Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN.
Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN.
J Cell Biol. 2019 Nov 4;218(11):3647-3662. doi: 10.1083/jcb.201902063. Epub 2019 Sep 27.
In patients with inactivating mutations in myosin Vb (Myo5B), enterocytes show large inclusions lined by microvilli. The origin of inclusions in small-intestinal enterocytes in microvillus inclusion disease is currently unclear. We postulated that inclusions in Myo5b KO mouse enterocytes form through invagination of the apical brush border membrane. 70-kD FITC-dextran added apically to Myo5b KO intestinal explants accumulated in intracellular inclusions. Live imaging of Myo5b KO-derived enteroids confirmed the formation of inclusions from the apical membrane. Treatment of intestinal explants and enteroids with Dyngo resulted in accumulation of inclusions at the apical membrane. Inclusions in Myo5b KO enterocytes contained VAMP4 and Pacsin 2 (Syndapin 2). Myo5b;Pacsin 2 double-KO mice showed a significant decrease in inclusion formation. Our results suggest that apical bulk endocytosis in Myo5b KO enterocytes resembles activity-dependent bulk endocytosis, the primary mechanism for synaptic vesicle uptake during intense neuronal stimulation. Thus, apical bulk endocytosis mediates the formation of inclusions in neonatal Myo5b KO enterocytes.
在肌球蛋白 VB（Myo5B）失活突变的患者中，肠细胞显示出由微绒毛排列的大包涵体。微绒毛包涵体病中小肠肠细胞中包涵体的起源目前尚不清楚。我们推测，Myo5b KO 鼠肠细胞中的包涵体是通过顶膜的内陷形成的。将 70kD FITC-葡聚糖添加到 Myo5b KO 肠外植体的顶端，会在细胞内包涵体中积累。Myo5b KO 衍生的类肠细胞的实时成像证实了顶膜的包涵体形成。用 Dyngo 处理肠外植体和类肠细胞会导致顶膜处的包涵体堆积。Myo5b KO 肠细胞中的包涵体含有 VAMP4 和 Pacsin 2（Syndapin 2）。Myo5b;Pacsin 2 双 KO 小鼠的包涵体形成明显减少。我们的结果表明，Myo5b KO 肠细胞中的顶端体大胞饮作用类似于活性依赖性大胞饮作用，这是神经元强烈刺激期间突触囊泡摄取的主要机制。因此，顶端体大胞饮作用介导了新生 Myo5b KO 肠细胞中包涵体的形成。
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