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（由申请方提供）：顶复门寄生虫是严重人类疾病的原因，包括疟原虫属。引起疟疾，弓形虫引起脑炎和出生缺陷，隐孢子虫引起腹泻。耐药性和/或特异性差不断破坏治疗这些疾病的治疗方案。为了确定新的药物靶点，P.I.他的实验室致力于提高对寄生虫与宿主不同的细胞生物学过程的理解。一个这样的过程是细胞分裂，因为它在形态上不同于哺乳动物细胞分裂，并且位于与顶复门疾病相关的病理学的核心。破译寄生虫细胞分裂也是理解不同真核细胞谱系中细胞分裂进化的兴趣所在。具体而言，根据这一建议，女儿出芽将研究使用弓形虫速殖子细胞分裂作为一个简单和方便的模型。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，随后伸长，作为细胞器发生和分配的支架。细胞骨架构件如何在中心体上组装尚不清楚。然而，现在已经确定，许多成分是寄生虫独有的，并且不与哺乳动物宿主共享。此外，在这种细胞骨架支架组装的中途，收缩力开始使子体朝向基端逐渐变细。负责这种收缩的基础复合物是哺乳动物收缩环的功能直系同源物，但有趣的是，它的收缩是独立的肌动蛋白聚合。事实上，驱动基底复合体的马达仍然是未知的。总而言之，尽管我们对驱动胞质分裂的结构成分有基本的了解，但我们仍然缺乏关于胞质分裂是如何被驱动的以及各个步骤是如何被控制和协调的详细信息。在这个提议下，研究人员将剖析胞质分裂的假定磷酸化控制。通过最近对胞质分裂的几项研究，已经确定了几种在不同细胞分裂步骤中具有明显关键作用的激酶和磷酸酶。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。独立于第一个目标，基底复合体收缩的神秘机制将被揭开。候选马达蛋白将在寻求候选独立方法之后进行实验验证。后者需要化学遗传Bio-ID方法，并与超分辨率显微镜相结合，将导致基础复杂结构的分子定义。在完成这项提案后，研究人员希望能够在细胞分裂过程的不同阶段表征关键的磷酸化控制，并确定驱动基底复合物收缩的机制。这两个里程碑将提供特定的药物靶点，作为未来工作的起点。