Evidence that the small GTPase Rab8 is involved in melanosome traffic and dendrite extension in B16 melanoma cells

Abstract

One of the major activities of melanocytes in skin is to produce melanin and transport it via dendrites to neighboring keratinocytes. Here, we present evidence that Rab8, a member of the small GTPase superfamily, is present in purified melanosomal fractions, and is upregulated by pigmentogenic agents like melanocyte-stimulating hormone/isobutylmethyl xanthine (MSH/IBMX) and ultraviolet radiation B (UVB). Confocal immunofluorescence microscopic studies revealed that Rab8 is colocalized with Mel5, a melanosomal protein, at the trans-Golgi area and in the cytoplasmic vesicles of B16 cells. During MSH/IBMX treatment, while a number of dendrites with numerous processes are formed, colocalization is extended towards the tips of protrusions. Since process formation is supported by cytoskeletal assembly as well as membrane transport, we tested the colocalization of Rab8 with actin filaments in B16 cells. Rab8, indeed, colocalized with phalloidin, mostly at the periphery, but when irradiated with UVB, cells were rounded instead of dendritic, and colocalization was found predominantly at the cytoplasmic area. Further, suppression of Rab8 expression by its antisense oligonucleotide revealed the reduction in staining intensity of Rab8 but not of Mel5, dendrite formation and melanosome transport towards the tips of the dendrites in B16 melanoma cells. Taken together, it is suggestive that Rab8, in B16 melanoma cells, might have a role in melanosome traffic and dendrite extension, both in constitutive and regulated fashion.

Inhibition of cell proliferation, migration and invasion of B16-F10 melanoma cells by α-mangostin

“…An additional observable parameter of melanoma cell differentiation is morphological change [29]. B16-F10 cells are usually polygonal but extension of dendritic processes is observed after induction of differentiation [30]. Surprisingly, cells treated with a-M displayed a non-classical differentiate phenotype and acquired an elongated morphology, hence this absence of a marked dendritogenesis requires further investigations.…”

Global Analysis of Host Cell Gene Expression Late during Cytomegalovirus Infection Reveals Extensive Dysregulation of Cell Cycle Gene Expression and Induction of Pseudomitosis Independent of US28 Function

“…Only RNA preparations reaching optical density at 260/280 nm (OD 260/280 ) ratio values of Ն2.0 were used. Labeled cDNA populations were synthesized from 2 g of mRNA per sample by reverse transcription and simultaneous incorporation of Cy3-dUTP (reference sample, green) or Cy5-dUTP (infected samples, red) (Amersham Biosciences, Piscataway, N.J.) with an oligo-dT 20 primer and the Superscript II Reverse Transcriptase (Invitrogen). Reference and sample cDNA populations were mixed, and unincorporated nucleotides were removed by using a CyScribe GFX purification kit (Amersham Biosciences).…”

“…Three of the six consistently upmodulated rabs transcripts encode proteins involved in melanosome transport and morphogenesis (rab8a, rab38, and rab27a). Although rab38 is necessary to target the melanin biosynthesis enzyme tyrp1 to endstage melanosomes (60), rab8 plays a role in melanosome trafficking (20) and rab27a is required for the recruitment of myosin Va to melanosomes, which then stimulates the microtubule-mediated transport of melanosomes to the tips of dendrites in melanocytes and their transfer to adjacent keratinocytes (48). Since fibroblasts do not possess melanosomes, it is tempting to speculate that the observed transcriptional upmodulation of these genes might be part of a virus-controlled strategy to promote its maturation and release through the specific vesicle traffic mechanisms active in melanocytes.…”

Rab8 Regulates the Actin-based Movement of Melanosomes

“…Because Rab8 has been linked to specific isoforms of myosin V and was detected in a subcellular fraction enriched in melanosomes (Pruyne et al, 1998;Rodriguez and Cheney, 2002;Chakraborty et al, 2003), we hypothesized that Rab8 may contribute to the motility of melanosomes. To examine this hypothesis, we first determined whether Rab8 was associated with melanosomes.…”

“…This movement can be considered as a paradigm for the motility of numerous intracellular organelles in eucaryotic cells. Two Rab proteins that have been detected on melanosomes are linked to myosin V. Rab8 in epithelial cells and its orthologue Sec4 in yeast interact with specific myosin V isoforms, whereas activated Rab27 recruits myosin V to melanosomes and thereby regulates the actin-based movement of melanosomes (Pruyne et al, 1998;Hume et al, 2001;Rodriguez and Cheney, 2002;Wu et al, 2002;Chakraborty et al, 2003). To analyze the contribution of Rab27 and Rab8 to the actinbased movement of melanosomes, we reconstituted the movement of isolated melanosomes on actin bundles in vitro.…”