Cell Tissue Res (2003) 314:381–388 DOI 10.1007/s00441-003-0773-6
REGULAR ARTICLE
Ashok K. Chakraborty · Yoko Funasaka · Keishi Araki · Tatsuya Horikawa · Masamitsu Ichihashi
Evidence that the small GTPase Rab8 is involved in melanosome traffic and dendrite extension in B16 melanoma cells Received: 9 March 2003 / Accepted: 8 July 2003 / Published online: 26 August 2003 Springer-Verlag 2003
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 This investigation was supported in part by Grants-In-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (grant 15591176) A. K. Chakraborty ()) Department of Dermatology, Yale University School of Medicine, New Haven, CT 06520, USA e-mail:
[email protected] Fax: +1-203-7857637 Y. Funasaka · K. Araki · T. Horikawa · M. Ichihashi ()) Department of Dermatology, Kobe University School of Medicine, 650-0017 Kobe, Japan e-mail:
[email protected] Fax: +1-78-3826149
melanoma cells, might have a role in melanosome traffic and dendrite extension, both in constitutive and regulated fashion. Keywords Rab8 · Melanosome transport · Actin filaments · Cell culture · Mouse
Introduction Melanosomes are melanocyte-specific membrane-bound organelles that arise from coated vesicles from the Golgi apparatus, and mature melanosomes are transported along the dendrites to neighboring keratinocytes, where they confer the intensity of skin color and protect the cells against possible assault from damaging radiation (for review, see Jimbow et al. 1993a; Hearing and King 1993). Melanosome biogenesis involves the transport of structural and enzymatic proteins, tyrosinase and tyrosinaserelated proteins (TRPs), Gp100 (Pmel 17), and lysosomalassociated membrane protein (LAMP), by vesicles from Golgi (trans-Golgi network, TGN) to the melanosomal compartment (Jimbow et al. 1997, 2000a, 2000b; Orlow 1998; Raposo et al. 2001). During the formation of transport vesicles, they assemble on the cytoplasmic face of the TGN to select cargo by interacting directly or indirectly with coat proteins (Jimbow et al. 2000a, 2000b). Following biogenesis of the melanosome, it is translocated along the dendrites for secretion (Jimbow and Sugiyama 1998). Two possible pathways for transferring melanin pigment outside the cytosol of melanocytes have been proposed: first, a phagocytic process during which the keratinocytes engulf the tip of the dendritic process of melanocytes including melanosomes (Klaus 1969; Wolff 1973). Second is a process where melanosomal membranes fuse to the cellular membranes and excrete the melanin pigment outside the melanocytes followed by uptake by keratinocytes (Moellmann et al. 1988; Yamamoto and Bhawan 1994; Virador et al. 2001). How-
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ever, the actual regulating mechanisms of melanosome transport have not yet been clarified. As it is already known that small GTP-binding proteins (SMG) of the Rab family are involved in the transport of many proteins in endoplasmic reticulum (ER)-Golgilysosome (endosome) systems (Nakano and Muramatsu 1989; Gravotta et al. 1990; Goud and McCaffrey 1991; Waters et al. 1991; Coleman and Sprang 1996), it is quite likely that SMG-binding proteins present in, or associated with, the melanosomal proteins play a critical role in mediating vesicular transport of proteins. Of the five Rab proteins, Rab7, Rab3a, Rab8, Rab27a, and Rab27b have so far been identified as being associated with or present in melanosomes (Araki et al. 2000; Scott and Zhao 2001; Gomez et al. 2001; Bahadoran et al. 2001; Hume et al. 2001), and Rab7 has been implicated in the transport of tyrosinase-related protein 1 (TRP-1) through the Golgi stack to the melanosome (Gomez et al. 1996, 2001). Rab3A has been suggested to play a role with SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) in targeting melanosomes to the plasma membrane, transfer of melanosomes to keratinocytes, or both (Scott and Zhao 2001). While Rab27a was shown to participate in melanosome transport and its defect leads to a clustering of melanin pigment in the hair shaft as well as defective melanosome transport in the melanocyte (Wu et al. 1998; Menasche et al. 2000; Wilson et al. 2000; Bahadoran et al. 2001), a dominant negative mutant of Rab27b has recently been shown to disrupt melanosomal movement (Chen et al. 2002). However, the localization of Rab8, though identified in melanosomes by immunoblotting, has not yet been characterized functionally (Gomez et al. 2001). Based on known effects of Rab8 on vesicular traffic between the trans-Golgi network (TGN) and the basolateral plasma membrane in different cell types including hippocampal neurons (Huber et al. 1993a, 1993b; Fischer et al. 1994; Mohrmann and van der Sluijs 1999), it can be speculated that Rab8 might have a role in melanocytes for melanosome secretion. Dendrite formation is critically important for melanosome transfer (Fitzpatrick et al. 1967), but pigment cell stimulators like melanocyte-stimulating hormone (MSH) and UV enhance melanogenesis via different mechanisms. MSH increases melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes, but does not stimulate the proliferation of melanocytes. UV irradiation increases pigmentation by increasing melanocyte proliferation as well as by increasing melanocyte activity (Gibbs et al. 2000). Determining the precise subcellular location of Rab protein(s) under basal and/or stimulated conditions may provide important insights into their possible functions and interacting partners. Here we present evidence that Rab8 is localized in melanosomal fractions, and the deficiency of Rab8 impairs the intracellular melanosome traffic and dendrite formation in B16 melanoma cells.
Materials and methods Cells and treatment B16 mouse melanoma cells were grown and passaged in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. For stimulation with MSH, cells were treated with b-MSH (2107 M) in conjunction with cyclic phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX) (104 M, IBMX) to potentiate the MSH action (Pawelek 1979). During ultraviolet radiation B (UVB), irradiation (50 mJ/cm2) from a bank of five fluorescent sunlamps (Toshiba FL20SE, Toshiba Medicals, Tokyo, Japan) emitting 280– 370 nm (mainly UVB energy with a peak at 305 nm) was used. Prior to UVB irradiation, the culture medium was replaced with Ca2+/Mg2+-free phosphate-buffered saline, PBS(-), and immediately afterwards PBS was removed and culture medium was added back to the cells. The cells were further incubated for 48 h at 37C in a 5% CO2 atmosphere. Flux intensity was measured with an UVR-305/365D digital radiometer (Opto-Electronic Measuring Instruments, Tokyo Optical, Tokyo, Japan). Antibodies and animals Polyclonal anti-Rab-8 antibody, which according to the manufacturer’s data sheet is highly specific and does not cross-react with other member proteins of the Rab family, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal anti-TRP1 antibody (Mel-5) and peroxidase conjugated goat antirabbit immunoglobulin antibody were purchased from Signet Laboratories, Inc. (Dedham, MA, USA), and Amersham-Pharmacia Biotech (Bucks., UK), respectively. Texas red-conjugated goat anti-rabbit immunoglobulins and FITC-conjugated goat anti-mouse immunoglobulins were from Becton Dickinson Immunocytometry Systems (San Jose, CA, USA), and Organon Teknika Co. (West Chester, NY, USA), respectively. FITC-conjugated phalloidin was purchased from Sigma (St. Louis, MO, USA). Isolation of vesicle fractions and melanosome-enriched fractions To obtain vesicles and a melanosome-enriched fraction from melanoma cell extracts, a sucrose density gradient was used as previously described (Chakraborty et al. 1989). Briefly, B16 melanoma cells were harvested from implanted tumors on the back of C57BL/6 mice and washed twice with cold PBS(-). Excess connective tissues were carefully trimmed off with small scissors and the tumors were cut into small pieces. The small pieces of the tumors were suspended in homogenizing buffer (0.25 M sucrose, 100 mM 2-N-morpholinoethanesulfonic acid, pH 6.5, 1 mM EGTA, 0.5 mM MgCl2, 100 mM phenylmethylsulfonyl fluoride), and homogenized using a Potter-Elvehjem grinder. The lysate was centrifuged at 700 g for 10 min, and the supernatant was recentrifuged at 11,000 g for 10 min. The pellet was saved for melanosome isolation, while the supernatant was centrifuged at 100,000 g for 1 h to pellet the small granule fractions. The small granule fractions, containing vesicles, were resuspended in homogenizing buffer and were recentrifuged through a discontinuous gradient of 5–60% sucrose for 1 h at 100,000 g. The vesicleenriched fractions were collected from the 10–30% sucrose interface. The 11,000-g pellet was resuspended in the homogenizing buffer and applied to a discontinuous gradient of 1.0–2.0 M sucrose and centrifuged for 90 min at 100,000 g. Melanosome-enriched fractions were collected at the 1.8–2.0 M interface. All the purification procedures were performed at 4C. Structural integrity of the isolated organelles was checked by electron microscopy.
383 Oligonucleotide treatment Antisense of Rab8 (ATG-antisense, 5’-CCATATTACACTCTC-3’; inner-antisense, 5-AATCGTAGGTCTTCG-3’) and Rab3A (ATGantisense, 5’-CCATCTTGCCCTCTC; inner-antisense, 5’-AGTCTGTGGCTGAGG-3’) used in this study were synthesized as described by Huber et al. (1995). The sequence sites selected were centered on the initiation ATG (ATG antisense) and on a nonoverlapping site located immediately downstream (inner region antisense), deduced from a canine Rab8 cDNA (Chavrier et al. 1990) and the rat Rab3A cDNA (Elferink et al. 1992). Pairs of control oligonucleotides were designed from identical regions by reversing the sequences of the ATG and inner-region oligonucleotides. Oligonucleotides were chosen short enough to enter living cells and long enough to be sequence specific (Akhtar and Juliano 1992; Marcus-Sekura 1988). Phosphothioate oligonucleotides were synthesized via phosphoramidite chemistry by sulfurization with tetraethylthiuram disulfide in acetonitrile (Vu and Hirschbein 1991), using an ABI 394 DNA/RNA synthesizer (Applied Biosystems), ethanol precipitated, and taken up in sterile water. Transfection of oligonucleotides (8 mg DNA) was carried out, and after 4 h the cells were plated in six-well plates. Superfect transfection reagent (Qiagen, Valencia, CA, USA) was used in this experiment, and the method was followed according to the manufacturer’s instructions. SDS-PAGE and immunoblot analysis Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 13% polyacrylamide gel by the method of Laemmli (1970), and transferred electrophoretically to polyvinyl difluoride (PVDF) membrane (Immobilon-P; Millipore Co., Bedford, MA, USA). After blocking non-specific binding with 5% BSA in TBS (50 mM TRIS/HCl, pH 7.5, 0.2 M NaCl) for 60 min, the membrane was incubated with rabbit anti-Rab8 antibody (1:500) for 60 min at room temperature followed by incubation with peroxidase conjugated goat anti-rabbit IgG (1:1000) for an hour. Subsequent visualization of antibody binding was carried out with enhanced chemiluminescence reagent (Amersham Pharmacia Biotech., Inc., Piscataway, NJ, USA) according to the manufacturer’s instructions. Confocal fluorescence microscopy Cells were fixed with cold acetone, and preincubated with 5% BSA in PBS(-) for 1 h to block non-specific binding before coincubation with rabbit anti-Rab 8 antibody (1:200) and mouse anti-TRP-1 monoclonal antibodies (Mel-5, 1:200) for 60 min. The cells were then washed 3 times with PBS(-), and incubated with Texas redconjugated goat anti-rabbit IgG (1:200) and FITC-conjugated goat anti-mouse immunoglobulin (1:200) for 60 min. In some experiments, Rab8 localization with actin was studied where actin was localized with FITC-conjugated phalloidin. Cells were washed 3 times with PBS(-) before mounting on a coverslip for microscopic analysis. Confocal images were obtained with an MRC-1024 confocal imaging system (Japan Bio-Rad, Tokyo, Japan).
Results Subcellular distribution of Rab8 in MSH and UVB exposed melanoma cells Electron-microscopic examination reveals that the vesicle fractions consist of numerous vesicular structures, many with full or partial coats, and melanosomal fractions consist of many ellipsoidal bodies having an oriented
Fig. 1 Immunoblot analysis of subcellular localization of Rab8 in B16 mouse melanoma cells. Homogenate (Ho), as well as purified vesicle fractions (V) and melanosome-enriched fractions (MS) from untreated (C control), MSH/IBMX (M) and ultraviolet-B-irradiated cells (U), were resolved on a 13% SDS-polyacrylamide gel, transblotted to PVDF (immobilon-P) membrane, and probed with rabbit anti-Rab8 antibody as described in “Materials and methods.” The same amount of total protein, 20 mg, was loaded in each lane. The arrowhead indicates Rab8, which was detected mostly in the melanosome fractions with a faint expression in vesicle fractions (lower panel). Note that the expression is upregulated by MSH and UVB in cell homogenate (upper panel), in vesicles, and in melanosomes (lower panel). Since the band intensities in melanosome fractions are much thicker, the two- to threefold increase, however, may not be very obvious as we see in the case of cell homogenate
array of membranes (not shown). SDS-PAGE followed by immunoblot analysis revealed that Rab8 is mainly present in melanosome-enriched fractions; however, a faint band is also seen in vesicles. Pretreatment of cells with MSH/ IBMX or UVB increased the expression of Rab8 in cell homogenate (Fig. 1, upper panel), in vesicles, and in melanosomes (Fig. 1, lower panel). The effect of MSH/ IBMX appears to be higher than that of UVB irradiation. Since band intensities in melanosome fractions are much thicker, the two- to threefold increase, however, may not be very obvious as we see in the case of cell homogenate. Confocal fluorescence microscopic images of Rab8 Confocal video-microscopic evidence suggests that in untreated B16 melanoma cells, Rab8 (red) is colocalized with Mel5 (green) at the trans-Golgi area and cytoplasmic vesicles (Fig. 2A–C). MSH/IBMX treatment for 2 days shows dendrite formation, with numerous processes containing Rab8-associated melanosomes (yellow) distributed along the tips of the dendrites (Fig. 2D–F). While UVB irradiation causes the cells to be mostly rounded instead of dendritic, Rab8 is mainly found to be restricted in association with Mel5 at the trans-Golgi and cytoplasmic area (Fig. 2G–I).
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Fig. 2A–I Confocal images of Rab8 and Mel-5 in B16 melanoma cells. B16 cells treated with or without (control, A–C) MSH/IBMX (D–F) or UVB (G–I) were fixed and double stained with rabbit anti-Rab8 polyclonal antibody (A, D, G; red) and MoAb Mel-5 (B, E, H; green) as described in “Materials and methods.” C, F and I (yellow fluorescence) indicate colocalization of Rab8 and Mel-5. In control cells, colocalization was observed in the trans-Golgi area (arrowheads) and cytoplasmic vesicles (double arrowheads). MSH treatment for 2 days (D–F) shows an increased amount of dendrite formation with numerous processes containing Rab8-associated melanosomes distributed along the tips of the dendrites (F, arrows). UVB irradiation (G–I) makes the cells mostly rounded rather than dendritic, while Rab8 is found to be restricted in association with Mel5 mainly at the trans-Golgi and cytoplasmic area (I, arrowheads). Bar 20 nm
Fig. 3A–I Confocal microscopy of B16 melanoma cells doublelabeled with rabbit anti-Rab8 antibody and phalloidin. Cells were labeled with anti-Rab8 antibody followed by second antibody conjugated with Texas red (red), and double labeled with fluorescein-conjugated phalloidin (green). Note that, in unstimulated cells (A–C) Rab8 is partially colocalized with phalloidin at the periphery (C, arrowhead) and along the dendrites (arrows). MSH/IBMX treatment (D–F) enhances their colocalization both at the cytoplasmic area (F, arrowhead) and at the ruffled membrane (arrows). UVB irradiation (G–I) causes their colocalization predominantly at the cytoplasmic area (long arrow), instead of at the cell membrane (arrowhead). Furthermore, no dendrites are seen in UV-treated cells; rather, cells are rounded. Bar 20 nm
Colocalization of Rab8 with actin filaments Since Rab8 is known to modulate polarized membrane transport through reorganization of actin and microtubules, and directs membrane transport to the cell surface (Peranen et al. 1996; Peranen and Furuhjelm 2000), we tested the localization of Rab8 and FITC-conjugated phalloidin in our studies. In unstimulated cells, Rab8 is colocalized with phalloidin at the periphery (Fig. 3A–C), but with much less intensity compared to MSH/IBMXtreated cells (Fig. 3D–F). In UVB-treated cells, no dendrites are formed; rather cells are rounded, and Rab8 is colocalized with actin predominantly at the cytoplasmic area, instead of the cell membrane (Fig. 3G–I). Rab8 antisense reduces the protein expression Western blot experiments revealed the level of expression of the Rab8 protein in control and antisense oligonucleotide-treated B16 melanoma cells (Fig. 4). More than a 70% reduction in Rab8 level was noted in the Rab8antisense-treated cells (Fig. 1, lane 3), but not in Rab3aantisense-treated cells (lane 2), when compared to reagent
Fig. 4 Immunoblot analysis of Rab8 in B16 mouse melanoma cells after treatment with antisense oligonucleotide. Cell homogenates were resolved on a 13% SDS-polyacrylamide gel, transblotted to PVDF (immobilon-P) membrane, and probed with rabbit anti-Rab8 antibody as described in “Materials and methods” (lane 1 no transfection, lane 2 Rab3A antisense oligonucleotide, lane 3 Rab8 antisense, lane 4 superfect transfection reagent only, no DNA). Expression of b-actin protein level was shown to evaluate the effect of Rab8 antisense on its protein level. Note that, although some inhibition on protein level by the reagent itself was noted, the effect of Rab8-antisense is obvious in reducing its protein level when compared with the effect of Rab3A antisense and/or reagent blank
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Fig. 5A–I Effect of Rab8 antisense on MSH/IBMX-induced intracellular melanosome distribution and dendrite formation. B16 melanoma cells after being transfected with Rab8 antisense or with reverse antisense (negative control) oligonucleotides were incubated with MSH/IBMX for 2 days before confocal immunofluorescence studies. A, D, G indicate staining with Rab8 (red), while B, E and H indicate staining with Mel5 (green). C, F and I indicate colocalization of Rab8 and Mel5 (yellow). In the case of no transfection, cells on incubation with MSH/IBMX (A–C) exhibit
formation of numerous dendrites as well as melanosome transport towards the tip of the dendrites (C, arrowheads). Antisense treatment significantly reduces Rab8 labeling, intensity of colocalization with Mel5 and dendrite formation, and the localization of Rab8 protein mainly remains in the cytoplasmic vesicles (arrows, D–F). However, reverse ATG-antisense did not affect either Rab8 expression or dendrite formation, and the colocalization pattern of Rab8 with Mel5 was similar to control cells, with no transfection (G–I)
blank (lane 4). Although we found some inhibition at the protein level by the reagent itself, which may be due to some toxicity, the effect of Rab8 antisense is obvious in the reduction of its protein level when compared with the effect of Rab3A antisense and/or reagent blank. Further, the effect is specific for Rab8 protein, since the level of expression of Rab3a after Rab8 antisense treatment was the same as in the control cells (data not shown).
changes, but also a significant reduction in Rab8 labeling of Rab8 in antisense-treated cells (Fig. 5D–F) compared to cells treated with MSH/IBMX but no transfection (Fig. 5A–C). Furthermore, the Rab8 (red) was found exclusively in the cell body, possibly in the perinuclear Golgi area, in all of the antisense oligonucleotide-treated cells (Fig. 5D–F); otherwise, it was extended towards the tip of the dendrites (Fig. 5A–C). The typical effect of Rab8 antisense, also seen, was either arrest in process formation or formation of one or two short dendrites. Treatment with control oligonucleotides (reversed ATGantisense) did not affect either Rab8 expression or dendrite formation, and the colocalization pattern of Rab8 with Mel5 was similar to MSH/IBMX-treated cells with no transfection (Fig. 5G–I). These results suggest that depletion of Rab8 specifically interferes with the membrane transport and dendrite formation in B16 melanoma cells.
Rab8 antisense reduces the staining intensity of Rab8, dendrite formation and melanosome transport To examine the effect of Rab8 on melanosome transport and/or dendrite formation, B16 melanoma cells, after antisense treatment, were incubated with MSH/IBMX for 2 days before confocal immunofluorescence studies. MSH/IBMX in control cells stimulates dendrite formation as well as melanosome transport towards the tip of the dendrites. Results show not only the morphological
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Discussion Melanin polymer begins to accumulate in melanosomes, leading to maturation from type I and II (partially melanized) to fully melanized type III and IV in which the internal structure is totally obscured (Seiji et al. 1963). Mature melanosomes are transported to the tip of dendritic processes of melanocytes before being transferred to surrounding keratinocytes. Thus proper pigmentation requires the melanosomes to be transported out from their site of synthesis at the perinuclear region to the cell periphery. The mechanisms underlying the melanosome transfer are less well understood and may occur through a combination of processes, including phagocytosis of the melanocyte dendrite, and exocytosis of the melanosome with uptake by keratinocytes, or both (Cohen and Szabo 1968; Wolff 1973; Moellmann et al. 1988; Yamamoto and Bhawan 1994; Virador et al. 2001). Membrane fusion between the melanosome and the plasma membrane of the melanocyte dendrite or the keratinocyte membrane during the process of transfer is likely to be involved. The movement of membranes and proteins through the secretory pathways involves their orderly progression through a series of intracellular compartments (Palade 1975) and is mediated by vesicles that bud from one compartment and fuse with the next (Ferro-Novick and Jahn 1994; Rothman and Weieland 1996). In many eukaryotic cells, the secretion of biomolecules is mediated through both the constitutive and regulated transport of vesicles (Pfeffer 1999). Accumulated evidence suggests that members of the Rab subfamily regulate vesiclemediated protein transport by recruiting tethering and docking factors to establish firm contact between the membranes to fuse, after which SNAREs become involved to complete the fusion process (Novick and Zerial 1997; Pfeffer 1999; Gerst 1999; Watson 1999). Each unique interorganelle transport pathway within the cell is regulated by a distinct Rab GTPase, and this is reflected in the localization of each Rab to distinct organelles (Seabra et al. 2002; Novick and Zerial 1997; Lazar et al. 1997). Our present study with immunoblot experiments demonstrates that melanosome fraction purified through discontinuous sucrose gradient (1.0–1.5–1.8–2.0 M) contains a significant amount of Rab8, whereas the vesicle fraction which contains much less protein in total, exhibits a faint expression of Rab8 (Fig. 1). Stimulation of cells by MSH/IBMX and UVB radiation upregulates the Rab8 expression; however, the effect of MSH/IBMX appears to be much higher than that of UVB irradiation. Confocal imaging showed that in untreated B16 cells Rab8 was colocalized with melanosomal protein, Mel5, at the trans-Golgi area and extended towards the dendrites (Fig. 2A–C). While MSH/IBMX treatment stimulates the formation of dendrites with numerous processes, it also exhibits extensive colocalization of Rab8 with Mel5 in these processes (Fig. 2D–F). A similar pattern of Rab8 localization from the perinuclear region to the plasma
membrane and to the tip of protrusions has been noted in different cells including hippocampal neurons (Hattula et al. 2002; Chen et al. 1993; Huber et al. 1993a, 1993b, 1995). UVB irradiation makes the cells rounded rather than dendritic, and Rab8 was found to be restricted in association with Mel5 mainly at the trans-Golgi and cytoplasmic area (Fig. 2G–I). In vivo, both UVB irradiation and MSH treatment increase pigmentation (Ortonne 1990; Levine et al. 1991). Since a number of dendrites are evident in MSH/IBMXtreated, but not UVB-irradiated, B16 cells, our present results with differential membrane localization of Rab8 during UVB and MSH stimulation of B16 mouse melanoma cells further distinguish their function in the process of melanosome exocytosis. A similar conclusion was derived from other laboratories that UVB induces an accumulation of melanosomes in melanocytes and enhances phagocytosis by keratinocytes, and treatment with MSH induces exocytosis of melanosomes accompanied by ruffling of the melanocyte membrane and also the phagocytic process of keratinocytes (Virador et al. 2001). Further support for this notion is evident from our confocal image analysis that demonstrates the colocalization of Rab8 with actin filaments (revealed by phalloidin) mostly at the periphery of B16 cells (Fig. 3A–C). During MSH/IBMX treatment, which changes the morphology of the cells with membrane ruffling followed by dendrite formation, abundant colocalization of Rab8 with phalloidin was noticed along the cytoplasmic area to the membrane periphery (Fig. 3D–F). In UVB-irradiated cells, while no dendrites are formed, cells are mostly rounded, and Rab8 assembly with actin filaments was not observed at the cell membrane, but mainly localized at the cytoplasmic area (Fig. 3G–I). Since membrane transport involves a complex process of cyclical translocation among the membrane protein, small GTPase, and cytoskeletal structure (Takai et al. 1996), we do not know at present exactly why Rab8-actin assembly is disrupted during UVB-irradiated arrest of melanosome transport followed by dendrite formation. However, from these differential effects of MSH and UVB, it is indicated that Rab8 interaction with actin filaments is crucial for membrane ruffling and dendrite formation. Previously, Rab8 was shown to regulate post-Golgi transport to the basolateral plasma membrane in epithelial cells or to the dendrites in the neurons (Huber et al. 1993a, 1993b, 1995; Peranen et al. 1996; Kobayashi 2002). Likewise, in photoreceptor cells, Rab8 was shown to regulate the attachment of rhodopsin-bearing vesicles to the microfilament bundles before their incorporation into rod outer segment disk membranes (Deretic et al. 1995). The depletion of Rab8 from mature polarized neurons with antisense oligonucleotides impaired the delivery of a viral glycoprotein to the dendritic plasma membrane during neuronal process outgrowth (Huber et al. 1995). Similarly, other workers have shown that Rab8 regulates post-Golgi transport, constitutive secretion, and elongation of both podocyte processes and neuronal dendrites (Chen et al. 1993; Peranen 1996; Simons et al. 1999;
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Kobayashi 2002; Hattula et al. 2002). Here, we show that Rab8 antisense, while it reduces the cellular content of Rab8, also reduces the staining intensity for Rab8 and reduces dendrite formation in B16 melanoma cells. Furthermore, the localization of Rab8 protein mainly remains in the cytoplasmic vesicles of antisense-treated cells rather than at the dendritic tips as in the case of control cells. Taken together, our present results indicate that Rab8 is involved in melanosome traffic in B16 mouse melanoma cells and hence in the dendrite formation, both in constitutive and regulated fashion. Acknowledgements We thank Miss M. Horie and M. Komoto for technical assistance and Mr. James Platt (Yale) for critical reading of the manuscript.
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