Traffic 2004; 5: 393–399
Blackwell Munksgaard
Copyright
#
Blackwell Munksgaard 2004
doi: 10.1111/j.1600-0854.2004.00190.x
Review
Rab GTPases and Myosin Motors in Organelle Motility
Miguel C. Seabra1,* and Evelyne Coudrier2
Myosin Motors in Organelle Trafficking
1
Cell and Molecular Biology, Division of Biomedical
Sciences, Faculty of Medicine, Imperial College Londyon,
London SW7 2AZ, UK,
2
Unitè de Morphogenèse et Signalisation Cellulaires.
Institut Curie, CNRS UMR144, 75248 Paris Cedex 05,
France
* Corresponding author: Miguel C. Seabra,
[email protected]
Advances in video-enhanced contrast microscopy in the
early 1980s enabled the visualization of cytoplasmic
movement of organelles. Pioneering experiments in algae
and the squid giant axon suggested that organelles moved
on actin filaments, in addition to long-distance displacements
along microtubules (4–6). These early studies suggested
that actin-based movements carried organelles or vesicles
directionally along actin tracks and involved myosins, the
molecular motors associated with actin filaments. The first
myosin proposed to move an organelle on actin filaments
was a myosin I from the amoeba Acanthamoeba castellanii
on the basis of an in vitro motility assay (5).
The actin cytoskeleton is essential to ensure the proper
location of, and communication between, intracellular
organelles. Some actin-based myosin motors have been
implicated in this process, particularly members of the
class V myosins. We discuss here the emerging role of
the Ras-like GTPases of the Rab family as regulators of
myosin function in organelle transport. Evidence from
yeast secretory vesicles and mitochondria, and mammalian melanosomes and endosomes suggests that Rab
GTPases are crucial components of the myosin organelle
receptor machinery. Better understood is the case of the
melanosome where Rab27a recruits a specific effector
called melanophilin, which in turn binds myosin Va. The
presence of a linker protein between a Rab and a myosin
may represent a general mechanism. We argue that Rabs
are ideally suited to perform this role as they are exquisite
organelle markers. Furthermore, the molecular switch
property of Rabs may enable them to regulate the timing
of the myosin association with the target organelle.
Key words: actin, endosome, GTP-binding proteins,
melanosome, myosin V, Rab27, secretory granule
Received 5 March 2004, revised and accepted for publication 12 March 2004
The actin-based movement of organelles within cells is a
general process that is essential to the proper distribution
and communication between cellular compartments. One
type of actin-based motility is based on actin polymerization propelling organelles within the cytoplasm, such as
some movements observed for endosomes and lysosomes (1,2). This type of actin-based movement was
reviewed recently in Traffic and will not be discussed
much further here (3). The second type of movement
depends on acto-myosin forces to move organelles or
vesicles on actin tracks. We will focus our discussion on
this type of motility, and on the emerging role of Rab
GTPases as regulators of this process.
Myosins share a common structure and are composed of
three modules: the head domain comprising the motor
domain, which hydrolyzes adenosine 5’ triphosphate (ATP)
to power movement along actin filaments; the neck
domain, which acts as a lever arm and binds regulatory
light-chains such as calmodulin; and the tail domain, which
is highly divergent among the different myosin classes and
serves to bind specific cargo. Despite their common motor
domain structures, myosins present fundamental differences in the kinetics of their ATP cycle and oligomeric
structure. Some myosins are processive, i.e. can perform
many consecutive steps along actin without dissociating,
whereas other myosins can exert tensions between actin
filaments, or actin filaments and membrane domains.
Among the 18 classes that constitute the myosin superfamily (7), four classes of myosins (I, II, V and VI) have been
implicated in the dynamics of a large variety of organelles
such as secretory vesicles, vacuoles, Golgi, endoplasmic
reticulum and mitochondria in yeast (8–12), and endoplasmic reticulum, recycling endosomes, lysosomes, secretory
granules and melanosomes in mammalian cells (13–18).
Myosin V may be ideally suited for organelle transport as it
is an efficient, processive motor that works in 37-nm steps
which correspond to the helical periodicity of the actin
filament (19). Indeed, several class V myosins have been
involved in organelle trafficking. In yeast, one of two class
V myosins, Myo2p, delivers various organelles to the bud
tip during cell division in Saccharomyces cerevisiae (8–12).
There are three myosin V-related genes in mammals, myosin Va, myosin Vb and myosin Vc (7). In addition, myosin
genes may be alternatively spliced, thus generating tissuespecific isoforms (20). These different myosin V isoforms
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Seabra and Coudrier
Table 1: Summary of reported interactions between Rab GTPases and myosin motors
Rabs
Motors
Mode of interaction
Organelle
Rab 11
Myosin Vb
Sec4p
Myo2 (class
V myosin)
Myo2
Myosin Va
Direct?
FIP2
?
Recycling
endosomes
Secretory
vesicles
Mitochondria
Skin melanocyte
melanosomes
RPE melanosomes
Endosomes
Ypt11p
Rab27a (Griscelli
syndrome, ashen mice)
Rab27a
Rab8?
Myosin VIIa
Myosin Vc
Direct?
Melanophilin
MyRIP
?
are also involved in the transport of multiple organelles
such as the endoplasmic reticulum, melanosomes, secretory granules and recycling endosomes (Table 1).
The divergent tail domain of myosins contains the information necessary for the targeting of myosins to specific intracellular compartments. However, until recently, little was
known concerning the link between myosin tails and their
cognate organelle. Genetic, morphologic and biochemical
analysis of three mouse coat color mutants, dilute, ashen
and leaden, gave some clues as to the mechanisms regulating the binding of one class V myosin to melanosomes, the
pigment-containing granules of skin melanocytes (21–23).
Rab27a recruits myosin Va to melanosomes
via an effector protein
Mercer et al. showed that the dilute locus in mice encodes
the murine ortholog of myosin Va (24). This coat color
pigment dilution phenotype, associated with neurologic
disease leading to early neonatal death in dilute-lethal
mice, is due to loss-of-function myosin Va mutations.
Less severe alleles such as dilute-viral that exhibit only
pigment dilution primarily affect the synthesis of the
melanocyte-specific alternatively spliced form (containing
exon F) of myosin Va (20).
Visible pigmentation in mammals requires the transfer of
pigment granules from the melanocytes to adjacent keratinocytes in the basal layers of the epidermis. Once
mature and fully pigmented, melanosomes must be transported to the distal end of the melanocyte dendrite extensions in order to be efficiently transferred to keratinocytes
(25,26). The pigment dilution in dilute mice results from
defects in melanosome transport within skin melanocytes,
such that dilute melanosomes appear clustered around
the perinuclear area and only a few melanosomes are
observed at the periphery of the cell, in contrast to what
is observed in a wild type melanocyte (Figure 1). Association of myosin Va with melanosomes has been revealed by
immuno-electron microscopy and subcellular fractionation
(27–29). Furthermore, dominant-negative protein expres394
References
(17,54)
(8)
(11)
(38,41,66)
(43,48)
(55)
sion and in vitro motility assay reconstituting the movements of melanosomes on actin bundles further
confirmed the role of myosin Va as the actin-based motor
involved in the transport of melanosomes (18,29).
Ten years after the initial description of myosin Va as the
dilute gene, several independent lines of investigation
focused on Rab27a as a component of the organelle
trafficking machinery acting in concert with myosin Va. Firstly,
Rab27a mutations were found in patients suffering from
Griscelli syndrome (30). Griscelli syndrome is a disease
that associates partial albinism with characteristic gray
hair and immunodeficiency due to loss of cytotoxic
T-lymphocyte killing. Concomitantly, the ashen gene was
identified as Rab27a, leading to the identification of ashen
as a model of Griscelli syndrome (31). Secondly, both
Rab27a and myosin Va colocalize on wild type melanosomes in skin melanocytes, and expression of Rab27a in
melanocytes from patients suffering from Griscelli syndrome restores the distribution of melanosomes to the
tips of the dendrites (32–34). Thirdly, the association of
Rab27a and myosin Va was confirmed by coimmunoprecipitation (33). However, Rab27a and myosin Va do not
interact directly. Instead, they require the product of the
leaden gene, which was called melanophilin (Mlph, also
called Slac-2a) (35). The ashen, leaden and dilute gene
products are thus believed to be responsible for the
recruitment of melanosomes to actin filaments at the cell
periphery. The absence of any one of these gene products
leads to striking clustering of melanosomes around the
nucleus as in dilute melanocytes (Figure 1).
Rab27a localizes to melanosomes irrespective of the presence of Mlph or myosin Va, suggesting that it represents
the organelle-recognition element in the system (36–38).
In wild type melanocytes, Rab27a associates with mature
melanosomes, and upon activation via GTP loading,
Rab27a-GTP recruits melanophilin. Melanophilin stabilized
at the melanosome surface then binds the tail of the
melanocyte-specific spliced form of myosin Va. The
motor domain of this myosin then allows association of
melanosomes with the actin network and leads to their
accumulation in dendrite extensions (Figure 2A).
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Rab GTPases in Organelle Transport
Figure 1: Melanosome transport
defects in mouse models of
Griscelli Syndrome. Phase contrast
photographs of primary cultures skin
melanocytes derived from wild type,
ashen (Rab27aash), leaden (Mlphln)
and dilute (Myo5ad) are shown.
Rab27a Recruits Two Distinct Myosins to
Melanosomes via Distinct Effectors
same type of organelle (melanosome) in different cell
types (melanocytes or RPE cells).
Rab27a and its close homolog Rab27b interact with at
least 10 different effectors. Most of the effectors interact
with Rab27 via a conserved Rab27-binding domain, which
is present in Mlph and the Synaptotagmin-like proteins
(Slp) (39–42). MyRIP (for Myosin and Rab Interacting
Protein, also called Slac-2c) was originally identified as a
myosin VIIa-binding protein and the presence of a canonical N-terminal Rab27-binding domain as well as its close
homology with Mlph suggested a role as a linker between
Rabs and myosins (43,44).
There are significant differences between RPE and melanocyte melanosomes both in terms of cellular distribution
and ability to undergo cell transfer (25,48). This discrepancy might reflect the different properties of the two
myosins with which they associate. Myosin Va is clearly
a processive motor and has been observed to move
melanosomes on actin filaments, while myosin VIIa has
been proposed to bridge membrane domains to actin
filaments rather than to act as a processive motor (19,49).
Myosin VIIa plays a critical role in the inner ear and the
retina, since mutations in the MYO7A gene lead to
the blindness–deafness Usher syndrome type 1B (45). In
the retina, myosin VIIa is required for the normal localization of melanosomes in the apical area of retinal pigment
epithelial (RPE) cells, for phagocytosis of photoreceptor
disks by RPE cells and for the transport of opsins through
the connecting cilium in photoreceptors (43,46,47).
Recent observations indicate that Rab27a also contributes
to the distribution of melanosomes in RPE cells (48).
Altogether, the available data suggest that a Rab27a/
MyRIP/myosin VIIa complex regulates melanosome distribution in the RPE (Figure 2B). Thus, Rab27 appears to be
able to recruit distinct myosins (Va or VIIa) via different
effectors (Mlph or MyRIP) to regulate the dynamics of the
Traffic 2004; 5: 393–399
Rab27a and MyRIP have also been implicated in the
regulation of exocytosis in pancreatic b cells and neuroendocrine PC12 cells (50,51). However, the Rab27/MyRIP
protein complex does not appear to require recruitment of
a myosin on the secretory granules for function (50,51).
The association of myosin Va with Rab27/MyRIP has not
been demonstrated in vivo, although it contributes to the
cytoplasmic distribution of secretory granules in PC12 cells
(13). Myosin VIIa is apparently not expressed in pancreatic
b cells (50).
Interestingly, the two known Rab27 and myosin linker proteins, Mlph and MyRIP, also contain a putative actin-binding
domain near the C-terminus (44,52) (Figure 2A and B).
Overexpression of an actin-binding domain mutant form
of Mlph leads to a dominant-negative effect in cultured
395
Seabra and Coudrier
Figure 2: Molecular mechanisms of Rab-dependent, myosin-driven organelle motility. See text for details.
melanocytes, i.e. induces perinuclear clustering of melanosomes, as observed in ashen, leaden or dilute (52) (Figure 1).
This domain might dock melanosomes on actin filaments
and facilitate the interaction between myosin Va and
Rab27a (Figure 2A and B). Alternatively, it may contribute
to the capture and immobilization of melanosomes on
actin filaments in the vicinity of the plasma membrane,
prior to transfer to adjacent keratinocytes (52).
Two Distinct Rabs Recruit One Class V Myosin
to Distinct Organelles in Yeast
The S. cerevisiae Myo2p is another class V myosin
involved in organelle motility. Two Rab proteins, Sec4p
and Ypt11, have been implicated in the docking of Myo2p
onto secretory vesicles and mitochondria, respectively.
Genetic evidence suggests that Sec4p interacts with the
tail domain of Myo2p, but a direct interaction has not been
demonstrated (53). Furthermore, mutations in Sec2p, a
guanine nucleotide exchange factor (GEF) and thus an
activator of Sec4p, uncouple Myo2p from secretory vesicles (53). These observations support a similar mode of
action of a Rab GTPase and a myosin motor in yeast,
whereby a class V myosin is recruited to the surface of an
organelle upon activation of a Rab protein (Sec4p) (Figure 2D).
396
Ypt11p has recently been implicated in the inheritance of
mitochondria (11). Deletion of Ypt11 induces a partial delay
in transmission of mitochondria to the bud, whereas its
overexpression results in the accumulation of mitochondria in the bud (11). The Ypt11 GTPase binds to the tail
domain of Myo2p, although it is unclear whether the interaction is direct or indirect. Furthermore, residues in
Myo2p, whose substitution induces defects in mitochondrial transport, do not affect transport of secretory vesicles
and vice versa, suggesting that Sec4p and Ypt11 bind
different motifs on the tail of Myo2p (11,53) (Figure 2D).
Rab11a Recruits Myosin Vb to Endosomes
In mammals, another member of the class V myosin,
myosin Vb, has been shown to regulate a membrane
traffic step (plasma membrane recycling) through its association with a distinct Rab GTPase (Rab11a) (17) (Figure
2C). It is not yet clear whether the interaction is direct or
whether it requires the Rab11a and myosin Vb-binding
protein, Rab11 family interacting protein-2 (Rab11-FIP2)
(54). The interaction of one Rab and one myosin via a linker
protein is reminiscent of the Rab27a/melanophilin/myosin
Va interaction.
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Rab GTPases in Organelle Transport
Myosin Vc, the major class V isoform in epithelial cells is
also involved in regulating the cellular distribution of endocytic compartments distinct from those exhibiting myosin
Vb (55). Overexpression of myosin Vc perturbs the distribution of compartments labeled for Rab8 and the transferrin receptor. Thus Rab8 is another potential candidate Rab,
in addition to Rab27, Rab11, Sec4 and Ypt11, which might
selectively recruit a class V myosin to a defined organelle.
Why Rabs as Organelle Receptors for
Myosins?
The available experimental evidence suggests that there is
great complexity in myosin-driven organelle dynamics. The
same class V myosin isoform transports multiple organelles with unique destinations. The generation of multiple
splice isoforms creates diversity in the myosin tail domain
and thus enables myosins to operate in multiple transport
steps. Furthermore, myosin V-driven motility of an organelle is differentially controlled during the cell cycle, as
shown for the endoplasmic reticulum in Xenopus egg
extracts (56). Therefore, the interaction of myosin with
organelles needs to be regulated in time and space. The
recent evidence suggesting that the interaction between
Rabs and myosins might be a general feature in organelle
motility raises important questions concerning the suitability
of Rab GTPases to perform the role required of a myosin
receptor in membrane traffic. Several of the known properties of Rabs provide some clues to the answers.
Rab GTPases represent the largest family within the Ras
superfamily and mammalian genomes contain more than
60 independent genes encoding Rab proteins (57,58).
Rabs are localized within cellular organelles and increasing
evidence suggests that Rabs may be important recognition
elements in the organelle identification machinery (59). As
such, Rabs are among the best known organelle markers
and myosins could thus connect precisely to the desired
organelle. In addition to their role as organelle markers,
Rabs function as GTP-dependent molecular switches. The
ability to control the activation and inactivation of any one
Rab in time and space may provide the means to regulate
the association and dissociation of myosins with the appropriate organelle.
Do Rabs Coordinate Myosin-dependent
Transport and Membrane Fusion?
The coordination of vesicle motility is just one of the roles
Rabs play. In fact, Rabs have been implicated in almost
every step of vesicular transport (57,60). The key to the
multiple roles of Rabs appears to reside in the ability to
interact with multiple effectors. For example, recombinant
activated Rab5-GTP interacts with more than 20 bovine
brain cytosolic proteins (61). Rabs and their effectors may play
multiple roles in transport pathways and may thus have
sequential functions, such as the recruitment of myosins
via one specific effector and recruitment of tethering facTraffic 2004; 5: 393–399
tors via other effectors. For Rab27, effector proteins of the
Slp family contain tandem Caþþ and phospholipid-binding C2
domains in addition to a Rab27-binding domain, and thus may
play tethering roles. Another recently identified effector of
Rab27 is Munc13–4, which is thought to regulate SNARE function in membrane fusion (62). This evidence raises the interesting possibility that Rabs recruit hierarchically a set of effectors
and thereby coordinate organelle transport with tethering and
fusion to their target compartment. However, the mechanistic
details of how this hierarchy of effects could be achieved remain
mysterious.
Do Rab Proteins Regulate Cooperation
between Myosin and Kinesin?
Microtubule-based and actin-based movements cooperate
in the transport of organelles from the perinuclear region to
the cell periphery, and vice versa. In skin melanocytes, the
bidirectional microtubule-dependent transport system
provides a way of transporting the melanosomes to the
cell periphery, where the actomyosin system acts to
ensure that the mature melanosomes are captured and
stay in position to be transferred to keratinocytes. This
system has been a fruitful one to study cooperation
between the cytoskeletal networks. Several models have
been proposed to describe the cooperation between
microtubule-based motors and myosins (21,63,64). Most
recently, the ‘tug-of-war’ model proposes a competition
between microtubule-based motors and myosins. In
Xenopus, myosin V contributes to the dispersion of melanosomes by counteracting the action of dynein, particularly
shortening the length of dynein-driven runs, and by ensuring the regular motion of melanosomes towards the microtubule plus-end (65).
The productive association of myosins with organelles
might therefore be coordinated with the inactivation of
microtubule motors. Genetic evidence in yeast suggests
that the kinesin-related protein, Smy1, stabilizes the protein complex formed by Sec4p, and Myo2p. As molecular
switches, Rabs are ideally suited to regulate this type of
coordination of events but a direct role for Rabs in this
process has yet to be uncovered.
In conclusion, the experimental evidence reviewed herein
strongly suggests that the recruitment of different isoforms
of the processive motor myosin V onto organelles is regulated by Rab proteins. Whether or not other myosins
involved in membrane trafficking such as class I, II or VI
myosins are also regulated by their specific recruitment by
Rab proteins remains an interesting possibility.
Acknowledgments
We thank Mimi Mules for providing the photographs in Figure 1 and Abul
Tarafder for help designing Figure 2.
397
Seabra and Coudrier
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