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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 1, 2004 as doi:10.1096/fj.03-1240fje. |
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INSERM U597, Biologie et Pathologie des Cellules Mélanocytaires, Faculté de Médecine, Nice cedex, France
2Correspondence: INSERM U597, Biologie et Pathologie des Celllules Mélanocytaires, Faculté de Médecine, Avenue de Valombrose, 06107, Nice cedex 2, France. E-mail: ballotti{at}unice.fr
SPECIFIC AIMS
The aim of the present study was to investigate the possible regulation of melanosome transport in mammalian melanocytes by 
-MSH, a physiologic melanocyte-differentiating agent that stimulates the cyclic AMP (cAMP) pathway. We studied, at the molecular level, the effect of the activation of the cAMP pathway on proteins involved in the control of melanosome transport.
PRINCIPAL FINDINGS
1. cAMP induces rapid melanosome docking at the cell periphery: role of the actin network
Using time-lapse video microscopy, we observed in basal conditions a bi-directional movement of melanosomes with no change in melanosome repartition after 70 min (Fig. 1
A). We added forskolin (20 µM) and observed the same cell an additional 100 min (Fig. 1B
, movie 1). As early as 14 min after forskolin addition, we noted that melanosomes started to gather at the extremities of dendrite outgrowths. This was more obvious after longer exposure to forskolin. MSH, a physiological cAMP-elevating agent, also induced melanosome docking at dendrite tips (Fig. 1C
). The docking of melanosomes induced by cAMP is a reversible phenomenon, since after forskolin removal, melanosomes left the dendrite tips and spread all over the cell (Fig. 1D
, movie 2). Thus, MSH and cAMP regulate melanosome transport to promote the accumulation of melanosomes at dendrite extremities.
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Disruption of the actin filaments by cytochalasin D had a tendency to decrease melanosome docking at the dendrite tips whereas disruption of the microtubule network by nocodazole led to an increase in melanosome accumulation. These observations suggest that the actin network appears to be required for melanosome docking at dendrite tips and that the retrograde transport of melanosomes on microtubules is more efficient than the anterograde transport.
2. RhoA and p160Rock, but not PKA, are involved in redistribution of melanosomes induced by cAMP
Next we investigated the role of protein kinase A (PKA) in the peripheral docking of melanosomes induced by cAMP elevating agents. Immunofluorescence studies with anti-Tyrp1 antibodies showed that PKA inhibition by H89 did not prevent the docking of melanosomes at dendrite tips induced by forskolin. In the same conditions, H89 completely blocked the forskolin-induced phosphorylation of CREB. Thus, PKA does not mediate the effect of cAMP on melanosome transport.
It has been shown that RhoA, a key regulator of actin cytoskeleton, is inhibited by cAMP; thus, we investigated the role of this Rho GTPase in regulating melanosome transport by cAMP. DIC images of B16 melanoma cells exposed for 300 min to C3 toxin showed that the inhibition of Rho induced an accumulation of melanosomes at dendrite tips. Similarly, inhibition of the RhoA-associated kinase (p160 Rock) by Y27632 promoted a peripheral docking of melanosomes (movie 3). These results indicate that inhibition of RhoA and p160 Rock is involved in the cAMP-induced relocalization of melanosomes.
3. cAMP does not regulate the interaction between Rab27a, melanophilin/Slac2-a, and myosin-Va
Rab27a, melanophilin/Slac2-a, and myosin-Va constitute a molecular tripartite complex that allows interaction of melanosomes with the actin network. Therefore, we studied the role of these proteins in the regulation of melanosome distribution by cAMP.
Western blot analysis of Rab27a, melanophilin/Slac2-a, and myosin-Va proteins showed that Rab27a, melanophilin/Slac2-a, and myosin-Va levels were not modified by short-term (2 h) treatment with forskolin. Longer exposure to forskolin (20 h) stimulated Rab27a and myosin-Va expression. Since the accumulation of melanosomes at dendrite tips induced by cAMP takes <2 h (Fig. 1B, C
), the effect of cAMP on Rab27a and myosin-Va expression cannot be involved in the rapid changes of melanosome localization evoked by cAMP.
We next studied the short-term effect of cAMP on Rab27a function and its interaction with melanophiln/Slac2-a. A pull-down experiment using GST-melanophilin/Slac2-a with extracts from basal or forskolin-exposed B16 melanoma cells showed that forskolin treatment did not increase the amount of Rab27a associated with melanophilin/Slac2-a. Since melanophilin/Slac2-a interacts with GTP-bound Rab27a, we conclude that cAMP does not modify the balance between GDP- and GTP-bound forms of Rab27a. We explored the possibility that forskolin increased the association of melanophilin/Slac2-a with myosin Va. We transfected B16 melanoma cells with a GFP-tagged myosin-Va tail construct; after exposure to forskolin, cell extracts were immunoprecipitated with the anti-melanophilin/Slac2-a antibody. Western blot analysis showed that treatment with forskolin did not modify the amount of melanophilin/Slac2-a and myosin-Va-GFP in total extract or the amount of GFP-tagged myosin-Va coprecipitated by the anti-melanophilin/Slac2-a antibody. Thus, cAMP does not increase interaction between melanophilin/Slac2-a and myosin Va.
4. cAMP increases the interaction between melanophilin/Slac2-a and actin
Although cAMP does not activate Rab27a or increase the formation of Rab27a/melanophilin/Slac2-a and melanophilin/Slac2-a/myosin Va complexes, we considered whether cAMP could regulate recruitment of these molecules to the melanosomes. We performed immunopurification of intact melanosomes. The right panel of Fig. 2
A shows Western blot experiments with antibodies to myosin-Va, melanophilin/Slac2-a, Tyrp1, and Rab27a performed after melanosome purification with anti-Tyrp1 antibodies. Forskolin did not increase the amount of myosin-Va, melanophilin/Slac2-a, and Rab27a associated with melanosomes. Left and middle panels show Western blot experiments performed on total extracts before immunopurification and after purification with preimmune serum, respectively.
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Rab27a/melanophilin/Slac2-a/myosin-Va complexes connect melanosomes with the actin filaments. We hypothesized that cAMP could favor the interaction of this complex with actin. To investigate this, B16 melanoma cells transfected with an actin-GFP encoding vector were exposed to forskolin. To verify the amount of melanophilin/Slac2-a and actin-GFP, total extracts were analyzed by Western blot with anti-GFP and anti-melanophilin/Slac2-a antibodies (Fig. 2
, upper panels). Proteins immunoprecipitated with anti-melanophilin/Slac2-a were analyzed by Western blot with the same antibodies (Fig. 2B
). The amount of actin-GFP coprecipitated with melanophilin/Slac2-a was increased by short-term exposure to forskolin, demonstrating that cAMP stimulates the interaction between actin and melanophilin/Slac2-a.
CONCLUSIONS AND SIGNIFICANCE
In the present study, using video microscopy, we demonstrate that -MSH and cAMP induce a rapid and active centrifugal transport, leading to an accumulation of the melanosomes at the extremity of the dendrites. The redistribution of the melanosomes within the cells is reversible and requires the integrity of the actin network.
PKA activation is not necessary for melanosome accumulation at dendrite tips, but inhibition of the Rho pathway by cAMP seems to be required for the peripheral docking of melanosome. Inhibition of Rho by cAMP, which could be the consequence of phosphatidylinositol 3-kinase inhibition by cAMP, has been reported in numerous cell lines, including B16 melanoma cells.
We hypothesized that cAMP might increase the expression of Rab27a, melanophilin/Slac2-a, or myosin-Va that links melanosomes to actin filaments. However, such effects were observed after 20 h of forskolin treatment and cannot account for the rapid cAMP-induced melanosome docking. cAMP does not increase the amount of GTP-bound Rab27a or favor interactions between members of the tripartite complex. cAMP does not affect the recruitment of these proteins to the melanosomes. Finally, we showed that cAMP rapidly stimulates the interaction between actin and melanophilin/Slac2-a and so could allow the capture and docking of melanosomes at the peripheral actin network. Melanophilin/Slac2-a and actin might interact indirectly through myosin-Va, which binds both proteins. However, we did not observe any stimulation of the interaction between actin and myosin-Va by cAMP. It has recently been shown that melanophilin/Slac2-a can interact directly with actin. In agreement, cAMP appears to increase the direct interaction between melanophilin/Slac2-a and actin.
We propose that stimulation of the interaction between melanophilin/Slac2-a and actin would allow rapid accumulation of melanosome in the actin-rich region of the dendrite extremities. However, we cannot rule out the possibility that cAMP also acts on microtubule-dependent transport by increasing anterograde, kinesin-mediated transport or decreasing the retrograde, dynein-mediated transport.
It remains to be determined how cAMP increases the association of melanophilin/Slac2-a and actin. Melanophilin/Slac2-a is heavily phosphorylated on serine and threonine, but cAMP does not change its global phosphorylation level. A tedious analysis of the site-specific phosphorylation (>45 potential phosphorylation sites) level upon cAMP treatment is required to elucidate the role of the melanophilin/Slac2-a phosphorylation in controlling its interaction with actin. The role of the Rho pathway in the control of melanophilin/Slac2-a and actin interaction needs to be elucidated.
The rapid stimulation of transport and docking of melanosomes through increased interaction between melanophilin/Slac2-a and actin could be a key molecular process accounting for the immediate skin tanning induced by sun exposure in humans. Up-regulation of myosin-Va and Rab27a expression could favor sustained melanosome relocalization to the dendrite tips and participate in delayed skin tanning. Both immediate and delayed skin tanning could be considered pivotal photo-protective processes against the carcinogenic effect of UV radiation of solar light.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1240fje; 
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