Dissecting the mechanism and regulation of Toxoplasma cytokinesis

描述（由申请人提供）：顶复虫是导致严重人类疾病的寄生虫，包括引起疟疾的疟原虫，引起脑炎和出生缺陷的刚地弓形虫和引起腹泻的小隐孢子虫。耐药性和/或特异性差不断破坏治疗这些疾病的治疗方案。为了确定新的药物目标，私家侦探他的实验室专注于加强对细胞生物学过程的理解，其中寄生虫与其宿主不同。一个这样的过程是细胞分裂，因为它在形态学上不同于哺乳动物的细胞分裂，是与顶复合体疾病相关的病理的核心。破译寄生虫细胞分裂对于理解不同真核生物谱系中细胞分裂的进化也很有意义。具体地说，在这个建议下，将利用弓形虫速殖子细胞分裂作为一种简单而容易获得的模型来研究子芽。速殖子通过内部出芽分裂，其中两个子细胞在母细胞内组装。子细胞骨架在复制的中心体周围形成，随后拉长作为细胞器发生和分裂的支架。细胞骨架是如何在中心体上组装的还不清楚。然而，现在已经确定，许多成分是寄生虫所特有的，并不与哺乳动物宿主共享。此外，在细胞骨架支架组装的中途，收缩力开始使子细胞向基端逐渐变细。负责这种收缩的基础复合体是哺乳动物收缩环的功能同源物，但有趣的是，它的收缩与肌动蛋白聚合无关。事实上，驱动基础复合体的动力机制仍然未知。总之，尽管我们对驱动细胞分裂的结构成分有基本的了解，但我们仍然缺乏关于它是如何被驱动的以及如何控制和协调各个步骤的详细信息。根据这一建议，研究人员将剖析假定的磷酸化控制细胞分裂。通过最近对细胞分裂的研究，已经确定了几种激酶和磷酸酶，它们在不同的细胞分裂步骤中起着明显的关键作用。这些酶的功能将通过敲除研究以及激酶底物鉴定研究来剖析。独立于这第一个目标，基础复杂收缩的神秘机制将被解开。候选运动蛋白将通过实验验证，然后寻求一种独立的候选方法。后者需要化学遗传Bio-ID方法，并与超分辨率显微镜相结合，将导致基础复合物结构的分子定义。在完成这一提议后，研究人员希望在细胞分裂过程的不同阶段描述关键的磷酸化控制，并确定驱动基础复合体收缩的机制。这两个里程碑都将提供具体的药物靶点，作为未来工作的起点。

DESCRIPTION (provided by applicant): Apicomplexan parasites are responsible for severe human diseases, including Plasmodium spp. causing malaria, Toxoplasma gondii causing encephalitis and birth defects, and Cryptosporidium parvum causing diarrhea. Drug resistance and/or poor specificity are constantly undermining therapeutic regimens to treat these diseases. In order to identify new drug targets, the P.I.'s lab focuses on enhancing the understanding of cell biological processes wherein the parasite differs from its host. One such process is cell division since it is morphologically distinct from mammalian cell division and lies at the core of the pathology associated with apicomplexan diseases. Deciphering parasite cell division is also of interest in understanding the evolution of cell division in different eukaryotic lineages. Specifically, under this proposal daughter budding will be studied using Toxoplasma tachyzoite cell division as a simple and accessible model. Tachyzoites divide by internal budding, wherein two daughter cells are assembled inside the mother cell. The daughter cytoskeletons form around the duplicated centrosomes, and subsequently elongate to serve as scaffold for organelle genesis and partitioning. How the cytoskeleton building blocks assemble on the centrosome is not well understood. However, it is now established that many components are unique to the parasite and are not shared with the mammalian host. Furthermore, halfway through assembly of this cytoskeleton scaffold a contractile force starts to taper the daughters toward the basal end. The basal complex responsible for this contraction is the functional ortholog of the mammalian contractile ring, but interestingly, its constriction is independent of actin polymerization. In fact, the motor that powers the basal complex is still unknown. In sum, despite our basic knowledge of the structural components driving cytokinesis, we still lack detailed information on how it is powered and how the various steps are controlled and coordinated. Under this proposal the researchers will dissect putative phosphorylation controls of cytokinesis. Through several recent studies of cytokinesis several kinases and phosphatases with apparent critical roles in different cell division steps have already been identified. The functions of these enzymes will be dissected by knock-out studies as well as kinase substrate identification studies. Independent of this first goal, the enigmatic mechanism underlying basal complex constriction will be unraveled. Candidate motor proteins will be experimentally validated next to the pursuit of a candidate independent approach. The latter entails the chemical genetic Bio-ID approach and, in conjunction with super-resolution microscopy, will lead to the molecular definition of the basal complex architecture. Upon completion of this proposal the researchers expect to have characterized critical phosphorylation controls at the different stages in the cell division process, and to have identified the mechanism driving basal complex constriction. Both these milestones will provide specific drug targets serving as jump off points for future work.