Role of myosin VIIa and Rab27a in the motility and localization of RPE melanosomes

Abstract

Myosin VIIa functions in the outer retina, and loss of this function causes human blindness in Usher syndrome type 1B (USH1B). In mice with mutant Myo7a, melanosomes in the retinal pigmented epithelium (RPE) are distributed abnormally. In this investigation we detected many proteins in RPE cells that could potentially participate in melanosome transport, but of those tested, only myosin VIIa and Rab27a were found to be required for normal distribution. Two other expressed proteins, melanophilin and myosin Va, both of which are required for normal melanosome distribution in melanocytes, were not required in RPE, despite the association of myosin Va with the RPE melanosome fraction. Both myosin VIIa and myosin Va were immunodetected broadly in sections of the RPE, overlapping with a region of apical filamentous actin. Some 70-80% of the myosin VIIa in RPE cells was detected on melanosome membranes by both subcellular fractionation of RPE cells and quantitative immunoelectron microscopy, consistent with a role for myosin VIIa in melanosome motility. Time-lapse microscopy of melanosomes in primary cultures of mouse RPE cells demonstrated that the melanosomes move in a saltatory manner, interrupting slow movements with short bursts of rapid movement (>1 μm/second). In RPE cells from Myo7a-null mice, both the slow and rapid movements still occurred, except that more melanosomes underwent rapid movements, and each movement extended approximately five times longer (and further). Hence, our studies demonstrate the presence of many potential effectors of melanosome motility and localization in the RPE, with a specific requirement for Rab27a and myosin VIIa, which function by transporting and constraining melanosomes within a region of filamentous actin. The presence of two distinct melanosome velocities in both control and Myo7a-null RPE cells suggests the involvement of at least two motors other than myosin VIIa in melanosome motility, most probably, a microtubule motor and myosin Va.

The unconventional myosin-VIIa associates with lysosomes

“…Fusion of phagosomes with melanosomes is most prominently observed in cells overloaded with ROS or undigestable products (Orlow 1995;Schraermeyer et al 1999). Recent studies show that a large fraction of myosin-VIIa in primary RPE cells is found on melanosomal membranes (Gibbs et al 2004). Our studies with a new myosin-VIIa antibody have found that a population of myosin-VIIa also associates specifically with lysosomes, an organelle known to be required for ROS digestion.…”

“…Lysosome organization is maintained by the microtubule network with its motors, as has been shown for other cell types (Matteoni and Kreis 1987). Gibbs et al (2004) suggested that myosin-VIIa may play a role in short range movements and temporary tethering of melanosomes. Perhaps this is also true for myosin-VIIa found on lysosomes.…”

“…Several studies have found that myosin-VIIa is enriched in the apical region of the RPE (Hasson et al, 1995;El-Amraoui et a., 1996;Gibbs et al, 2004). Both traditional light microscopy and confocal microscopy of rat retina cryosections revealed that, like rabbit antibodies to myosinVIIa, the 138-1 monoclonal anti-myosin-VIIa antibody (mαmyo7a) specifically recognized myosin-VIIa in RPE (Fig.…”

“…Higher magnification revealed that myosin-VIIa was localized to apical punctate structures resembling a vesicular compartment directly above the nucleus. The apical region of the RPE contains abundant melanosomes, lysosomes and phagosomes (Bosch et al, 1993) and indeed myosin-VIIa has been shown to be associated with melanosomes in mouse RPE (Gibbs et al, 2004). However, myosin-VIIa has also been implicated in the late stages of ROS phagocytosis (Gibbs et al, 2003).…”

“…Instead of being transported to the apical projections, in shaker-1 RPE the melanosomes were retained in a perinuclear region . Electron microscopy data suggested that myosin-VIIa was on a subset of melanosomes (ElAmraoui et al 2002;Gibbs et al 2004), but the association of myosin-VIIa with a definitive digestion organelle has not been rigorously shown.…”