The Apical Complex Provides a Regulated Gateway for Secretion of Invasion Factors in <i>Toxoplasma</i>

The apical complex is the definitive cell structure of phylum Apicomplexa, and is the focus of the events of host cell penetration and the establishment of intracellular parasitism. Despite the importance of this structure, its molecular composition is relatively poorly known and few studies have experimentally tested its functions. We have characterized a novel Toxoplasma gondii protein, RNG2, that is located at the apical polar ring—the common structural element of apical complexes. During cell division, RNG2 is first recruited to centrosomes immediately after their duplication, confirming that assembly of the new apical complex commences as one of the earliest events of cell replication. RNG2 subsequently forms a ring, with the carboxy- and amino-termini anchored to the apical polar ring and mobile conoid, respectively, linking these two structures. Super-resolution microscopy resolves these two termini, and reveals that RNG2 orientation flips during invasion when the conoid is extruded. Inducible knockdown of RNG2 strongly inhibits host cell invasion. Consistent with this, secretion of micronemes is prevented in the absence of RNG2. This block, however, can be fully or partially overcome by exogenous stimulation of calcium or cGMP signaling pathways, respectively, implicating the apical complex directly in these signaling events. RNG2 demonstrates for the first time a role for the apical complex in controlling secretion of invasion factors in this important group of parasites.顶复体（apical complex）是顶复门（Apicomplexa）物种的标志性细胞结构，亦是宿主细胞侵入与胞内寄生建立过程中核心事件的发生位点。尽管该结构具有重要生物学功能，但其分子组成仍相对不明，相关功能实验研究亦较为匮乏。本研究对刚地弓形虫（Toxoplasma gondii）的新型蛋白RNG2开展了表征工作，该蛋白定位于顶极环（apical polar ring）——顶复体的共有结构元件。在细胞分裂过程中，RNG2会在中心体复制完成后即刻被招募至中心体，这证实新顶复体的组装起始于细胞复制的早期事件之一。RNG2随后形成环状结构，其羧基末端与氨基末端分别锚定在顶极环与可移动的顶极锥体上，进而连接这两种结构。超分辨率显微镜（super-resolution microscopy）可分辨这两个末端，并揭示在宿主入侵过程中顶极锥体伸出时，RNG2的取向会发生翻转。对RNG2进行诱导性敲降可显著抑制宿主细胞侵入。与此一致，RNG2缺失会阻断微线体（micronemes）的分泌。不过，该分泌阻滞可分别通过外源激活钙信号通路或环磷酸鸟苷（cGMP）信号通路完全或部分解除，这表明顶复体直接参与了此类信号传导事件。本研究首次证实了顶复体在调控这类重要寄生虫入侵因子分泌过程中的功能。