Dissecting the mechanism and regulation of Toxoplasma cytokinesis

剖析弓形虫胞质分裂的机制和调控

• 批准号: 9128297
• 负责人: Marc-Jan Gubbels
• 金额: $ 48.37万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9128297
• 关键词: Actins; Alveolar; Architecture; Automobile Driving; Biochemical; Biological Process; Biology; Biotin; Biotinylation; Cell Division Process; Cell division; Cells; Centrosome; Clinical; Complement; Complex; Congenital Abnormality; Cryptosporidium parvum; Cytokinesis; Cytoskeleton; Data; Daughter; Development; Diarrhea; Disease; Dissection; Drug Targeting; Drug resistance; Encephalitis; Enzymes; Eukaryota; Event; Evolution; Family; Future; Gatekeeping; Genes; Goals; Health; Intermediate Filaments; Knock-out; Knowledge; Lead; Ligase; Lytic; Lytic Phase; Malaria; Mammalian Cell; Mass Spectrum Analysis; Mediating; Membrane; Microscopy; Mitosis; Modeling; Modification; Molecular; Mothers; Motor; Mutation; Myosin ATPase; Organelles; Orthologous Gene; Parasites; Pathogenesis; Pathology; Pharmaceutical Preparations; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Plasmodium; Process; Proteins; Regimen; Regulation; Research Personnel; Resolution; Role; Severities; Specificity; Staging; Structure; Sum; Tetracyclines; Therapeutic; Therapeutic Intervention; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Validation; Variant; Vesicle; Work; analog; chemical genetics; constriction; daughter cell; dynein light chain; human disease; in vitro Assay; inhibitor/antagonist; insight; interest; kinase inhibitor; mutant; polymerization; promoter; recombinase; research study; scaffold; thiophosphate; tool

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.

描述（由申请人提供）：Apicomplexan寄生虫造成严重的人类疾病，包括疟原虫属。引起疟疾，弓形虫引起脑炎和出生缺陷，以及孢子虫的疟原虫引起腹泻。耐药性和/或特异性不断破坏治疗治疗这些疾病的治疗方案。为了识别新药靶标，P.I.的实验室重点是增强对寄生虫与宿主不同的细胞生物过程的理解。这样一个过程就是细胞分裂，因为它在形态上与哺乳动物细胞分裂不同，并且位于与Apicomplexan疾病相关的病理学的核心。解密的寄生虫细胞分裂也很感兴趣地了解不同真核谱系中细胞分裂的演变。具体而言，在此提案下，将使用弓形虫tachyzoite细胞分裂作为一种简单且可访问的模型来研究女儿的出现。 tachyzoites除以内部出芽，其中两个子细胞组装在母细胞内。女儿的细胞骨架在重复的中心体周围形成，随后伸长成为细胞器创世纪和分隔的支架。如何在中心体上组装细胞骨架的构建块尚不清楚。但是，现在已经确定，许多成分是寄生虫所独有的，并且与哺乳动物宿主没有共享。此外，在这种细胞骨架支架组装的一半中，收缩力开始逐渐缩小女儿的基础端。负责这种收缩的基底复合物是哺乳动物收缩环的功能性直系同源物，但有趣的是，其收缩与肌动蛋白聚合无关。实际上，为基础配合物提供动力的电动机仍然未知。总而言之，尽管我们对驱动细胞因子的结构成分的基本了解，但我们仍然缺乏有关其如何供电以及如何控制和协调各种步骤的详细信息。根据该建议，研究人员将剖析细胞因子的推定磷酸化控制。通过对细胞因子的几项研究，已经鉴定出了几种激酶和磷酸酶在不同的细胞分裂步骤中具有明显关键作用的研究。这些酶的功能将通过敲除研究以及激酶底物鉴定研究来解剖。独立于第一个目标，将揭示基础复合物收缩的神秘机制。候选运动蛋白将在追求候选独立方法旁边的实验验证。后者需要化学遗传生物ID方法，并结合超分辨率显微镜，将导致基底复合体结构的分子定义。该提案完成后，研究人员期望在细胞分裂过程的不同阶段表征临界磷酸化控制，并确定了驱动基础复合物收缩的机制。这两个里程碑都将提供特定的药物靶标，可作为未来工作的跳跃点。