Rodríguez A, Rioult M G, Ora A, Andrews N W
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
J Cell Biol. 1995 Jun;129(5):1263-73. doi: 10.1083/jcb.129.5.1263.
Lysosomes are recruited to the invasion site during host cell entry by Trypanosoma cruzi, an unusual process suggestive of the triggering of signal transduction mechanisms. Previous studies showed that trypomastigotes, but not the noninfective epimastigotes, contain a proteolytically generated trypomastigote factor (PGTF) that induces intracellular free Ca2+ transients in several mammalian cell types. Using confocal time-lapse imaging of normal rat kidney (NRK) fibroblasts loaded with the Ca(2+)-sensitive dye fluo-3, we show that the initial intracellular free Ca(2+) concentration ([Ca2+]i) transient detected a few seconds after exposure to trypomastigote extracts is a result of Ca2+ release from intracellular stores. Removal of Ca2+ from the extracellular medium or inhibition of Ca2+ channels with NiCl2 did not affect the response to PGTF, while depletion of intracellular stores with thapsigargin abolished it. [Ca2+]i transients induced by PGTF were shown to be coupled to the activity of phospholipase C (PLC), since the specific inhibitor U73122 completely blocked the response, while its inactive analogue U73343 had no effect. In addition, polyphosphoinositide hydrolysis and inositol 1,4,5-trisphosphate (IP3) were detected upon cell stimulation with PGTF, suggesting the participation of IP3-sensitive intracellular Ca2+ channels. An immediate effect of the signaling induced by PGTF and live trypomastigotes was a rapid and transient reorganization of host cell microfilaments. The redistribution of F-actin appeared to be a direct consequence of increased [Ca2+]i, since thrombin and the Ca2+ ionophore ionomycin produced a similar effect, with a time course that corresponded to the kinetics of the elevation in [Ca2+]i. These observations support the hypothesis that PGTF-induced disassembly of the cortical actin cytoskeleton may play a role in T. cruzi invasion, by facilitating lysosome access to the invasion site. Taken together, our findings suggest that the proteolytically generated trypomastigote factor PGTF is a novel agonist that acts through the PLC/phosphoinositide signaling pathway of mammalian cells.
在克氏锥虫侵入宿主细胞的过程中，溶酶体会被招募到侵入位点，这一不同寻常的过程提示信号转导机制被触发。先前的研究表明，感染性锥鞭毛体含有一种经蛋白水解产生的锥鞭毛体因子（PGTF），而非感染性的上鞭毛体则没有，该因子能在几种哺乳动物细胞类型中诱导细胞内游离Ca2+瞬变。利用共聚焦延时成像技术，对负载了Ca(2+)敏感染料fluo-3的正常大鼠肾（NRK）成纤维细胞进行观察，我们发现，在暴露于锥鞭毛体提取物后几秒检测到的初始细胞内游离Ca(2+)浓度（[Ca2+]i）瞬变是细胞内储存的Ca2+释放的结果。从细胞外培养基中去除Ca2+或用NiCl2抑制Ca2+通道并不影响对PGTF的反应，而用毒胡萝卜素耗尽细胞内储存则消除了该反应。由PGTF诱导的[Ca2+]i瞬变显示与磷脂酶C（PLC）的活性相关，因为特异性抑制剂U73122完全阻断了反应，而其无活性类似物U73343则没有作用。此外，在用PGTF刺激细胞后检测到多磷酸肌醇水解和肌醇1,4,5-三磷酸（IP3），这表明IP3敏感的细胞内Ca2+通道参与其中。PGTF和活的锥鞭毛体诱导的信号的直接作用是宿主细胞微丝的快速和短暂重组。F-肌动蛋白的重新分布似乎是[Ca2+]i增加的直接结果，因为凝血酶和Ca2+离子载体离子霉素产生了类似的效果，其时间进程与[Ca2+]i升高的动力学相对应。这些观察结果支持这样一种假设，即PGTF诱导的皮质肌动蛋白细胞骨架的解体可能通过促进溶酶体到达侵入位点而在克氏锥虫侵入中发挥作用。综上所述，我们的研究结果表明，经蛋白水解产生的锥鞭毛体因子PGTF是一种通过哺乳动物细胞的PLC/磷酸肌醇信号通路起作用的新型激动剂。
