Copyright ª 2006 The Author
Journal compilation ª 2006 Blackwell Munksgaard
The molecular regulation of organelle transport in
mammalian retinal pigment epithelial cells
Clare E. Futter*
Division of Cell Biology, Institute of Ophthalmology, University
College London, 11-43 Bath Street, London EC1V 9EL, UK
*Address correspondence to Clare Futter,
e-mail: c.futter@ucl.ac.uk
Summary
Retinal pigment epithelial cells contain large num-
bers of melanosomes that can enter the apical pro-
cesses extending between the outer segments of
the overlying photoreceptors. Every day the distal
portion of the photoreceptor outer segment is shed
and phagocytosed by the retinal pigment epithelial
cell. The phagosome is then transported into the
cell body and the contents degraded by lysosomal
enzymes. This review focuses on recent progress
made in the identification of molecules that regulate
the transport of melanosomes into the apical pro-
cesses and the transport of phagosomes into the
cell body. Myosin VIIa is a key player in both pro-
cesses and, at least in the case of melanosome
movement, myosin VIIa is recruited to the melano-
some via the GTPase, Rab27a. The possible role
played by defects in the transport of melanosomes
and phagosomes in the development of retinal
degenerative diseases is discussed.
Key words: Melanosome/phagosome/Rab27a/myosin
Vlla/retina
Received 20 January 2006, revised and accepted for
publication 6 February 2006
Retinal pigment epithelial (RPE) cells are a pigmented
monolayer of cells that form part of the blood/retina bar-
rier. Their apical surface forms numerous long proces-
ses that partially envelop the outer segments of the
photoreceptors. This close interaction between the RPE
and photoreceptors is critical for the maintenance of vis-
ual function. It facilitates (i) the transfer of retinal
between photoreceptors and RPE which is required for
the visual cycle of retinal and the maintenance of photo-
receptor excitability; and (ii) the phagocytosis by RPE
cells of the daily shed tips of the outer segments of
photoreceptors, a process essential for photoreceptor
104
Review
doi: 10.1111/j.1600-0749.2006.00303.x
survival. The basolateral surface of the RPE lies on
Bruch’s membrane, which separates the RPE from the
choriocapillaris, a layer of fenestrated capillaries. The
RPE transports nutrients from the blood to the retina
and transports ions, water and waste products from the
sub-retinal space to the blood. This review will focus on
two aspects of RPE cell biology; the regulation of mel-
anosome transport and phagosome transport.
Engulfment of the distal tip of the photoreceptor outer
segment is regulated by light and circadian rhythm and
requires alphavbeta5 integrins, CD36 and the Mer tyro-
sine kinase (reviewed in Strauss, 2005). Less is known
about the molecular regulation of the subsequent pro-
cessing of the phagosome which must move into the cell
body to fuse with lysosomes before degradation of the
phagosome content (see Figure 1) (Bosch et al., 1993;
Herman and Steinberg, 1982a,b). However, it is becom-
ing clear that defects in the processing of the products of
photoreceptor phagocytosis are likely to contribute to the
pathogenesis of many retinal degenerative diseases.
Another organelle within the RPE whose motility is
regulated by a light and/or circadian rhythm is the mel-
anosome. Melanin within melanosomes of the RPE
plays an important role in the development of the neural
retina. Albino animals show abnormal patterns of cell
proliferation during development and at maturity have
reduced numbers of photoreceptors and abnormal chi-
asmatic pathways (Jeffery, 1997). Melanin within mel-
anosomes of RPE cells also absorbs light scatter and is
likely to aid in protection against photo-oxidation. Mel-
anosomes within the RPE of fish and amphibians exhibit
a dramatic redistribution from the cell body to the apical
processes upon light onset, which is reversed in the
dark (Burnside and Laties, 1979). Until recently the mel-
anosomes of mammalian RPE cells were thought not to
move but have now been shown to undergo a modest
redistribution into the apical processes of murine RPE
cells after light onset (Futter et al., 2004) and to exhibit
motility in isolated primary murine RPE cells (Gibbs
et al., 2004). The regulation of melanosome motility has
been extensively studied in melanocytes of the skin
where melanosomes move to the cell periphery along
microtubules and are trapped in the cell periphery by
interaction with the cortical actin cytoskeleton, a pro-
cess which is essential for their transfer to neighbouring
keratinocytes (Wu et al., 1998). Interaction of the mel-
anosome with the actin cytoskeleton is mediated by a
tripartite complex of the small Ras-like GTPase, Rab27a,
Pigment Cell Res. 19; 104–111

Figure 1. Phagocytosis of rod outer
segments in RPE cells. (A) A possible
model for phagosome maturation and
fusion with the lysosome, indicating
where myoVIIa and Rabs might act. (B) A
phagosome (P) where the engulfment by
apical processes (arrowheads) is almost
complete. Arrows indicate the distension
of the membrane of the apical process
around the melanosome. (C) Phagosomes
both newly engulfed and deep in the cell
body. (D) A putative phagolysosome (PL)
and a possible product of melanosome–
phagosome fusion (asterisk). Bar ¼ 0.5lm.
melanophilin and myosin Va, where Rab27a binds to the
melanosome, myosinVa to the actin cytoskeleton, and
melanophilin acts as a linker (Wu et al., 2001, 2002;
Fukuda et al., 2002; Hume et al., 2001, 2002; Provance
et al., 2002; Strom et al., 2002).
Recent studies on the molecular regulation of melano-
some movement in RPE cells have revealed both paral-
lels and differences with skin melanocytes. In addition,
apparently some of the same molecular machinery that
regulates melanosome movement may also regulate
phagosome movement. That some of these molecular
regulators are defective in human diseases that exhibit
retinal degenerations suggests that a fuller understand-
ing of organelle motility in RPE cells may shed light on
the pathogenesis of these diseases.
Myosin VIIa – a regulator of melanosome
and phagosome distribution in RPE cells
Myosin VIIa was the first molecular regulator of melano-
some distribution to be identified in mammalian RPE
cells. The shaker-1 mouse is deficient in myosin VIIa
(Gibson et al., 1995; Mburu et al., 1997), a plus-end-
directed motor (Inoue and Ikebe, 2003) that within the
retina has been localized to the cilium of the photore-
ceptors, to the apical region of RPE cells (Wolfrum
Pigment Cell Res. 19; 104–111
Organelle transport in retinal pigment epithelium
et al., 1998), and to melanosomes within RPE cells (El-
Amraoui et al., 2002; Gibbs et al., 2004). Three functions
of myosin VIIa have been identified within the retina: (i)
the regulation of the distribution of melanosomes within
the apical processes of RPE cells (Liu et al., 1998); (ii)
the transport of opsin from the inner to the outer seg-
ment of the photoreceptors (Liu et al., 1999); and (iii) the
transport of phagosomes from the apical region to the
cell body in RPE cells (Gibbs et al., 2003).
Within the RPE of shaker-1 mice melanosomes are
found exclusively in the cell body and are excluded from
the apical processes, indicating that myosin VIIa is
required for movement of melanosomes into, or retent-
ion of melanosomes within, the apical processes (Liu
et al., 1998). In RPE cells, F-actin is found just beneath
the apical plasma membrane, in the circumferential actin
filaments extending from the adherens junctions, and
within the apical microvilli, and is largely absent from
the cell body (Figure 2). In the absence of myosin VIIa
melanosomes reside exclusively in the microtubule rich
cell body, and so myosin VIIa may be required for trans-
fer from microtubules to the actin rich apical region and
for transport through it. Microtubules are completely
absent from the apical processes (Figure 2) (Burnside
and Laties, 1979; Futter et al., 2004), and so the actin
cytoskeleton is likely to be involved in the subsequent
105
Futter
Figure 2. Transfer of melanosomes from the microtubule rich cell
body to the actin rich apical processes. Confocal microscopy of
sections through wild-type retina shows a meshwork of
microtubules (green) in the cell body of RPE cells but microtubules
are absent from the apical processes. Phalloidin staining (red)
shows F-actin associated with the infoldings of the basal plasma
membrane, with the circumferential actin ring extending from the
adherens junctions (visible in 0.5 lm sections as short stretches,
indicated by arrows) and within the apical processes.
Melanosomes (artificially coloured blue) are in the microtubule rich
cell body but are also found associated with the actin-rich apical
processes. Transmission EM shows that in wild type mice the
melanosomes are oriented parallel to the longitudinal axis of the
RPE cell and enter the apical processes that extend between the
rod outer segments (ROS). BM, basement membrane. In the
ashen mouse, the melanosomes are excluded from the apical
processes. Bar ¼ 2.0lm. Reproduced from Futter et al. (2004).
movement up the apical processes. Whether myosin
VIIa or other motor proteins are required for this pro-
cess has yet to be shown. The narrow diameter of the
apical process, compared with the diameter of the mel-
anosome, and the distortion of the plasma membrane
106
of the apical process that occurs as the melanosome
moves up it (see example in Figure 1) suggests that
force is required for this movement.
Thus, myosin VIIa plays a key role in regulating mel-
anosome distribution in RPE cells which may be equival-
ent to the role of myosin Va in skin melanocytes.
Although myosin Va is present on melanosomes in RPE
cells the distribution of melanosomes within RPE cells
of the dilute mouse (lacking myosin Va) is normal (Gibbs
et al., 2004).
The shaker-1 mice also exhibit a defect in the uptake
and processing of phagocytosed rod outer segments
(Gibbs et al., 2003). It is not clear whether the reduced
phagocytosis is due to a defect in shedding of outer
segments or in the uptake process. However, there is a
very clear inhibition in the subsequent movement of the
phagosome from the apical region to the cell body and
in the degradation of the phagosome content (Gibbs
et al., 2003). This at first appears somewhat paradoxical
as myosin VIIa is required for the transport of melano-
somes in a basal to apical direction and of phagosomes
in the opposite direction. Possibly the role of myosin
VIIa in phagosome transport is indirect and may be
required for efficient interaction of the phagosome with
the endocytic pathway, as has been proposed for myo-
sin Va (Al-Haddad et al., 2001).
In macrophages myosin V colocalises with the fully
internalized phagosome (Swanson et al., 1999) and in
macrophages of dilute lethal mice (lacking myosin Va)
phagosomes show altered motility. Their accumulation
in the perinuclear region of the cell is accelerated
because short-range movements to the cell periphery
are prevented (Al-Haddad et al., 2001). These authors
proposed that the short-range movements to the cell
periphery are necessary for interaction of the phago-
some with the endocytic pathway. Phagosome matur-
ation, at least in macrophages where it has been
studied extensively, involves interaction first with the
early endosome and then with late endosomes and
lysosomes (Desjardins et al., 1994). These interactions
require sequentially Rab5 and then Rab7. Rab5 recruits
the phosphatidylinositol-3-kinase, Vps34, the activity of
which recruits EEA1 (Fratti et al., 2001; Vieira et al.,
2003). EEA1 is a phosphatidylinositol-3P-binding protein
localized to early endosomes that acts as a docking fac-
tor required for early endosome–endosome fusion
(Christoforidis et al., 1999). EEA1 recruitment to the
phagosome is required for subsequent phagosome mat-
uration, whilst Rab7 is required for fusion with lyso-
somes and degradation of phagosome content (Harrison
et al., 2003). Although phagosome maturation has been
less extensively studied in RPE cells phagosomes con-
taining latex beads stain for Rab5 at early time points
and markers of the lysosome at later time points (Hoppe
et al., 2004). A crucial question is where within RPE
cells do phagosomes acquire the markers of phago-
some maturation, such as Rab 5, EEA1 and Rab 7 (see
Pigment Cell Res. 19; 104–111

Figure 1), and what is the effect of loss of myosin Va
and myosin VIIa on their acquisition by the phagosome?
Inhibitors of both the microtubule and actin cytoskele-
tons inhibit phagosome maturation (Desjardins et al.,
1994) and phagosome-lysosome fusion (Funato et al.,
1997). Rab5 regulates early endosome association with
and movement along microtubules (Nielsen et al.,
1999). If myosin VIIa is required for association of the
phagosome with early endosomes, and/or the acquisi-
tion of Rab5, then loss of myosin VIIa could indirectly
affect microtubule-dependent transport into the cell
body and subsequent lysosome fusion.
Rab27a and its effectors in the regulation
of melanosome distribution in RPE cells
Studies using cells from the ashen mouse, which lacks
Rab27a, have shown that in melanocytes of the skin
this GTPase binds to the melanosome and mediates
interaction with the actin cytoskeleton via myosin Va
(Wu et al., 2001, 2002; Hume et al., 2001). Rab27a also
binds to the melanosome in RPE cells and in the RPE
of the ashen mouse the melanosomes remain in the
cell body (Figure 2), failing to move beyond the level of
the adherens junctions and thus have a phenotype with
respect to melanosome distribution indistinguishable
from the RPE of the shaker mouse (Futter et al., 2004;
Gibbs et al., 2004).
Further studies have shown that Rab27a is a marker
of mature secretory organelles and may play an import-
ant role in regulated exocytosis from both conventional
secretory cells (such as endocrine and exocrine glands)
and haematopoietic cells (Tolmachova et al., 2003).
Rab27a appears to be important for actin-based motility
of both melanosomes and secretory granules via the
recruitment of unconventional myosin motors. The inter-
action with unconventional myosins is indirect, via linker
myosin and Rab-interacting proteins. One such protein
acting in skin melanocytes is melanophilin, which is
required for the peripheral capture of melanosomes
(Fukuda et al., 2002; Provance et al., 2002; Strom et al.,
2002). Another rab effector, MyRIP, has been implicated
in RPE melanosome motility because of its expression
in RPE cells and its ability to bind both Rab27a and myo-
sin VIIa (El-Amraoui et al., 2002). Based on these obser-
vations, it was proposed that a tripartite complex of
Rab27a-MyRIP-myosin VIIa regulates melanosome dis-
tribution in RPE cells, in a manner analogous to the
Rab27a–melanophilin–myosin Va complex that operates
in melanocytes (El-Amraoui et al., 2002). However, in
the absence of a MyRIP knockout mouse no direct evi-
dence for a role for MyRIP in the maintenance of mel-
anosome distribution in RPE cells has been obtained.
Furthermore, MyRIP has been shown in vitro to bind
myosin Va as well as myosin VIIa, and to bind actin
directly (Fukuda and Kuroda, 2002). MyRIP also has a
role in secretory granule exocytosis that may be inde-
Pigment Cell Res. 19; 104–111
Organelle transport in retinal pigment epithelium
pendent of myosin V or VIIa (Desnos et al., 2003;
Waselle et al., 2003). It has been reported that MyRIP/
myosin VIIa can rescue the normal peripheral distribu-
tion of melanosomes in melanocytes lacking melanophi-
lin (Kuroda and Fukuda, 2005), indicating that in an
intact cell MyRIP can bind myosin VIIa and act as a lin-
ker between Rab27a on melanosomes and the actin
cytoskeleton. MyRIP, therefore, is a likely candidate for
playing a role in maintaining melanosome distribution in
RPE cells but at which step remains to be shown.
Rab27a is able to recruit at least 11 different effector
proteins (Fukuda, 2005) which fall into four groups: (i)
Slps (Slp1 through 5), for ‘synaptotagmin-like proteins’
which contain two C2 domains (Ca2+ and phospholipid-
binding domains); (ii) melanophilin and MyRIP which bind
Rab27a and myosins but lack C2 domains (also referred
to as Slacs for ‘Slps lacking C2 domains’); (iii) Rabphilin
and Noc2 which also contain C2 domains and bind pro-
miscuously to both Rab3 and Rab27 proteins; and (iv)
Munc13–4, a putative regulator of SNARE complexes. A
recent study showed that, in addition to melanophilin, a
second Rab27a effector, Slp2-a, is bound to the melano-
some and regulates melanosome distribution in melano-
cytes (Kuroda and Fukuda, 2004). Therefore, possibly in
RPE cells more than one Rab27a effector protein regu-
lates the distribution of melanosomes, and multiple
Rab27a effectors are expressed in the RPE (Gibbs et al.,
2004). The fact that melanosomes are unable to access
the actin-rich apical region within RPE cells in both ashen
and shaker mice suggests that Rab27a–MyRIP (prob-
ably)–myosin VIIa regulates transfer of melanosomes
from the microtubule-rich cell body to the apical actin
cytoskeleton, although other Rab27a effectors could be
involved in subsequent transport steps.
A recent study has indicated that another Rab protein,
Rab8, is also involved in actin-dependent movement of
melanosomes in melanocytes, but is unlikely to operate
through myosin V (Chabrillat et al., 2005). Rab8 may
directly regulate transient interactions with the actin
cytoskeleton. Rab27a and Rab8 can localize to the same
melanosome but how the activities of the two Rab pro-
teins is co-ordinated is as yet unclear. A role for Rab8 in
regulating melanosome distribution in RPE cells has not
been reported.
Whether Rab27a, like myosin VIIa, plays a role in
uptake of photoreceptor outer segments or in their sub-
sequent intracellular transport and degradation has yet
to be determined. As described above for myosin VIIa,
Rab27a could play an indirect role in phagosome pro-
cessing by either regulating interactions with endo-
somes or even by regulating interactions with
melanosomes. A link between phagosomes and mel-
anosomes has been suggested by the frequently
observed fusion profiles between melanosomes and
phagosomes (see Figure 1 for a possible example).
Indeed following phagocytosis of gold labelled outer
segments which have been injected into the subretinal
107
Futter
space, gold particles are found within melanosomes
within the RPE (Schraermeyer et al., 1999). Melano-
somes are lysosome-related organelles and contain
some degradative enzymes. Whether they play any role
in the degradation of the phagosome content, the deg-
radation products of which could be used for generation
of more melanin pigment, is not clear. It is interesting
to note, however, that mature melanosomes within
melanocytes are not accessible to endocytic probes
(Raposo et al., 2001), suggesting a potential functional
specialisation of melanosomes within RPE cells.
The potential roles of defects in organelle
transport within RPE cells in the
pathology of eye diseases
Defects in the degradation of phagocytosed outer
segments
The shaker-1 mouse, deficient in myosin VIIa, is the
mouse model for the human disease, Usher syndrome
1B (Weil et al., 1995). Patients with this disease suffer
from progressive retinal degeneration, are profoundly
deaf and exhibit vestibular defects (Petit, 2001). The find-
ing that RPE cells of shaker-1 mice have a defect in both
the initial uptake of outer segments and in their subse-
quent transport raises the possibility that this could cause
the retinal degeneration observed in Usher syndrome 1B.
A major defect in phagocytosis of outer segments
would be expected to promote photoreceptor degener-
ation, as has been observed in the RCS rat (Bok and
Hall, 1971; Dowling and Sidman, 1962; Herron et al.,
1969) which lacks functional mer tyrosine kinase
(D’Cruz et al., 2000), in the mer knockout mouse (Dun-
can et al., 2003) and in the b5 knockout mouse (Nandrot
et al., 2004). A defect in the processing of phagocyto-
sed photoreceptor outer segments might be expected
to lead to the progressive accumulation of toxic degra-
dation products of photoreceptor outer segments within
the RPE. The accumulation of lipofuscin within RPE
cells that occurs with age, and is increased in patients
suffering from age-related macular disease (AMD), is
believed to originate from incompletely digested outer
segments (Kennedy et al., 1995). A severe inhibition of
degradation of photoreceptor outer segments in trans-
genic mice expressing a mutant form of cathepsin D,
the most critical enzyme for the digestion of rhodopsin,
leads to accumulation of lipofuscin and basal deposits
that have some, but not all, the characteristics of the
basal deposits (Drusen) observed in AMD (Rakoczy
et al., 2002). The shaker-1 mice exhibit some electro-
retinographic abnormalities, namely a reduction in the
wave response to light (Libby and Steel, 2001), but do
not show any retinal degeneration. The accumulation of
lipofuscin and basal deposits have not been described in
the shaker-1 mice, but studies on aging mice have not
been reported. They do, however, exhibit hearing and
vestibular defects. The reason for this difference
108
between the effects of loss of function of myosin VIIa
between mice and humans is not clear. If the retinal
degeneration seen in Usher Syndrome 1B is caused by
a reduction in the ability of the RPE to take up or
degrade outer segments, it may be that the mice simply
do not live long enough to exhibit these effects. Each
RPE cell is exposed to approximately 40 photoreceptors
and as there is a little or no proliferation of adult RPE
cells a single human RPE cell can phagocytose an enor-
mous number of photoreceptor discs during a 70 year
life span. Even a minor defect in the processing of the
phagocytosed discs could lead to a considerable accu-
mulation of undigested material within the RPE over this
time scale, but possibly not during the more limited life
time of a mouse.
Defects in melanosome movement in RPE cells
The dilute, ashen and leaden mice are models of Gris-
celli syndrome types 1, 2 and 3, respectively (Menasche
et al., 2000, 2003; Pastural et al., 1997). All three types
are characterized by pigment dilution but mutations in
myosin V also lead to a severe neurological impairment
(Pastural et al., 1997), mutations in rab27a lead to a hae-
mophagocytic syndrome (Menasche et al., 2000), whilst
the disease caused by mutations in melanophilin is
restricted to hypopigmentation (Menasche et al., 2003).
Patients do not suffer retinal degeneration but this could
be due to their early death. The ashen mice also do not
exhibit retinal degeneration (Futter et al., 2004) but, as
suggested above for the shaker-1 mouse, the comparat-
ively short life of the mouse may prevent this. Several
potential roles for melanosome movement in protection
from retinal degeneration can be envisaged. It has been
proposed that the presence of melanosomes within the
apical processes might reduce disc shedding and their
phagocytosis by shielding the photoreceptor outer seg-
ments from light (Sarangarajan and Apte, 2005). The
rate of disc shedding has not been measured in the
ashen or shaker-1 mice. Phagocytosis, at least in the
shaker-1 mouse, is reduced but this could be due to a
direct role for myosin VIIa in phagocytosis. Phagocytosis
itself produces peroxides and having melanin granules
surrounding the newly formed phagosome may rapidly
absorb peroxides and prevent their damaging effects.
Rab27a has been implicated in retinal degeneration
through studies of X-linked choroideraemia (CHM) (Se-
abra et al., 2002), which is caused by mutations in the
Rab Escort protein, REP1 (Cremers et al., 1990; Merry
et al., 1992). The disease affects not only the RPE, but
also the adjacent photoreceptors and the choroid and
affected males exhibit slow retinal degeneration and are
blind by middle age (Heckenlively and Bird, 1988;
McCulloch, 1988). Rab escort proteins are required for
Rab prenylation, which is an absolute requirement for
Rab function (Seabra and Wasmeier, 2004). Many Rabs
are prenylated normally in X-linked CHM patients
through the action of REP2 (Cremers et al., 1994;
Pigment Cell Res. 19; 104–111

Seabra, 1996). However, Rab27a appears to be a poor
substrate for REP2 and lympoblasts from CHM patients
have an excess of unprenylated Rab27a (Seabra et al.,
1995). This observation, together with the high expres-
sion of Rab27a in the RPE and the choroid, lead to the
suggestion that defects in the function of Rab27a might
be responsible for CHM. As other tissues where
Rab27a has important functions are comparatively unaf-
fected in CHM it now appears that at least some
Rab27a must be prenylated in these patients. Indeed, in
recently developed conditional mouse models of choroi-
deremia in which the degeneration of photoreceptors
and RPE cells has been shown to arise independently,
different subsets of Rabs show defective prenylation in
the two cell types (Tolmachova et al., 2006).
Conclusion
This review serves to highlight as much what we do
not know as what we do know about the molecular
regulation of melanosome movement and phagosome
maturation within RPE cells. This is partly because
immortalized RPE cell lines and isolated RPE cells in cul-
ture do not readily maintain their pigmentation or their
extensive apical processes and, although they can phag-
ocytose rod outer segments, aspects of their in vivo
regulation by interaction with photoreceptors and
Bruch’s membrane, are lost. Recently, mouse models
have allowed the delineation of the roles of proteins like
myosin VIIa and Rab27a in phagocytosis and melano-
some motility. Differences between mice and humans
in terms not only of their retinal architecture, but also of
their longevity, has so far made the roles of defects in
these processes in human retinal degenerative diseases
difficult to determine. In addition, elucidation of the role
of other molecular components regulating organelle
transport within the RPE is limited by the lack of suit-
able mouse models. The development of systems to
manipulate primary RPE cells in culture, under condi-
tions where they more closely reproduce their in vivo
phenotype, should allow further progress in our under-
standing of the processes regulating organelle transport
in a cell that plays a central role in maintaining the
neural retina and hence vision.
Acknowledgements
The author would like to thank Miguel Seabra and Steve Moss for
critical reading of this review. Work in the author’s laboratory is
supported by Cancer Research UK, Wellcome Trust, BBSRC, Fight
for Sight and the Special Trustees of Moorfields Eye Hospital.
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