1 Introduction

In eukaryotic cells endocytosis is essential for the uptake of many nutrients, for defence against invading microorganisms and for the regulated uptake of cell surface receptors, thus linking endocytosis to cell signalling [1, 2]. Following internalisation the endocytosed material is transferred to early endosomes from where it is either recycled back to the cell surface or transported further into the cell, where it can be found in late endosomes or lysosomes. The site of endocytosis is the plasma membrane, which is directly linked to the underlying actin network in the cell cortex. Genetic evidence in yeast links the organisation of the actin cytoskeleton to both receptor mediated and fluid phase endocytosis [3, 4]. In higher eukaryotic cells the link between endocytosis and the actin cytoskeleton is less certain and the evidence conflicting, being dependent on the cell type and the assay used [5, 6]. However, since an increasing number of proteins associated with the endocytic machinery also interact with the actin cytoskeleton a link is strongly suggested (reviewed in [7]). The filamentous actin network could either provide a structural framework for the spatial organisation of the endocytic machinery and/or serve a more indirect function by providing the tracks for the movement of myosin motor proteins involved in force generation and vesicle movement.

2 Myosins

The myosins are a diverse superfamily of actin activated mechanoenzymes that use the energy from ATP hydrolysis to generate force and movement along actin filaments. Each myosin can be divided into two functional domains: (1) an N-terminal motor domain, which is comprised of a catalytic domain that binds actin and ATP, a ‘converter’ region and a regulatory neck region (‘lever arm’) that binds light chains or calmodulin and (2) a C-terminal tail domain, which anchors the myosin and may carry cargo [8]. The myosin superfamily comprises at least 18 different classes and so far only myosins in classes I, V, VI and VII have been directly implicated in membrane traffic [9].

3 Myosin VI, a motor protein with unique properties

In this review we focus on myosin VI and discuss its possible role(s) in receptor mediated endocytosis. Myosin VI is ubiquitously expressed in higher eukaryotic tissues, but is absent in Dictyostelium and yeast. It has been shown to be important for hearing in mouse and human [10, 11] and for embryogenesis and spermatogenesis in Drosophila [12, 13]. Myosin VI is a dimeric molecule with two motor domains, a short region of coiled coil and two globular tails. We have shown that myosin VI is phosphorylated in vivo and that it can be phosphorylated in vitro in the motor domain by a p21 activated kinase (PAK) [14]. Although the phosphorylation site has not yet been directly identified, considerable circumstantial evidence indicates it is T₄₀₆ in agreement with the TEDS rule (consensus sequence position in the motor domain present in nearly all myosins which is either a threonine (T), glutamate (E), aspartate (D) or serine (S)) proposed by [15]. Recent kinetic data [16] measuring the rates of the steps in the ATPase cycle of myosin VI have shown that a mutant (T₄₀₆E) mimicking phosphorylation at the T₄₀₆ site has an accelerated rate of P_i release in the presence of actin. Yoshimura et al. [17] however find that phosphorylation of myosin VI at T₄₀₆ is not required for actin activated ATPase activity but is essential for generating actin filament sliding. How phosphorylation activates the motile activity of myosin VI is not known. Both groups [16, 17] agree that myosin VI has a high affinity for ADP in the presence of MgATP and remains strongly attached to actin during most of its ATPase cycle. Unlike all the other myosins that have so far been studied, which move towards the plus end of actin filaments, myosin VI is unique in moving towards the minus ends of actin filaments [18]. Myosin VI has a unique 53 amino acid insert in the converter region of the catalytic domain between the motor domain and the lever arm, which was thought to be at least partly responsible for the movement in the reverse direction. However it has recently been demonstrated [19] using chimaeric myosins composed of motor domains and lever arm domains from myosins that move in opposite directions on actin, that it appears to be the core motor domain that determines the direction of myosin movement. The minus end directionality would support a role of myosin VI as an endocytic motor protein, since inside cells the actin filaments which are present in filopodia, lamellipodia, microvilli, stereocilia and in the actin cortical network have their plus ends inserted into or pointed towards the plasma membrane and their minus ends pointing into the cell. Myosin VI moving towards the minus ends of actin filaments would thus move endocytic cargo vesicles into the cell (see Fig. 3).

4 Myosin VI in polarised cells

Myosin VI is expressed as three different splice variants containing either a large, a small or no insert in the tail domain, as first described in Drosophila [20] and more recently in fish [21]. In mammalian cells these splice isoforms display a tissue specific distribution with the splice variant containing the large insert specifically found in tissues containing polarised cells with microvilli at their apical surface [22]. In polarised cells from the intestinal brush border, myosin VI is concentrated in the terminal web [22, 23] with small amounts in the microvilli themselves. Myosin VI present at the apical domain is localised to clathrin coated pits/vesicles. In non-polarised cells the myosin VI isoforms without the large insert are expressed and these colocalise to a much lesser extent with clathrin coated pits/vesicles [22]. Overexpression of GFP tagged myosin VI containing the large insert, cloned from a polarised tissue, in unpolarised fibroblastic cells results in colocalisation with clathrin coated pits/vesicles (see Fig. 1 and [22] for more detail). The association of myosin VI with the endocytic machinery in polarised cells appears to be one of the key functions of myosin VI. Absence of myosin VI in Snell's Waltzer mice (myosin VI knock out) results in defects which are most apparent in polarised cells; for example the lack of myosin VI leads to degeneration of the stereocilia, which resemble highly specialised microvilli at the apical domain of the hair cells of the inner ear, and this results in a loss of hearing [24]. In addition, in these mutant mice the microvilli on intestinal brush border cells are shorter when compared to WT mice ([24], Buss and Stewart, unpublished observation). Since the Snell's Waltzer mice survive there must be motors and possibly other proteins, which are able to at least partially compensate for the loss of myosin VI in most cells apart from those in the inner ear.

5 What precise role(s) does myosin VI have in receptor mediated endocytosis?

The clear association of myosin VI with clathrin coated pits/vesicles in polarised cells, raises the question at which step in the formation and the transport of a clathrin coated pit or vesicle is myosin VI involved? Is myosin VI required for (1) the spatial organisation of the endocytic machinery in the plasma membrane; (2) sequestering of receptors and coated pit formation; (3) invagination of the plasma membrane; (4) pinching off newly formed vesicles or (5) the transport of vesicles away from the plasma membrane into the cell?

The specific localisation of the myosin VI isoform with the large tail insert to endocytic clathrin coated vesicles concentrated at the apical domain of polarised cells compared to non-polarised fibroblastic cells may provide us with a clue as to which step in the pathway myosin VI is involved. As mentioned above actin may not be obligatory for receptor mediated endocytosis per se in all cell types. For example depolymerisation of the actin cytoskeleton using drugs like cytochalasin D and latrunculin A, which cause F-actin filament disassembly, does not appear to inhibit receptor mediated endocytosis in non-polarised, adherent A431 or COS-7 cells [5]. In contrast, in polarised cells containing apical microvilli with their highly specialised actin cytoskeleton and plasma membrane organisation, as for example in human intestinal brush border Caco-2 cells or MDCK cells (Madin–Darby canine kidney cells), the requirement for actin in clathrin coated vesicle uptake from the apical domain is unambiguous. Treating these cells with cytochalasin D blocks clathrin coated vesicle uptake specifically at the apical domain and leads to an increase in the number of coated pits at the apical surface (Fig. 2a ), indicating that actin may be important for pinching off vesicles from the plasma membrane [25-27]. We therefore believe that actin, in association with motor proteins like myosin VI, is needed for receptor mediated endocytosis in certain cells under specific physiological conditions or in cells with highly specialised plasma membrane domains.

6 Sequential steps in endocytosis

Clathrin mediated uptake at the plasma membrane can be divided into a number of steps (see Fig. 3 ) and in this review we will discuss the evidence for myosin VI involvement in each. At the EM level myosin VI can be found at a very early stage of clathrin coated pit formation, at the step where coated pits are deeply invaginated and it is also present in clathrin coated vesicles, which appear to have pinched off from the plasma membrane (Fig. 2b).

7 What kind of motor is myosin VI? Is it processive or non-processive?

Recent work by a number of groups [16, 17, 19] probing the kinetic mechanism of myosin VI activity provide us with further clues to the potential role(s) of this myosin in the cell or at least may exclude certain functions. They demonstrate that ADP release was the rate limiting step in the ATPase cycle and that the myosin VI ADP state bound strongly to actin. Furthermore their kinetic data suggested that myosin VI could be a processive, dimeric (two motor domain) motor with at least one motor domain always attached to actin; in other words it has a high duty ratio (it spends a significant proportion of its ATPase cycle strongly bound to actin) and is a motor ideally adapted for maintaining tension. De La Cruz et al. [16] propose an ‘alternating site’ model for the stepping and processivity of the dimeric myosin VI along actin filaments which may allow us to further define the role of this motor in endocytic pathways; for example myosin VI by exerting tension could anchor the endocytic machinery to the underlying actin cytoskeleton or pull membrane into the cell to form a clathrin coated pit or transport endocytic vesicles processively along actin filaments into the cell. Myosin V has similar properties to myosin VI [16] and has been shown to move processively along actin filaments but in the opposite direction [38, 39]. Myosin V has a long lever arm with six IQ (calmodulin binding) repeats and can achieve a step size of 36 nm ‘walking’ along an actin filament (equivalent to the helical repeat of the actin filament) whereas myosin VI has a short lever arm with only a single IQ repeat. So if myosin VI is processive and needs a large step size to move vesicles in a straight path along an actin filament then it would require that either the coiled coil domain of the tail unfolds possibly involving the region of charged repeats observed in the middle of this domain [14] and/or the flexibility of the insert between the motor domain and lever arm changes or some other still to be identified mechanism acts to achieve such a step. Future studies promise to yield exciting results.

8 Myosin VI as an endocytic motor in differentiation

If myosin VI works as an endocytic motor, how can we explain its key role in hearing, embryo development and spermatogenesis? In Snell's Waltzer mice, which are missing myosin VI, the major phenotype affects the sensory hair cells of the inner ear. At birth the rows of microvilli in the hair cells which form the stereocilia have a normal appearance but 3 days after birth as the stereocilia elongate and mature, they start fusing together. In wild type mice this phase of stereocilia maturation is accompanied by extensive membrane recycling at the apical domain of these hair cells and clathrin coated pits can easily be visualised at the base of immature stereocilia [35]. Since myosin VI has been shown to specifically associate with clathrin coated pits/vesicles at the apical domain of polarised cells, it is likely that myosin VI plays a vital role in endocytosis at the apical domain during hair cell maturation. In Drosophila, myosin VI is required at various developmental stages including imaginal disc morphogenesis, follicle cell epithelial morphogenesis [36], formation of membrane invagination in the syncytial blastoderm [12] and sperm individualisation during spermatogenesis [13]. The latter process involves the formation of a complex machinery bringing together cytoskeleton and membrane remodelling. The only proteins identified so far as being crucial in this process are myosin VI [13], actin and the coat protein clathrin [37]. This evidence indicates that endocytosis and membrane recycling form an important step during sperm individualisation and provide further support for the proposal that myosin VI works as an endocytic motor.