Dissection of formin functions in cell edge protrusion and migration

细胞的迁移过程和任何类型的形状变化都依赖于肌动蛋白细胞骨架细丝的动态重组。为此，许多细胞类型采用网状、扁平和指状的尖状结构，分别称为板足和丝状足。这些结构的形成是由大量的信号分子和肌动蛋白细胞骨架的调节因子直接或间接驱动的，但所有这些因素的复杂相互作用尚不完全清楚。肌动蛋白细丝的直接调节是通过胞质分子结合肌动蛋白单体或细丝，甚至催化单体向细丝的转变（称为成核）来完成的，formin家族的蛋白质就是一个突出的例子。哺乳动物表达15个formin家族成员，其确切的个体功能仍未得到充分研究。这组蛋白的一些成员，如mDia和FMNL，已知会影响细胞突起的形成，如板足和丝足，但它们如何精确地做到这一点，15个formin家族成员中有多少人可以共享这一功能，以及它们对这些过程的精确相对贡献在很大程度上仍然未知。特别是，在迁移细胞的突出前部形成丝状足，这被认为是感觉功能，被认为是由formin活性调节的，但是formin家族的哪些成员或是否有任何成员对哺乳动物细胞中这些结构的形成至关重要，迄今为止还不能确定。本文提出的项目将澄清这些问题，从使用CRISPR/Cas9在实验室中已经建立的细胞系开始，例如单独或组合缺乏FMNL2和FMNL3的细胞系，并将其扩展到缺乏formin家族成员的新细胞系，这些成员构成参与丝状足形成的有希望的成员，如mDia2， mDia3和DAAM。所有这些formin成员都将被遗传定位，并分析它们对丝状足形成的贡献，无论是单独的还是与先前鉴定的品系相结合。为了做到这一点，将优化各自细胞系的生长和信号条件，允许在计算机辅助的方式下形成稳健且易于量化的丝状足。此外，在至少两种来源不同的互补亲本细胞系中建立丝足形成的已定义分子机制的共性，将保证选择与广泛的细胞类型和/或发育阶段相关的一般机制和途径。最后，已建立的细胞系也将被用于鉴定迄今为止未知的形成蛋白，这些形成蛋白可能涉及所谓的母丝的产生，这些母丝是指板足肌动蛋白网络中Arp2/3复合物依赖的丝分支。所有这些问题的澄清将大大改善我们对细胞肌动蛋白重组和运动过程的理解。

Migratory processes and any type of shape change in cells critically depend on dynamic reorganization of filaments of the actin cytoskeleton. For this, many cell types employ web-like, flat and fingerlike, pointed structures termed lamellipodia and filopodia, respectively. Formation of these structures is directly and indirectly driven by a large number of signalling molecules and regulators of the actin cytoskeleton, but the intricate interplay of all these factors is incompletely understood. Direct regulation of actin filaments is accomplished by cytosolic molecules binding either actin monomers or filaments, or even catalysing the transition of monomer to filament, called nucleation, as prominently exemplified by the formin family of proteins. Mammals express 15 formin family members, the precise individual functions of which are still understudied. Some members of this group of proteins, such as mDia and FMNL are known to affect the formation of cellular protrusions such as lamellipodia and filopodia, but how precisely they do this, how many of the 15 formin family members can share this function, as well as their precise relative contributions to these processes have largely remained unknown. In particular, the formation of filopodia at the protrusive front of migrating cells, which have been ascribed sensory functions, are proposed to be regulated by formin activity, but which members of the formin family or if any at all are essential for the formation of these structures in mammalian cells could not be established to date. The project proposed here will clarify these questions, starting from cell lines already established in the lab using CRISPR/Cas9, for instance lines lacking FMNL2 and FMNL3 individually and in combination, as well as extending those to novel cell lines lacking formin family members constituting promising members concerning involvement in filopodia formation, such as mDia2, mDia3 and DAAM. All these formin members will be genetically targeted and analysed for their contribution to filopodia formation individually and in combination with previously characterised lines. To do these, growth and signalling conditions of respective cell lines will be optimized allowing robust and easily quantifiable filopodia formation in a computer-aided fashion. Moreover, establishment of commonalities of defined molecular mechanisms of filopodia formation in at least two complementary parent cell lines of distinct origin will guarantee to select for general mechanisms and pathways, relevant for a broad range of cell types and/or developmental stages. Finally, established cell lines will also be employed for identification of hitherto unknown formins potentially involved in the generation of so called mother filaments for Arp2/3 complex-dependent branching of filaments in lamellipodial actin networks. Clarification of all these issues will significantly refine our understanding or cellular actin reorganization and motility processes.