Commentary
3453
Myosin VI: two distinct roles in endocytosis
Tama Hasson
Division of Biological Sciences, Section of Cell and Developmental Biology, University of California at San Diego, 2129 Bonner Hall, MC 0368,
9500 Gilman Drive, La Jolla, CA 92093-0368, USA
(e-mail: [email protected])
Journal of Cell Science 116, 3453-3461 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00669
Summary
Actin is found at the cortex of the cell where endocytosis
occurs, but does it play a role in this essential process?
Recent studies on the unconventional myosin, myosin VI,
an actin-based molecular motor, provide compelling
evidence that this myosin and therefore actin is involved in
two distinct steps of endocytosis in higher eukaryotes: the
formation of clathrin-coated vesicles and the movement of
nascent uncoated vesicles from the actin-rich cell periphery
Introduction
Endocytosis is an essential function in all cells and is required
for nutrient uptake, receptor internalization and synaptic
transmission. Immediately under the plasma membrane where
endocytosis occurs is a cytoskeletal layer made up primarily of
F-actin and associated proteins. You might predict that actin
plays a role in endocytosis; however, the role of actin in this
process has been controversial (reviewed by Qualmann et al.,
2000), given evidence both for and against its importance,
depending primarily on the cell type. Only in lower eukaryotes,
such as yeast, have genetic and biochemical studies tightly
linked the actin cytoskeleton to the endocytic process
(reviewed by Qualmann and Kessels, 2002). Recent studies on
the unconventional myosin, myosin VI, an actin-based
molecular motor expressed only in higher eukaryotes, now
provide compelling evidence that this myosin, and therefore
actin, is involved in two distinct steps in endocytosis. Here I
highlight these findings and discuss mechanisms by which a
myosin might be recruited to actin filaments to facilitate
endocytic traffic in mammalian cells.
Myosins
Myosins are a large family of structurally diverse molecular
motors. All myosins share a conserved domain that binds to Factin microfilaments and hydrolyzes ATP to produce
movement along the filament. At least 18 distinct myosin
classes have been identified to date (Berg et al., 2001), each
having a unique C-terminus that presumably targets it to
distinct cargo and subcellular regions.
Myosin function can be inferred in part from the direction
of motor movement on actin filaments. At the cell cortex,
where endocytosis occurs, polarized actin filaments are
anchored with their barbed ends at the plasma membrane and
the pointed ends facing inwards. Conventional myosins as well
as unconventional myosins, such as myosins I and V, travel
towards the barbed end of the actin filament. Thus, most
to the early endosome. Three distinct adapter proteins –
GIPC, Dab2 and SAP97 – that associate with the cargobinding tail domain of myosin VI have been identified.
These proteins may recruit myosin VI to its sites of action.
Key words: Unconventional myosin, Myo6, Actin, Receptormediated endocytosis, Clathrin-coated Pits, Vesicle trafficking
myosins function in exocytosis or outward movement of
organelles and not endocytosis. Only one newly identified
unconventional myosin, myosin VI, has been shown to travel
along actin filaments towards the pointed end (Wells et al.,
1999). Its role as the only identified pointed-end directed
molecular motor has been reviewed elsewhere (Cramer, 2000).
Below I focus on its potential roles in endocytosis.
Myosin VI and endocytic regions
In polarized cells, myosin VI is associated with endocytic
domains. In kidney proximal tubule cells and intestinal
enterocytes, it is enriched at specialized clathrin-coated
invaginations at the base of the brush-border microvilli
(Biemesderfer et al., 2002; Buss et al., 2001; Heintzelman et
al., 1994) (Fig. 1). These clathrin-rich invaginations are
encased in the actin-rich terminal web, and within this region
myosin VI overlaps with both clathrin and the clathrin-adapter
protein AP-2 (Biemesderfer et al., 2002; Buss et al., 2001). In
a very different polarized cell type, the hair cells of the inner
ear, myosin VI is enriched in the pericuticular necklace, a
vesicle rich region that is the primary site of endocytosis in this
cell type (Hasson et al., 1997). Taken together these very
divergent localization studies implicate myosin VI in endocytic
processes.
Three distinct stages of endocytosis in polarized cells might
require an actin-based motor like myosin VI (Fig. 2). The first
is clustering of ligand-bound receptors into clathrin-coated pits
(Fig. 2A). In the kidney, for example, the brush border
microvilli are enriched in endocytic receptors. These receptors
are involved in the uptake of amino acids, vitamins and other
components from the filtered urine. Although these receptors
are present along the length of the microvillus, they are also
concentrated in the clathrin-rich apical invaginations between
the microvilli (reviewed by Christensen et al., 1998). Pulsechase studies following uptake of cationic ferritin in MDCK
cells revealed that ferritin associates first with microvilli and
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Journal of Cell Science 116 (17)
receptor-mediated endocytosis in polarized cells as well as
cells that have a dense cortical actin network, a mechanism
must be in place to facilitate movement of the uncoated vesicle
from the peripheral region of the cell, through the actin
meshwork, towards the more central early endosomes and
microtubule networks for further transport. As a pointed-end
directed motor present in the terminal web and peripheral actin
networks, myosin VI is a good candidate for a vesicle motor
at the heart of such a mechanism.
Of these three potential roles, studies in cultured cell models
have provided strong evidence for two – formation of clathrincoated vesicles (Fig. 2B) and transport of uncoated vesicles
(Fig. 2C). The evidence implicating myosin VI in these two
steps is based on studies of splicing variants that allow myosin
VI to target to two distinct endocytic compartments
(Aschenbrenner et al., 2003; Buss et al., 2001).
Fig. 1. Myosin VI is located at the base of microvilli. Confocal
image of a frozen section of mouse kidney stained with antibodies to
myosin VI (visualized with a FITC-conjugated secondary antibody)
and rhodamine-conjugated phalloidin to stain F-actin. Actin (red) is
most prominent in apical microvilli in the proximal tubules of the
kidney. Myosin VI (green) is enriched in the endocytic region at the
base of the microvilli. Bars, 10 µm.
later with apical intermicrovillar invaginations and clathrincoated pits, suggesting that ligand-bound receptors are actively
transported to the base of microvilli (Gottlieb et al., 1993).
Microvilli are composed primarily of bundled actin filaments
polarized with their barbed ends towards the tips of the
microvilli (Mooseker et al., 1982). Regulated movement of
ligand-bound receptors down the microvilli would require a
pointed-end-directed myosin motor to bind and transport
components directly down the microvillus. Because myosin VI
is present at low levels along microvilli (Biemesderfer et al.,
2002) as well as at their base, it may serve this role in directed
receptor transport.
A second potential role for myosin VI is in the formation of
clathrin-coated vesicles (Fig. 2B). Studies in proximal tubule
cells following the scavenger receptor megalin after ligand
binding revealed that the ligand is first concentrated in the
clathrin-rich invaginations before traversing through an
endosomal compartment and reaching the lysosome (Birn et
al., 1997; Christensen and Nielsen, 1991). The intermicrovillar
invaginations are enmeshed in a dense actin cytoskeleton (the
terminal web). This actin may act as a barrier to vesicle
formation or as an active player in the vesicle formation
process. Indeed, four distinct models for a positive role for
actin in clathrin-coated vesicle formation have been proposed
(Qualmann et al., 2000) (see below). Because myosin VI is
concentrated in the clathrin-rich invaginations, it might
function in any or all of these actin-dependent processes,
facilitating the creation of clathrin-coated vesicles.
Finally, a third potential role for myosin VI is at a later step
of endocytosis; the transport of uncoated vesicles through the
actin-rich terminal web towards the early endosome
(Aschenbrenner et al., 2003) (Fig. 2C). During the process of
Myosin VI domain organization
Myosin VI has four functional domains (Fig. 3). The conserved
motor domain of myosin VI is at the N-terminus of the protein,
and is followed by a single IQ motif, which serves as a lightchain-binding site for the calcium-binding protein calmodulin
(Hasson and Mooseker, 1994). The motor–IQ domain is
sufficient for pointed-end directed movement (Homma et al.,
2001; Wells et al., 1999). Following the motor is the tail
domain, which is made up of two regions: a 200-residue coiledcoil region and a C-terminal globular region (Hasson and
Mooseker, 1994). The coiled-coil region mediates dimerization
(De La Cruz et al., 2001). The globular region contains no
recognizable motifs but is highly conserved across species.
The tail of class VI myosins is alternatively spliced in both
insects and vertebrates (Breckler et al., 2000; Buss et al.,
2001; Buss et al., 1998; Kellerman and Miller, 1992), leading
to insertions between the coiled-coil domain and the globular
domain, as well as additional residues at the C-terminus.
Although the significance of the alternative splicing has not
been fully elucidated, in vertebrates alternative splicing
generates two predominant forms of myosin VI, a longer and
a shorter form, which differ by a 23-residue insert between
the coiled-coil and the globular region (Fig. 3). Both the long
and short forms of myosin VI have been implicated in
endocytosis.
Myosin VI and clathrin-coated vesicle association
The longer splice form of myosin VI has been directly
implicated in the formation of clathrin-coated vesicles during
endocytosis (Buss et al., 2001) (Fig. 3). This form of myosin
VI colocalizes with clathrin-coated pits when expressed in
cultured cells, and PCR analysis suggests that it is the major
form expressed in rat kidney and polarized CaCo-2 intestinal
epithelial cells (Buss et al., 2001). Analysis of myosin VI
fragments revealed that the tail domain alone does not target
myosin VI to clathrin-coated pits, but the tail domain plus the
splice site insert is sufficient for coated-pit targeting. Cultured
NRK cells expressing a myosin VI fragment containing the tail
domain with the splice insert exhibit a dramatic reduction
(~70%) in transferrin endocytosis (Buss et al., 2001).
Therefore, alternative splicing of myosin VI generates a form
of myosin VI unique to polarized cell types that can be
Role of myosin VI in endocytosis
3455
recruited to function in the formation of clathrincoated vesicles.
Myosin VI and uncoated vesicle
association
Epithelial cells cultured under non-polarizing
conditions express the shorter version of myosin VI
(Fig. 3) (Buss et al., 2001; Hasson and Mooseker,
1994). Although these cells lack microvilli, they do
exhibit dense cortical actin networks. In these cells,
myosin VI is found on peripheral vesicles that
pulse-chase analysis confirms are recently uncoated
endocytic vesicles (Aschenbrenner et al., 2003).
Analysis of myosin VI fragments has revealed that
the globular tail region alone is sufficient to target
myosin VI to uncoated vesicles (Aschenbrenner et
al., 2003).
Cultured epithelial cells overexpressing the
myosin VI globular tail region exhibit a dramatic
reduction in transferrin uptake (~20% of normal)
(Aschenbrenner et al., 2003) confirming that the
shorter myosin VI isoform also has a role in
endocytosis. Remarkably, under these conditions
initial rates of transferrin uptake into clathrin-coated
vesicles are normal and instead the block is at
the uncoated vesicle stage of endocytosis
(Aschenbrenner et al., 2003). The uncoated vesicles
remained ‘stuck’ at the cell periphery, dramatically
delaying delivery of the transferrin cargo to the early
endosome. Therefore, depending on the splice
version present, myosin VI may act at an early or
late stage of endocytosis.
Targeting of myosin VI to distinct endocytic
compartments
The tail domain of myosin VI must associate with
distinct cargo proteins that differentially localize the
motor to clathrin-coated pits and/or uncoated
endocytic vesicles. Three distinct linker proteins
have been identified that associate with the tail of
myosin VI: disabled 2 (Dab2), GAIP-interacting
protein-C-terminus (GIPC) and synapse-associated
protein 97 (SAP97).
DAB2
Dab2 (also called DOC-2) is a putative tumor
suppressor protein implicated in cell surface
receptor turnover, endocytosis and cell signaling
pathways. It is a complex molecule containing
several well-characterized protein-binding motifs
(Fig. 4). Near its N-terminus is a phosphotyrosinebinding (PTB) domain, which binds to multiple
cell-surface receptors of the low-density lipoprotein
receptor (LDLR) family, all of which contain a
conserved NPXY motif (Morris and Cooper, 2001;
Oleinikov et al., 2000). PTB domains are
structurally similar to plekstrin homology (PH)
domains and, like PH domains, the PTB domain of
Fig. 2. Three models for myosin VI function in endocytosis. Depicted are two
microvilli filled with polarized actin filaments (arrows) and the endocytic region
found between them. See text for details.
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Journal of Cell Science 116 (17)
Myosin VI
Tail domain
Motor domain
IQ
Coiledcoil
Motor domain
IQ
Coiledcoil
Fig. 3. Myosin VI splice forms.
Globular
domain
Targets to uncoated vesicles
Globular
domain
Targets to clathrin-coated pits
Tail insert
Dab2 binds to phosphoinositides and Dab2 can simultaneously
2002; Morris et al., 2002a). Overexpression of Dab2
associate
with
NPXY-containing
proteins
and
reorganizes surface AP-2, and myosin VI that contains the
phosphoinositide-containing lipids (Mishra et al., 2002).
splice insert is recruited to these structures (Morris et al.,
Centrally located in Dab2 are a series of DPF motifs (Fig.
2002a). Therefore, Dab2 probably serves as the bridge that
4), binding sites for the clathrin adapter AP-2, and these motifs
links the longer form of myosin VI to clathrin-coated pits.
are sufficient for targeting of Dab2 to clathrin-coated pits
Indeed, antibodies to myosin VI co-immunoprecipitate Dab2,
(Morris and Cooper, 2001). Also in this region are type I and
AP-2 and megalin from the proximal tubule, confirming the in
type II binding sites for clathrin heavy chain (Mishra et al.,
vivo association of these proteins (Biemesderfer et al.,
2002). The presence of these binding sites implicates Dab2 in
abstract). It is interesting to speculate that Dab2 might recruit
regulating clathrin-coated vesicle formation. Indeed, an Nmyosin VI to ligand-bound megalin; this complex would then
terminal fragment containing the PTB and the central APbe primed to transport the ligand-bound receptor down the
2/clathrin-binding domain is sufficient to initiate the formation
microvillus, where it would be anchored into clathrin-coated
of clathrin-coated vesicles from phosphoinositide-containing
pits through association of Dab2 with clathrin, AP-2, and other
lipids in vitro, a process that is further accelerated in the
accessory proteins.
presence of AP-2 adapters (Mishra et al., 2002). Dab2 also
One unanswered question is how the association of Dab2
contains five NPF motifs, which in other proteins are sufficient
with myosin VI is regulated. In in vitro binding assays, Dab2
for association with Eps15 homology (EH) domains found in
can associate with both splice forms of myosin VI (Inoue et
a variety of accessory proteins involved in endocytosis.
al., 2002; Morris et al., 2002a); however in vivo, the tail insert
Therefore, Dab2 exhibits all the features characteristic of an
is required for targeting to clathrin-coated pits (Buss et al.,
endocytic adapter protein. Because it can also associate with
2001; Morris et al., 2002a). Constructs lacking this insert do
LDLR family members, Dab2 may be involved in linking
not target to clathrin-coated pits even if Dab2 is present
specific cargo with clathrin polymerization on the membrane.
(Aschenbrenner et al., 2003). An as-yet-uncharacterized
Analysis of Dab2-knockout mice has confirmed that Dab2
mechanism must exist that regulates myosin VI targeting to
functions in endocytosis. Renal
proximal tubule cells from these
knockout mice have fewer clathrinMyosin-VI-binding domain
Dab2
coated pits and show defects in
Amino acids SYF
amino acid and vitamin uptake, a
essential for
PTB
characteristic of defects in megalin
p96
myosin VI binding
endocytosis (Morris et al., 2002b).
Proline-rich
Megalin is a member of the LDLR
PTB
gene family and associates directly
p67
NPF/DPF
Clathrin box
with the Dab2 PTB domain
(Oleinikov et al., 2000).
Dab2 associates with myosin VI
GIPC
through its C-terminal serine- and
Required for myosin VI binding
proline-rich region (Fig. 4) and
binds to the C-terminal globular tail
of myosin VI in vitro (Inoue et al.,
PDZ
Fig. 4. The three linker proteins that
associate with the globular tail domain
of myosin VI: Dab2, GIPC and Sap97.
Dab2 is alternatively spliced and so
only the major splice forms are shown.
See text for an explanation of domains
found in each protein. The myosin VIbinding domains for all three linker
proteins are shown in light blue.
Proline-rich
Sap97
Myosin-VI-binding domain
PDZ PDZ
PDZ
SH3
GUK
Role of myosin VI in endocytosis
Dab2 in clathrin-coated pits, specifically recruiting the longer
splice version. Because Dab2 is a linker protein capable of
multiple simultaneous associations, perhaps another protein in
the complex binds the insert sequence and confers this
specificity.
Alternatively,
splicing
or
differential
phosphorylation of Dab2 might regulate the integration of
myosin VI into Dab2-containing clathrin-coated pits. Dab2
was first characterized as an alternatively spliced mitogenregulated phosphoprotein with two predominant splice forms
(Xu et al., 1995) (Fig. 4). Both identified splice forms of Dab2
can associate with myosin VI (Inoue et al., 2002; Morris et al.,
2002a). However, the shorter Dab2 isoform might associate
with AP-2 and clathin to a lesser extent because the region
containing the important association motifs is missing (Mishra
et al., 2002; Morris et al., 2002a).
GIPC
GIPC is a PDZ-domain-containing protein known under a
variety of monickers depending on the yeast-two hybrid screen
bait used to identify it (Fig. 4). Its centrally located PDZ
domain binds to proteins that have at their C-termini either a
conserved type I PDZ-binding site (S/T)-X-(V/A) (Songyang
et al., 1997), or a similar C-terminal sequence such as S-Y-S.
This binding flexibility may explain the abundance of
published GIPC associations.
The list of identified binding partners for GIPC includes
many transmembrane proteins, such as multiple members of
the LDL receptor family (e.g. megalin) (Gotthardt et al., 2000),
the glucose transporter GLUT1C (Bunn et al., 1999), receptor
tyrosine kinases [insulin-like growth factor-1 (IGF-1) receptor
(Ligensa et al., 2001); TrkA and TrkB (Lou et al., 2001)], and
the receptor serine/threonine kinase transforming growth factor
β (TGFβ) receptor type III (Blobe et al., 2001). It has also been
identified as a binding partner for many different cell surface
molecules involved in adhesion, including 5T4 [a protein
highly expressed in transformed cells that correlates with
metastatic phenotypes (Awan et al., 2002)], integrins α5, α6a
and α6b (El Mourabit et al., 2002; Tani and Mercurio, 2001),
the adhesion regulator syndecan-4 (Gao et al., 2000), the
semaphorins M-SemaF and SemC (Wang et al., 1999) and the
semaphorin receptor neuropilin-1 (Cai and Reed, 1999).
Although this list continues to grow, there is little in vivo
evidence for association of GIPC with any of these proteins at
the plasma membrane.
There is evidence for a role for GIPC in trafficking of
transmembrane proteins through the Golgi stacks, however.
GIPC associates with GAIP, a membrane-anchored GTPaseactivating protein for Gαi3 subunits (De Vries et al., 1998b).
GAIP localizes to clathrin-coated vesicles in the Golgi region,
which implicates GIPC in membrane trafficking (De Vries et
al., 1998a). GIPC also associates with gp75 tyrosinase related
protein 1, a melanosomal membrane protein (Liu et al., 2001),
but only with newly synthesized gp75 as it traverses the Golgi.
Perhaps GIPC functions in the sorting of the other
transmembrane receptors. It may also be involved in recruiting
myosin VI to the Golgi, given that a fraction of myosin VI is
reported to be Golgi associated (Buss et al., 1998).
Myosin VI was first identified as a binding partner of GIPC
in a yeast two-hybrid screen (Bunn et al., 1999). The precise
myosin VI-binding region on GIPC was not defined; however
3457
the PDZ domain of GIPC was found to not be sufficient for
binding in vitro. GIPC is present on small vesicles near the
plasma membrane in cultured cell lines (De Vries et al., 1998b)
and myosin VI colocalizes with GIPC on these peripherally
located vesicles (Aschenbrenner et al., 2003). Pulse-chase
experiments confirmed that the vesicles are uncoated
transferrin-containing endocytic vesicles, implicating GIPC in
endocytosis (Aschenbrenner et al., 2003). In vivo, GIPC is
enriched at both the clathrin-rich invaginations and the
endocytic compartments found between microvilli in proximal
tubule kidney cells, where it overlaps with GAIP (Lou et al.,
2002), clathrin, AP-2 and myosin VI (Biemesderfer et al.,
2002). Therefore, GIPC, in common with Dab2, might
associate with myosin VI to cluster megalin or other receptors
that have a type I PDZ-binding motif into clathrin-coated
intermicrovillar regions. Unlike Dab2, however, GIPC can
remain associated with myosin VI after the clathrin-coated
vesicle is formed and uncoated, perhaps serving a role in later
stages of vesicle trafficking.
Sap97
SAP97 is a member of the PSD-95 family of membraneassociated guanylate kinase homologues (MAGUKs)
(reviewed by Fujita and Kurachi, 2000). Unlike other SAPs,
SAP97 is also expressed in non-neuronal cells and is present
at cadherin-based cell-cell adhesions in epithelial cells (Muller
et al., 1995; Reuver and Garner, 1998). It has three centrally
located PDZ domains, as well as a Src-Homology 3 (SH3)
domain and a C-terminal guanylate kinase (GUK) homology
domain (Fig. 4), all domains classically involved in proteinprotein interactions. The N-terminal domain of SAP97 is
required for targeting of SAP97 to adhesion sites in epithelial
cells (Wu et al., 1998). This domain associates with multiple
binding partners, including three MAGUK scaffolding proteins
[Lin-2, DLG2 and DLG3 (Karnak et al., 2002)] and myosin VI
(Wu et al., 2002). The association with the MAGUKs likely
mediates the targeting of SAP97 to adhesion sites in epithelial
cells. An association between myosin VI and SAP97 is not
seen in epithelial cells (Karnak et al., 2002; Wu et al., 2002),
and evidence for a SAP97 – myosin VI association has only
been reported in brain (Wu et al., 2002).
SAP97 is present throughout neurons as well as at the
synapse. It is implicated in localization of the AMPA-type
glutamate receptor subunit, GluR1. The first PDZ domain of
SAP97 associates with a type I PDZ-binding motif found at the
C-terminus of GluR1 (Leonard et al., 1998) and facilitates its
trafficking through the Golgi to the plasma membrane (Sans et
al., 2001). Since a fraction of cellular myosin VI resides in the
Golgi (Buss et al., 1998), this could be associated with SAP97.
Although SAP97 is a synaptic protein, thus far there is no
evidence for association of SAP97 with GluR1 or myosin VI
at the synapse. Moreover, there is also as yet no evidence for
a role for SAP97 in endocytosis.
Actin, myosin VI and formation of clathrin-coated
vesicles
Dab2, GIPC or both may mediate recruitment of myosin VI to
clathrin-coated pits, but what function is myosin VI serving
here? The answer probably lies with actin, which has several
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Journal of Cell Science 116 (17)
potential roles in early stages of endocytosis (Apodaca, 2001;
Qualmann and Kessels, 2002; Qualmann et al., 2000) (Fig.
2B), each of which could require myosin.
By associating with both actin and linker proteins, myosin
VI might cluster receptors onto actin networks and thereby
spatially organize the endocytic machinery. Such an
arrangement would be particularly important in the
intermicrovillar clathrin-rich endocytic regions of polarized
epithelial cells, regions rich in myosin VI. In cultured cells,
clathrin-coated pits are often aligned with the underlying actin
cytoskeleton, particularly on basal cell surfaces (Puszkin et al.,
1982). Movement of clathrin-coated pits within the plasma
membrane has also been shown to require actin (Gaidarov et
al., 1999). These mechanisms for clathrin-coated pit
positioning within the plasma membrane have not been
characterized but could involve a myosin such as myosin VI.
Alternatively, rather than having a strictly structural
function, as a two-headed motor myosin VI may provide the
force necessary for deformation of the plasma membrane seen
at sites of pits or serve as a force generator during or after
vesicle fission. In support of the latter hypothesis,
overexpression of myosin VI tail fragments containing the tail
insert does not alter the morphology of the clathrin-coated pits
but does cause a defect in clathrin-coated vesicle formation
(Buss et al., 2001). Higher-resolution analysis of clathrincoated vesicle formation should distinguish between these two
potential functions.
Studies of other actin-binding proteins implicated in
endocytosis suggest that a mechanism to dissolve the cortical
actin barrier is essential for endocytosis in some cell types,
and this may involve myosin VI (reviewed by Qualmann and
Kessels, 2002). Specifically, the actin-spectrin network must
be dissolved for coated-pit budding in fibroblasts. The loss of
spectrin depends on cleavage by an activated calpain
protease, a calmodulin-dependent enzyme (Kamal et al.,
1998). As a calmodulin-associated protein, myosin VI might
be involved.
Evidence also indicates a need for actin polymerization
during endocytosis (Qualmann and Kessels, 2002; Qualmann
et al., 2000). Several actin-binding proteins that can recruit the
polymerization machinery have been implicated in early steps
of endocytosis (Olazabal and Machesky, 2001; Qualmann and
Kessels, 2002), and actin polymerization has been observed at
the neck of invaginating clathrin-coated pits (Merrifield et al.,
2002). Actin polymerization might accelerate vesicle fission or
in the creation of an actin ‘tail’ that provides the first push
moving the vesicle away from the plasma membrane surface.
Studies that focus on the budding step have shown that drugs
that depolymerize actin have little effect on endocytosis in
permeabilized fibroblasts or nonpolarized cells in vitro
(Fujimoto et al., 2000; Lamaze et al., 1997), but significantly
affect clathrin-mediated uptake in hepatoma cells and
enterocytes (Durrbach et al., 1996; Jackman et al., 1994).
Therefore, there is a potential role for polymerization in apical
endocytosis particularly in polarized cells.
How does myosin VI fit into existing models for actin
polymerization in clathrin-coated vesicle formation? Studies
of the Drosophila myosin VI homologue, Jaguar (also known
as 95F myosin), have implicated it in an actin polymerization
process that occurs during spermatogenesis (Rogat and Miller,
2002). Jaguar is highly homologous to mammalian myosin VI,
with the highest levels of sequence similarity being in the
cargo binding tail domains suggesting conserved function
(Hasson and Mooseker, 1994). Jaguar mutations affect testes
myosin VI gene expression and jaguar flies have a defect in
the individualization stage of spermatogenesis. During this
stage, membranes are laid down between each spermatid,
separating each from its neighbors. A cone of actin precedes
the addition of membrane, and myosin VI is enriched at the
leading edge of this cone. Myosin VI is required at this
position to bring in the actin polymerization machinery,
including the ARP2/3 complex (Hicks et al., 1999; Rogat and
Miller, 2002). Given the high levels of homology between fly
and mammalian myosin VI, mammalian myosin VI might
similarly be required to recruit the actin polymerization
machinery to the newly forming clathrin-coated vesicle during
endocytosis.
Myosin VI as a vesicle motor
Because overexpression of the tail domain of myosin VI
(lacking any spliced inserts) causes an accumulation of
uncoated vesicles containing endocytosed transferrin, myosin
VI might be involved in the movement of newly formed
vesicles inwards to the early endosome (Fig. 2C)
(Aschenbrenner et al., 2003). Under these conditions, the
vesicles are competent for fusion with the early endosome and
contain the fusion factor Rab5, but remain trapped in the actin
meshwork at the periphery of the cell (Aschenbrenner et al.,
2003). What is the myosin’s role? It might simply transport the
vesicles along actin filaments. This type of model has been
suggested for other myosins involved in membrane trafficking
(reviewed by Langford, 2002; Tuxworth and Titus, 2000),
although, in all these cases, the myosin is transporting its cargo
towards the cell periphery. Although straightforward, a simple
transport model for myosin VI may not be sufficient to explain
the dramatic delay in vesicle trafficking seen upon disruption
of myosin VI function. The actin filaments within the cortical
actin network found at the cell periphery are not all oriented
with pointed ends inwards, as is seen at the plasma membrane.
Therefore direct transport inward by myosin VI may not be
feasible, and other possible roles for the motor must be
considered.
Myosin VI might facilitate actin reorganization necessary
for fusion of vesicles with the early endosome. Kinetic studies
of myosin VI suggest that during most of its duty cycle it
maintains a tight association with F-actin (De La Cruz et al.,
2001). Because of this property, as a two-headed myosin,
myosin VI could potentially associate with two distinct
filaments and mediate filament sliding necessary to extricate
the vesicle from the dense actin mesh found in peripheral
regions of cells and the terminal web. Such rearrangements
may be necessary for the fusion machinery to access the early
endosome.
Alternatively, as described in the previous section, myosin
VI could act as a regulator of actin dynamics. Actin
polymerization has been implicated as a mechanism for
movement of endocytic vesicles and actin comet tails have
been seen on endocytic vesicles in mast cells (Merrifield et al.,
1999). Therefore, myosin VI may function in the movement of
endocytic vesicles by recruiting components necessary for
actin polymerization, thereby pushing the uncoated vesicles
Role of myosin VI in endocytosis
out of the peripheral actin-rich domains and towards the early
endosome.
Ultimately, a defect in any of these three processes could
explain the accumulation of vesicles seen at the cell periphery
after disruption of myosin VI function and further studies will
be required to distinguish between them.
Analysis of myosin VI mutants suggests trafficking
roles
Analysis of phenotypes associated with myosin VI mutations
has not directly implicated myosin VI in endocytosis, but does
suggest roles in actin cytoskeleton assembly, and membrane
trafficking. In mammals, myosin VI mutations cause profound
neurosensory deafness and balance disorders (Avraham et al.,
1995; Melchionda et al., 2001). Both phenotypes are due to
defects in the inner ear hair cells, a polarized cell type with
unique actin-based projections called stereocilia. Stereocilia
are similar in structure to microvilli and are required for
sensing sound and balance signals. Myosin VI is found at the
base of the stereocilia in an actin rich structure called the
cuticular plate, occupying a position similar to that seen in
kidney microvilli (Hasson et al., 1997) (Fig. 1). Snell’s waltzer
(sv) mice, which lack myosin VI, have defects in stereocilia
development (Self et al., 1999). Although microvilli are
initially present on the surface of the hair cells, they do not go
through the usual elongation and widening steps associated
with stereocilia development and instead aberrant actincontaining protrusions are assembled on the hair cell surface.
This specific developmental defect could be due to one of the
many potential actin-associated activities of myosin VI (Fig.
2) including errors in actin polymerization or defects in the
coordinated regulation of membrane trafficking.
In the inner ear, unlike the kidney, endocytosis does not
occur between the actin projections of the stereocilia. Instead,
endocytosis occurs in an apical domain outside the actin-rich
stereocilia and cuticular plate called the pericuticular necklace
(Hasson et al., 1997; Kachar et al., 1997). In keeping with a
role in endocytosis, myosin VI is enriched within this
necklace; however studies of hair cell fluid phase uptake in sv
mice suggest that unregulated endocytosis at least is normal
in these animals (Self et al., 1999). Analysis of clathrinmediated uptake has not been evaluated in sv mice, but this
result is in keeping with the observation that fluid-phase
uptake is not affected when dominant-negative versions of
myosin VI are expressed in cultured cells (Aschenbrenner et
al., 2003; Buss et al., 2001). Therefore, myosin VI may have
regulated functions in hair-cell endocytosis, or it may have
other functions distinct from those proposed here in
membrane trafficking. For example, studies in C. elegans have
implicated myosin VI in the asymmetric movement of
organelles seen during spermatogenesis (Kelleher et al.,
2000). Furthermore, studies in Drosophila have similarly
implicated myosin VI in the directed movement of
intracellular particles, presumed to be membrane vesicles,
during the syncytial blastoderm stage of embryogenesis
(Mermall et al., 1994) and during oogenesis (Bohrmann,
1997). Therefore, myosin VI may participate in the movement
of many types of membrane organelles along the actin
cytoskeleton and not just the vesicle populations discussed
here.
3459
Conclusions and future directions
Studies in a variety of organisms and cell types have revealed
universal roles for myosin VI in membrane trafficking events.
Recent data using mammalian cultured cell models has
suggested that myosin VI plays a role in clathrin-coated vesicle
formation and the trafficking of uncoated nascent vesicles. It
is likely that in both processes, myosin VI plays an accessory
role, perhaps increasing the efficiency of endocytosis. Myosin
VI cannot play an indispensible role in either process as sv
mice are viable and fertile and do not exhibit defects in
endocytic function at a gross level (Avraham et al., 1995).
Therefore, mechanisms must be in place in mammals to
compensate for the lack of myosin VI, perhaps recruiting
another as-yet-unidentified pointed-end directed myosin.
The fact that myosin VI has been implicated in two very
distinct steps of the endocytic process is remarkable:
depending on the cell type myosin VI is either recruited to
uncoated vesicles or clathrin-coated pits. The difference in
myosin VI targeting may be due to local differences in the actin
cytoskeleton, to alternative splicing or to differences in
components that bind to the C-terminal tail domain of myosin
VI. Another possibility is that the differences in myosin VI
targeting reflect different signaling pathways that are activated
to recruit myosin VI to distinct regions. The best such
candidates are p21-activated kinase (PAK) family members,
which have been shown to phosphorylate myosin VI in vitro
(Buss et al., 1998; Yoshimura et al., 2001). Myosin VI is indeed
phosphorylated in vivo (Buss et al., 1998) (our unpublished
data) but the significance of myosin-VI phosphorylation
remains to be studied.
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