A low- to super-resolution imaging pipeline to dissect the molecular mechanisms underpinning mitotic spindle assembly

低至超分辨率成像管道，用于剖析支持有丝分裂纺锤体组装的分子机制

• 批准号: RGPIN-2019-06753
• 负责人: Pelletier, Laurence
• 金额: $ 3.06万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=688423
• 关键词: low; super; resolution; imaging; pipeline

The mitotic spindle is an essential cellular structure that physically carries out the task of accurately transmitting your genetic material (chromosomes) to progeny cells as they grow and divide. The accurate segregation and transmission of genetic material to progeny cells is essential for cell viability and the maintenance of genome integrity. Failure to do so faithfully leads to gains or losses of individual chromosomes, a condition known as aneuploidy, a hallmark of cancer cells.******A number of cellular "machines" participate in the formation of robust mitotic spindles. For example, centrosomes play an important part in mitotic spindle assembly by nucleating MTs and organizing the two mitotic spindle poles. MT nucleation also occurs from distinct cellular locales including chromosomes, kinetochores and within the mitotic spindle itself. Together these different MTs all contribute to the assembly of robust mitotic spindles. A specialized group of MTs, called astral microtubules emanate from the two mitotic spindle poles and interact with the cell cortex to ensure proper spindle positioning (e.g. during the development of the neocortex). MT nucleation mechanisms work in concert with motor (kinesins and dynein) and non-motor proteins to ensure the stability of the mitotic spindle and the precise movement of chromosomes. Defects in any of the above-described processes can have deleterious effect on mitotic spindle assembly and can therefore contribute to deleterious mitotic errors. It is therefore of importance to better understand the molecular mechanism underpinning these cellular processes.******Here, we will develop a next-generation discovery platform built around single-cell, phenotype-based genome editing screens (using the CRISPR-Cas9 system). This automated platform will be compatible with single cell isolation / analytics in high-throughput format from low- all the way to super-resolution cellular imaging. Photolithography-based micropatterning will be used to impose geometrical constraints on individual cells to allow more a more robust detection of phenotypes and to limit cell-cell variability issues that commonly plague functional screens. Once established this platform will be used in large- to genome-scale screens to identify novel regulators of mitotic spindle assembly, with emphasis on PCM organization, MT nucleation and mitotic spindle positioning.******Overall this work will lead to the development of a next generation gene-discovery pipeline compatible with phenotypic profiling at unprecedented spatiotemporal-resolution and provide a better mechanistic understanding of mitotic spindle assembly and in the long run other cellular processes.*****

有丝分裂纺锤体是一种基本的细胞结构，当你的遗传物质（染色体）在后代细胞生长和分裂时，它在物理上执行准确地传递给后代细胞的任务。遗传物质向后代细胞的准确分离和传递对细胞活力和基因组完整性的维持至关重要。如果不能忠实地做到这一点，就会导致单个染色体的增加或减少，这种情况被称为非整倍体，这是癌细胞的一个标志。******许多细胞“机器”参与有丝分裂纺锤体的形成。例如，中心体在有丝分裂纺锤体组装中起着重要的作用，它使mt成核并组织两个有丝分裂纺锤体极点。MT成核也发生在不同的细胞区域，包括染色体、着丝点和有丝分裂纺锤体内部。这些不同的mt一起都有助于强健的有丝分裂纺锤体的组装。一组特殊的mt，称为星体微管，从两个有丝分裂纺锤极发出，并与细胞皮层相互作用，以确保正确的纺锤定位（例如在新皮层的发育过程中）。MT成核机制与运动蛋白（运动蛋白和动力蛋白）和非运动蛋白协同工作，以确保有丝分裂纺锤体的稳定性和染色体的精确运动。上述过程中的任何缺陷都可能对有丝分裂纺锤体组装产生有害影响，因此可能导致有害的有丝分裂错误。因此，更好地理解支撑这些细胞过程的分子机制是很重要的。******在这里，我们将围绕单细胞、基于表型的基因组编辑屏幕（使用CRISPR-Cas9系统）开发下一代发现平台。该自动化平台将兼容从低到超分辨率细胞成像的高通量格式的单细胞隔离/分析。基于光刻的微图案将用于对单个细胞施加几何约束，以允许更可靠的表型检测，并限制通常困扰功能屏幕的细胞-细胞变异性问题。一旦建立，该平台将用于大到基因组规模的筛选，以确定有丝分裂纺锤体组装的新调节器，重点是PCM组织，MT成核和有丝分裂纺锤体定位。******总的来说，这项工作将导致在前所未有的时空分辨率下与表型分析兼容的下一代基因发现管道的发展，并为有丝分裂纺锤体组装和长期其他细胞过程提供更好的机制理解*****