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: tama{at}ucsd.edu)
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Summary |
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Key words: Unconventional myosin, Myo6, Actin, Receptor-mediated endocytosis, Clathrin-coated Pits, Vesicle trafficking
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Introduction |
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Myosins |
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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 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.
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Myosin VI and endocytic regions |
---|
|
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). Pulse-chase studies following uptake of cationic ferritin
in MDCK cells revealed that ferritin associates first with microvilli and
later with apical intermicrovillar invaginations and clathrin-coated 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 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 clathrin-coated 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
).
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Myosin VI domain organization |
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|
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.
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Myosin VI and clathrin-coated vesicle association |
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Myosin VI and uncoated vesicle association |
---|
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.
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Targeting of myosin VI to distinct endocytic compartments |
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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 phosphotyrosine-binding (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 Dab2 binds to phosphoinositides and Dab2 can
simultaneously associate with NPXY-containing proteins and
phosphoinositide-containing lipids (Mishra
et al., 2002
).
|
Centrally located in Dab2 are a series of DPF motifs
(Fig. 4), binding sites for the
clathrin adapter AP-2, and these motifs are sufficient for targeting of Dab2
to clathrin-coated pits (Morris and
Cooper, 2001). Also in this region are type I and type II binding
sites for clathrin heavy chain (Mishra et
al., 2002
). The presence of these binding sites implicates Dab2 in
regulating clathrin-coated vesicle formation. Indeed, an N-terminal fragment
containing the PTB and the central AP-2/clathrin-binding domain is sufficient
to initiate the formation of clathrin-coated vesicles from
phosphoinositide-containing lipids in vitro, a process that is further
accelerated in the presence of AP-2 adapters
(Mishra et al., 2002
). Dab2
also contains five NPF motifs, which in other proteins are sufficient for
association with Eps15 homology (EH) domains found in a variety of accessory
proteins involved in endocytosis. Therefore, Dab2 exhibits all the features
characteristic of an endocytic adapter protein. Because it can also associate
with LDLR family members, Dab2 may be involved in linking specific cargo with
clathrin polymerization on the membrane.
Analysis of Dab2-knockout mice has confirmed that Dab2 functions in
endocytosis. Renal proximal tubule cells from these knockout mice have fewer
clathrin-coated pits and show defects in amino acid and vitamin uptake, a
characteristic of defects in megalin endocytosis
(Morris et al., 2002b).
Megalin is a member of the LDLR gene family and associates directly with the
Dab2 PTB domain (Oleinikov et al.,
2000
).
Dab2 associates with myosin VI through its C-terminal serine- and
proline-rich region (Fig. 4)
and binds to the C-terminal globular tail of myosin VI in vitro
(Inoue et al., 2002;
Morris et al., 2002a
).
Overexpression of Dab2 reorganizes surface AP-2, and myosin VI that contains
the splice insert is recruited to these structures
(Morris et al., 2002a
).
Therefore, Dab2 probably serves as the bridge that links the longer form of
myosin VI to clathrin-coated pits. Indeed, antibodies to myosin VI
co-immunoprecipitate Dab2, AP-2 and megalin from the proximal tubule,
confirming the in vivo association of these proteins (Biemesderfer et al.,
abstract). It is interesting to speculate that Dab2 might recruit myosin VI to
ligand-bound megalin; this complex would then be primed to transport the
ligand-bound receptor down the microvillus, where it would be anchored into
clathrin-coated pits through association of Dab2 with clathrin, AP-2, and
other accessory proteins.
One unanswered question is how the association of Dab2 with myosin VI is
regulated. In in vitro binding assays, Dab2 can associate with both splice
forms of myosin VI (Inoue et al.,
2002; Morris et al.,
2002a
); however in vivo, the tail insert is required for targeting
to clathrin-coated pits (Buss et al.,
2001
; Morris et al.,
2002a
). Constructs lacking this insert do not target to
clathrin-coated pits even if Dab2 is present
(Aschenbrenner et al., 2003
).
An as-yet-uncharacterized mechanism must exist that regulates myosin VI
targeting to 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 mitogen-regulated 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 ß
(TGFß) 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
5,
6a and
6b
(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 GTPase-activating protein for
Gi3 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 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 membrane-associated 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 protein-protein 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.
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Actin, myosin VI and formation of clathrin-coated vesicles |
---|
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 clathrin-coated 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.
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Myosin VI as a vesicle motor |
---|
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 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.
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Analysis of myosin VI mutants suggests trafficking roles |
---|
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 clathrin-mediated 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.
![]() |
Conclusions and future directions |
---|
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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