Department of Microbiology and Molecular Genetics, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas, USA.
mSphere. 2022 Apr 27;7(2):e0004722. doi: 10.1128/msphere.00047-22. Epub 2022 Mar 17.
Cytokinesis in Trypanosoma brucei occurs unidirectionally from the anterior toward the posterior through mechanisms distinct from those of its human host and is controlled by a signaling pathway comprising evolutionarily conserved and trypanosome-specific regulatory proteins. The mechanistic roles and the functional interplay of these cytokinesis regulators remain poorly understood. Here, we investigate the requirement of the structural motifs in the trypanosome-specific cytokinesis regulator CIF3 for the initiation of cytokinesis, the interaction with other cytokinesis regulators, and the recruitment of CIF3-interacting proteins to the cytokinesis initiation site. We demonstrate that the internal and C-terminal coiled-coil motifs, but not the N-terminal coiled-coil motif, of CIF3 play essential roles in cytokinesis and interact with distinct cytokinesis regulators. CIF3 interacts with TbPLK, CIF1, CIF4, and FPRC through the N-terminal and C-terminal coiled-coil motifs and with KAT80 through all three coiled-coil motifs. The C-terminal coiled-coil motif of CIF3 is required for the localization of CIF3 and all of its interacting proteins, and additionally, the internal coiled-coil motif of CIF3 is required for KAT80 localization. Conversely, all the CIF3-interacting proteins are required to maintain CIF3 at the cytokinesis initiation site at different cell cycle stages. These results demonstrate that CIF3 cooperates with multiple interacting partner proteins to promote cytokinesis in T. brucei. Cytokinesis is the final stage of cell division and is regulated by a signaling pathway conserved from yeast to humans. Cytokinesis in Trypanosoma brucei, an early-branching protozoan parasite causing human sleeping sickness, is regulated by mechanisms that are distinct from those of its human host, employing a number of trypanosome-specific regulatory proteins to cooperate with evolutionarily conserved regulators. The functional interplay of these cytokinesis regulators is still poorly understood. In this work, we investigated the structural requirement of the trypanosome-specific cytokinesis regulator CIF3 for the initiation of cytokinesis, the interaction with other cytokinesis regulatory proteins, and the recruitment of CIF3-interacting proteins. We demonstrated that different structural motifs of CIF3 played distinct roles in cytokinesis, interacted with distinct cytokinesis regulatory proteins, and were required for the recruitment of distinct cytokinesis regulatory proteins. These findings provided novel insights into the cooperative roles of cytokinesis regulators in promoting cytokinesis in T. brucei.
在布氏锥虫中，有丝分裂从前向后单向发生，通过与人类宿主不同的机制发生，并且由一个信号通路控制，该信号通路包括进化保守和锥虫特异性的调节蛋白。这些细胞分裂调节剂的机械作用和功能相互作用仍知之甚少。在这里，我们研究了锥虫特异性细胞分裂调节剂 CIF3 的结构基序在有丝分裂起始、与其他细胞分裂调节剂相互作用以及招募 CIF3 相互作用蛋白到细胞分裂起始位点中的要求。我们证明了 CIF3 的内部和 C 端卷曲螺旋基序，但不是 N 端卷曲螺旋基序，在有丝分裂中起重要作用，并与不同的细胞分裂调节剂相互作用。CIF3 通过 N 端和 C 端卷曲螺旋基序与 TbPLK、CIF1、CIF4 和 FPRC 相互作用，并通过所有三个卷曲螺旋基序与 KAT80 相互作用。CIF3 的 C 端卷曲螺旋基序对于 CIF3 和其所有相互作用蛋白的定位是必需的，此外，CIF3 的内部卷曲螺旋基序对于 KAT80 的定位也是必需的。相反，所有 CIF3 相互作用蛋白都需要在不同的细胞周期阶段将 CIF3 维持在细胞分裂起始位点。这些结果表明，CIF3 与多个相互作用的伴侣蛋白合作，促进了布氏锥虫的有丝分裂。有丝分裂是细胞分裂的最后阶段，由从酵母到人都保守的信号通路调控。布氏锥虫（一种早期分支的原生动物寄生虫，引起人类昏睡病）的有丝分裂受与人类宿主不同的机制调控，利用许多锥虫特异性调节蛋白与进化保守的调节蛋白合作。这些细胞分裂调节剂的功能相互作用仍知之甚少。在这项工作中，我们研究了锥虫特异性有丝分裂调节剂 CIF3 对有丝分裂起始、与其他有丝分裂调节蛋白相互作用以及 CIF3 相互作用蛋白募集的结构要求。我们证明了 CIF3 的不同结构基序在有丝分裂中发挥不同的作用，与不同的有丝分裂调节蛋白相互作用，并招募不同的有丝分裂调节蛋白。这些发现为细胞分裂调节剂在促进布氏锥虫有丝分裂中的合作作用提供了新的见解。
