A novel super-resolution microscopy approach to investigate the role of actin filament branching in cancer cell migration.

大多数癌症患者死于转移，这需要癌细胞迁移。细胞迁移的力量是由肌动蛋白聚合提供的。电子显微镜显示，肌动蛋白细丝在固定细胞的前沿被Arp2/3复合体从现有的细丝一侧成核，形成一个分支阵列。然而，这还没有在活的迁移细胞中得到证实，可以用来提供癌基因在癌细胞迁移过程中激活Arp2/3的关键信息。在这个项目中，我们正在研究SCAR/WAVE复合体的三个新的结合伙伴。它们与迁移细胞前沿的疤痕/波浪复合体共定位。我的研究问题是确定这些蛋白质是否调节SCAR/WAVE复合体的招募和活性，已知的是，SCAR/WAVE复合体通过分支核因子Arp2/3复合体控制板脂体内肌动蛋白网络的分支。我还试图确定这些蛋白质如何在定向网络中发挥作用，以及这些网络如何相互作用以产生振荡突起--先前研究中已经看到的前沿的收缩。然后，我希望展示这一调控机制的变化如何表现为细胞迁移机制的变化，从而寻求确定这三种蛋白质在细胞运动背景下的功能和重要性。各种功能和超分辨率显微镜技术结合前沿和单细胞跟踪数据将被用于解决这些问题和假设。后者将促进分析从分子相互作用的水平转移到细胞迁移机制和表型的水平。与生物驱动的实验相结合，将开发新的超分辨率技术，如PAINT/IRIS(单分子定位显微镜的瞬时结合方法)，并将使用最近建立的软件来分析高密度超分辨显微图像。在超分辨率和FRET-FILM中的新的新型探针将被设计来探测结构和信号蛋白质，以及确定它们的活性和相互作用。将改进用于分析细胞迁移的MatLab程序的数学实现，重点是确定迁移限制、速度、持久性和稳定性等参数。这些参数将更好地定量描述细胞迁移的特征，并将产生更具统计学意义的、无偏见的结论。FRET-Flim显微镜将使用我们实验室开发的一种新型Arp2/3生物传感器在细胞中研究Arp2/3复合体的活性，在该传感器中，使用CRISPR/Cas9技术敲除新的SCAR/波结合蛋白的基因。通过重新表达这些SCAR/WAVE结合位点突变的蛋白的cDNA来测试这一调控的功能意义。除了FRET-FLIM，还将开发一种新的超分辨率技术来成像活细胞中的肌动蛋白网络及其分支点：将跟踪Arp2/3复合体的位置，并根据肌动蛋白的全球运动进行校正。此外，计算技术将被用来监测前沿的收缩-突出，并将其与从先前的功能和超分辨率图像中确定的信息相关联，以确定前沿振荡和稳定性的分子起源。最后，细胞追踪分析将确定前沿动力学的变化如何导致细胞运动的变化。最终，目的是汇编这些信息以构建一幅图景：涉及上述三种蛋白质的信号网络的复杂相互作用；这些蛋白质如何改变片状脂体的物理结构；以及这种结构变化如何影响迁移特征。

Most cancer patients die from metastasis which require cancer cells to migrate. The force for cell migration is provided by actin polymerisation. Electron microscopy showed that actin filaments are nucleated at the leading edge of fixed cells by the Arp2/3 complex from the side of existing filaments creating a branched array. However, this has not been proven in live migrating cells and could be used to provide crucial information on Arp2/3 activation by oncogenes during cancer cell migrationIn this project we are studying three novel binding partners of the Scar/WAVE complex. They co-localise with the Scar/WAVE complex at the leading edge of migrating cells. My research question is to determine whether these proteins regulate the recruitment and activity of the Scar/WAVE complex, which is known to control branching of actin networks in lamellipodia via the branch-nucleator, Arp2/3 complex. I am also seeking to determine how these proteins function in directed networks, and how these networks interact to generate oscillatory protrusion-retractions of the leading edge which has been seen in previous research. I hope to then show how changes to this regulation manifest as changes in the mechanisms of cell migration, and thus seek to determine the function and importance of these three proteins in the context of cell motility.A variety of functional and super-resolution microscopy techniques combined with leading-edge and single-cell tracking data will be used to address these questions and hypotheses. The latter will facilitate the analysis to move from the level of molecular interactions to the level of cell-migration mechanisms and phenotype.In conjunction with the biological-driven experimentation, novel techniques in super-resolution such as PAINT/IRIS (transient binding approaches to single molecule localisation microscopy) will be developed and recently established software for the analysis of highly dense super-resolved micrographs will be employed. New novel probes in both super-resolution and FRET-FLIM will be designed to probe both structural and signalling proteins, as well as determine their activity and interactions. Improvements will be made to the mathematical implementations of Matlab programs designed to analyse cell migration, with emphasis on determining parameters such as migratory confinement, speed, persistence, and stability. These parameters will better enable the characterisation of cell migration in a quantitative way, and will result in more statistically robust, unbiased conclusions.FRET-FLIM microscopy will be used to study the activity of the Arp2/3 complex using a novel Arp2/3 biosensor developed in our laboratory in cells in which the genes for the novel Scar/WAVE binding proteins are knocked out using the CRISPR/CAS9 technique. Re-expression of cDNA of these proteins mutant for Scar/WAVE binding sites will be employed to test the functional significance of this regulation. In addition to FRET-FLIM, a novel super-resolution technique will be developed to image the actin network and its branch points in live cells: the positions of the Arp2/3 complex will be tracked and corrected against the global motion of the actin. Furthermore, computational techniques will be employed to monitor the retraction-protrusion of leading edges, and correlate this with information ascertained from prior functional and super-resolution images to determine the molecular origin of leading-edge oscillations and stability. Finally, cell tracking analysis will determine how changes in leading edge dynamics result in changes to cell motility.Ultimately, the aim is to compile this information to build a picture of: the complex interactions of the signalling networks involving the three aforementioned proteins; how these proteins alter the physical structure of lamellipodia; and how this change in structure effects the characteristics of migration.