Department of Dermatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
Author for correspondence (e-mail: Glynis_Scott{at}urmc.rochester.edu )
Accepted 4 January 2002
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Summary |
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Key words: Melanosome, Melanocyte, Cdc42, Filopodia, Keratinocyte
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Introduction |
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It is now known that melanosome trafficking is mediated in part by
microtubular motor myosin Va, the product of the dilute locus, which
traps melanosomes at the actin-rich periphery of the dendrite, and rab27a, the
product of the ashen locus
(Mercer et al., 1991;
Provance et al., 1996
;
Wu et al., 1997
;
Wu et al., 1998
;
Wei et al., 1997
;
Wilson et al., 2000
;
Bahadoran et al., 2001
;
Wu et al., 2001
). Rab27a is
involved in the transport of melanosomes through its ability to recruit myosin
Va to the tip of the melanocyte dendrite
(Hume et al., 2001
). These
important and relatively recent insights into melanosome trafficking were made
possible through the use of mutant mouse strains and time-lapse video
microscopy of cultured cells, which allowed direct visualization of melanosome
movement and modifiers of actin, microtubules and their motor proteins. In
contrast with melanosome trafficking, much less is known about melanosome
transfer. A major hurdle that has severely limited progress in understanding
the molecular basis of melanosome transfer has been the lack of a model
system. The majority of studies of melanosome transfer to keratinocytes have
been based on co-cultures of non-human cells observed by electron microscopy.
Studies performed utilizing time-lapse video microscopy have been limited by
the relatively poor resolution achieved
(Mottaz and Zelickson, 1967
;
Cohen and Szabo, 1968
;
Wolff, 1973
). Other more
recent studies have utilized gold particle uptake by keratinocytes, melanin
uptake or transfer of cytoplasmic dyes from melanocytes to keratinocytes to
measure transfer (Seiberg et al.,
2000a
; Seiberg et al.,
2000b
; Sharlow et al.,
2000
; Minwalla et al.,
2001
). In toto, these prior studies led to important observations
that suggested phagocytosis of melanocyte dendrites by keratinocytes as the
major mode of melanosome transfer, although exocytosis of melanosomes into the
extracellular space with uptake by keratinocytes and insertion of melanocyte
dendrites and transfer of melanosomes to keratinocytes have also been proposed
(Yamamoto and Bhawan, 1994
).
Although the more recent studies using particle uptake provide insight into
the role of the keratinocyte in granule uptake, the use of a model system in
which melanosome transfer is being studied directly provides an opportunity to
examine the potential role of the melanocyte in melanosome transfer.
It is well established that Cdc42, a member of the Rho family of
GTP-binding proteins, is involved in filopodia and microspike formation in
many cell types. Filopodia are actin-based structures that arise from neuronal
growth cones and function in neuronal pathfinding
(Davenport et al., 1993;
Rosentreter et al., 1998
). The
recent demonstration that Cdc42 is associated with coatamer proteins in the
Golgi apparatus, that it regulates exit of apical and basolateral proteins
from the Golgi network and is involved in exocytosis of secretory granules in
mast cells is indicative of the diverse roles that Cdc42 plays in cells
(Brown et al., 1998
;
Hong-Geller and Cerione, 2000
;
Wu et al., 2000
;
Müsch et al., 2001
). The
downstream effectors of Cdc42 fall into six families most of which contain a
CRIB-binding domain and include Cdc42-binding kinase, myotonic dystrophy
kinase-related Cdc42-binding kinase, mixed lineage kinase, p21-activated
kinase (PAK), WASP (Wiscot-Aldrich Syndrome Protein), IQGAP and MSE55/BORG/CEP
(Burbelo et al., 1995
). At
least four closely related isoforms of PAK (PAK1, PAK2, PAK3 and PAK4) exist
in mammalian cells (Manser et al.,
1994
; Manser et al.,
1995
; Martin et al.,
1995
; Dan et al.,
2001
). PAK-family kinases are activated by GTP-Cdc42 or GTP-Rac1
as well as G-protein-coupled receptors and cytokines and phosphotidyl-inositol
3-kinase (PI3-kinase); this leads to a change in conformation of the kinase
inducing autophosphorylation on multiple serine and threonine residues and
activation (Knaus et al.,
1995
; Manser et al.,
1997
; Wang et al.,
1999
; Chung et al.,
2001
). Activation of PAK results in effects that mimic Rac1 and
Cdc42 and include lamellipodia and filopodia formation, activation of the
c-Jun N-terminal kinase MAP kinase cascade and NK
B, alteration in cell
motility and inhibition of apoptosis and stimulation of macropinocytosis
(Sells 1997; Sells et al.,
1999
; Frost et al.,
1998
; Frost et al.,
2000
; Dharmawardhane,
2000
). Non-kinases that interact with Cdc42 include the WASP
family, which consist of WASp, N-WASP and related Scar proteins isolated in
Dictyostelium. WASP, in concert with WIP (WASP-interacting protein)
participates with the Arp2/3 complex to induce actin nucleation and filopodia
formation (Symons et al.,
1996
; Miki et al.,
1996
; Miki et al.,
1998
; Rohatgi et al.,
1999
; Banzai et al.,
2000
; Martinez-Quiles et al.,
2001
). WASP is only expressed in hematopoietic cells and is
mutated in patients with Wiscot-Aldrich syndrome, whereas N-WASP is
ubiquitously expressed but is enriched in the brain
(Fukuoka et al., 1997
). In a
cell-free system, addition of active Cdc42 significantly stimulates
neuronal-WASP (N-WASP) by exposure of N-WASPs' actin depolymerizing region,
creating free barbed ends from which actin polymerization can take place
(Suzuki et al., 1998
).
In this report we used have high resolution movies made from digital images to directly observe melanosome transfer to keratinocytes in human cells. These movies, along with electron microscopy of cells in vitro and skin in vivo provide evidence that suggests that melanosome delivery to keratinocytes occurs along filopodia. We show that expression of activated Cdc42 in human melanocytes accentuates filopodia formation and melanosome transport and that melanosomes are enriched in PAK1 and N-WASP, Cdc42-effector proteins. In combination with previous data showing SNARE and rab proteins on melanosomes (Scott and Zhao, 2001), these observations suggest a novel model for melanosome transfer to keratinocytes.
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Materials and Methods |
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Cell culture
Neonatal foreskins were obtained according to the University of Rochester's
Research Subject Review Board. Co-cultures of human melanocytes and
keratinocytes were initiated from human foreskins as previously described
(Scott and Haake, 1991) and
maintained in Keratinocyte Growth Media (KGM, Gibco BRL, Gaithersburg, PA). In
primary skin cultures this media sustains melanocyte growth through the
production of melanocyte growth factors by proliferating keratinocytes
(Halaban et al., 1988
). For
growth of melanocytes, cells were cultured in Melanocyte Growth Media (MGM,
Gibco-BRL).
Time-lapse digital microscopy and image processing
Co-cultures of melanocytes and keratinocytes (approximately 105
cells total) were subcultured on vitrogen-coated 25 mm glass coverslips for
1-2 days and placed in a closed heated chamber (Warner Instruments, New Haven,
CT) maintained at 37°C. The cells were viewed on a Nikon Eclipse
Microscope 800 under differential interference contrast (DIC) optics with a
100x objective. The chamber was perfused with KGM maintained at a
constant temperature of 37°C by an in-line heater (Warner Instruments)
using gravity flow. The rate of flow was approximately 166 µl/min and
imaging lasted 45 minutes. Cell viability was checked following experiments
with trypan blue and no cytotoxicity was observed.
Sequential images were obtained at 8 second intervals using the green filter of a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI). The resulting 8 bit/pixel megapixel (1315X1033) images yielded a resolution of 10 pixels/micron when combined with the 100x microscope objective. A series of operations to reduce noise and artefacts were performed on the images using the Matlab GUI facility (Mathworks, Natick, MA). To further reduce size, the movies were created using QuickTime Pro (Apple Computer Inc, Cupertino, CA).
Labeling of melanocyte and keratinocyte membranes and evaluation of
membrane fusion
Melanocytes grown in MGM and keratinocytes grown in KGM were labeled with
DiI (0.6 µM) and DiO (0.2 µM) respectively for 10 minutes at 37°C
followed by extensive washing. 24 hours later, melanocytes were trypsinized
from the dish and added to keratinocytes on vitrogen-coated 100 mm glass
coverslips at an approximate ratio of 1:1 in KGM. To stimulate melanosome
transfer, co-cultures were irradiated with a single dose of ultraviolet (UV)
irradiation using a solar simulator at a dose of 4 J/cm2 as
previously described (Scott and Zhao, 2001). 24 hours after irradiation, live
cells were viewed on a Nikon Eclipse Microscope 800 and images were captured
with a Spot digital camera. To arrive at an approximate percentage of cells
with membrane fusion, the number of keratinocytes with yellow fluorescence
viewed under a filter to detect both DiI and DiO was counted in 10 random
fields (100x objective). Experiments were repeated three times. Digital
images were postprocessed using Adobe PhotoShop 5.0.
Melanosome isolation and western blotting
Melanosomes were isolated from human melanocytes essentially as described
for isolation of melanosomes from cultured B16F1 cells, with a few
modifications (Scott and Zhao, 2001). The postnuclear supernatant was
centrifuged for 10 minutes at 10,000 g at 4°C to obtain a
large granule and a small granule fraction. The large granule fraction, which
is enriched in melanosomes, was then layered onto a sucrose gradient and
centrifuged at 85,000 g at 4°C for 1 hour. The
melanosome-rich fraction was collected from the 2M layer of the gradient and
lysed in buffer (150 mM NaCl, 10 mM Tris-HCL, pH 7.8, 1% Triton-X) plus
protease inhibitors (Complete TM Mini, Boehringer Mannheim, GmbH, Germany).
Protein samples were quantified using the Bio-Rad Dc protein assay kit
(Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as standard.
Equal amounts of protein were electrophoresed on 10% or 15% precast SDS gels
(Jule Inc, New Haven, CT) and blotted to nitrocellulose membranes (Bio-Rad
Laboratories) using standard procedures. Full range rainbow molecular weight
markers were purchased from Amersham Life Sciences (Arlington Heights, Ill).
Visualization of the immunoreactive proteins was accomplished using an
enhanced chemiluminescence reaction (Amersham Life Sciences). Positive
controls for Cdc42 and N-WASP consisted of mouse brain extracts (StresGen
Biotechnologies, Victoria, Canada); positive controls for PAK1 consisted of
Jurkat cell lysates (Upstate Biotechnology, Lake Placid, NY).
Immunofluorescence staining
Melanocytes grown in MGM were subcultured onto vitrogen-coated 2-well glass
chamber slides (Nalge Nunc International Corp., Naperville, IL). Cell
monolayers were fixed in cold methanol/acetone (1/1) followed by
permeabilization in 0.5% triton-X-100 in stabilization buffer (PBS, 100 mM
MgCl2, 1 mM CaCl2) for 15 minutes, and non-specific
binding of antibody was blocked by incubation of the slides in 10% normal goat
serum. Primary antibodies were applied overnight at 4°C followed by
incubation with appropriate Texas-Red- or fluorescein-conjugated secondary
antibodies for one hour at room temperature. For double labeling experiments,
the second primary antibody was applied for one hour at room temperature
followed by the appropriate secondary antibody. DAPI (Vector Laboratories,
Burlingame, CA) was used to stain nuclei. To stain actin, cells were fixed in
formalin, permeabilized as described above and incubated with Alexa Fluor 595
for 2 hours at room temperature. Images were captured with a Spot digital
camera and post-processed using Adobe PhotoShop 5.0.
Electron microscopy
Melanocyte-keratinocyte co-cultures were grown on vitrogen-coated glass
chamber slides, as described above, in KGM. Cells were fixed for 30 minutes in
2.5% glutaraldehyde in Sorensen's phosphate buffer pH 7.4 and were post-fixed
in 1.0% osmium tetroxide in Sorensen's phosphate buffer. Cell membranes were
enhanced by incubation of cells for 45 minutes in 0.5% uranyl acetate diluted
in 25% ethanol. After dehydrating in a graded series of ethanol, the cells
were infiltrated for 30 minutes with a 1:1 solution of 100% ethanol and 100%
Spurr epoxy resin and were then infiltrated overnight in 100% Spurr epoxy
resin. The next day the slides were inverted onto Spurr epoxy resin filled
BEEM capsules and allowed to polymerize. Capsules were trimmed and
thin-sectioned with 2.0% uranyl acetate and Reynolds lead citrate and viewed
with a Hitachi 7100 electron microscope. To assess purity of melanosome
fractions, melanosomes were fixed in glutaraldehyde and post-fixed in osmium
tetroxide as described above. Melanosomes were captured in 4% warm agarose and
were embedded in Spurr epoxy resin and thin sectioned as described above
except that thin sections were not stained with uranyl acetate or lead
citrate. Electron microscopy of human skin was accomplished by fixation of a 2
mm punch biopsy of skin from a male subject in glutaraldehyde in Sorensen's
buffer overnight. The tissue was processed identically to the cells in culture
except that cell membranes were not enhanced by uranyl acetate.
Infection of cells with adenovirus
Recombinant adenovirus capable of expressing constitutively active Cdc42
(Cdc42V12) and green fluorescence protein (GFP) in the AdEasy
vector (Quantum Biotechnologies, Montreal, Canada), and empty vector
expressing GFP alone, were a kind gift of Dr Bambera (Colorado State
University, CO) and have been described previously
(Brown et al., 2000). Infection
efficiency was monitored by viewing the cells in an inverted phase microscope
(Nikon Diaphot) equipped with a filter to detect GFP. To assess the effect of
Cdc42V12 on melanosome transfer to keratinocytes, pure populations
of melanocytes (105 cells) grown in MGM were infected with either
Adeasy Cdc42V12 or empty vector with a multiplicity of infection
(MOI) of 30. 18 hours later keratinocytes were added (105) and the
co-culture was allowed to grow in KGM for at least 5 days prior to
imaging.
Cdc42 GTPase activity assay
Cell lysates were incubated with GST-PAK-PBD fusion protein according to
the manufacturer's instructions (Cytoskeleton Inc., Boulder, CO), and
GTP-bound Cdc42 was captured by incubation of the lysate with glutathione
beads (BD pharMingen, San Diego, CA). Positive controls consisted of lysates
pre-loaded with GTPS (200 µM). The beads and proteins bound to the
fusion protein were washed in lysis buffer, eluted in Laemmli sample buffer,
resolved on 15% gels and blotted with antibodies against Cdc42.
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Results |
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Melanosome transfer to keratinocytes in culture is an uncommon event; however Fig. 2a shows a sequence of images in which melanosome transfer to keratinocytes has occurred. A melanocyte and keratinocyte viewed at 100x magnification is shown; next to it are enlarged views of sequential images of an area of melanocyte-keratinocyte contact (images 2-5). A filopodia arising from the lateral aspect of the tip of the dendrite overlays or is attached to the keratinocyte membrane. Sequential images demonstrate melanosomes moving upward towards the keratinocyte in single file over the course of 80 seconds. Melanosomes were also frequently observed in filopodia that arose from the body of the dendrite, even in the absence of contact with a keratinocyte (Fig. 2a; image 6). In addition to filopodia, which were easily recognizable owing to their dynamic motion and thin diameter, we also observed shorter thicker projections arising predominantly from the sides of melanocyte dendrites, which contacted keratinocytes (Fig. 2b). Melanosomes were transported along these structures singly or in pairs towards the keratinocyte membrane.
|
Because the optical properties of the filopodia and the keratinocyte
membrane are similar, we were unable to definitively determine whether
membrane fusion occurred using this technique. In an initial attempt to
address this question we utilized two lipophilic fluorescent membrane dyes to
separately label melanocyte and keratinocytes, followed by co-culture of the
cells after a single dose of irradiation to stimulate melanosome transfer.
Lipophilic dyes have been commonly used to assess cell fusion in other cell
types (Sowers, 1985;
Spotl et al., 1995
) and show
little leakage of one dye to another. DiI absorbs maximally at 546 nm and has
a maximum emission at 563 nm. DiO, a closely related compound, absorbs
maximally at 489 nm and its peak emission is at 499 nm
(Sims et al., 1974
;
Montecucco et al., 1979
; Honig
and Hune, 1986). Our preliminary experiments showed that these lipophilic dyes
are rapidly incorporated into melanocyte and keratinocyte cell membranes where
they persist for weeks in culture and show little if any cytotoxicity. 24
hours after irradiation of co-cultures that had been separately labeled with
DiI and DiO, approximately 10% of keratinocytes exhibited yellow fluorescence
when viewed with a filter to detect both dyes
(Fig. 2c). Yellow fluorescence
was observed in an intracellular vesicular pattern, which resembled endosomes,
as well as in larger deposits in the Golgi area. The presence of yellow
fluorescence within endosome-like structures is consistent with membrane
fusion between melanocytes and keratinocytes, with subsequent recycling of the
fused membranes into recycling endosomes and transport to the Golgi apparatus.
We cannot exclude, however, the possibility that keratinocyte-phagocytosis of
melanocyte dendrites resulted in the presence of yellow fluorescence in
keratinocytes. In sham-irradiated cells evidence of membrane fusion was
observed in approximately 1% of keratinocytes, indicating membrane fusion even
in unstimulated cells.
Electron microscopy performed on human melanocyte-keratinocyte co-cultures demonstrated thin projections consistent with filopodia arising from the tips and sides of melanocytes (Fig. 3a). In many cases filopodia were cut in cross section; a longitudinal section of a filopodia is shown in Fig. 3b. Melanosomes aligned themselves near the base of the filopodia and osmophilic membrane-bound bodies consistent with melanosomes were observed within the filopodia (Fig. 3c,d). In some cases we observed direct connection between melanocytes and keratinocytes in the form of thin projections that spanned a small space between the two cells (Fig. 3e,f). Melanosomes appeared to be passing between the melanocyte and the keratinocyte along these projections (Fig. 3f). In human skin in vivo we detected structures consistent with filopodia arising from the sides and tips of dendritis which contained melanosomes within their lumina (Fig. 3g-j).
|
Expression of a Cdc42V12 in human melanocytes results in
melanocyte dendricity and filopodia formation
Because of the well known role of Cdc42 in mediating filopodia formation,
we next examined the effect of expression of constitutively active Cdc42 on
melanocyte morphology, filopodia formation and melanosome transfer to
keratinocytes. To investigate whether the virus-expressed Cdc42V12
would exhibit the expected properties of a constitutively active mutant
construct in melanocytes, in vitro binding assays of lysates of cells infected
with Adeasy virus expressing Cdc42V12 and empty vector-infected
cells with the PBD were performed (Fig.
4a). The PBD is conserved in several effector proteins of Cdc42
and Rac1 and mediates their interactions in a GTP-dependent manner
(Sander et al., 1998). Five
days after infection of melanocytes (1x106 cells) with 30 MOI
of Cdc42V12 AdEasy vector, levels of PBD-bound Cdc42 were increased
in Cdc42V12 expressing cells compared with empty vector-infected
cells. These results indicate that Cdc42V12 expressed from
adenovirus is functionally active in melanocytes. The amount of Cdc42
activation is likely to be underestimated in this experiment because infection
efficiency was only 60% and therefore Cdc42 from non-infected cells diluted
the amount of activated Cdc42. Because the GFP and Cdc42V12 cDNAs
are driven from separate CMV promoters, we confirmed that GFP-expressing cells
also overexpressed Cdc42 by staining infected cells with antibodies to Cdc42.
Virtually all cells that expressed GFP also overexpressed Cdc42 (not
shown).
|
Melanocytes were infected with 30 MOI of Cdc42V12 Adeasy virus or 30 MOI of Adeasy virus (empty vector), and 1 day later keratinocytes were added at a ratio of 1:1. The morphological features of melanocytes 5 days after infection with Cdc42V12 Adeasy vector, viewed under immunofluorescence microscopy, are shown in Fig. 4b (images 1-3). Melanocytes exhibited multiple arborizing dendrites and some cells displayed a growth-cone like morphology. Cells infected with empty vector maintained a bipolar morphology typical of melanocytes grown in the absence of phorbol esters (image 4). To determine whether expression of Cdc42V12 resulted in increased numbers of melanosome-containing filopodia, infected cells were stained with antibodies against TRP-1 (Fig. 4c; images 1 and 2). Filopodia were clearly visible in infected cells owing to the presence of numerous melanosomes within them. Vector-expressing cells stained for TRP-1 showed some melanosomes in filopodia but they were less numerous than in Cdc42V12-expressing cells. (Fig. 4c; images 3 and 4). Time-lapse digital microscopy of these cultures viewed under DIC optics confirmed that Cdc42V12-infected cells exhibited multiple long filopodia arising from dendrite tips (Fig. 4d, images 1-3) and in many cases filopodia contained melanosomes. Cdc42V12 expressing melanocytes showed more extensive contacts with keratinocytes through filopodia. As expected, empty vector expressing melanocytes viewed under DIC optics showed filopodia arising from the tips of dendrites, but the number of filopodia was dramatically fewer than in Cdc42V12-expressing cells (images 4 and 5).
Melanosomes express the Cdc42 effector proteins PAK1 and N-WASP
We next examined the expression and localization of Cdc42 and PAK1 in human
melanocytes by immunofluorescence microscopy
(Fig. 5). We were unable to
perform immunofluorescence staining with the antibodies to N-WASP available to
us. Cdc42 was present in a vesicular pattern with prominent localization to
the melanocyte cell membrane, as well as in the peri-nuclear area
(Fig. 5a; image 1). The
localization of Cdc42 to the peri-nuclear area (presumed to be the Golgi
apparatus) is consistent with previous reports showing that Cdc42 localizes to
the Golgi (Erickson et al., 1996). PAK1 was heavily concentrated in the
peri-nuclear area with some vesicular staining in the dendrites
(Fig. 5a; image 2). Staining of
cells with normal rabbit serum instead of a primary antibody failed to show
any labeling (not shown). To determine if Cdc42 or PAK1 colocalized with
melanosomes, double labeling with antibodies to the melanosome-specific
protein mel-5 (TRP-1) and either Cdc42 or PAK1 was performed
(Fig. 5b). PAK1 and TRP-1
colocalized in the perinuclear region as well as focally in the melanocyte
dendrites (images 1-3). Because melanosomes are heavily concentrated in the
peri-nuclear area, it is difficult to determine whether this staining pattern
represents true colocalization or an artefact of overlay of melanosomes and
other PAK1-expressing structures in this area. Higher power images of
melanocyte dendrites from double-labeled cells (lower panel,
Fig. 5b) shows clear
colocalization of PAK1 and TRP-1 within the dendrites; however colocalization
was not 100%. Cdc42 did not colocalize with melanosomes but did colocalize
with the transferrin receptor, indicating a component of Cdc42 in recycling
endosomes (not shown). Myosin Va in melanocytes colocalizes with melanosomes,
the endoplasmic reticulum, Golgi apparatus and mitochondria
(Nascimento et al., 1997;
Tabb et al., 1998
), and myosin
Va has been shown to play an important role in filopodia extension in neuronal
cells, and in murine melanocytes myosin Va has been identified at the tips of
filopodia (Wang et al., 1996
;
Tsakraklides et al., 1999
).
Myosin Va was also present in a punctate or dot-like pattern at the tips of
filopodia and along the length of the filopodia
(Fig. 5c; image 1 and 2).
|
Western blots were performed for analysis of expression of Cdc42, PAK1 and N-WASP in melanosome fractions (Fig. 6). The purity of the melanosome isolate was assessed by electron microscopy, which demonstrated a relatively homogeneous population of stage III and stage IV melanosomes with few if any contaminating elements such as mitochondria (Fig. 6a). Transferrin receptor expression, used as a marker for recycling endosomes, was not detected in melanosome-enriched fractions, indicating low amounts of contaminating recycling endosomes in the preparation (Fig. 6b). PAK1 was heavily enriched in melanosome extracts; a single strong immunoreactive band was detected (Fig. 6c; 20 µg/lane). Western blotting for N-WASP showed an immunoreactive band at the expected molecular weight for N-WASP in melanosome extracts (Fig. 6c; 70 µg/lane). Cdc42 was not detected in melanosome-enriched fractions even when large amounts of protein (up to 80 µg) were loaded onto the gel (not shown).
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Discussion |
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The well known role of Cdc42 in filopodia formation, as well as data
showing increased filopodia formation in melanocytes expressing a
constiutively active Cdc42 mutant, are consistent with a role for Cdc42 in
mediating filopodia formation in human melanocytes. Although at this point we
can only speculate on the motors involved in moving melanosomes along the
length of the filopodia, the presence of myosin Va at the tips of filopodia
and the fact that filopodia contain actin but not microtubules makes myosin Va
a strong candidate. The rapid movement and attachment of melanocyte filopodia
to keratinocyte membranes is highly analogous to growth cone filopodia, which
contact nearby axons with subsequent synapse formation and synaptic vesicle
transmission. In neuronal cells the primary role of growth cone filopodia is
to sample the immediate environment and translate environmental cues to the
growth cone, which in turn affects growth cone behavior
(Rosentreter et al., 1998;
Harris, 1999
). Recent reports
show that neuronal filopodia respond to exogenous growth factors such as
fibroblast growth factor by doubling their length
(Szebenyi et al., 2001
). In a
similar manner, melanocytes respond to keratinocyte-derived growth factors
through dendrite extension and possibly melanosome transfer
(Imokawa et al., 1995
;
Hara et al., 1995
). It is
likely that melanocyte filopodia respond to a gradient of keratinocyte-derived
growth factors that direct filopodia growth and attachment and subsequent
transfer of melanosomes, although further experiments are needed to establish
this. Sabry et al. (Sabry et al.,
1991
) showed that microtubules invade neuronal filopodia during
growth cone steering events subsequent to filopodia attachment to guidepost
cells. Therefore, another potential function of melanocyte filopodia may be to
serve as a vanguard for dendrite extension. We also frequently observed blunt,
short projections arising from the shafts of melanocyte dendrites, which also
functioned as conduits for melanosome transfer to keratinocytes. These
projections may be analogous to dendritic spines, which, in neuronal cells,
are actincontaining, short bulbous projections that arise from the sides of
neuronal dendrites (Harris,
1999
; Kaech et al.,
2001
).
Activated Cdc42 induced a markedly dendritic morphology in human
melanocytes. Because activation of Rac1 results in dendrite extension in
melanocytes (Scott and Cassidy,
1998) and because others have shown that inhibition of RhoA
results in dendrite extension in melanocytes
(Busca et al., 1998
), signaling
for melanocyte dendrite extension may be analogous to N1E-115 cells in which
activated Rac1 inhibits RhoA with subsequent dendrite extension
(Altun-Gultekin and Wagner,
1996
; Kozma et al.,
1997
). A hierarchical, unidirectional cascade of activation of
Cdc42, Rac and Rho has been described in a variety of cell types (for a
review, see Kjoller and Hall,
1999
). In most cell types, Cdc42 activates Rac1, which leads to
inhibition of Rho activity (Sander et al.,
1999
; Reid et al.,
1999
). These studies suggest that in melanocytes Cdc42 may be
upstream of Rac1 in dendrite formation through activation of Rac1, which in
turn inhibits Rho A. Cdc42 is activated by the inflammatory cytokines tumor
necrosis factor-
and interleukin-1
(Wojciak-Stothard et al.,
1998
; Puls et al.,
1999
), both of which are released by keratinocytes following UV
irradiation, a potent stimulus for melanosome transfer
(Pathak et al., 1978
;
Sturm, 1998
;
Kondo, 1999
). Therefore the
well known effect of UV irradiation on melanocyte dendrite formation and
melanosome transfer may be mediated in part by interleukin-1- and
tumor-necrosis-
-induced activation of Cdc42.
The role of PAK1 and N-WASP, which were enriched in melanosome fractions,
in melanosome movement is unclear and must await further experiments analyzing
the effect of mutants of these proteins on melanosome transport and transfer.
A strong association between PAK1 activation and lamellipodia formation, loss
of stress fibers, disassembly of focal adhesions and increased cell motility
has been demonstrated (Frost et al.,
1998; Sells et al.,
1999
). Daniels et al. (Daniels et al., 1998) have shown that
nerve-growth-factor-induced neurite outgrowth in PC10 cells is mediated by
PAK1. We have shown previously that Rac1 mediates melanocyte dendrite
formation in response to growth factors and to UV irradiation
(Scott and Cassidy, 1998
). It
is possible that in melanocytes, which, similar to PC10 cells, are neuronally
derived cells, PAK1 is a downstream effector for Rac1-mediated dendrite
extension. Although PAK1 is heavily enriched on melanosomes, activated PAK1,
as identified by antibodies to phosphorylated PAK1 (a generous gift of Dr
Chernoff, Fox Chase Cancer Center, Philadelphia, PA) was not associated with
melanosomes but with the small granule fraction of melanocytes as demonstrated
by western blotting (G.S., unpublished). Therefore activation of PAK1 is
unlikely to occur on the melanosome membrane.
It is likely that melanosome transfer is accomplished through multiple
mechanisms, including phagocytosis of dendrite tips and possibly exocytosis of
the melanosome into the extracellular space with uptake by keratinocytes
(Yamamoto and Bhawan, 1994).
Although we did not directly observe phagocytosis of melanocyte dendrites in
time-lapse movies, this may have been due to the relative infrequency of
melanosome transfer in culture, lack of appropriate stimulus or both. Although
the digital movies presented (jcs.biologists.org/supplemental or
www.urmc.rochester.edu/derm/scottmovies.html
) provide intriguing evidence of a role for filopodia in melanosome transport,
we are unable to definitively conclude that melanosome transfer occurred
because of the optical properties of the melanocyte and keratinocyte
membranes. Our initial attempt to circumvent this problem using membrane dyes
is suggestive of keratinocyte-melanocyte membrane fusion but is not
conclusive. We believe that definitive evaluation of melanosome transfer will
require in vivo labeling of melanosomes with a marker that would allow one to
observe movement of melanosomes from the melanocyte to the keratinocyte, in
combination with high resolution digital movies. This would allow one to
assess transferred melanosomes within keratinocytes both through direct
visualization, and through biochemical means, in response to expression of
mutants of a variety of candidate proteins, including PAK1, N-WASP and Cdc42.
At the present time we are evaluating the ability of a GFP-Pmel17 fusion
protein to label human melanocyte melanosomes.
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Altun-Gultekin, Z. F. and Wagner, J. A. (1996). Src, Ras, and Rac mediate the migratory response elicited by NGF and PMA in PC12 cells. J. Neuroscience Res. 44,308 -327.[Medline]
Bagrodia, S., Taylor, S. J., Creasy, C. L., Chernoff, J. and
Cerione, R. A. (1995). Identification of a mouse p21Cdc42/Rac
activated kinase. J. Biol. Chem.
270,22731
-22737.
Bahadoran, P., Aberdam, E., Mantoux, F., Busca, R., Bille, K.,
Yalman, N., de Saint-Basile, G., Casaroli-Marano, R., Ortonne, J. P. and
Ballotti, R. (2001). Rab27a: A key to melanosome transport in
human melanocytes. J. Cell Biol.
152,843
-850.
Banzai, Y., Miki, H., Yamaguchi, H. and Takenawa, T.
(2000). Essential role of neural Wiskott-Aldrich syndrome protein
in neurite extension in PC12 cells and rat hippocampal primary culture cells.
J. Biol. Chem. 275,11987
-11992.
Brown, A., O'Sullivan, A. J. and Gomperts, B. D.
(1998). Induction of exocytosis from permeabilized mast cells by
the guanosine triphosphatases Rac and Cdc42. Mol. Biol.
Cell 9,1053
-1063.
Brown, M. D., Cornejo, B. J., Kuhn, T. B. and Bamburg, J. R. (2000). Cdc42 stimulates neurite outgrowth and formation of growth cone filopodia and lamellipodia. J. Neurobiol. 43,352 -364.[Medline]
Burbelo, P. D., Dreschsel, D. and Hall, A.
(1995). A conserved binding motif defines numerous candidate
target proteins for both Cdc42 and Rac GTPases. J. Biol.
Chem. 270,29071
-29074.
Busca, R., Bertolotto, C., Abbe, P., Englaro, W., Ishizaki, T.,
Narumiya, S., Boquet, P., Ortonne, J. P. and Ballotti, R.
(1998). Inhibition of Rho is required for cAMP-induced melanoma
cell differentiation. Mol. Biol. Cell
9,1367
-1378.
Chung, C. Y., Potikyan, G. and Firtel, R. A.
(2001). Control of cell polarity and chemotaxis by Akt/PKB and
PI3 kinase through the regulation of PAK. Mol.
Cell 7,937
-947.[Medline]
Cohen, J. and Szabo, G. (1968). Study of pigment donation in vitro. Exp. Cell. Res. 50,418 -434.
Corcuff, P., Chaussepied, C., Madry, G. and Hadjur, C. (2001). Skin optics revisited by in vivo confocal microscopy: Melanin and sun exposure. J. Cosmet Sci. 52, 91-102.[Medline]
Dan, C., Kelly, A., Bernard, O. and Minden, A.
(2001). Cytoskeletal changes regulated by the PAK4
serine/threonine kinase are mediated by LIMK and cofilin. J. Biol.
Chem. 276,32115
-32121.
Davenport, R. W., Dou, P., Rehder, V. and Kater, S. B. (1993). A sensory role for neuronal growth cone filopodia. Nature 361,721 -724.[Medline]
Dharmawardhane, S., Schurmann, A., Sells, M. A., Chernoff, J.,
Schmid, S. L. and Bokoch, G. M. (2000). Regulation of
macropinocytosis by p21-activated kinase-1. Mol. Biol.
Cell 11,3341
-3352.
Erickson, J. W. and Cerione, R. A. (2001). Multiple roles for Cdc42 in cell regulation. Curr. Opin. Cell Biol. 13,153 -157.[Medline]
Espreafico, E. M., Cheney, R. E., Matteoli, M., Nascimento, A. A., De Camilli, P. V., Larson, R. E. and Mooseker, M. S. (1992). Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains. J. Cell Biol. 119,1541 -1557.[Abstract]
Frost, J. A., Khokhlatchev, A., Stippec, S., White, M. A. and
Cobb, M. H. (1998). Differential effects of PAK1-activating
mutations reveal activity-dependent and -independent effects on cytoskeletal
regulation. J. Biol. Chem.
273,28191
-28198.
Frost, J. A., Swantek, J. L., Stippec, S., Yin, M. J., Gaynor,
R. and Cobb, M. H. (2000). Stimulation of NFkappa B activity
by multiple signaling pathways requires PAK1. J. Biol.
Chem. 275,19693
-19699.
Fukuoka, M., Miki, H. and Takenawa, T. (1997). Identification of N-WASP homologs in human and rat brain. Gene 196,43 -48.[Medline]
Halaban, R., Langdon, R., Birchall, N., Cuono, C., Baird, A., Scott, G., Moellmann, G. and McGuire, J. (1988). Paracrine stimulation of melanocytes by keratinocytes through basic fibroblast growth factor. Ann. NY Acad. Sci. 548,180 -190.[Abstract]
Hara, M., Yaar, M. and Gilchrest, B. A. (1995). Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity. J. Invest. Dermatol. 105,744 -748.[Abstract]
Harris, K. M. (1999). Structure, development, and plasticity of dendritic spines. Curr. Opin. Neurobiol. 9,343 -348.[Medline]
Hong-Geller, E. and Cerione, R. A. (2000).
Cdc42 and Rac stimulate exocytosis of secretory granules by activating the
IP3/calcium pathway in RBL-2H3 mast cells. J. Cell
Biol. 148,481
-493.
Honig, M. G. and Hume, R. I. (1986). Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures. J. Cell Biol. 103,171 -187.[Abstract]
Hume, A. N., Collinson, L. M., Rapak, A., Gomes, A. Q., Hopkins,
C. R., Seabra, M. C. (2001) Rab27a regulates the peripheral
distribution of melanosomes in melanocytes. J. Cell
Biol. 152,795
-808.
Imokawa, G., Miyagishi, M. and Yada, Y. (1995). Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis. J. Invest. Derm. 105,32 -37.[Abstract]
Kaech, S., Parmar, H., Roelandse, M., Bornmann, C. and Matus,
A. (2001). Cytoskeletal microdifferentiation: A mechanism for
organizing morphological plasticity in dentrites. Proc. Natl. Acad.
Sci. USA 98,7086
-7092.
Kjoller, L. and Hall, A. (1999). Signaling to Rho GTPases. Exp. Cell Res. 253,166 -179.[Medline]
Knaus, U. G., Morris, S., Dong, H., Chernoff, J. and Bokoch, G. M. (1995). Regulation of human leukocyte p21-activated kinases through G protein-coupled receptors. Science 269,221 -223.[Medline]
Kondo, S. (1999). The roles of keratinocyte-derived cytokines in the epidermis and their possible responses to UVA-irradiation. J. Invest. Dermatol. Symp. Proc. 4, 177-183.
Kozma, R., Sarner, S., Ahmed, S. and Lim, L. (1997). Rho family GTPases and neuronal growth cone remodeling: Relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid. Mol. Cell. Biol. 17,1201 -1211.[Abstract]
Kushimoto, T., Basrur, V., Valencia, J., Matsunaga, J., Vieira,
W. D., Ferrans, V. J., Muller, J., Appella, E. and Hearing, V. J.
(2001). A model for melanosome biogenesis based on the
purification and analysis of early melanosomes. Proc. Natl. Acad.
Sci. USA 98,10698
-10703.
Manser, E., Leung, T., Salihuddin, H., Zhao, S.-S. and Lim, L. (1994). A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature 367, 40-46.[Medline]
Manser, E., Chong, C., Zhao, Z.-S., Leung, T., Michael, G.,
Hall, C. and Lim, L. (1995). Molecular cloning of a new
member of the p21-Cdc42/Rac-activated kinase (PAK) family. J. Biol.
Chem. 270,25070
-25078.
Manser, E., Huang, H. Y., Loo, T. H., Chen, X. Q., Dong, J. M.,
Leung, T. and Lim, L. (1997). Expression of constitutively
active -PAK reveals effects of the kinase on actin and focal complexes.
Mol. Cell. Biol. 17,1129
-1143.[Abstract]
Marks, M. S. and Seabra, M. C. (2001) The melanosome: membrane dynamics in black and white. Nat. Rev. Mol. Cell Biol. 2,738 -748.[Medline]
Martin, G. A., Bollag, G., McCormick, F. and Abo, A. (1995). A novel serine kinase activated by rac1/CDC42Hs-dependent autophosphorylation is related to PAK65 and STE20. EMBO J. 14,1970 -1978.[Abstract]
Martinez-Quiles, N., Rohatgi, R., Anton, I. M., Medina, M.,
Saville, S. P., Miki, H., Yamaguchi, H., Takenawa, T., Hartwig, J. H. and
Geha, R. S. (2001). WIP regulates N-WASP-mediated actin
polymerization and filopodium formation. J. Cell Sci.
114,1801
-1809.
Mercer, J. A., Seperack, P. K., Strobel, M. C., Copeland, N. G. and Jenkins, N. A. (1991). Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature 349,709 -713.[Medline]
Miki, H., Miura, K. and Takenawa, T. (1996). N-WASP, a novel actin depolymerizing protein, regulates the cortical cytoskeletal., rearrangement in a PIP2-dependent manner downstream of tyrosine kinases. EMBO J. 15,5326 -5335.[Abstract]
Miki, H., Sasaki, T., Takai, Y. and Takenawa, T. (1998). Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP. Nature 391, 93-96.[Medline]
Minwalla, L., Zhao, Y., Cornelius, J., Babcock, G. F., Wickett, R. R., LePoole, I. C. and Boissy, R. E. (2001). Inhibition of melanosome transfer from melanocytes to keratinocytes by lectins and neoglycoproteins in an in vitro model system. Pigment Cell Res. 14,185 -194.[Medline]
Montecucco, C., Pozzan, T. and Rink, T. (1979) Dicarbocyanine fluorescent probes of membrane potential block lymphocyte capping, deplete cellular ATP and inhibit respiration of isolated mitochondria. Biochim. Biophys. Acta. 552,552 -557.[Medline]
Mottaz, J. H. and Zelickson, A. S. (1967). Melanin transfer: a possible phagocytic process. J. Invest. Dermatol. 49,605 -610.[Medline]
Müsch, A., Choen, D., Kreitzer, G. and Rodriguez-Boulan,
E. (2001). Cdc42 regulates the exit of apical and basolateral
proteins from the trans-Golgi network. EMBO J.
20,2171
-2179.
Nascimento, A. A. C., Amaral, R. G., Bizario, J. C. S., Larson,
R. E. and Espreafico, E. M. (1997). Subcellular localization
of myosin-V in the B16 melanoma cells, a wild-type cell line for the dilute
gene. Mol. Biol. Cell 8,1971
-1988.
Pathak, M. A., Jimbow, K. and Szabo, G. (1978). Sunlight and melanin pigmentation. In Smith KC, ed. Photochem Photobiol Reviews, Vol. 1. New York: Plenum Press, 221-239.
Provance, D. W., Wei, M., Ipe, V. and Mercer, J. A.
(1996). Cultured melanocytes from dilute mutant mice exhibit
dendritic morphology and altered melanosome distribution. Proc.
Natl. Acad. Sci USA 93,14554
-14558.
Puls, A., Eliopoulos, G. G., Nobes, C. D., Bridges, T., Young,
L. S. and Hall, A. (1999). Activation of the small GTPase
Cdc42 by the inflammatory cytokines TNF() and IL-1, and by the
Epstein-Barr virus transforming protein LMP1. J. Cell
Sci. 112,2983
-2992.
Raposo, G., Tenza, D., Murphy, D. M., Berson, J. F. and Marks,
M. S. (2001). Distinct protein sorting and localization to
premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.
J. Cell Biol. 152,809
-824.
Reid, T., Bathoorn, A., Ahmadian, M. R. and Collard, J. G.
(1999). Identification and characterization of hPEM-2, a guanine
nucleotide exchange factor specific for Cdc42. J. Biol.
Chem. 274,33587
-33593.
Rohatgi, R., Ma, L., Miki, H., Lopez, M., Kirchhausen, T., Takenawa, T. and Kirschner, M. W. (1999). The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 97,221 -231.[Medline]
Rosentreter, S. M., Davenport, R. W., Loschinger, J., Huf, J., Jung, J. and Bonhoeffer, F. (1998). Response of retinal ganglion cell axons to striped linear gradients of repellent guidance molecules. J. Neuroscience 37,541 -562.
Sabry, J. H., O'Connor, T. P., Evans, L., Toroian-Raymond, A., Kirschner, M. and Bentley, M. (1991). Microtubule behavior during guidance of pioneer neuron growth comes in situ. J. Cell Biol. 115,381 -395.[Abstract]
Sander, E. E., Van Delft, S., Ten Klooster, J. P., Reid, T., Van
Der Kammen, A., Michiels, F. and Collard, J. G. (1998).
Matrix-dependent Tiam/Rac signaling in epithelial cells promotes either
cell-cell adhesion or cell migration and its regulate by phosphatidylinositol
3-kinase. J. Cell Biol.
143,1385
-1398.
Sander, E. E., ten Klooster, J. P., van Delft, S., van der
Kammen, R. A. and Collard, J. G. (1999). Rac downregulates
Rho activity: Reciprocal balance between both GTPases determines cellular
morphology and migratory behavior. J. Cell Biol.
147,1009
-1021.
Scott, G. A. and Haake, A. R. (1991). Keratinocytes regulate melanocytes number in human fetal and neonatal skin equivalents. J. Invest. Dermatol. 97,776 -781.[Abstract]
Scott, G. A. and Cassidy, L. (1998). Rac1 mediates dendrite formation in response to melanocytes stimulating hormone and ultraviolet light in a murine melanoma model. J. Invest. Dermatol. 111,243 -250.[Abstract]
Scott, G. A. and Zhou, Q. (2001). Rab3a and
SNARE proteins: potential regulators of melanosome movement. J.
Invest. Dermatol. 116,296
-304.
Seiberg, M., Paine, C., Sharlow, E., Andrade-Gordon, P., Costanzo, M., Eisinger, M. and Shapiro, S. S. (2000a). The protease-activated receptor-2 regulates pigmentation via keratinocyte-melanocyte interactions. Exp. Cell Res. 254, 25-32.[Medline]
Seiberg, M., Paine, C., Sharlow, E., Andrade-Gordon, P.,
Costanzo, M., Eisinger, M. and Shapiro, S. S. (2000b).
Inhibition of melanosome transfer results in skin lightening. J.
Invest. Dermatol. 115,162
-167.
Sells, M. A., Knaus, U. G., Bagrodia, S., Ambrose, D. M., Bokoch, G. M. and Chernoff, J. (1997). Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr. Biol. 7,202 -210.[Medline]
Sells, M. A., Boyd, J. T. and Chernoff, J.
(1999). P21-activated kinase 1 (PAK1) regulates cell motility in
mammalian fibroblasts. J. Cell Biol.
145,837
-849.
Sharlow, E. R., Paine, C., Babiarz, L., Eisinger, M., Shapiro,
S. S. and Seiberg, M. (2000). The protease activated
receptor-2 upregulates keratinocyte phagocytosis. J. Cell
Sci. 113,3093
-3101.
Sims, P. J., Waggoner, A. S., Wang, C. H. and Hoffman, J. F. (1974). Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry 13,3315 -3330.[Medline]
Sowers, A. E. (1985). Movement of a fluorescent lipid label from a labeled erythrocyte membrane to an unlabeled erythrocyte membrane following electric-field-induced fusion. Biophys. J. 47,519 -525.[Abstract]
Spotl, L., Sarti, A., Dierich, M. P. and Most, J. (1995). Cell membrane labeling with flourescent dyes for the demonstration of cytokine-induced fusion between monocytes and tumor cells. Cytometry 21,160 -169.[Medline]
Sturm, R. A. (1998). Human pigmentation genes and their response to solar UV irradiation. Mutation Res. 422,69 -76.[Medline]
Suzuki, T., Miki, H., Takenawa, T. and Saskawa, C.
(1998). Neural Wiskott-Aldrich syndrome protein is implicated in
the actin-based motility of Shigella flexneri. EMBO J.
17,2767
-2776.
Symons, M., Derry, J. M., Karlak, B., Jiang, S., Lemahieu, V., McCormick, F., Francke, U. and Abo, A. (1996). Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization. Cell 84,723 -734.[Medline]
Szebenyi, G., Dent, E. W., Callaway, J. L., Seys, C., Leuth, H.
and Kalil, K. (2001). Fibroblast growth factor-2 promotes
axon branching of cortical neurons by influencing morphology and behavior of
the primary growth cone. J. Neuroscience
21,3932
-3941.
Tabb, J. S., Molyneaux, B. J., Cohen, D. L., Kuznetsov, S. A. and Langford, G. M. (1998). Transport of ER vesicles on actin filaments in neurons by myosin V. Science 111,3221 -3234.
Tsakraklides, V., Krogh, K., Wang, L., Bizario, J. C. S.,
Larson, R. E., Espreafico, E. M. and Wolenski, J. S. (1999).
Subcellular localization of GFP-myosin-V in live mouse melanocytes.
J. Cell Sci. 112,2853
-2865.
Wang F.-S., Wolenski, J. S., Cheney, R. E., Mooseker, M. S. and Jay, D. G. (1996). Function of myosin V in filopodia extension of neuronal growth cones. Science 273,660 -663.[Abstract]
Wang, J., Frost, J. A., Cobb, M. H. and Ross, E. M.
(1999). Reciprocal signaling between heterotrimeric G proteins
and the p21-stimulated protein kinase. J. Biol. Chem.
274,31641
-34147.
Wei, Q., Wu, X. and Hammer, J. A., III (1997). The predominant defect in dilute melanocytes is in melanosome distribution and not cell shape, supporting a role for myosin V in melanosome transport. J. Muscle Res. Cell Mot. 18,517 -527.[Medline]
Wilson, S., Yip, R., Swing, D. A., O'Sullivan, N., Zhang, Y.,
Novak, E. D., Swank, R. T., Russell, L. B., Copeland, N. G. and Jenkins, N.
A. (2000). A mutation in the rab27a causes the vesicle
transport defects observed in ashen mice. Proc. Natl. Acad.
Sci. 97,7933
-7938.
Wojciak-Stothard, B., Entwistle, A., Garg, R. and Ridley, A. J. (1998). Regulation of TNF-alpha-induced reorganiztion of the actin cytoskelton and cell-celljunctions by Rho, Rac, and Cdc42 in human endothelial cells. J. Cell. Physiol. 179,150 -165.
Wolff, K. (1973). Melanocyte-keratinocyte interactions in vivo: the fate of melanosomes. Yale J. Biol. Med. 46,384 -396.[Medline]
Wu, W. J., Erickson, J. W., Lin, R. and Cerione, R. A.
(2000). The -subunit of the coatomer complex binds Cdc42
to mediate transformation. Nature
405,800
-803.[Medline]
Wu, X., Bowers, B., Wei, Q., Kocher, B. and Hammer, J. A.
(1997). Myosin V associates with melanosomes in mouse
melanocytes: evidence that myosin-V is an organelle motor. J. Cell
Sci. 110,847
-859.
Wu, X. F., Bowers, B., Rao, K., Wei, Q. and Hammer, J. A.,
III (1998). Visualization of melanosome dynamics within
wild-type and dilute melanocytes suggests a paradigm for myosin V function in
vivo. J. Cell Biol. 143,1899
-1918.
Wu, X., Rao, K., Bowers, M. B., Copeland, N. G., Jenkins, N. A.
and Hammer, J. A. (2001). Rab27a enables myosin Va-dependent
melanosome capture by recruiting the myosin to the organelle. J.
Cell Sci. 114,1091
-1100.
Yamamoto, O. and Bhawan, J. (1994). Three modes of melanosome transfer in Caucasian facial skin: hypothesis based on an ultrastructural study. Pig. Cell Res. 7, 158-169.
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