已知弓形虫细胞外信号调节激酶7(ERK7)有助于顶端复合物的完整性,并参与圆锥形生物发生的最后一步。在没有ERK7的情况下,成熟的寄生虫会失去其圆锥形复合物,并且无法滑行,入侵,或从宿主细胞出口。与以前的报告相比,我们在这里表明,ERK7表型的消耗复制了顶端帽蛋白AC9或AC10的消耗。ERK7的缺失导致顶端极环(APR)的丢失,细胞下微管(SPMT)篮子的解体,微丝分泌严重受损。超微结构扩展显微镜(U-ExM),偶联到细胞内寄生虫的N-羟基琥珀酰亚胺酯(NHS-酯)染色,提供了前所未有的分辨率水平,并突出了在没有ERK7的情况下,根尖部以及扩张的质膜的混乱。野生型和ERK7耗竭寄生虫的比较蛋白质组学分析证实了已知的顶端复合蛋白的消失,包括顶端极环的标记和一个名为AC11的新顶端帽。同时,ERK7的缺乏导致微丝蛋白的积累,由于细胞器的胞吐缺陷。AC9耗尽的寄生虫作为对照,表现出内膜复合蛋白的增加,有两种新的蛋白质分配给这个隔间,即,IMC33和IMC34。重要性圆锥是个谜,位于顶孔丛球虫亚群顶端的动态细胞器,靠近根尖极环(APR),膜下微管(SPMT)从中出现,分泌细胞器(微粒和rhoptries)通过它到达质膜进行胞吐。在弓形虫中,圆锥形突出伴随着微丝分泌,在外出期间,运动性,和入侵。根尖帽结构蛋白AC9或AC10的条件性消耗导致SPMT的解体以及APR和圆锥形的丧失,导致微丝分泌缺陷和运动受阻,入侵,和出口。我们在这里表明,激酶ERK7的耗竭表型AC9和AC10突变体。超微结构扩展显微镜和NHS酯染色的结合显示,ERK7耗尽的寄生虫表现出扩张的顶端质膜和改变的根茎定位,而电子显微镜图像明确地突出了APR的损失。
Toxoplasma gondii extracellular signal-regulated kinase 7 (ERK7) is known to contribute to the integrity of the apical complex and to participate in the final step of conoid biogenesis. In the absence of ERK7, mature parasites lose their conoid complex and are unable to glide, invade, or egress from host cells. In contrast to a previous report, we show here that the depletion of ERK7 phenocopies the depletion of the apical cap protein AC9 or AC10. The absence of ERK7 leads to the loss of the apical polar ring (APR), the disorganization of the basket of subpellicular microtubules (SPMTs), and a severe impairment in microneme secretion. Ultrastructure expansion microscopy (U-ExM), coupled to N-hydroxysuccinimide ester (NHS-ester) staining on intracellular parasites, offers an unprecedented level of resolution and highlights the disorganization of the rhoptries as well as the dilated plasma membrane at the apical pole in the absence of ERK7. Comparative proteomics analysis of wild-type and ERK7-depleted parasites confirmed the disappearance of known apical complex proteins, including markers of the apical polar ring and a new apical cap named AC11. Concomitantly, the absence of ERK7 led to an accumulation of microneme proteins, resulting from the defect in the exocytosis of the organelles. AC9-depleted parasites were included as controls and exhibited an increase in inner membrane complex proteins, with two new proteins assigned to this compartment, namely, IMC33 and IMC34. IMPORTANCE The conoid is an enigmatic, dynamic organelle positioned at the apical tip of the coccidian subgroup of the Apicomplexa, close to the apical polar ring (APR) from which the subpellicular microtubules (SPMTs) emerge and through which the secretory organelles (micronemes and rhoptries) reach the plasma membrane for exocytosis. In Toxoplasma gondii, the conoid protrudes concomitantly with microneme secretion, during egress, motility, and invasion. The conditional depletion of the apical cap structural protein AC9 or AC10 leads to a disorganization of SPMTs as well as the loss of the APR and conoid, resulting in a microneme secretion defect and a block in motility, invasion, and egress. We show here that the depletion of the kinase ERK7 phenocopies AC9 and AC10 mutants. The combination of ultrastructure expansion microscopy and NHS-ester staining revealed that ERK7-depleted parasites exhibit a dilated apical plasma membrane and an altered positioning of the rhoptries, while electron microscopy images unambiguously highlight the loss of the APR.