Biology subjects

Nightingale, T. D.

Publications and source records attributed to Nightingale, T. D..

6 recordsLinked to original sources

bioRxiv2025

Force sensing by Piezo1 regulates endothelial secretory granule exocytosis

Endothelial cells (ECs) rapidly alter their phenotype to support haemostasis and inflammation through the exocytosis of specialised storage organelles, known as Weibel-Palade bodies (WPBs). This exocytic response is stimulated by a variety of physiological and pathological triggers which act through well-described transmembrane receptors. However, the influence of mechanical forces and how they shape this fundamental endothelial response remains relatively unexplored. Here we demonstrate that opening of the mechanosensitive cation channel Piezo1 on the EC surface triggers rapid WPB exocytosis. The dynamics and mechanisms were investigated using the Piezo1 agonist (Yoda1) and by modelling mechanical opening through EC-ICAM-1 ligation by THP-1 cells under low shear flow. In vitro, localised von Willebrand Factor (VWF) secretion occurred at THP-1 adhesion sites, suggesting a reciprocity of VWF release alongside leukocyte capture. In vivo, Yoda1 stimulation of tissues resulted in a dramatic increase in neutrophil and platelet adhesion to postcapillary venular walls, spatially aligned with cargo release (VWF). The rapid dynamics of this response was revealed by the application of high resolution confocal intravital microscopy to transgenic EGFP-Rab27a mice that exhibit fluorescent WPBs and neutrophils. These data reveal a previously unrecognised instigator of WPB exocytosis and uncover a mechanosensitive molecular pathway coupling inflammation with haemostasis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/678766v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@d8ed6org.highwire.dtl.DTLVardef@1cbc0corg.highwire.dtl.DTLVardef@7dd6a9org.highwire.dtl.DTLVardef@197f286_HPS_FORMAT_FIGEXP M_FIG Graphical abstract: Activation of Piezo1 by pharmacological agonism or leukocyte adherence to ICAM-1 under low flow promotes VWF secretion and platelet capture through a protein kinase C-dependent mechanism. C_FIG SUMMARYThis research describes how the mechanosensitive cation channel Piezo1 elicits the rapid exocytosis of endothelial secretory organelles, provoking platelet capture to sites of leukocyte adherence. The EGFP-Rab27a transgenic mouse demonstrates a technical advance, facilitating intravital imaging of endothelial secretory organelles.

cell biology↗

bioRxiv2025

Endothelial β-PIX (ARHGEF7) drives exocytosis through enabling the dynamic reorganisation of the cytoskeleton

Endothelial cells rapidly respond to blood vessel injury or infection by releasing haemostatic and inflammatory proteins from their secretory organelles called Weibel-Palade Bodies (WPBs). Upon stimulation, WPBs traffic to the cell surface to secrete cargo such as von Willebrand factor (VWF), essential for platelet recruitment, and P-selectin, which facilitates the capture and subsequent rolling of immune cells. While this process is critical, many aspects of WPB trafficking and exocytosis remain unclear--particularly how release sites are selected across the cell surface and how fusion is directed to either the apical (lumen-facing) or basolateral (basement membrane-facing) side. These decisions directly impact the biological consequences and effectiveness of the secreted proteins. Using live-cell imaging, fibronectin micropatterning, loss-of-function and biochemical assays, we identified trafficking and release at hot spots near focal adhesions (FAs). We also discovered that the FA-resident guanine nucleotide exchange factor (GEF), {beta}-PIX (ARHGEF7), regulates VWF secretion. {beta}-PIX is known to activate Rho GTPases (Cdc42/Rac), triggering downstream p21-activated kinase 2 (PAK2) signalling and cytoskeletal remodelling. Here, depletion of {beta}-PIX impaired VWF secretion and delayed its release by perturbing cytoskeletal reorganisation. Knockdown and rescue experiments using truncated mutants further revealed which domains of {beta}-PIX are necessary for exocytosis and cytoskeletal reorganisation. This is the first demonstration of {beta}-PIXs role in VWF secretion from endothelial cells and our data provides new insights into spatial targeting of WPB exocytosis. Such targeting may be essential for guiding leukocyte transmigration or platelet binding, thereby maintaining vascular integrity. SchematicEndothelial secretory organelles, known as Weibel-Palade bodies (WPBs), store von Willebrand factor (VWF), and their regulated exocytosis is critical for blood clotting and immune responses. This process can be stimulated experimentally by phorbol esters as well as physiological stimuli such as adrenaline and histamine. Cytoskeletal remodelling is required for both pre- and post-fusion events in this secretory pathway. Here, we present evidence supporting the involvement of focal adhesion (FA)-associated exocytosis in the release of WPBs. For the first time, we demonstrate a regulatory role for the FA-resident guanine nucleotide exchange factor (GEF) {beta}-PIX in VWF secretion. Through its GEF activity toward Cdc42 and Rac, {beta}-PIX activates p21-activated kinases, driving the necessary cytoskeletal dynamics. Disruption of {beta}-PIX function results in abnormal cytoskeletal architecture that fails to respond appropriately to phorbol ester stimulation. Consequently, F-actin, nonmuscle myosin IIA (NMIIA), and septins become mislocalised, forming prominent filaments above the nucleus. These data provide a novel insight into the spatial targeting and molecular events underpinning the exocytosis of endothelial secretory organelles. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/652370v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1dbc615org.highwire.dtl.DTLVardef@1a9a959org.highwire.dtl.DTLVardef@1ad31aforg.highwire.dtl.DTLVardef@1990000_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

The unconventional Myosin-1C augments endothelial secretion of von Willebrand factor by linking contractile actomyosin machinery to the plasma membrane

Blood endothelial cells control the hemostatic and inflammatory response by secreting von Willebrand factor (VWF) and P-selectin from storage organelles called Weibel-Palade bodies (WPB). Actin-associated motor proteins regulate this secretory pathway at multiple points. Prior to fusion, myosin Va forms a complex that anchors WPBs to peripheral actin structures allowing maturation of content. Post-fusion, an actomyosin ring/coat is recruited and compresses to forcibly expel the largest VWF multimers. Here we provide the first evidence for the involvement of class I myosins during regulated VWF secretion. We show that unconventional myosin-1C (Myo1c) is recruited post fusion via its pleckstrin homology domain in an actin-independent process providing a link between the actin ring and phosphatidylinositol 4,5-bisphosphate (PIP2) at the membrane of the fused organelle. This is necessary to ensure maximal VWF secretion in response to secretagogue stimulation. Inhibition of class I myosins using the inhibitor Pentachloropseudilin alters the kinetics of the exocytic actin ring. These data offer new insight into the control of an essential physiological process and provide a new potential way in which it might be therapeutically controlled. SIGNFICANCE STATEMENTMyosin motors play diverse roles in regulated secretion. In endothelial cells, the role of conventional myosins (e.g. non-muscle myosin II) are well described however little is known about the requirement of unconventional myosins. Our data identify an important function of the class 1 myosin, Myosin-1C, in the actomyosin mediated expulsion of an essential blood clotting factor (von Willebrand factor) from endothelial cells. This is the first description of how class 1 myosins contribute to primary hemostasis and is therefore greatly improves our understanding of a fundamental physiological process.

cell biology↗

bioRxiv2021

The coagulation factor IX (F9) loss of function prevents the cell cycle arrest induced by CDK4/6 inhibitors treatment

During this last decade the development of pro-senescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and have showed efficacy in reducing tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their anti-tumour activity. Here, using a functional genome wide CRISPR/Cas9 genetic screen we found several genes that synergistically participate in the proliferation arrest induced by the CDK4/6 inhibitor, Palbociclib. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout reduces senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitors response that will be useful to design new therapeutic strategies in personalized medicine in order to increase their efficiency, stratify patients and avoid drug resistance.

cancer biology↗

bioRxiv2021

Proximity interactome of LC3B in normal growth conditions

LC3 (Light Chain 3) is a key player of autophagy, a major stress-responsive proteolysis pathway promoting cellular homeostasis. It coordinates the formation and maturation of autophagosomes and recruits cargo to be further degraded upon autophagosome-lysosome fusion. To orchestrate its functions, LC3 binds to multiple proteins from the autophagosomes inner and outer membranes, but the full extent of these interactions is not known. Moreover, LC3 has been increasingly reported in other cellular locations than the autophagosome, with cellular outcome not fully understood and not all related to autophagy. Furthermore, novel functions of LC3 as well as autophagy can occur in cells growing in a normal medium thus in non-stressed conditions. A better knowledge of the molecule in proximity to LC3 in normal growth conditions will improve the understanding of LC3 function in autophagy and in other cell biology function. Using an APEX2 based proteomic approach, we have detected 407 proteins in proximity to the well-characterised LC3B isoform in non-stress conditions. These include known and novel LC3B proximity proteins, associated with various cell localisation and biological functions. Sixty-nine of these proteins contain a putative LIR (LC3 Interacting Region) including 41 not reported associated to autophagy. Several APEX2 hits were validated by co-immunoprecipitation and co-immunofluorescence. This study uncovers the LC3B global interactome and reveals novel LC3B interactors, irrespective of LC3B localisation and function. This knowledge could be exploited to better understand the role of LC3B in autophagy and non-autophagy cellular processes.

cell biology↗

bioRxiv2019

Dynamic trafficking and turnover of Jam-C is essential for endothelial cell migration

Junctional complexes between endothelial cells form a dynamic barrier that hinder passive diffusion of blood constituents into interstitial tissues. Re-modelling of junctions is an essential process during leukocyte trafficking, vascular permeability and angiogenesis. However, for many junctional proteins the mechanisms of junctional remodelling have yet to be determined. Here we used receptor mutagenesis, HRP and APEX-2 proximity labelling, alongside light and electron microscopy, to map the intracellular trafficking routes of junctional adhesion molecule-C (Jam-C). We found that Jam-C co-traffics with receptors associated with changes in permeability, such as VE-Cadherin, NRP-1 and 2, but not with junctional proteins associated with the transmigration of leukocytes. Dynamic Jam-C trafficking and degradation is necessary for junctional remodelling during cell migration and angiogenesis. By identifying new trafficking machinery we show that a key point of regulation is the ubiquitylation of Jam-C by the E3 ligase CBL, this controls the rate of recycling versus lysosomal degradation.

cell biology↗