crucial first steps of phagosome maturation. We find that, although early Rab5 dynamics are unaffected, loss of PIKfyve prevents phagosomes from acquiring Rab7 by fusion with a pool of Rab7 and V-ATPase positive endosomes. By following PIP dynamics using our recently characterised PI(3,5)P2-probe SnxA, we delineate multiple subpopulations of Rab7-positive endosomes that fuse sequentially with phagosomes. We identify one of these as PI(3,5)P2-positive macropinosomes, which dock and fuse with phagosomes in a PIKfyve-dependent manner. We therefore show that Dictyostelium phagosomes primarily accumulate Rab7 by vesicular fusion rather than from a cytosolic pool, and that this requires PIKfyve. In particular PI(3,5)P2 defines a specific subset of fusogenic macropinosomes, which we propose enables content mixing and the efficient bulk delivery of lysosomal components to phagosomes.

cell biology↗

bioRxiv2021

A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C driven phosphoinositide conversion

Polarized trafficking is necessary for the development of eukaryotes and is regulated by a conserved molecular machinery. Late steps of cargo delivery are mediated by the exocyst complex, which integrates lipid and protein components to tether vesicles for plasma membrane fusion. However, the molecular mechanisms of this process are poorly defined. Here, we reconstitute functional octameric human exocyst, demonstrating the basis for holocomplex coalescence and biochemically stable subcomplexes. We determine that each subcomplex independently binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which is minimally sufficient for membrane tethering. Through reconstitution and epithelial cell biology experiments, we show that Arf6-mediated recruitment of the lipid kinase PIP5K1C rapidly converts phosphatidylinositol 4-phosphate (PI(4)P) to PI(4,5)P2, driving exocyst recruitment and membrane tethering. These results provide a molecular mechanism of exocyst-mediated tethering and a unique functional requirement for phosphoinositide signaling on latestage vesicles in the vicinity of the plasma membrane.

cell biology↗