Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic Center for Cell Signaling, Mayo Clinic, Rochester, Minnesota 55905, USA.
Infect Immun. 2010 Jul;78(7):2927-36. doi: 10.1128/IAI.00077-10. Epub 2010 May 10.
Internalization of the obligate intracellular apicomplexan parasite, Cryptosporidium parvum, results in the formation of a unique intramembranous yet extracytoplasmic niche on the apical surfaces of host epithelial cells, a process that depends on host cell membrane extension. We previously demonstrated that efficient C. parvum invasion of biliary epithelial cells (cholangiocytes) requires host cell actin polymerization and localized membrane translocation/insertion of Na(+)/glucose cotransporter 1 (SGLT1) and of aquaporin 1 (Aqp1), a water channel, at the attachment site. The resultant localized water influx facilitates parasite cellular invasion by promoting host-cell membrane protrusion. However, the molecular mechanisms by which C. parvum induces membrane translocation/insertion of SGLT1/Aqp1 are obscure. We report here that cultured human cholangiocytes express several nonmuscle myosins, including myosins IIA and IIB. Moreover, C. parvum infection of cultured cholangiocytes results in the localized selective aggregation of myosin IIB but not myosin IIA at the region of parasite attachment, as assessed by dual-label immunofluorescence confocal microscopy. Concordantly, treatment of cells with the myosin light chain kinase inhibitor ML-7 or the myosin II-specific inhibitor blebbistatin or selective RNA-mediated repression of myosin IIB significantly inhibits (P < 0.05) C. parvum cellular invasion (by 60 to 80%). Furthermore ML-7 and blebbistatin significantly decrease (P < 0.02) C. parvum-induced accumulation of SGLT1 at infection sites (by approximately 80%). Thus, localized actomyosin-dependent membrane translocation of transporters/channels initiated by C. parvum is essential for membrane extension and parasite internalization, a phenomenon that may also be relevant to the mechanisms of cell membrane protrusion in general.
专性细胞内顶复门寄生虫隐孢子虫的内化导致在宿主上皮细胞的顶表面上形成独特的膜内但细胞外生态位，这一过程依赖于宿主细胞膜的延伸。我们之前的研究表明，隐孢子虫有效入侵胆管上皮细胞（胆管细胞）需要宿主细胞肌动蛋白聚合和钠/葡萄糖共转运蛋白 1（SGLT1）和水通道蛋白 1（Aqp1）的局部膜转位/插入，在附着部位。由此产生的局部水流入促进了寄生虫的细胞入侵，通过促进宿主细胞膜的突出。然而，隐孢子虫诱导 SGLT1/Aqp1 膜转位/插入的分子机制尚不清楚。我们在这里报告，培养的人胆管细胞表达几种非肌肉肌球蛋白，包括肌球蛋白 IIA 和 IIB。此外，隐孢子虫感染培养的胆管细胞导致肌球蛋白 IIB 的局部选择性聚集，但不是肌球蛋白 IIA 在寄生虫附着区，如通过双标记免疫荧光共焦显微镜评估。一致地，用肌球蛋白轻链激酶抑制剂 ML-7 或肌球蛋白 II 特异性抑制剂 blebbistatin 或肌球蛋白 IIB 的选择性 RNA 介导抑制处理细胞显著抑制（P <0.05）隐孢子虫细胞入侵（60-80%）。此外，ML-7 和 blebbistatin 显著降低（P <0.02）隐孢子虫诱导的 SGLT1 在感染部位的积累（约 80%）。因此，由隐孢子虫引发的局部肌动蛋白依赖性膜转运体/通道对于膜延伸和寄生虫内化是必不可少的，这一现象可能也与一般细胞膜突出的机制有关。
