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

有丝分裂纺锤体是一种基本的细胞结构，它在生长和分裂时将遗传物质（染色体）准确地传递给后代细胞。遗传物质的准确分离和传递到后代细胞对于细胞活力和维持基因组完整性是必不可少的。如果不能忠实地做到这一点，就会导致个体染色体的增加或丢失，这种情况被称为非整倍体，这是癌细胞的标志。许多细胞“机器”参与了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作用，它通过使MT成核和组织两个有丝分裂纺锤体极。MT成核也发生在不同的细胞区域，包括染色体、动粒和有丝分裂纺锤体本身。这些不同的MT一起都有助于组装强大的有丝分裂纺锤体。一组特殊的MT，称为星形微管，从两个有丝分裂纺锤体极发出，并与细胞皮质相互作用，以确保纺锤体的正确定位（例如在新皮质的发育过程中）。 MT成核机制与马达（驱动蛋白和动力蛋白）和非马达蛋白协同工作，以确保有丝分裂纺锤体的稳定性和染色体的精确运动。任何上述过程中的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，更好地理解这些细胞过程的分子机制是很重要的。在这里，我们将开发一个围绕单细胞、基于表型的基因组编辑筛选（使用CRISPR-Cas9系统）构建的下一代发现平台。这种自动化平台将与高通量格式的单细胞分离/分析兼容，从低分辨率到超分辨率细胞成像。基于光刻的微图案化将用于对单个细胞施加几何约束，以允许对表型进行更稳健的检测，并限制通常困扰功能筛选的细胞间变异性问题。 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因子，重点是PCM组织，MT成核和有丝分裂纺锤体定位。总的来说，这项工作将导致下一代基因发现管道的开发，该管道与前所未有的时空分辨率下的表型分析兼容，并提供对有丝分裂纺锤体组装和长期其他细胞过程的更好的机械理解。

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.*****