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联肌动蛋白丝、非肌肉肌球蛋白II和相关蛋白。该层的组装、收缩和力学是细胞形状的决定性因素，因此在包括迁移、细胞分裂或组织形态发生的各种过程中起着核心作用。到目前为止，我们可以证明，在Dictyosteoblastoma细胞的细胞皮质的完整性是由三个皮质formins的保障。细胞皮质调节中的保守功能现在也可以归因于mDia 1和mDia 3在哺乳动物细胞中的协同活性。然而，有趣的是，对所有这些突变体的分析清楚地表明，与粘附性较低的细胞类型相比，粘附性较强的NIH 3 T3细胞中的细胞皮质缺陷在更大程度上影响相对迁移率。因此，表征与强粘附NIH 3 T3成纤维细胞中这些皮质形成蛋白丢失相关的机械特性和机制是本后续提案的主要目标之一。为了充分了解细胞功能和协同formin的建立和维护细胞皮层，它是绝对不可或缺的，此外有关于这些肌动蛋白组装因子的完整库存的知识。由于在mDia 1/3-KO细胞中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由至少一个额外的线粒体支持。最可能的候选者是Rho效应子mDia 2。因此，我们还旨在使用CRISPR/Cas9技术产生mDia 2缺陷型小鼠成纤维细胞，以评估来自mDia-subfamily的这种剩余mRNA在细胞迁移和皮质调节中的功能。在动物细胞中的细胞皮层力学中解剖细胞功能无疑是一项具有挑战性的任务，但我们预计，通过各种互补方法进行检查，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，如工作计划中所述，将对我们理解细胞皮层组装和细胞力学产生重大而广泛的影响。

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.