| The large yellow croaker(Larimichthys crocea)is an important mariculture fish in China,which is famous for its golden body color.Its golden body color has become an important economic trait,and the price of large yellow croaker is closely related to its body color.The golden body color of large yellow croaker comes from xanthophores on its body surface.Interestingly,large yellow croaker exists the phenomenon of "whitening when exposed to light".In the daytime or under light irradiation,the golden body color of large yellow croaker quickly turns to silver white in 2-3 minutes.At night or in the dark,they appear golden yellow.In recent years,our research group has shown that the golden color of large yellow croaker is mainly dependent on the action of melanocyte-stimulating hormone(MSH)on its MC5R receptor of xanthophores,thus causing the dispersion of intracellular pigment granules.While the body color becomes silvery white,it is because the specific wavelength in the light stimulates the xanthophores’ photoreceptor.And then,it causes the intracellular pigment granules to aggregation.As we all know,the aggregation and dispersion of pigment granules in pigment cells depend on the intracellular transport system.Current studies on the transport of melanosomes in melanocytes/melanophores indicate that it is mainly related to the transport system based on microtubules and microfilaments(also known as actin filaments).In this transport system,microtubules and microfilaments mainly function as rails,and the power of transport is derived from motor proteins,such as kinesin and dynein,which bind to microtubules,and myosin,which binds to microfilaments.In this study,in order to further elucidate the molecular mechanism of the intracellular transport of pigment granules in large yellow croaker,the xanthophores in the abdominal scales of large yellow croaker were used as the object for the study.We studied the functions of microtubules,microfilaments and their motor proteins in the movement of intracellular pigment granules by means of specific drug treatment and morphological observation.The results from this paper are as follows:1.When the dispersed xanthophores were pretreated with 1 μM Nocodazole for 12 hours in darkness,which is a microtubule-specific depolymerization agent,light cannot cause the aggregation of intracellular pigment granules.And the fully aggregated xanthophores were not able to maintain their complete aggregated states after being pretreated with 1 μM Nocodazole under light,the dispersion occurred about 5 minutes after the treatment with Nocodazole.On the contrary,the dispersed xanthophores pretreated with Latrunculin A at 1 μM for 12 hours in darkness,which is a specific inhibitor of actin polymerization,light could still cause aggregation.And treatment with Latrunculin A(1 μM)did not affect the maintenance of fully aggregated states of xanthophores under light,which initial state is complete aggregation.In addition,although exogenous MSH(1 μM,30 minutes)could induce the dispersion of xanthophores,their microfilaments were depolymerized by the treatment with Latrunculin A at 1 μM for 12 hours,the degree of dispersion was significantly lower than that of control group(p<0.05).When pretreated with Latrunculin A(final concentration of 1 μM)and Nocodazole(final concentration of 1μM),the degree of dispersion of xanthophores induced by exogenous MSH(1μM,30 minutes)was significantly lower than that of xanthophores pretreated with 1 μM Latrunculin A alone(p<0.05).These results indicated that the aggregation process of pigment granules in xanthophores depended on microtubules,while the dispersion process was associated with both the microtubules and microfilaments.2.The aggregation of xanthophores is dependent on microtubules,in which dynein is the main motor protein that plays the role of centripetal transport.To verify whether dynein plays a role in the aggregation of xanthophores in large yellow croaker,we treated the xanthophores with Sodium Orthovanadate,an inhibitor of dynein,and found that Sodium Orthovanadate could inhibit the aggregation of xanthophores induced by light in a time-dependent and dose-dependent manner.Dispersed xanthophores pretreated with 100 μM Sodium Orthovanadate for 30 minutes,71.77±1.61%of which could be caused to aggregation by light.However,extending the treatment time to 60 minutes,only 25.20±1.27%of xanthophores could be aggregated by light.The dispersed xanthophores pretreated with Sodium Orthovanadate at different concentrations(1000 μM,100 μM,50 μM)for 60 minutes in dark,the light induced aggregation was inhibited in different degrees at 100 μM and 50 μM,and at 1000 μM almost all xanthophores aggregation was inhibited.In addition,dynein played an important role in maintaining the completely aggregated morphology of xanthophores.Compared with the control group(0 μM),the proportion of xanthophores that changed their initial aggregated states was significantly increased after treating with different concentrations of Sodium Orthovanadate for 60 minutes.Treatment with 1000 μM inhibited almost all xanthophores aggregation(95.61±1.23%),and the treatment with 100 μM and 50μM inhibited the xanthophores aggregation by 43.83±10.04%and 23.68±2.17%,respectively.These results suggested that dynein was involved in the aggregation of pigment granules in xanthophores.3.Our previous studies have shown that the aggregation of pigment granules in xanthophores is related to Ca2+related signaling pathways.Therefore,we further investigated whether PKC,the downstream signaling of Ca2+,is involved in the aggregation of xanthophores.We treated xanthophores with PMA,an activator of PKC.After pretreatment with PMA(0,1,10,100 μM)for 1 hours in darkness,xanthophores in the 10 μM PMA treatment group showed semi-aggregation,while xanthophores in the 100 μM PMA treatment group were completely aggregated.These results suggested that the aggregation of xanthophores was related to the activation of PKC.Our study showed that microtubules,the microtubule-dependent motor protein dynein and the Ca2+-dependent protein kinase PKC were all involved in the aggregation of pigment granules in xanthophores.We further investigated whether there was a potential association between dynein and PKC.In the dark,we first treated xanthophores with Latrunculin A(1 μM)for 12 hours to depolymerize their intracellular microfilaments,and then treated them with PKC inhibitor Sotrastaurin(10 μM)for 2 hours followed by light stimulation.We found that Sotrastaurin(10 μM)inhibited the aggregation of xanthophores with or without depolymerizing microfilaments.Additionally,we also found that treatment with PKC activator PMA(100 μM)for 60 minutes did not induce the aggregation of dispersed xanthophores,which were pretreated with dynein inhibitor Sodium Orthovanadate(1000 μM)for 60 minutes in darkness.This result suggested that PKC was an upstream regulator of dynein.4.Previous studies of our group have shown that increasing the intracellular cAMP level can cause the dispersion of pigment granules,but we have not explored whether the aggregation of xanthophores depends on the decrease of cAMP level.For this purpose,we used theophylline,an inhibitor of phosphodiesterase(PDE4)that specifically hydrolyzes cAMP,to study the effect of cAMP level on the aggregation of xanthophores in vivo and in vitro experiments.We found that abdominal skin of large yellow croaker after being immersed in 10 μM theophylline in dark for 30 minutes could not whiten completely even if exposed to the light for 10 minutes.In the in vitro experiments,compared with the control group(0 mM),the ratio of aggregated xanthophores in theophylline treatment group(0.1 mM,1 mM,10 mM)was significantly decreased,and the aggregated ratio was decreased with the increase of theophylline concentration.The inhibition effect at 10 mM was the most significant(p<0.05),which could reduce the aggregated ratio of xanthophores to 23.04±2.12%.In summary,both microfilaments and microtubules are involved in the dispersion of xanthophores in large yellow croaker,and the force of dispersion on microfilaments is stronger than that on microtubules.The light-induced aggregation of pigment granules in xanthophores was microtubule-dependent rather than microfilament-dependent.Dynein,a motor protein responsible for centripetal movement on microtubules,is involved in the aggregation process of pigment granules.Furthermore,the intracellular Ca2+-PKC signaling pathway is involved in the aggregation of pigment granules,and dynein is a downstream regulator of this pathway.Meanwhile,the aggregation of pigment granules also depends on the decrease of intracellular cAMP level.This study has a certain significance for revealing the molecular mechanism of the movement of pigment granules in xanthophores of large yellow croaker. |
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