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    • To address this we developed

    2021-10-11

    To address this, we developed approaches to image at high spatial and temporal resolution live WPB fusions as well as the release of VWF and other WPB cargo by spinning disk microscopy and combined laser ablation and total internal reflection fluorescence (TIRF) microscopy. Our live cell imaging revealed different modes of WPB fusions and post-fusion morphologies that differ in kinetics of VWF release and in participation of the chlortetracycline cytoskeleton. Furthermore, we show that actin rings/coats form during histamine-evoked WPB exocytosis. However, such actin structures are only recruited to a subset of WPB fusion sites and their formation is significantly reduced when the cortical actin cytoskeleton is weakened by expression of a permanently active INF2 mutant that favors actin filament assembly at the ER. Actin ring/coat formation is RhoA dependent and is also observed when intracellular Ca2+ is elevated by laser ablation-induced cell wounding.
    Results
    Discussion Using live cell microscopy approaches, we identified and characterized different types of WPB fusion events that are characterized by a differential recruitment of F-actin. In endothelial cells stimulated by Ca2+-raising agonists approximately 70% of WPBs undergoing exocytosis produce a diffuse VWF signal, whereas the remaining 30% of Ca2+-dependent fusion events are characterized by a stable dot-like post-fusion appearance of the major WPB cargo, VWF. Moreover, we correlated the appearance of the round WPB post-fusion structures with a recruitment of F-actin to these fusion sites which was greatly reduced following sequestration of actin to the ER by a permanent CaAR peak-like reaction or inhibition of Rho activity. We suggest that the recruitment of actin rings/coats to a subset of WPB post-fusion structures is required for the stabilization of these structures and/or a more efficient expulsion of large WPB cargo in the presence of a dense actin cortex.
    Materials and methods
    Conflict of interest statement
    Transparency document
    Acknowledgements We thank Martin Bähler and Alexander Schmidt (University of Münster) for the TAT-C3 and TAT constructs. This work was supported by grants from the German Research Foundation to VG (DFG GE514/6-2 and SFB 1348).
    Introduction Pompe disease is a progressive form of muscular dystrophy caused by a deficiency in the lysosomal enzyme α-glucosidase, which leads to glycogen accumulation in the autolysosomes of affected cells [1], [2]. There is no effective cure for this lysosomal storage disorder, but clinical approval has been obtained for enzyme replacement therapy (ERT), which reduces the amount of glycogen in a number of affected tissues, including skeletal muscle and heart [3]; significantly reducing the morbidity and prolonging patient survival [4]. However, the inability of ERT to access glycogen stored in specific autolysosome compartments [5], [6] warrants the development of novel or adjunct therapeutic options capable of more effectively treating the disease. The relocation of glycogen from autolysosome storage compartments of affected cells, and into circulation, where extracellular glycogen is degraded by other amylases, could provide an effective adjunct treatment strategy. Under certain culture conditions, up to 80% of stored glycogen is exocytosed from cultured Pompe skin fibroblasts within 2 h [7], suggesting this therapeutic strategy is feasible. However, the culture conditions used to exocytose this large amount of glycogen corresponded to rapid cell division [7], and tissues having high amounts of glycogen storage in Pompe patients like heart and muscle are terminally differentiated and undergo minimal cell division. The challenge is therefore to find a drug capable of exocytosing the glycogen without the need for extensive cell division. Exocytosis of stored substrate from Pompe and other lysosomal storage disorder cells has been achieved by modulating the exocytic process, including the over-expression of proteins involved in the exocytic machinery. Kidney cells derived from metachromatic leucodystrophy patients treated with ionomycin demonstrated increased sulphatide release compared to untreated controls, with a concomitant 5-fold increase in β-hexosaminidase (β-hex) release [8]. In another study, the transcription factor EB (TFEB), a regulator of exocytosis, was over-expressed in cells derived from Pompe, multiple sulphatase deficiency, MPS IIIA and neuronal ceroidlipofuscinoses, leading to increased lysosomal exocytosis; as measured by lysosomal enzyme release and cell surface LAMP-1 staining [9]. Increased exocytosis led to reduced intracellular storage, ranging from 20% (MPS IIIA) to 60% (multiple sulphatase deficiency), compared to control cells. Other studies involving TFEB over-expression showed reduced glycogen storage in cultured muscle cells [10], [11] and Pompe mouse muscle [10]. It was hypothesized glycogen exocytosis could be induced with compounds previously shown to increase exocytosis [12], [13].