Review

Rab GTPases and Myosin Motors in Organelle Motility

Miguel C. Seabra1,* and Evelyne Coudrier2

1Cell and Molecular Biology, Division of BiomedicalSciences, Faculty of Medicine, Imperial College Londyon,London SW7 2AZ, UK,2Unite de Morphogenese et Signalisation Cellulaires.Institut Curie, CNRS UMR144, 75248 Paris Cedex 05,France* Corresponding author: Miguel C. Seabra,m.seabra@imperial.ac.uk

The actin cytoskeleton is essential to ensure the properlocation of, and communication between, intracellularorganelles. Some actin-based myosin motors have beenimplicated in this process, particularly members of theclass V myosins. We discuss here the emerging role ofthe Ras-like GTPases of the Rab family as regulators ofmyosin function in organelle transport. Evidence fromyeast secretory vesicles and mitochondria, and mamma-lian melanosomes and endosomes suggests that RabGTPases are crucial components of the myosin organellereceptor machinery. Better understood is the case of themelanosome where Rab27a recruits a specific effectorcalled melanophilin, which in turn binds myosin Va. Thepresence of a linker protein between a Rab and a myosinmay represent a general mechanism. We argue that Rabsare ideally suited to perform this role as they are exquisiteorganelle markers. Furthermore, the molecular switchproperty of Rabs may enable them to regulate the timingof 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 publica-tion 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 polymeriza-

tion propelling organelles within the cytoplasm, such as

some movements observed for endosomes and lyso-

somes (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.

Myosin Motors in Organelle Trafficking

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).

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 differ-

ences 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 super-

family (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 endoplas-

mic 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,myo-

sin Va, myosin Vb and myosin Vc (7). In addition, myosin

genes may be alternatively spliced, thus generating tissue-

specific isoforms (20). These different myosin V isoforms

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Blackwell Munksgaard doi: 10.1111/j.1600-0854.2004.00190.x

393

are also involved in the transport of multiple organelles

such as the endoplasmic reticulum, melanosomes, secre-

tory granules and recycling endosomes (Table 1).

The divergent tail domain of myosins contains the informa-

tion necessary for the targeting of myosins to specific intra-

cellular 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 regulat-

ing the binding of one class V myosin to melanosomes, the

pigment-containing granules of skin melanocytes (21–23).

Rab27a recruits myosin Va to melanosomesvia 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 kera-

tinocytes in the basal layers of the epidermis. Once

mature and fully pigmented, melanosomes must be trans-

ported to the distal end of the melanocyte dendrite exten-

sions 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). Associ-

ation of myosin Va withmelanosomes has been revealed by

immuno-electron microscopy and subcellular fractionation

(27–29). Furthermore, dominant-negative protein expres-

sion and in vitro motility assay reconstituting the move-

ments 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

traffickingmachinery acting in concertwithmyosin 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 melano-

somes in skin melanocytes, and expression of Rab27a in

melanocytes from patients suffering from Griscelli syn-

drome restores the distribution of melanosomes to the

tips of the dendrites (32–34). Thirdly, the association of

Rab27a and myosin Va was confirmed by coimmunopre-

cipitation (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 dilutemelanocytes (Figure 1).

Rab27a localizes to melanosomes irrespective of the pre-

sence 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).

Table 1: Summary of reported interactions between Rab GTPases and myosin motors

Rabs Motors Mode of interaction Organelle References

Rab 11 Myosin Vb Direct? Recycling (17,54)

FIP2 endosomes

Sec4p Myo2 (class ? Secretory (8)

V myosin) vesicles

Ypt11p Myo2 Direct? Mitochondria (11)

Rab27a (Griscelli Myosin Va Melanophilin Skin melanocyte (38,41,66)

syndrome, ashen mice) melanosomes

Rab27a Myosin VIIa MyRIP RPE melanosomes (43,48)

Rab8? Myosin Vc ? Endosomes (55)

Seabra and Coudrier

394 Traffic 2004; 5: 393–399

Rab27a Recruits Two Distinct Myosins toMelanosomes via Distinct Effectors

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 canon-

ical 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).

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 localiza-

tion 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 distri-

bution 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

same type of organelle (melanosome) in different cell

types (melanocytes or RPE cells).

There are significant differences between RPE and mela-

nocyte melanosomes both in terms of cellular distribution

and ability to undergo cell transfer (25,48). This discrep-

ancy 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).

Rab27a and MyRIP have also been implicated in the

regulation of exocytosis in pancreatic b cells and neuro-

endocrine 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 pro-

teins, 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

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.

Rab GTPases in Organelle Transport

Traffic 2004; 5: 393–399 395

melanocytes, i.e. induces perinuclear clustering of melano-

somes, 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 Myosinto 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 vesi-

cles (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).

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 mitochon-

dria in the bud (11). The Ypt11 GTPase binds to the tail

domain of Myo2p, although it is unclear whether the inter-

action is direct or indirect. Furthermore, residues in

Myo2p, whose substitution induces defects in mitochon-

drial 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 asso-

ciation 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.

Figure2:Molecularmechanisms of Rab-dependent, myosin-driven organellemotility. See text for details.

Seabra and Coudrier

396 Traffic 2004; 5: 393–399

Myosin Vc, the major class V isoform in epithelial cells is

also involved in regulating the cellular distribution of endo-

cytic compartments distinct from those exhibiting myosin

Vb (55). Overexpression of myosin Vc perturbs the distri-

bution of compartments labeled for Rab8 and the transfer-

rin 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 forMyosins?

The available experimental evidence suggests that there is

great complexity in myosin-driven organelle dynamics. The

same class V myosin isoform transports multiple organ-

elles 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 organ-

elle 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 proper-

ties 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 appro-

priate organelle.

Do Rabs Coordinate Myosin-dependentTransport 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 fac-

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 isMunc13–4, which is thought to regulate SNARE func-

tion inmembrane fusion (62). This evidence raises the interest-

ing 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

detailsofhowthishierarchyofeffectscouldbeachieved remain

mysterious.

Do Rab Proteins Regulate Cooperationbetween 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 mela-

nosomes by counteracting the action of dynein, particularly

shortening the length of dynein-driven runs, and by ensur-

ing the regular motion of melanosomes towards the micro-

tubule 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 pro-

tein 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 regu-

lated 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.

Rab GTPases in Organelle Transport

Traffic 2004; 5: 393–399 397

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