Morphological Studies on Microfilaments and their Organizing Center in Killifish (Fundulus heteroclitus L.) Melanophores

Abstract

Fish chromatophores serve as excellent study models for cytoskeleton-dependent organelle translocations because the distribution of pigmentary organelles can be observed against a time frame by microscopy. In this study the distribution of microfilaments along with microtubules in cultured melanophores of the killifish (Fundulus heteroclitus Linneaus) are examined using whole-cell transmission electron microscopy (WCTEM), fluorescence, and laser scanning confocal microscopy. Dispersing, dispersed, aggregating and aggregated states of pigment are induced by adding either caffeine (for dispersion) or epinephrine (for aggregation) to the cells in a standard culture medium. The cells that exhibited a random melanosome distribution in the standard culture media without these two reagents, served as the control. The results indicate that: (i) a structure considered to be the actin-filament organizing center (AFOC) is in close proximity to the microtubule-organizing center (MTOC); (ii) the radial layout of microfilaments remains similar over four physiological states of pigmentary response with the exception of epinephrine-aggregated pigment, in which the aggregate blocks the viewing of the AFOC and central microfilament rays, yet radial microfilaments, whether central and/or peripheral, are apparent in all physiological states of distribution; and (iii) microfilaments serve, together with microtubules, as scaffolding for melanosomes which migrate in bi-directional rows on cross-bridges, thus shedding light on the mechanisms for orderly melanosome translocations in a structural continuum.

Morphological studies on the mechanisms of pigmentary organelle transport in fish xanthophores and melanophores

“…These melanosomes appear to be connected to microtubules by tiny cross bridges by thin-section TEM (Murphy and Tilney, 1974), and by WCTEM (Haddad et al, 1995;Kimler and Vallarapu, 1997;Kimler et al, 2002), supporting the argument of the structural continuum. The identity of the cross bridges is yet to be determined, however, the suggestion of motor molecules is plausible.…”

“…ϭ 4; P.O.I. ϭ 4) by confocal microscopy (for review see Kimler and Vallarapu, 1997;Kimler et al, 2002). When melanophores were doublelabeled with rhodamine-phalloidin and FITC-conjugated anti ␤-tubulin, both radial microfilaments, as well as radial microtubules were seen, following the same pattern in three out of the four physiologic states of pigment distribution.…”

“…Based on the evidence of the actin polymerization/depolymerization and the existence of the MTOC, we sought to determine the nature of that central "starburst-like" region in the center of a cultured melanophore, by labeling cells with rhodamine-phalloidin for fluorescence microscopy. We proposed the existence of an actin filament-organizing center (AFOC) by WCTEM, fluorescence (Haddad et al, 1995;Kimler and Vallarapu, 1997), and laser scanning confocal microscopic examination (Kimler et al, 2002). Confocal imaging of melanophores with dispersed pigment appears to have solved the limitation of two-dimensional viewing of the AFOC by serial optical sectioning of the melanophores.…”

“…Recently, microfilaments were found to emanate from the cytocenter, which followed the radial pattern of the microtubules in the killifish melanophore. We are calling this central region of actin labeling the AFOC (Haddad et al, 1995;Kimler and Vallarapu, 1997;Kimler et al, 2002), and melanosomes may also translocate upon its radial microfilament tracks. Fluorescence and confocal microscopy indicated that the AFOC is close to the MTOC, and that the radial distribution of microfilaments remains similar in three of the four states of pigmentary distribution.…”

VDR Potentiation and NMDA R Inhibition Facilitates Axo-Dendritic Process Formation in Melanocyte Model for Pigmented Cells in Parkinsonism

“…To further elaborate on MT polymerization and depolymerization, epinephrine, norepinephrine and dopamine (adrenergic transmitters) are capable of inducing polymerization of microtubules through secondary messenger mechanism involving cyclic adenosine monophosphate (cAMP) [24,25]. In the melanocytes isolated from the Tilapia, epinephrine and cAMP drives assembly of motor proteins dyenin and kinesin from the Microtubule Organization Center (MTOC) for counter shading and adaptive coloration (aggregation versus dispersal of melanosomes) in the Tilapia [26]. We are thus anticipating that dopaminergic neurons may contain intrinsic pathways of generating epinephrine to drive a similar movement of melanin pigment in response to impulse from the association areas; similar to the findings of Nilsson and co-workers [27].…”