Our results demonstrate the presence of many potential effectors of melanosome motility and localization in the RPE, with a possible role for myosin Va, and a specific requirement for Rab27a and myosin VIIa. The majority (70–80%) of myosin VIIa in the RPE was found to be associated with the membrane of melanosomes, consistent with a role in melanosome transport. Time-lapse microscopy of cultured RPE cells showed that the motility of melanosomes was far greater in the absence of myosin VIIa. Fluorescence microscopy of RPE double-labeled with antibodies and phalloidin shows that the apical distribution of myosin VIIa and myosin Va overlaps with the apical actin filament network. It is this region of overlap from which melanosomes are absent in Rab27a- and Myo7a-null RPE. Our analysis is consistent with Rab27a and myosin VIIa transporting and capturing melanosomes in this region of the RPE actin network.
Melanosomes in teleost and amphibian RPE undergo quite large movements in response to changes in ambient lighting to effect a light-dark adaptive mechanism (Back et al., 1965
; Burnside, 2001
). Such changes do not occur in mammalian RPE. Indeed, there have been only a few indications that melanosomes in mammalian RPE move at all. One is implied from the mislocalization of melanosomes in the RPE of Myo7a
-mutant mice (Liu et al., 1998
) (and the present study). Another, more definitively, comes from a detailed analysis of melanosome distribution with respect to time of day in mouse RPE. A small, but significant displacement of melanosomes was detected during the first hours after the lights went on (Futter et al., 2004
). Here, by live-cell imaging, we have observed that melanosomes do move in mouse RPE cells (especially in the absence of myosin VIIa).
A role for myosin VIIa in tethering melanosomes compares to its proposed role in inner ear sensory hair cells, where it appears to function in tethering the cell membrane to the actin cytoskeleton of stereocilia (Kros et al., 2002
). Moreover, the finding that myosin VIIa is required to constrain the motility and orientation of RPE melanosomes makes the comparison between myosin VIIa in RPE and myosin Va in melanocytes and melanophores quite striking. In melanocytes, the role of myosin Va is to ‘capture’ melanosomes from dendritic microtubules in the periphery of the cell (Wu et al., 1998a
). Melanosomes are transported to the periphery by a microtubule motor, at a similar velocity to the rapid movements we measured for RPE melanosomes. In the absence of myosin Va, they are not retained in the periphery and travel back to the cell body (Wu et al., 1998a
; Wu et al., 1998b
; Wu and Hammer, 2000
). In Xenopus
melanophores, myosin Va has been implicated in promoting the cyclic AMP-sensitive dispersal of melanosomes by acting as a molecular ratchet, increasing the relative contribution of kinesin II-mediated movement over that of cytoplasmic dyenin (Rodionov et al., 1998
; Rogers and Gelfand, 1998
; Gross et al., 2002
The movement of RPE melanosomes occurred with at least two distinct velocities, indicating the participation of at least two motor proteins. The measured velocities were of a similar range in control and Myo7a
-null RPE, indicating that these motor proteins are in addition to myosin VIIa; although slower movements were less evident in the mutant cells, some were still detected. A possible hypothesis is that the more rapid movements (>1 µm/second) are effected by a microtubule motor. Similar to the role of myosin Va in melanocytes (above), myosin VIIa may take delivery of a melanosome from a microtubule motor, and thus normally restrict faster microtubule-based movement. Myosin Va is likely to be involved in the slower movements (11–250 nm/seconds) of melanosomes seen in both control and Myo7a
-null cells. We found that myosin Va is not required for the correct localization of RPE melanosomes, however, it was enriched in the subcellular fraction containing melanosomes, suggesting a role in melanosome transport, even though it is not essential for the localization of the melanosomes. The movements of melanosomes in teleost RPE in response to lighting changes requires an intact actin cytoskeleton (King-Smith et al., 1997
), and the melanosomes have been shown recently, in elegant motility studies, to contain a plus-end directed myosin(s) (McNeil et al., 2004
). Interestingly, however, light-dependent movements of melanosomes still occur in the RPE of mariner zebrafish (Perkins et al., 2004
), which lack myosin VIIa (Ernest et al., 2000
), indicating that myosin VIIa is not required for these movements. We suggest that myosin Va is a more likely candidate for light-dependent movements.
Myosin VIIa may also participate in the slower movements since relatively fewer melanosomes moved at these velocities in Myo7a
-null RPE. In vitro, myosin VIIa was found to move along actin filaments at around 200 nm/second (Udovichenko et al., 2002
; Inoue and Ikebe, 2003
). Of course, the reduction in slower movements could be simply a result of the identification of more melanosomes moving at the fast velocity. However, we have never observed even a single melanosome in an apical process of a Myo7a
-mutant RPE, suggesting that myosin VIIa is responsible for transporting melanosomes into this region. If it were only responsible for tethering melanosomes, we would expect, at least occasionally, to observe a melanosome in an apical process of a Myo7a
-mutant RPE. Hence, myosin VIIa appears to move melanosomes in addition to tethering them.
With the finding that Myrip binds both myosin VIIa and Rab27a (El-Amraoui et al., 2002
; Fukuda and Kuroda, 2002
), together with the mislocalized melanosomes in shaker1 RPE (Liu et al., 1998
), it has been suggested that a Rab27a-Myripmyosin VIIa complex might localize RPE melanosomes just as a Rab27a-melanophilin-myosin Va complex is responsible for the peripheral localization of melanosomes in melanocytes (El-Amraoui et al., 2002
). Our results support this hypothesis. The finding of the same melanosome phenotype in the RPE of Rab27a
-mutant mice as in the RPE of Myo7a
-mutant mice demonstrates the requirement of Rab27a as well as myosin VIIa. This observation has now also been described by another group (Futter et al., 2004
). Hence, Rab27a regulation of melanosome localization is conserved between melanocytes and the RPE, yet different myosins are used to tether these organelles in the different cells.
It is not known at present whether Myrip is an obligate member of the localization complex in RPE, as melanophilin is in melanocytes (Provance et al., 2002
; Hume et al., 2002
). Many different exophilins, which might link myosins to Rab27a on the melanosome membrane, were detected in the RPE – including melanophilin. What seems likely is that the combined tasks of transporting and tethering melanosomes in the RPE may involve many different proteins. Further analysis of melanosome motility is needed to dissect the different functions of the various motors and linkers.
The last point for discussion concerns the requirement of myosin VIIa to maintain vision. As we have discussed previously, it seems unlikely that a mislocalization of RPE melanosomes per se would lead to the photoreceptor degeneration found in Usher 1B patients. A delay in transporting and degrading phagosomes appears to be a more serious RPE defect, caused by the loss of myosin VIIa (Gibbs et al., 2003
). However, the present study shows that melanosomes are not only misplaced, but are more highly mobile in the absence of myosin VIIa. Melanosomes ‘on the loose’ may have deleterious consequences for the health of the RPE cells, and thus, in turn, contribute to photoreceptor degeneration.