A NIR-Fluorochrome for Live Cell Dual Emission and Lifetime Tracking from the First Plasma Membrane Interaction to Subcellular and Extracellular Locales.

Molecular probes with the ability to differentiate between subcellular variations in acidity levels remain important for the investigation of dynamic cellular processes and functions. In this context, a series of cyclic peptide and PEG bio-conjugated dual near-infrared emissive BF2-azadipyrromethene fluorophores with maxima emissions at 720 nm (at pH > 6) and 790 nm (at pH < 5) have been developed and their aqueous solution photophysical properties determined. Their inter-converting emissions and fluorescence lifetime characteristics were exploited to track their spatial and temporal progression from first contact with the plasma membrane to subcellular locales to their release within extracellular vesicles. A pH-dependent reversible phenolate/phenol interconversion on the fluorophore controlled the dynamic changes in dual emission responses and corresponding lifetime changes. Live-cell confocal microscopy experiments in the metastatic breast cancer cell line MDA-MB-231 confirmed the usability of the dual emissive properties for imaging over prolonged periods. All three derivatives performed as probes capable of real-time continuous imaging of fundamental cellular processes such as plasma membrane interaction, tracking endocytosis, lysosomal/large acidic vesicle accumulation, and efflux within extracellular vesicles without perturbing cellular function. Furthermore, fluorescence lifetime imaging microscopy provided valuable insights regarding fluorophore progression through intracellular microenvironments over time. Overall, the unique photophysical properties of these fluorophores show excellent potential for their use as information-rich probes.

O-glycan determinants regulate VWF trafficking to Weibel-Palade bodies.

ABSTRACT: von Willebrand factor (VWF) undergoes complex posttranslational modification within endothelial cells (ECs) before secretion. This includes significant N- and O-linked glycosylation. Previous studies have demonstrated that changes in N-linked glycan structures significantly influence VWF biosynthesis. In contrast, although abnormalities in VWF O-linked glycans (OLGs) have been associated with enhanced VWF clearance, their effect on VWF biosynthesis remains poorly explored. Herein, we report a novel role for OLG determinants in regulating VWF biosynthesis and trafficking within ECs. We demonstrate that alterations in OLGs (notably reduced terminal sialylation) lead to activation of the A1 domain of VWF within EC. In the presence of altered OLG, VWF multimerization is reduced and Weibel-Palade body (WPB) formation significantly impaired. Consistently, the amount of VWF secreted from WPB after EC activation was significantly reduced in the context of O-glycosylation inhibition. Finally, altered OLG on VWF not only reduced the amount of VWF secreted after EC activation but also affected its hemostatic efficacy. Notably, VWF secreted after WPB exocytosis consisted predominantly of low molecular weight multimers, and the length of tethered VWF string formation on the surface of activated ECs was significantly reduced. In conclusion, our data therefore support the hypothesis that alterations in O-glycosylation pathways directly affect VWF trafficking within human EC. These findings are interesting given that previous studies have reported altered OLG on plasma VWF (notably increased T-antigen expression) in patients with von Willebrand disease.