RESUMEN
Cytotoxic T lymphocytes (CTLs) kill target cells through the polarized release of lytic molecules from secretory lysosomes. Loss of munc13-4 function inhibits this process and causes familial hemophagocytic lymphohistiocytosis type 3 (FHL3). munc13-4 binds rab27a, but the necessity of the complex remains enigmatic, because studies in knockout models suggest separate functions. In the present study, we describe a noncanonical rab27a-binding motif in the N-terminus of munc13-4. Point mutants in this sequence have severely impaired rab27a binding, allowing dissection of rab27a requirements in munc13-4 function. The munc13-4-rab27a complex is not needed for secretory lysosome maturation, as shown by complementation in CTLs from FHL3 patients and in a mast cell line silenced for munc13-4. In contrast, fusion of secretory lysosomes with, and content release at the plasma membrane during degranulation, strictly required the munc13-4-rab27a complex. Total internal reflection fluorescence microscopy imaging revealed that the complex corrals motile secretory lysosomes beneath the plasma membrane during degranulation and controls their docking. The propensity to stall motility of secretory lysosomes is lost in cells expressing munc13-4 point mutants that do not bind rab27. In summary, these results uncovered a mechanism for tethering secretory lysosomes to the plasma membrane that is essential for degranulation in immune cells.
Asunto(s)
Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Exocitosis , Células HEK293 , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/metabolismo , Linfohistiocitosis Hemofagocítica/patología , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía Confocal , Microscopía Fluorescente , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Mutación , Unión Proteica , Homología de Secuencia de Aminoácido , Linfocitos T Citotóxicos/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTPRESUMEN
Systemic sclerosis (SSc or scleroderma) is an auto-immune disease characterized by skin fibrosis. While primary cells from patients are considered as a unique resource to better understand human disease biology, the effect of in vitro culture on these cells and their evaluation as a platform to identify disease regulators remain poorly characterized. The goal of our studies was to provide insights into the utility of SSc dermal fibroblast primary cells for therapeutic target discovery. The disease phenotypes of freshly isolated and in vitro cultured SSc dermal fibroblasts were characterized using whole transcriptome profiling, alpha smooth muscle actin (ASMA) expression and cell impedance. SSc dermal fibroblasts retained most of the molecular disease phenotype upon in vitro culture for at least four cell culture passages (approximatively 10 cell doublings). We validated an RNA interference high throughput assay that successfully identified genes affecting the myofibroblast phenotype of SSc skin fibroblasts. These genes included MKL1, RHOA and LOXL2 that were previously proposed as therapeutic anti-fibrotic target, and ITGA5, that has been less studied in fibrosis biology and may be a novel potential modifier of SSc fibroblast biology. Together our results demonstrated the value of carefully-phenotyped SSc dermal fibroblasts as a platform for SSc target and drug discovery.