Function of cortical formins in the motility of strongly adherent cells

肌动蛋白皮质是真核细胞质膜下的一层薄薄的交叉连接的肌动蛋白细丝、非肌肉肌球蛋白II和相关蛋白。这一层的组装、收缩和力学是细胞形态的决定性因素，因此在包括迁移、细胞分裂或组织形态形成在内的各种过程中发挥着核心作用。到目前为止，我们可以证明Dictyostelius细胞中的细胞皮质的完整性是由三种皮质福尔马林保护的。在细胞皮质调节中的保守功能现在也可以归因于哺乳动物细胞中mDia1和mDia3的协同作用。然而，有趣的是，对所有这些Form in突变体的分析清楚地表明，与粘附性较弱的细胞类型相比，粘附性强的NIH3T3细胞中的细胞皮质缺陷对相对迁移率的影响要大得多。因此，研究NIH3T3成纤维细胞的力学特性和与这些皮质福尔马林丢失相关的机制是这项后续计划的主要目标之一。为了充分了解福尔马林在建立和维持细胞皮质中的细胞功能和协同作用，了解这些肌动蛋白组装因子的完整清单是绝对必要的。由于mDia1/3-KO细胞缺乏明显的胞质分裂缺陷，动物细胞中细胞皮质的功能很可能是由至少一个额外的福尔马林支持的。最有可能的候选者是Rho效应器mDia2。因此，我们的目标也是使用CRISPR/Cas9技术来培养mDia2缺陷的小鼠成纤维细胞，以评估mdia2亚家族中的这个剩余的Forin在细胞迁移和皮质调节中的功能。在动物细胞中对Forin功能的剖析无疑是一项具有挑战性的任务，但我们预计，工作计划中规定的各种补充方法的检查，包括先进的生物物理和全面的细胞生物学分析以及敲除细胞系的高分辨率成像，将对我们对细胞皮质组装和细胞力学的理解产生实质性和广泛的影响。

The actin cortex is a thin layer of cross-linked actin filaments, nonmuscle myosin II, and associated proteins beneath the plasma membrane of eukaryotic cells. Assembly, contraction and mechanics of this layer is the decisive factor of cell shape and therefore plays a central role in various processes including migration, cell division or tissue morphogenesis. As yet, we could demonstrate that integrity of the cell cortex in Dictyostelium cells is safeguarded by three cortical formins. Conserved functions in cell cortex regulation can now also be ascribed to synergistic activities of mDia1 and mDia3 in mammalian cells. Interestingly, however, analyses of all these formin mutants clearly showed that cell cortex defects in the strongly adhering NIH 3T3 cells affect relative migration rate to a much larger extent as compared to less adhesive cell types. The characterization of the mechanical properties and the mechanisms associated with loss of these cortical formins in the strongly adhering NIH 3T3 fibroblasts is therefore one of the main objectives in this follow-up proposal. In order to fully understand the cellular function and synergy of formins in the establishment and maintenance of the cell cortex, it is furthermore absolutely indispensable to have knowledge about the complete inventory of these actin-assembly factors. Due to the lack of a noticeable cytokinesis defect in mDia1/3-KO cells, it is more than likely that functionality of the cell cortex in animal cells is backed up by at least one additional formin. The most likely candidate is the Rho effector mDia2. Thus, we also aim to generate mDia2-deficient mouse fibroblast using CRISPR/Cas9 technology to assess the function of this remaining formin from the mDia-subfamily in cell migration and cortex regulation. The dissection of formin function in cell cortex mechanics in animal cells is undoubtedly a challenging task, but we anticipate that examination by various complementary approaches including advanced biophysical and comprehensive cell biological analyses as well as high-resolution imaging of knockout cell lines, as specified in the work programme, will have substantial and wide reaching implications on our understanding on cell cortex assembly and cell mechanics.