Michael Danielsen E, Hansen Gert H
Department of Cellular and Molecular Medicine, The Panum Institute, Faculty of Health Sciences, University of Copenhagen, Denmark.
Department of Cellular and Molecular Medicine, The Panum Institute, Faculty of Health Sciences, University of Copenhagen, Denmark.
Biochim Biophys Acta. 2016 Feb;1858(2):233-43. doi: 10.1016/j.bbamem.2015.11.022. Epub 2015 Nov 24.
Pinocytosis at the small intestinal brush border was studied in postweaned porcine cultured mucosal explants, using the fluorescent polar probes Alexa hydrazide (AH, MW 570), Texas red dextran (TRD, MW ~ 3000), and Cascade blue dextran (CBD, MW ~ 10,000). Within 1 h, AH appeared in a string of subapical punctae in enterocytes, indicative of an ongoing constitutive pinocytosis. By comparison, TRD was taken up less efficiently into the same compartment, and no intracellular labeling of CBD was detectable, indicating that only small molecules are pinocytosed from the postweaned gut lumen. AH remained in the terminal web region in EEA-1-positive endosomes (“TWEEs”) for at least 2 h, implying that the pinocytic uptake does not proceed towards a transcytic pathway. Like AH, cholera toxin B subunit (CTB) was readily internalized, but the two probes appeared in completely non-overlapping subapical compartments, indicating the existence of two different uptake mechanisms operating simultaneously at the brush border. CTB is internalized by clathrin-dependent receptor mediated endocytosis, but surprisingly the toxin also caused a rapid disappearance from the apical cell surface of two major brush border enzymes, alkaline phosphatase and aminopeptidase N, demonstrating the disruptive effect of this pathway. By immunofluorescence, caveolin-1 was hardly detectable in enterocytes, arguing against a caveolae-mediated uptake of AH, whereas the pinocytosis/phagocytosis inhibitors dimethyl amiloride and cytochalasin D both arrested AH uptake. We propose that the constitutive pinocytic mechanism visualized by AH contributes to maintenance of membrane homeostasis and to enrich the contents of lipid raft constituents at the brush border.
利用荧光极性探针Alexa酰肼（AH，分子量570）、德克萨斯红葡聚糖（TRD，分子量约3000）和级联蓝葡聚糖（CBD，分子量约10000），对断奶后猪培养的黏膜外植体小肠刷状缘的胞饮作用进行了研究。在1小时内，AH出现在肠细胞顶端下的一串点状结构中，表明存在持续的组成型胞饮作用。相比之下，TRD进入同一区室的效率较低，且未检测到CBD的细胞内标记，这表明断奶后肠腔中只有小分子被胞饮。AH在早期内体抗原1（EEA-1）阳性的内体（“TWEEs”）的终末网区域中至少保留2小时，这意味着胞饮摄取不会进入转胞吞途径。与AH一样，霍乱毒素B亚基（CTB）很容易被内化，但这两种探针出现在完全不重叠的顶端下区室中，表明在刷状缘同时存在两种不同的摄取机制。CTB通过网格蛋白依赖性受体介导的内吞作用被内化，但令人惊讶的是，该毒素还导致两种主要刷状缘酶，即碱性磷酸酶和氨肽酶N从顶端细胞表面迅速消失，证明了该途径的破坏作用。通过免疫荧光法，在肠细胞中几乎检测不到小窝蛋白-1，这表明AH不是通过小窝介导的摄取方式，而胞饮作用/吞噬作用抑制剂二甲胺氯吡嗪和细胞松弛素D均能阻止AH的摄取。我们认为，AH显示的组成型胞饮机制有助于维持膜稳态，并丰富刷状缘脂筏成分的含量。
