Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis

During endocytosis in S. cerevisiae, actin polymerization is proposed to provide the driving force for invagination against the effects of turgor pressure. In previous studies, Ysc84 was demonstrated to bind actin through a conserved N-terminal domain. However, full length Ysc84 could only bind actin when its C-terminal SH3 domain also bound to the yeast WASP homologue Las17. Live cell-imaging has revealed that Ysc84 localizes to endocytic sites after Las17/WASP but before other known actin binding proteins, suggesting it is likely to function at an early stage of membrane invagination. While there are homologues of Ysc84 in other organisms, including its human homologue SH3yl-1, little is known of its mode of interaction with actin or how this interaction affects actin filament dynamics. Here we identify key residues involved both in Ysc84 actin and lipid binding, and demonstrate that its actin binding activity is negatively regulated by PI(4,5)P2. Ysc84 mutants defective in their lipid or actin-binding interaction were characterized in vivo. The abilities of Ysc84 to bind Las17 through its C-terminal SH3 domain, or to actin and lipid through the N-terminal domain were all shown to be essential in order to rescue temperature sensitive growth in a strain requiring YSC84 expression. Live cell imaging in strains with fluorescently tagged endocytic reporter proteins revealed distinct phenotypes for the mutants indicating the importance of these interactions for regulating key stages of endocytosis

A review of non-destructive testing techniques for the in-situ investigation of fretting fatigue cracks

© 2020 The Authors Fretting fatigue can significantly reduce the life of components, leading to unexpected in-service failures. This phenomenon has been studied for over a century, with significant progress being made during the past decade. There are various methods that have been used to study fretting fatigue cracks in order to gain a greater understanding of the effects of fretting fatigue. Destructive methods are traditionally used to observe fretting fatigue cracks. Although useful in determining crack location, crack length, crack propagation modes, crack path and shape, it is not efficient or reliable for time based measurements. Non-destructive testing has developed in recent years and now in-situ monitoring can be used during testing in order to increase the understanding of fretting fatigue. This paper presents a review of non-destructive testing techniques used in-situ during fretting fatigue testing, which are compared in order to conclude the suitability of each technique. Recent developments in non-destructive techniques that could be also applied for fretting fatigue tests are also discussed, as well as recommendations for future research made