The myosins in class VI are distinguished in their role as intracellular motor proteins by their unique ability to move toward the minus end of actin filaments (1
). This directional bias, determined by the presence of a 53-amino acid insert (reverse gear) proximal to the motor domain (2
), renders myosin VI a critical functional component of several, direction-specific intracellular processes. For example, in the secretory pathway, myosin VI maintains Golgi complex morphology (3
) and is required for the sorting of cargo to the leading edge of motile cells (4
) and to the basolateral domain of polarized epithelial cells (5
). In this pathway, myosin VI has also been shown to modulate the fusion of secretory vesicles with the plasma membrane during the final stages of exocytosis (6
Myosin VI has also been broadly implicated in endocytosis, the critical cellular pathway by which essential proteins and nutrients are transferred from the external environment to the interior of a cell. The mammalian myosin VI isoform with a 21–31-amino acid “large insert” (LI)3
just before the C-terminal cargo-binding portion of the tail domain (7
) is specifically linked with the early stages of the clathrin-mediated endocytic pathway, a subcategory of endocytosis involving uptake into pits and vesicles coated with the protein clathrin (“clathrin-coated structures”). This myosin VI LI splice variant is selectively expressed in polarized epithelial cells, where it co-localizes at the apical domain with clathrin-coated structures (7
) and has been shown by total internal reflection fluorescence microscopy to be actively recruited to clathrin-coated structures (8
). Studies from the myosin VI KO mouse, the Snell's waltzer mouse, have demonstrated that myosin VI is essential for cystic fibrosis transmembrane conductance regulator endocytosis in enterocytes (9
), as well as for the endocytic uptake of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPARs) in hippocampal neurons (10
). Myosin VI is also involved in the later stages of endocytosis. The myosin VI no insert (NI) isoform has been implicated in the transport of uncoated endocytic vesicles through the cortical actin network to early endosomes (11
) and is required for the sorting of cargo at early endosomes and the delivery of cargo from early endosomes to endocytic recycling compartments (12
It is crucial to understand the specifics of these varied functional roles for myosin VI, as abnormalities in myosin VI function have been linked to such diseases as prostate and ovarian cancer (14
), hypertrophic cardiomyopathy (16
), neurodegeneration (10
), and deafness (17
). Perhaps the most critical target for such directed studies of myosin VI function is the well established role for myosin VI in the vesicle transport processes of the endocytic uptake pathway, as it is likely that this function makes a major contribution to the motor disease involvement. For example, defects in myosin VI-driven endocytic membrane transport processes in stereocilia may cause deafness, absence of myosin VI causes accumulation of cystic fibrosis transmembrane conductance regulator in enterocytes leading to secretory diarrhea, and the lack of endocytosis of specific glutamate receptors in myosin VI-depleted cells may result in neurodegeneration (7
). Thus, a thorough understanding of the roles of myosin VI in endocytosis is critical for identifying the endocytic malfunctions that may cause specific diseases and the subsequent development of new targeted treatments to correct these malfunctions.
Although some of the features of myosin VI involvement in endocytosis are known (e.g.
the binding partners involved), the specifics of its function on this pathway still require direct investigation. It has been suggested that the myosin VI LI isoform in polarized epithelial cells plays a role in the clustering of cell surface receptors into a clathrin-coated pit, in the formation of clathrin-coated vesicles, and in clathrin-coated vesicle transport away from the plasma membrane (21
), but no studies have so far demonstrated directly whether the myosin VI LI isoform plays a short or long term role in such tethering/transport processes or investigated the turnover kinetics of its association with clathrin-coated structures. On a more fundamental level, it is not clear whether myosin VI functions in endocytosis as a monomer or a dimer, a question of great importance in the field given the widespread dissent as to whether myosin VI has a natural tendency toward self-dimerization (23
). In particular, it has not been addressed whether myosin VI might transfer dynamically between an inactive, folded, monomeric conformation in the cytosol (comparable with that of myosin VII) (25
) and an active, unfolded, dimeric conformation (e.g.
when actively bound via Dab2 to clathrin-coated vesicles) (24
In this study, we investigate the specifics of the dynamic association of myosin VI with clathrin-coated structures and early endosomes using the microscopic method of fluorescence recovery after photobleaching (FRAP). As an initial probe into the basic kinetics of myosin VI involvement in endocytosis, we demonstrate the dynamic exchange and characteristic half-life of the myosin VI NI isoform on early endosomes and myosin VI LI isoform on clathrin-coated structures. We then demonstrate that the characteristic half-life of myosin VI is shared by Dab2, the binding partner known to recruit myosin VI to clathrin-coated structures. Our FRAP assay is then used to examine the turnover dynamics of myosin VI mutant constructs, a means of gaining insight into the behavior of myosin VI on the endocytic pathway. The results indicate that myosin VI is likely to behave as a functional monomer or as a dynamic monomer/dimer during endocytosis, because a novel artificial dimer construct of myosin VI designed for live cell expression displays strikingly different turnover dynamics from wild type myosin VI. The observation that a tail construct of myosin VI turns over at the same rate as the full-length molecule targets the tail domain as a critical determinant of turnover dynamics. Taken together, these results provide insight into the dynamics and monomer/dimer nature of myosin VI during endocytosis.