Melanosomes In Cells Research Articles

Structural analysis of melanosomes in living mammalian cells using scanning electron-assisted dielectric microscopy with deep neural network

Melanins are the main pigments found in mammals. Their synthesis and transfer to keratinocytes have been widely investigated for many years. However, analysis has been mainly carried out using fixed rather than live cells. In this study, we have analysed the melanosomes in living mammalian cells using newly developed scanning electron-assisted dielectric microscopy (SE-ADM). The melanosomes in human melanoma MNT-1 cells were observed as clear black particles in SE-ADM. The main structure of melanosomes was toroidal while that of normal melanocytes was ellipsoidal. In tyrosinase knockout MNT-1 cells, not only the black particles in the SE-ADM images but also the Raman shift of melanin peaks completely disappeared suggesting that the black particles were really melanosomes. We developed a deep neural network (DNN) system to automatically detect melanosomes in cells and analysed their diameter and roundness. In terms of melanosome morphology, the diameter of melanosomes in melanoma cells did not change while that in normal melanocytes increased during culture. The established DNN analysis system with SE-ADM can be used for other particles, e.g. exosomes, lysosomes, and other biological particles.

Study of melanin distribution in the hair cells of Karakul lambs of different colours

The relevance of this study is explained by the need to have a more accurate instrument for assessing the phenotype of Karakul lambs by colour. Despite the fact that the colouring of Karakul lambs is an important sign for obtaining a high-quality krimmer skin, its determination occurs only visually, which leads to large errors. This fact indicates the need for a more objective method for assessing the colour of Karakul lambs. In this regard, this article is aimed at studying melanin distribution type in cortical layer cells of Karakul lambs’ hair of different colours and evaluating the possibility of using this trait as an additional criterion for identifying the phenotype by lear. After the preparation of smears from hair macerates, light microscopy was performed with a visual count of cells with a particular distribution pattern of melanosomes in cells, according to the chosen classification. The absence of class zero cells (complete absence of melanin) for black lambs was revealed, however, there were unexpectedly quite few cells of the fifth class (with superpigmentation). The largest number of cells of grey lambs were assigned to the third or zero classes. With an increase in the cells of the zero class, the colour intensity of the taken hair also decreases. In lambs of medium-blue and dark-blue colours, cells of the third class prevailed. The materials of the article are of practical value for breeding work with Karakul sheep, providing more accurate control of the selection of sheep to achieve the desired result using a quantitative parameter.

Ultrastructural features of skin pigmentation in the lizardHeloderma suspectumwith emphasis on xanto-melanophores

The morphological origin of the dark and pink-orange areas in the skin of the venomous lizard Heloderma suspectum is not known. Histology and electron microscopy show that dark-grey areas of the skin derived from dermal chromatophores localized in specific areas present underneath the epidermis. A dynamic chromatophoric unit in the dermis is absent. In the darkest areas of the skin, the accumulation of melanosomes in cells of the beta-layer contributes to increase the black intensity. In the orange-pink areas, the superficial dermis contains xantophores storing numerous carotenoid vesicles, rare or absent lamellated pterinosomes and a variable number of melanosomes. These xanto-melanophores predominate over the remaining chromatophores and form a continuous stratum underneath the epidermis. Beneath this lipoid-rich stratum, iridophores are infrequent and do not form a continuous layer in the dermis. In the paler areas of the skin, melanophores are sparse in both superficial and deeper part of the dermis where irregularly oriented bundles of collagen fibrils are present. The prevalent xanto-melanophores localized in the pink-orange areas of the skin contribute to an effective sunlight protection in desert conditions in addition to the darker regions occupied by melanophores.

Anomalous Dynamics of Melanosomes Driven by Myosin-V in Xenopus laevis Melanophores

The organization of the cytoplasm is regulated by molecular motors, which transport organelles and other cargoes along cytoskeleton tracks. In this work, we use single particle tracking to study the in vivo regulation of the transport driven by myosin-V along actin filaments in Xenopus laevis melanophores. Melanophores have pigment organelles or melanosomes, which, in response to hormones, disperse in the cytoplasm or aggregate in the perinuclear region. We followed the motion of melanosomes in cells treated to depolymerize microtubules during aggregation and dispersion, focusing the analysis on the dynamics of these organelles in a time window not explored before to our knowledge. These data could not be explained by previous models that only consider active transport. We proposed a transport-diffusion model in which melanosomes may detach from actin tracks and reattach to nearby filaments to resume the active motion after a given time of diffusion. This model predicts that organelles spend ∼70% and 10% of the total time in active transport during dispersion and aggregation, respectively. Our results suggest that the transport along actin filaments and the switching from actin to microtubule networks are regulated by changes in the diffusion time between periods of active motion driven by myosin-V.

Inulavosin, a Melanogenesis Inhibitor, Leads to Mistargeting of Tyrosinase to Lysosomes and Accelerates its Degradation

The melanosome is a highly specialized organelle where melanin is synthesized. Tyrosinase and tyrosinase-related protein-1 (Tyrp1) are major melanosomal membrane proteins and key enzymes for melanin synthesis in melanocytes. Inulavosin, a melanogenesis inhibitor isolated from Inula nervosa (Compositae), reduced the melanin content without affecting either the enzymatic activities or the transcription of tyrosinase or Tyrp1 in B16 melanoma cells. To our knowledge, this inhibitor is previously unreported. Electron-microscopic analyses revealed that inulavosin impaired late-stage development of melanosomes (stages III and IV), in which melanin is heavily deposited. However, it did not alter the early stages of melanosomes (stages I and II), when filamentous structure is observed. Immunofluorescence analyses showed that tyrosinase, but not Tyrp1, was specifically eliminated from melanosomes in cells treated with inulavosin. Unexpectedly, inulavosin specifically accelerated the degradation of tyrosinase but not other melanosomal/lysosomal membrane proteins (Tyrp1, Pmel17, and LGP85). The degradation of tyrosinase induced by inulavosin associated with lysosomes but not the proteasome. Interestingly, lysosomal protease inhibitors restored the melanogenesis but not the targeting of tyrosinase to melanosomes in the cells treated with inulavosin. Instead, colocalization of tyrosinase with lysosome-associated membrane protein-1 at late endosomes/multivesicular bodies and lysosomes was accentuated. Taken together, inulavosin inhibits melanogenesis as a result of mistargeting of tyrosinase to lysosomes.

Pigmentation, impression hardness and the presence of melanosomes in bovine claw tissue

The physical properties of hardness and dry matter and the histological characteristics of dermal and epidermal tissues were investigated in lateral and medial claws obtained post mortem from mature crossbred female cattle. Claws containing pigmented (n=22) or non-pigmented (n=22) coronary wall horn were studied. Impression hardness, measured on the dorsal border of claws, proximo-distal at intervals of 5 mm from the lower perioplic line was shown to increase to the 20 mm lower site. Similarly, in comparison with values for pigmented horn, pooled measurements for non-pigmented horn showed greater hardness from the lower perioplic line to the 40 mm lower site but were not different for subsequent measurements towards the distal edge. Differences in values recorded for sole and heel and for coronary wall horn measured under the distal edge were not different for claws varying in coronary horn pigmentation. The absence of differences in dry matter at measurement sites suggested that differences in impression hardness values were not caused by variation in water content of horn. Histological examination of pigmented claw wall horn and underlying soft tissue showed pigment-containing cells in the coronary epidermis but not coronary dermis or in dermis or epidermis of laminae. Pigmentation appeared concentrated in melanosomes in cells along the basement membrane and was present uniformly throughout the epidermis and in cells lining horn tubules. There was no pigmentation detected in the soft tissue epidermis of claws selected visually for non-pigmented wall horn. The presence of melanosomes in epidermal cells from the pigmented coronary region was demonstrated by electron microscopy. The results suggest that pigmentation in the coronary region is associated with reduced initial development of hardness. The possible antagonism between antioxidant properties of melanins and requirement for an oxidative environment for disulphide bond formation in the cytoskeleton of horn cells is discussed.

Melanosomes Transported by Myosin-V in Xenopus Melanophores Perform Slow 35 nm Steps

We studied the motion of pigment organelles driven by myosin-V in Xenopus melanophores using a tracking technique with precision of 2 nm. The organelle trajectories showed occasional steps with a distribution centered at 35 nm and a standard deviation of 13 nm, in agreement with the step size of myosin-V determined in vitro. In contrast, trajectories of melanosomes in cells expressing a dominant negative form of myosin-V did not show steps. The step duration was in the range 20–80 ms, slower than what it would be expected from in vitro results. We speculate that the cytoplasm high viscosity may affect significantly the melanosomes’ motion.

Characterization of melanophore morphology by fractal dimension analysis.

Fractal or focal dimension (FD) analysis is a valuable tool to identify physiologic stimuli at the cellular and tissue levels that allows for quantification of cell perimeter complexity. The FD analysis was determined on fluorescence images of caffeine- or epinephrine-treated (or untreated control) killifish Fundulus heteroclitus (Linneaus) melanophores in culture. Cell perimeters were indicated by rhodamine-phalloidin labeling of cortical microfilaments using box-counting FD analysis. Caffeine-treated melanophores displayed dispersed melanosomes in cells with less serrated edges and reduced FD and complexity. Complexity in epinephrine-treated cells was significantly higher than the caffeine-treated cells or in the control. Cytoarchitectural variability of the cell perimeter is expected because cells change shape when cued with agents. Epinephrine-treated melanophores demonstrated aggregated melanosomes in cells with more serrated edges, significantly higher FD and thus complexity. Melanophores not treated with caffeine or epinephrine produced variable distributions of melanosomes and resulted in cells with variably serrated edges and intermediate FD with a larger SE of the regression and greater range of complexity. Dispersion of melanosomes occurs with rearrangements of the cytoskeleton to accommodate centrifugal distribution of melanosomes throughout the cell and to the periphery. The loading of melanosomes onto cortical microfilaments may provide a less complex cell contour, with the even distribution of the cytoskeleton and melanosomes. Aggregation of melanosomes occurs with rearrangements of the cytoskeleton to accommodate centripetal distribution of melanosomes. The aggregation of melanosomes may contribute to centripetal retraction of the cytoskeleton and plasma membrane. The FD analysis is, therefore, a convenient method to measure contrasting morphologic changes within stimulated cells.

Rab27a enables myosin Va-dependent melanosome capture by recruiting the myosin to the organelle

The peripheral accumulation of melanosomes characteristic of wild-type mouse melanocytes is driven by a cooperative process involving long-range, bidirectional, microtubule-dependent movements coupled to capture and local movement in the actin-rich periphery by myosin Va, the product of the dilute locus. Genetic evidence suggests that Rab27a, the product of the ashen locus, functions with myosin Va in this process. Here we show that ashen melanocytes, like dilute melanocytes, exhibit normal dendritic morphology and melanosome biogenesis, an abnormal accumulation of end-stage melanosomes in the cell center, and rapid, bidirectional, microtubule-dependent melanosome movements between the cell center and the periphery. This phenotype suggests that ashen melanocytes, like dilute melanocytes, are defective in peripheral melanosome capture. Consistent with this, introduction into ashen melanocytes of cDNAs encoding wild-type and GTP-bound versions of Rab27a restores the peripheral accumulation of melanosomes in a microtubule-dependent manner. Conversely, introduction into wild-type melanocytes of the GDP-bound version of Rab27a generates an ashen/dilute phenotype. Rab27a colocalizes with end-stage melanosomes in wild-type cells, and is most concentrated in melanosome-rich dendritic tips, where it also colocalizes with myosin Va. Finally, neither endogenous myosin Va nor an expressed, GFP-tagged, myosin Va tail domain fusion protein colocalize with melanosomes in ashen melanocytes, in contrast to that seen previously in wild-type cells. These results argue that Rab27a serves to enable the myosinVa-dependent capture of melanosomes delivered to the periphery by bidirectional, microtubule-dependent transport, and that it does so by recruiting the myosin to the melanosome surface. We suggest that Rab27a, in its GTP-bound and melanosome-associated form, predominates in the periphery, and that it is this form that recruits the myosin, enabling capture. These results argue that Rab27a serves as a myosin Va 'receptor', and add to the growing evidence that Rab GTPases regulate vesicle motors as well as SNARE pairing.