Myosin II and Rho kinase activity are required for melanosome aggregation in fish retinal pigment epithelial cells

Abstract

In the retinal pigment epithelium (RPE) of fish, melanosomes (pigment granules) migrate long distances through the cell body into apical projections in the light, and aggregate back into the cell body in the dark. RPE cells can be isolated from the eye, dissociated, and cultured as single cells in vitro. Treatment of isolated RPE cells with cAMP or the phosphatase inhibitor, okadaic acid (OA), stimulates melanosome aggregation, while cAMP or OA washout in the presence of dopamine triggers dispersion. Previous studies have shown that actin filaments are both necessary and sufficient for aggregation and dispersion of melanosomes within apical projections of isolated RPE. The role of myosin II in melanosome motility was investigated using the myosin II inhibitor, blebbistatin, and a specific rho kinase (ROCK) inhibitor, H-1152. Blebbistatin and H-1152 partially blocked melanosome aggregation triggered by cAMP in dissociated, isolated RPE cells and isolated sheets of RPE. In contrast, neither drug affected melanosome dispersion. In cells exposed to either blebbistatin or H-1152, then triggered to aggregate using OA, melanosome aggregation was completely inhibited. These results demonstrate that (1) melanosome aggregation and dispersion occur through different, actin-dependent mechanisms; (2) myosin II and ROCK activity are required for full melanosome aggregation, but not dispersion; (3) partial aggregation that occurred despite myosin II or ROCK inhibition suggests a second component of aggregation that is dependent on cAMP signaling, but independent of ROCK and myosin II.

Orientation of actin filaments in teleost retinal pigment epithelial cells, and the effect of the lectin, Concanavalin A, on melanosome motility

“…The different motility characteristics during aggregation and dispersion, and the assumption that actin was uniformly oriented with barbed ends distal, prompted a hypothesis that dispersion of melanosomes is effected by a barbed-end directed myosin motor, and aggregation occurs by passive association of melanosomes with actin fi laments undergoing retrograde actin fl ow (King-Smith et al, 1997 ;Barsoum & King-Smith, 2007 ). While actin dynamics in living RPE cells have not been observed, the notion that actin fi laments in apical projections undergo retrograde fl ow is supported by the fi nding that latex beads attached to the dorsal surface of isolated RPE cells are constitutively transported from the distal regions of apical projections toward the CB (Basciano & King-Smith, 2002 ).…”

“…While Arp2/3-dependent polymerization of actin fi laments powers retrograde movement at the cell edge, myosin II-dependent contraction of actin fi laments proximally is believed to effect retrograde actin fl ow (Verkhovsky et al, 1997 ;Ponti et al, 2004 ;Medeiros et al, 2006 ). Blebbistatin blocked pigment granule aggregation but had no effect on granule dispersion (Barsoum & King-Smith, 2007 ). Blebbistatin blocked pigment granule aggregation but had no effect on granule dispersion (Barsoum & King-Smith, 2007 ).…”

“…Isolated cells on coverslips were mounted on a glass slide with doublestick tape spacers to form a perfusion chamber and viewed using phase contrast microscopy with a 100× oil objective, as described previously (Barsoum & King-Smith, 2007 ). RPE sheets were placed in perfusion chambers in a drop of medium containing 1 mM cAMP to trigger aggregation.…”

“…Previous research using the myosin-II inhibitor, blebbistatin, and rho kinase (ROCK) inhibitors demonstrated a role for myosin II in melanosome aggregation, but not dispersion (Barsoum & King-Smith, 2007 ). These fi ndings, together with careful observations of melanosome motility characteristics during aggregation and dispersion, led to an hypothesis for melanosome transport, where dispersion occurs via myosin motor transport along actin fi laments, whereas aggregation occurs by binding of melanosomes to actin fi laments undergoing myosin-II-dependent retrograde fl ow (King Smith et al, 1997 ;Barsoum & King-Smith, 2007 ).…”

New Insights into the Regulation of Myosin Light Chain Phosphorylation in Retinal Pigment Epithelial Cells

“…Among these, myosin II has been shown to participate in the contractility of the CMB (Burnside and Bost-Usinger, 1998) and also to be required, together with ROCK, for melanosome aggregation (Barsoum and King-Smith, 2007). Recently, NMHC-IIA and NMHC-IIB involvement in the phagocytosis of (Breckler and Burnside, 1994); fish (Breckler and Burnside, 1994;Lin-Jones et al, 2009) II IIA IIA, IIB Abundant in the apical region associated with the circumferential microfilament bundle…”