Relating specific biochemical activities of ADF/cofilin family members to their physiological functions

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The dynamic turnover of actin filament networks is essential for a number of important cellular processes including establishment and maintenance of cell morphology, endocytosis and cell migration. Key players known to catalyze subunit turnover by severing and depolymerizing filaments belong to a family of small and ubiquitous proteins jointly called actin depolymerization factor (ADF)/cofilin. Vertebrates express three ADF/cofilin family members: ADF, cofilin1 and cofilin2. The inactivation of any of the three ADF/cofilin members in the mouse leads to discrete phenotypes in spite of continuous expression of the remaining two family members. Based on these findings, we hypothesize individual ADF/cofilin family members to exert distinct biochemical activities, i.e. concerning binding to G or F-actin, concerning severing or nucleation of actin filaments, either by themselves or in alliance with the cofilin-potentiating proteins coronin, Aip1 or CAP. The objective of the proposed work is to directly compare and quantitate the activities of the three mouse proteins and of phosphomimetic mutants, either in the absence or presence of these accessory disassembly factors, in order to uncover these differences and relate them to physiology. Additionally, we seek to examine the biochemical properties of as yet uncharacterized mutations in human cofilin2 causing nemaline and myofibrilar myopathies, with the aim to directly correlate them with the development of disease. To accomplish these ambitious goals, we will first and foremost employ advanced in vitro TIRF microscopy in conjunction with other biochemical and cell culture-based assays. Owing to rapid technological progress in the field and high demand from this and eight other SPP1464 projects, we here apply for the project-relevant acquisition of an advanced state-of-the-art TIRF microscope allowing single molecule detection combined with simultaneous multi-color imaging. The requested TIRF system will be exclusively used by the project partners of the SPP1464 to study the dynamics of actin-based processes on isolated proteins or protein complexes. These analyses will focus on the functions of various formins, WH2- domain-based nucleators and other actin-binding proteins in the assembly or disassembly of actin filaments. Single molecule multi-color TIRF imaging will allow us to address numerous, contemporary scientific questions and monitor actin assembly or disassembly reactions with supreme precision and at a high temporal resolution. We are confident therefore that these studies will have significant implications for our understanding of the multitude of mechanisms underlying actin-based motility.