The MYO6 interactome: selective motor-cargo complexes for diverse cellular processes

Myosins of class VI (MYO6) are unique actin-based motor proteins that move cargo towards the minus ends of actin filaments. As the sole myosin with this directionality, it is critically important in a number of biological processes. Indeed, loss or overexpression of MYO6 in humans is linked to a variety of pathologies including deafness, cardiomyopathy, neurodegenerative diseases as well as cancer. This myosin interacts with a wide variety of direct binding partners such as the selective autophagy receptors optineurin, TAX1BP1 and NDP52 and also Dab2, GIPC, TOM1 and LMTK2, which mediate distinct functions of different MYO6 isoforms along the endocytic pathway. Functional proteomics has recently been used to identify the wider MYO6 interactome including several large functionally-distinct multi-protein complexes, which highlight the importance of this myosin in regulating the actin and septin cytoskeleton. Interestingly, adaptor-binding not only triggers cargo attachment, but also controls the inactive folded conformation of MYO6. Thus, the C-terminal tail domain mediates cargo recognition and binding, but is also crucial for modulating motor activity and regulating cytoskeletal track dynamics.This work was funded by grants from the BBSRC (BB/R001316/1) and Medical Research Council (MR/N000048/1 and MR/S007776/1) to F.B. and a PhD studentship to J.J. de J. from the CIMR departmental funds

Quantitative characterization of the auxin-inducible degron: a guide for dynamic protein depletion in single yeast cells

Perturbations are essential for the interrogation of biological systems. The auxin-inducible degron harbors great potential for dynamic protein depletion in yeast. Here, we thoroughly and quantitatively characterize the auxin-inducible degron in single yeast cells. We show that an auxin concentration of 0.25 mM is necessary for fast and uniform protein depletion between single cells, and that in mother cells proteins are depleted faster than their daughters. Although, protein recovery starts immediately after removal of auxin, it takes multiple generations before equilibrium is reached between protein synthesis and dilution, which is when the original protein levels are restored. Further, we found that blue light, used for GFP excitation, together with auxin results in growth defects, caused by the photo-destruction of auxin to its toxic derivatives, which can be avoided if indole-free auxin substitutes are used. Our work provides guidelines for the successful combination of microscopy, microfluidics and the auxin-inducible degron, offering the yeast community an unprecedented tool for dynamic perturbations on the single cell level