Myosin V

Myosin V

Updated 4/9/02

The gene product of the murine “Dilute” mutant turns out to be a myosin V protein. These mice die about three weeks after birth and display a variety of neurological defects, eventually leading to seizures. The name comes from the fact that the coat colour has a washed out appearance due to the failure of melanin containing vesicles to be transported into the hair. Myosin V is a two-headed motor protein whose heavy chains dimerise through a coiled-coil motif (Cheney et al, 1993). Cheney et al, 1993). It is very likely that the dilute protein (myosin V) is involved with actin dependent transport of material down the axons of nerve cells as well as melanin vesicles in other cell type (Nascimento et al, 1997). Myosin V decorated vesicles have been found to be associated with both actin filaments and microtubule in nerve growth cones (Evans et al, 1997) leading to the speculation that such vesicles may be transported by both filament systems. Myosin Va in a variety of cell types is seen to co-localise with microtubule rich regions such as the spindle, MTOC, midbody (Wu et al, 1998), and the centrosome (Espreafico et al, 1998). In addition, the human myosin V (also called Myoxin) appears to co-localise with intermediate filaments in some cultured cells, and microtubules in others (Engle & Kennett, 1994). Two distinct myosin-Vs are involved with cytoplasmic inheritance of various organelles in yeast (Titus, 1997) this is discussed below.

Figure 1.

A) Myosin-V consist of two heavy chains containing the myosin head and a neck with six IQ motifs each which bind a total of four calmodulin and one each of an additional 25kDa and a 17kDa light chain (Chenney et al, 1993). Presently, the function of the 25 and the 17kDa proteins are not known nor is their position relative to the myosin V molecule, but brain myosin V may bind calmodulin, myosin-II essential light chains and the 8kDa light chain of dynein (Benashski et al, 1997; Espindola et al, 2000). The length of the lever arm constructed by the IQ motifs is thought to determine the overall transport velocity (Schott et al, 2002). Experiments in which the natural myosin V gene of Saccharomyces was replaced by cDNAs encoding myosin Vs with reduced or increased numbers of IQ repeats (and thus lever arm length) concluded that the longer this arm the faster the transport velocity in vivo (Schott et al, 2002). It will be interesting to test the myosin V IQ series of mutants generated by the Bretscher group in the sophisticated apparatus used by the Molloy group (Veigel et al, 2001) . The reports that suggest that myosin V binds the 8kDa light chain of dynein (Benashski et al, 1997; Espindola et al, 2000), may mean that both motors are targeted to the same vesicle by this same light chain. The motility of myosin-V is inhibited by calcium and moves at a speed of 300nm/sec (Cheney et al, 1993). Myosin V is a highly processive motor protein (it remains tightly bound to the filament as it moves along, even in the presence of ATP) and so seems ideally suited to its task of moving vesicles along filaments.

B). Myosin V is known to transport mitochondria to the bud site in growing yeast Saccharomyces cerevisiae (Titus et al, 1997), it is assumed that the globular tail regions specify cargo binding. In the yeast there are two myosin V genes, myo2 and myo4. Deletion of myo2 produces cells that are unable to bud (Johnston et al, 1991), while myo4 deletion ablates mating switching (Brown, 1997), a process that requires specific mRNA localisation at the daughter cell bud site. One of these mRNA is ASH1 mRNA and myo4 binds this message via an intermediate proteins She2p and She3p (Bohl et al, 2000;Takizawa et al, 2000).

Mechanical Properties of Myosin V
Myosin V is highly processive, moving along a filament without falling of (Mehta et al,1999;Sakamoto et al, 2000;Walker et al, 2000), this property was assumed to result from its having two heads (one of which would be connected to the filament at any time), however other single headed myosins are also processive. It is suggested that the processivity of myosin V results from a high duty cycle (the time in contact with actin throughout the myosin motion cycle). This is estimated to be in the region of 50% (Moore et al, 2001), compared to that of muscle myosin II of less than 5% (with no load) (Harris & Warshaw, 1993). Myosin V proceeds along actin filaments towards the barbed end with step sizes of 36 nm (Rief et al, 2000; Veigel et al, 2002), which is coincident with the heads binding to actin 36 nm apart (Walker et al, 2000). High quality electron microscopy clearly showed myosin V dimers in the act of F-actin binding (Walker et al, 2000), resembling skiers in the "telemark" stance. When moving along actin filaments myosin V travels in a spiral fashion, however it does not follow the natural right-handed helix of the actin filament but rather follows a left handed spiral as is binds every 11^th or 13^th actin monomer, neatly in register with its 36 nm step size (Ali et al, 2002). It should be noted that step sizes of 20 nm with the occasional 37 step have also been reported (Moore et al, 2001).

Figure 2. Linear structure of two Myosin VI members. In epithelial cells, a myosin-Vc isoform (Rodriguez & Cheney, 2002) exists that differs significantly in the C-terminus.

The vesicle receptor for Myosin Vs
A synaptotagmin-like protein, Slac2-a more recently renamed "melanophilin" has been shown to bind directly to Rab27 (through melanophilin's N-terminal SHD domain) and to myosin Va via the C terminal "globular tail" domain (Fukada et al, 2002). The importance of the tail region of myosin V for melanosome binding has also been demonstrated in vivo by expressing domains of the motor in melanocytes (da Silva Bizario et al, 2002). Rab27 is known to be associated with a number of myosin V cargo vesicles including melanosomes. The affinity of Rab27a for melanocytes is reduced fourfold in the presence of GDP rather than GTP (Wu et al, 2002). Some forms of Griscelli syndrome are caused by mutations in Rab27 gene in humans (Menasche et al, 2000), while others are due to mutations in the myosin V motor itself (see below). Myosin Va binds melanosomes depending on the particular isoform expressed, Myosin Va from brain without exon F does not bind melaoncytes (Wu et al, 2002a; Wu et al, 2002b). A protein called JFC1 also has a Rab27 binding domain homologous to melanophilin and is proposed to act as a similar adaptor protein in non-melanocytic cells (Hume et al, 2002). JFC1 is targeted to membranes via a C2A domain that binds 3'-phosphorylated phosphoinositides (Catz et al, 2002).

Figure 3. The attachment of myosin Va to melanosomes via melanophilin. Phosphorylation of myosin V by Calcium/calmodulin -dependent kinase II (CaMKII) at a single site (ser 1650, in the globular tail region) releases it from vesicles (Karcher et al, 2001). In addition to being a substrate for CaMKII, myosin V binds to the kinase and activates it (Costa et al, 1999).

Regulation of Myosin V.
In addition to regulation by docking partners (Figure 3), the enzymatic activity of myosin V is regulated by light chains and phosphorylation.

Binding partner	Function	Reference
rab11	A monomeric G-protein	Lapierre et al, 2001; Hales et al, 2001
BERP	a RING finger protein	El-din-Husseini & Vincent, 1999
NARF	a RING finger protein	Ohkawa et al, 2001
VAMP/synaptobrevin	A component of the secretory machinery	Ohyama et al, 2001
Calmodulin dependent protein kinase II	A kinase with many targets	Costa et al, 1999
Table 1 Myosin V binding proteins

Myosin V and disease
Ultra-structural studies in both the dilute mouse (Takagishi et al, 1996) and the dilute rat (Dekker-Ohno et al, 1996) indicate that ER is absent from the dendritic spines of Purkinje cells. As the ER is often a source of IP₃ releasable calcium, this may reduce the cells excitability. This makes sense of a human disease “Griscelli disease” in which mutations in the human myosin V gene result in ataxia, light pigmentation and a variety of immuno-deficiencies and neurological based symptoms (Hurvitz et al, 1993). Myosin V may function in the dendritic spines to pull loops of ER from the cell body into the spine in order to produce local Ca²⁺ fluxes via IP₃ receptors. This may work merely by the failure in dragging (Tabb et al, 1998) the ER localised in the dendrite to provide a calcium source and sink (Takagishi et al, 1996) or, conceivably by direct modulation of the

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