Effects of acrylamide, latrunculin, and nocodazole on intracellular transport and cytoskeletal organization in melanophores

Abstract

The effects of acrylamide (ACR), nocodazole, and latrunculin were studied on intracellular transport and cytoskeletal morphology in cultured Xenopus laevis melanophores, cells that are specialized for regulated and bidirectional melanosome transport. We used three different methods; light microscopy, fluorescence microscopy, and spectrophotometry. ACR affected the morphology of both microtubules and actin filaments in addition to inhibiting retrograde transport of melanosomes but leaving dispersion unaffected. Using the microtubule-inhibitor nocodazole and the actin filament-inhibitor latrunculin we found that microtubules and actin filaments are highly dependent on each other, and removing either component dramatically changed the organization of the other. Both ACR and latrunculin induced bundling of microtubules, while nocodazole promoted formation of filaments resembling stress fibers organized from the cell center to the periphery. Removal of actin filaments inhibited dispersion of melanosomes, further concentrated the central pigment mass in aggregated cells, and induced aggregation even in the absence of melatonin. Nocodazole, on the other hand, prevented aggregation and caused melanosomes to cluster and slowly disperse. Dispersion of nocodazole-treated cells was induced upon addition of alpha-melanocyte-stimulating hormone (MSH), showing that dispersion can proceed in the absence of microtubules, but the distribution pattern was altered. It is well established that ACR has neurotoxic effects, and based on the results in the present study we suggest that ACR has several cellular targets of which the minus-end microtubule motor dynein and the melatonin receptor might be involved. When combining morphological observations with qualitative and quantitative measurements of intracellular transport, melanophores provide a valuable model system for toxicological studies.

Effects of Hydroquinone on Cytoskeletal Organization and Intracellular Transport in Cultured Xenopus laevis Melanophores and Fibroblasts

“…At 50 μM HQ dispersion could be induced by addition of α-MSH in spite of the lack of actin filaments, showing that melanosomes can be transported along the reorganized microtubules. This effect differs from what is previously known from removal of actin-filaments by latrunculin, which induces aggregation of melanosomes in Xenopus [13,15], and a marked bundling of microtubules [13]. No dispersion can be induced by α-MSH in latrunculintreated cells, even if microtubules are present.…”

“…Adhesion is well known to be actin dependent [16], and its decrease implies an interaction of HQ with actin or actinbinding proteins, which has been shown in other cells as well [1][2][3]. In cells with preaggregated pigment, addition of 100 μM HQ induced a loosening of the central pigment mass, similar to what can be seen when microtubules are depolymerised [13,15]. However, since we were unable to perform immunocytochemistry on cells incubated with 100 μM HQ, no clear conclusions can be drawn about the reason for this.…”

“…HQ induced reorganization of microtubules into thin bundles, while all actin filaments and stress fibres disassembled. We have previously found that the toxic substance acrylamide can induce microtubule bundles, and the same effect is seen when using the actin-depolymerizing drug latrunculin, showing interdependence between microtubules and actin filaments [13]. However, microtubule bundles induced by acrylamide and latrunculin were thicker than those seen after incubation with HQ.…”

“…Movements of the black melanosomes can be easily visualized with light microscopy and the direction of transport can be regulated. We have recently established that these cells can be valuable for toxicological studies [13,14].…”

Electro-mechanical manipulation of mammalian cells in suspension

“…Treatments of cells with acrylamide (ACR) induced the formation of vimentin aggregates (Fig. 3.7 C), similar to what can be seen in PtK2 cells [100], and in melanophores following treatment with either ACR or nocodazole [107]. However, no significant changes in measured mechanical properties were found to occur without some concomitant disruption of MF networks (top-left images in Fig.…”