1 Department of Biological Science, Florida State University, Tallahassee, FL
32306, USA
2 MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, UK
* Author for correspondence (e-mail: roberts{at}bio.fsu.edu)
Accepted 10 March 2003
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Summary |
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Key words: Cytoskeleton, Major sperm protein, Nematoda, Phosphorylation
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Introduction |
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The amoeboid sperm of nematodes such as Ascaris offer a novel
perspective for investigating the biochemical basis of localized cytoskeletal
assembly and its relationship to motility
(Italiano et al., 2001;
Roberts and Stewart, 2000
). In
these cells locomotion is generated by a dynamic cytoskeleton constructed from
major sperm protein (MSP) rather than actin, but the fundamental physical
principles underlying motility have been retained. Consequently, nematode
sperm provide a simple and specialized system in which to probe the mechanism
of amoeboid cell locomotion (reviewed by
Roberts and Stewart, 2000
).
Ascaris sperm contain a robust system of MSP filaments that are
organized primarily into discrete, branched meshworks called fiber complexes
that span from the leading edge to the base of the lamellipod
(Sepsenwol et al., 1989
). The
fiber complexes elongate as a result of localized MSP polymerization along the
advancing front of the cell, and the rate of their extension exactly matches
the rate of protrusion of the leading edge
(Roberts and King, 1991
). This
protrusive activity has been reconstituted in vitro in cell-free extracts of
sperm in which columnar meshworks of MSP filaments, called fibers, assemble at
the surface of vesicles derived from the plasma membrane at the front of the
lamellipod. As the fiber grows, it pushes its vesicle forward at rates
comparable to that of leading edge protrusion in crawling sperm
(Italiano et al., 1996
).
Identifying the mechanism by which cytoskeletal assembly is targeted to
specific regions of the lamellipod is central to understanding the molecular
basis of cell locomotion. The pattern of MSP polymerization in vivo and in
vitro has led to the hypothesis that the leading edge of the lamellipodial
membrane of Ascaris sperm plays a central role in directing
cytoskeletal construction (Italiano et
al., 1999; Roberts et al.,
1998
). Here, we have tested this hypothesis and describe the
identification and characterization of a 48 kDa integral membrane protein that
is necessary for fiber formation and which appears to be the only membrane
component required to trigger MSP assembly under physiological conditions.
This protein is distributed throughout the lamellipodial membrane, but
nucleates MSP polymerization only at sites where it undergoes a pH-sensitive
tyrosine phosphorylation that appears to require an additional cytosolic
component rather than an autophosphorylation of the p48 protein itself.
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Materials and Methods |
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Preparation of sperm extracts for in vitro motility assays and
membrane protein isolation
Frozen sperm were thawed on ice and centrifuged at 10,000 g
for 10 minutes. The supernatant was then centrifuged at 100,000
g for 1 hour in a TLA100.3 rotor in a tabletop ultracentrifuge
(Beckman Instruments, Palo Alto, CA). The supernatant (S100) was then used for
motility assays or diluted 1:4 in KPM buffer (0.5 mM MgCl2, 10 mM
potassium phosphate, pH 6.8) and centrifuged for an additional 1 hour at
100,000 g at 4°C in a TLA 100.3 rotor to separate membrane
vesicles by sedimentation from soluble cytosolic components.
Proteins were extracted from membrane vesicles in 10 mM CHAPS (3-[3-(cholamidopropyl)dimethylammonio]-1-proanesulfonate) or 2.5% Triton X-100 in 150 mM NaCl, 10 mM potassium phosphate and 0.5 mM MgCl2 for 1 hour at 4°C. Detergent-insoluble material was removed by centrifugation at 100,000 g for 1 hour. Gel filtration chromatography was performed on the CHAPS-soluble protein fraction using a Beckman high-performance liquid chromatography (HPLC) setup with a Pharmacia Superdex 200 FPLC column equilibrated with 5 mM CHAPS, 250 mM NaCl in KPM buffer.
Amino acid sequencing
For internal amino acid sequence, protein bands were excised from
Coomassie-stained SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel
electrophoresis) gels, equilibrated with 2.5 µg endoproteinase lys-C (Roche
Pharmaceuticals, Nutley, NJ), 25 mM Tris-HCl, pH 8.5, and incubated for 8
hours at 37°C. The resulting peptide mixture was extracted from the gel
slices with two successive washes with 0.1% trifluoroacetic acid (TFA)/60%
acetonitrile. Washes were combined and individual peptides were separated with
an Applied Biosystems (Foster City, CA) 173A capillary HPLC system equipped
with a reversed-phase C18 column. Peptides were spotted onto polyvinylidene
fluoride (PVDF) and sequenced directly from the membrane using an ABI Procise
cLC 492 protein sequencer.
SDS-PAGE and western blotting
SDS-PAGE was performed according to the method of Laemmli
(Laemmli, 1970), using 12% or
4-15% gradient gels. For immunoblotting, gels were transferred to
nitrocellulose membranes (Schleicher and Schuell, Keene, NH) by the method of
Towbin et al. (Towbin et al.,
1979
), blocked in TBS-T (0.1% Tween-20, 137 mM NaCl, 20 mM Tris,
pH 7.6) with 1% bovine serum albumin (BSA) for 4 hours, and probed with
primary antibody and then with appropriate secondary antibody conjugated to
horseradish peroxidase (Jackson Immuno Research Laboratories, West Grove,
PA).
Antibodies and antiserum
Affinity-purified polyclonal antibody to phosphotyrosine was purchased from
BD Transduction Laboratories (Los Angeles, CA) and monoclonal
antiphosphotyrosine from Cell Signaling (Beverly, MA). Monoclonal antibody to
MSP, AZ10, was generated and purified as described
(Sepsenwol et al., 1989).
Synthetic peptides were synthesized by Sigma Genosys (The Woodlands, TX) and
engineered with an N-terminal cysteine for crosslinking to maleimide-activated
keyhole limpet hemacyanin (KLH) (Pierce, Rockford, IL). Antigens were combined
with RIBI Adjuvant (Corixa, Hamilton, MT) and administered to New Zealand
White rabbits in six intradermal sites, two intramuscular sites and one
subcutaneous site. Rabbits were boosted at 28 days and bled 10 days later.
Whole blood was clotted and centrifuged to collect the anti-serum.
Anti-peptide antibodies were affinity purified using the respective peptide
linked to agarose. Antibodies were stored at -20°C until use.
Immunolabeling
Live cells or fibers assembled in vitro were pipetted into chambers
assembled on glass slides then fixed using 1.25% glutaraldehyde and 0.1%
Triton X-100 (cells) or 1% glutaraldehyde (fibers). Samples were then blocked
in 20 mM NaBH4 for 20 minutes and 1% BSA for 6 hours. Preparations
were treated overnight at 4°C with primary antibodies diluted to 5
µg/ml in 1% BSA, 10 mM sodium phosphate and 150 mM NaCl, pH 7.4, washed and
treated with secondary antibodies (AlexaFluor 568 conjugated goat anti-mouse
or AlexaFluor 488 conjugated goat anti-rabbit; Molecular Probes, Eugene, OR)
at 5 µg/ml for 2 hours at 25°C. Cells were imaged with a Zeiss 410
laser scanning confocal microscope equipped with a dual HeNe laser with
appropriate filters for AlexaFluor 488 and 568 dyes. Fibers assembled in vitro
were examined with a Zeiss Axioskop microscope equipped with a 40x
acroplan/phase objective with appropriate filters and imaged with a Hamamatsu
Orca 12-bit digital camera. All images were processed using Metamorph software
(Universal Imaging, Downingtown, PA) and prepared for figures using Adobe
Photoshop.
Electron microscopy
Filaments assembled in vitro were grown on 22x22 mm ethanol-washed
glass coverslips and fixed for 30 minutes in 1% glutaraldehyde, dehydrated in
ethanol and critical-point dried as described previously
(Ris, 1985). After
sputter-coating with gold palladium, samples were imaged in a JEOL 840
scanning electron microscope operated at 20 kV. Platinum replicas were
prepared by the method of Svitkina and Borisy
(Svitkina and Borisy, 1998
)
and were imaged using a Phillips CM120 transmission electron microscope
operated at 80 kV.
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Results |
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Both the vesicles that assemble fibers in vitro and the plasma membrane at
the leading edge of the lamellipod of crawling sperm label with
antiphosphotyrosine antibodies (Fig.
2A) (see Italiano et al.,
1996). We found that a protein tyrosine phosphatase, YOP from
Yersenia enterocolitica, blocked fiber assembly when added to S100 at
800 U/ml (Fig. 2B). When we
added YOP together with 1 mM sodium orthovanadate, a potent inhibitor of
tyrosine phosphatases (Gordon,
1991
), fiber assembly was rescued and the rate of fiber growth was
indistinguishable from that in control extracts to which neither enzyme or
inhibitor had been added. Western blot analysis using antiphosphotyrosine
antibody showed that S100 contained a single major labeled band at
Mr
48 kDa. Labeling of this band could not be
detected in S100 treated with YOP, but reappeared in samples treated with YOP
plus orthovanadate (Fig. 2C).
Moreover, antiphosphotyrosine detected this band in the vesicles harvested
from S100 but not in the soluble cytosolic fraction
(Fig. 3A). Thus, the vesicles
from which fibers grow in the reconstituted motility system appear to require
a 48 kDa integral membrane protein that requires tyrosine phosphorylation for
activity.
|
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Isolation of the integral membrane protein required to induce MSP
polymerization
To isolate the 48 kDa protein (p48) from the membrane, we treated the
vesicles with either 1% Triton X-100 or 10 mM CHAPS followed by centrifugation
at 10,000 g for 5 minutes. Because the supernatant did not
contain intact vesicles, this material failed to produce fibers when added
back to cytosol. However, the membrane extract did induce the assembly of
extensive meshworks of filaments, readily detectable by scanning electron
microscopy (SEM), when recombined with cytosol
(Fig. 3B) (see
Italiano et al., 1996).
The low critical micelle concentration of CHAPS allowed solublized membrane proteins to be fractionated by gel permeation chromatography (Fig. 3C). The fractions were added back individually to cytosol in the presence of ATP to test for filament assembly and were also assayed for the presence of phosphorylated p48 by western blot analysis. Only those fractions that contained phosphorylated p48 (fractions 38-40 in the sample shown in Fig. 3C) were able to trigger filament assembly in cytosol detectable by SEM (Fig. 3D).
We further purified p48 by immunoprecipitation from the assembly inducing
chromatographic fractions with antiphosphotyrosine antibody. SDS-PAGE gels of
the immunoprecipitated material contained a band with a mobility that was
slightly faster than that of immunoglobulin G (IgG) heavy chain
(Fig. 4A). To confirm that this
band was p48 and not a degradation product of IgG heavy chain, we repeated the
immunoprecipitation after incubating the p48-enriched column fractions with
cytosol and 32P--ATP. Autoradiography of SDS-PAGE gels of
the immunoprecipitate revealed 32P labeling of the faster migrating
p48 but not of IgG heavy chain (Fig.
4A). When p48 obtained by immunoprecipitation was combined with
cytosol and ATP, we detected filaments by SEM
(Fig. 4B). Immunoprecipitations
of fractions lacking p48 yielded no detectable filaments.
|
In summary, the ability to initiate MSP polymerization in cytosolic fractions lacking vesicles paralleled the presence of tyrosine-phosphorylated p48 in fractionated detergent-solubilized membrane fractions.
Characterization of p48
We obtained two amino acid sequences from p48 isolated by
immunoprecipitation with antiphosphotyrosine antibody. One (RIVPSFLENREVFYK)
was obtained from the N-terminus of the intact p48. The same sequence, as well
as a second (KMHISQFYGFP), were obtained from peptides isolated by
reversed-phase HPLC following in-gel digestion of the protein with
endoproteinase Lys-C. BLAST searches failed to detect significant homology of
either of these peptide sequences to known proteins from other types of
organisms, to predicted proteins in the Caenorhabditis elegans
database, or to predicted sequences from a set of testis-enriched expressed
sequence tags (ESTs) from Ascaris. Several attempts to obtain a cDNA
encoding p48, by both reverse-transcriptase polymerase chain reaction (RT-PCR)
of mRNA isolated from Ascaris testis and by screening testis-specific
cDNA libraries, have been unsuccessful.
We generated a polyclonal rabbit antibody to p48 using a KLH-conjugated
synthetic peptide corresponding to the sequence CHISQYGFP of one of the
peptides isolated from the digest. On western blots, the antipeptide antibody
recognized p48 immunoprecipitated with antiphosphotyrosine
(Fig. 5A). In
immunofluorescence assays, the antipeptide antibody labeled
detergent-permeablized cells, resulting in a ring of fluorescence around the
lamellipod, and also stained the cell body
(Fig. 5B). The ring-like or
dotted labeling pattern in the cell body corresponds in number and location to
the membranous organelles, unique components of nematode sperm that contain
membrane proteins and fuse with the cell surface in the cell body
(Roberts et al., 1986). We
detected no labeling above background in sperm that were not permeabilized
with detergent before antibody treatment
(Fig. 4C).
|
We compared the membrane labeling pattern of the antipeptide antibody with
that of antiphosphotyrosine by confocal fluorescence microscopy and found that
the two antibodies colocalized only at the tips of the fiber complexes at the
leading edge of the lamellipod (Fig.
6). This labeling pattern suggests that p48 is distributed
throughout the sperm plasma membrane but its active phosphorylated form is
located only at the leading edge. To test this hypothesis, we examined the
labeling of cells incubated in weak organic acids to lower the intracellular
pH. This treatment stops MSP polymerization at the leading edge and causes the
cytoskeleton to disassemble so that the lamellipod rounds up
(King et al., 1994). As shown
in Fig. 7A, antipeptide
antibody labeled the membrane of acid-treated cells uniformly, whereas
antiphosphotyrosine failed to label these cells. When the acid is washed out,
cytoskeletal assembly resumes around the entire periphery of the lamellipod
(King et al., 1994
) and, in
these cells, antiphosphotyrosine antibody labeled discrete spots around the
periphery of the lamellipod. Within 60 seconds after removing the acid, these
cells complete the reconstruction of their cytoskeleton and locomotion
resumes. In these cells, the entire lamellipod membrane labeled with
antipeptide antibody but antiphosphotyrosine labeling was again restricted to
the leading edge (Fig. 7A).
|
|
The fluorescence labeling pattern observed in response to alteration of
intracellular pH correlates with the pH sensitivity of labeling of p48 with
32P--ATP in S100. As shown in
Fig. 7B, phosphorylation of p48
is readily detectable in S100 at pH 7. By contrast, when the pH of S100 was
lowered to 5.5 before addition of 32P-
-ATP, labeling of p48
was barely detectable.
Phosphorylation of p48 requires cytosol
To examine the pattern of phosphorylation of p48, we incubated vesicles
isolated from S100 with 32P--ATP under a range of different
conditions and monitored phosphorylation by autoradiography. As shown in
Fig. 8, when membrane vesicles
alone were incubated with 32P-
-ATP for 30 minutes, labeling
of p48 was barely detectable. However, adding cytosol to these vesicles
resulted in rapid phosphorylation of p48 so that within 5 minutes the protein
became heavily labeled. No significant protein phosphorylation was detected
when 32P-
-ATP was incubated with cytosol. Therefore, it
appears that phosphorylation of p48 requires a cytosolic factor.
|
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Discussion |
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p48 is the integral membrane protein required for MSP
polymerization
Direct observation of cytoskeletal dynamics identified the leading edge of
the lamellipod as the principal site of MSP polymerization in crawling
Ascaris sperm (Roberts and King,
1991; Sepsenwol et al.,
1989
; Sepsenwol and Taft,
1990
). Reconstitution of leading edge motility in vitro indicated
that the plasma membrane in this region of the cell had an important role in
directing MSP assembly (Italiano et al.,
1996
). By removing peripheral membrane proteins from membrane
vesicles and altering the vesicles with enzymes, we have now provided direct
evidence that the MSP assembly activity of these membrane vesicles derives
from a tyrosine-phosphorylated integral membrane protein.
The polymerization-inducing activity of the p48 integral membrane protein
does not require an intact bilayer, and so we were able to harvest vesicles
from S100, solubilize the membrane proteins with detergents, separate them by
size exclusion chromatography and recombine fractions at each step with
cytosol to assay for filament formation. The requirement for tyrosine
phosphorylation provided a convenient, complementary biochemical assay. At
each step in fractionation, we found that fractions that induced MSP
polymerization in cytosol also contained phosphorylated p48 on western blots.
In most preparations of vesicles, p48 was the only protein that labeled with
antiphosphotyrosine. Some preparations also contained a minor reactive band at
Mr 68 kDa. The sequence of a 20 amino acid peptide
fragment of this protein matched that of flavoprotein subunit II of fumarate
reductase, a 67.9 kDa mitochondrial enzyme from Ascaris that contains
phosphotyrosine residues (Kuramochi et
al., 1994
). Thus, the labeling of the Mr
68 kDa band protein is probably due to occasional mitochondrial
contamination in the vesicle fraction of S100. The presence of this protein in
mitochondria also explains the labeling of organelles in the cell body, where
the mitochondria are located, in antiphosphotyrosine immunofluorescence
assays. Because the 68 kDa protein was not present in the size exclusion
chromatography fractions that induced polymerization, we were able to use
immunoprecipitation with antiphosphotyrosine for further purification of p48.
The protein isolated in this way retained the ability to trigger
polymerization of MSP in cytosol.Because p48 specifies sites of sperm
cytoskeletal assembly, we have named it MSP polymerization organizing protein
(MPOP).
The MSP motility system is found exclusively in nematode sperm, and MSP itself is a unique protein. Thus, it is not unexpected that the protein that organizes MSP polymerization has no homologs in other types of organisms. C. elegans sperm also use an MSP-based motility system but the two peptide sequences that we identified from MPOP do not match the sequences of any of the known or predicted proteins in the C. elegans genome. Moreover, the antibody generated against one of the MPOP peptide sequences fails to label C. elegans sperm by immunofluorescence or by western blot analysis (data not shown). Thus, either there is no C. elegans analog to MPOP or the sequences we obtained come from regions that are not highly conserved in a C. elegans protein.
Function and regulation of p48
On the basis of analysis of the reconstituted MSP motility system, Roberts
et al. (Roberts et al., 1998)
proposed a model for the biochemical basis of site-directed cytoskeletal
assembly in which a vesicle protein, VP, recruits cytosolic proteins to the
membrane surface to create nucleation complexes. These factors were proposed
to operate by converting MSP into a polymerization-competent form that
produces filaments by a nucleation-elongation mechanism. By identifying MPOP
as VP, our data provide direct evidence supporting this hypothesis and we can
now incorporate additional details about how MPOP functions. In particular,
our data indicate that the activity of p48 is regulated by tyrosine
phosphorylation. Thus, conditions such as low pH or phosphatase treatment that
block cytoskeletal assembly also inhibit the labeling of MPOP by
32P-
-ATP in phosphorylation assays. Moreover, the effects of
these agents on both cytoskeletal assembly and phosphorylation of MPOP can be
reversed by elevation of pH or by inhibition of the phosphatase with
orthovanadate.
The pattern of phosphorylation of MPOP in vitro, whereby 32P
labeling of the protein occurs only in the presence of cytosol, suggests that
MPOP is not capable of autophosphorylation (as seen, for example, in growth
factor receptors) but instead is the target of a soluble protein kinase. The
activity of that kinase appears to be pH sensitive. Crawling sperm exhibit a
lamellipodial pH gradient such that the highest pH (6.8) is at the
leading edge where MPOP is phosphorylated
(King et al., 1994
).
Acidification of the cytoplasm stops cytoskeletal assembly. Under these
conditions MPOP is dephosphorylated. When the acid is removed lamellipodial pH
rebounds but is uniform and, under these conditions, cytoskeletal assembly and
antibody labeling occur around the entire periphery of the lamellipod
(Fig. 6) (see
King et al., 1994
). The
phosphorylation state of MPOP exhibits a similar sensitivity to pH in vitro.
These data are consistent with a regulatory mechanism in which MPOP is
phosphorylated and triggers MSP polymerization at pH 6.8 or higher, but is
dephosphorylated and inactivated under more acidic conditions by a phosphatase
that is active at pH <6.8.
Implications for the mechanism of amoeboid cell motility
The motile behavior of nematode sperm is remarkably similar to that of
conventional actin-based cells. Both cell types extend a lamellipod and
locomotion results from a coordinated cycle of leading edge protrusion,
substrate attachment and cell body retraction (reviewed by
Roberts and Stewart, 2000). In
both MSP and actin-based cells, protrusion of the leading edge is driven by
localized vectorial assembly of filaments that bundle and flux rearward
through the lamellipod before disassembling towards the rear of the cell.
Thus, both cell types contain a large recycling pool of cytoskeletal subunits,
and motility depends on establishing a leading edge as a specialized
compartment where conditions favor cytoskeletal assembly. To direct
polymerization to specific areas of the plasma membrane, actin-based cells
frequently utilize membrane receptors to recruit the components required to
assemble filaments and organize them into networks
(Pantaloni et al., 2001
;
Small et al., 2002
). For
example, in many of these cells WASP family proteins are key intermediaries
that bind to the plasma membrane and then activate the Arp2/3 complexes in the
vicinity (Higgs and Pollard,
2001
). Arp2/3 then binds to the side of an existing filament and
nucleates the assembly of a new filament with its barbed end extending away
from the nucleation complex. In this dendritic nucleation process, the
polarity of actin filaments plays a key role in maintaining persistence of
directed extension of the cytoskeleton
(Borisy and Svitkina,
2000
).
Ascaris sperm exhibit the same persistence with the MSP
cytoskeleton assembled continuously along the leading edge of the lamellipod
or at the vesicle surface in the in vitro system at a rate that matches that
of membrane extension. However, unlike actin, MSP filaments are assembled from
symmetric dimers arranged so that the filament has no overall polarity
(Bullock et al., 1998).
Ascaris sperm appear to compensate for the lack of MSP filament
polarity by using pH-regulated site-specific phosphorylation of MPOP to guide
cytoskeletal assembly. Like actin-based systems, the membrane protein involved
in MSP-based motility is necessary, but not sufficient, to nucleate filament
assembly, and other cytosolic components are required.
That two motility systems comprised of such different components use the
same general mechanism to establish and maintain the leading edge emphasizes
the importance of precise transmission of information from the membrane to the
cytoskeleton for amoeboid cell motility. Actin is a versatile protein used by
crawling cells for a range of other membrane-related functions such as
endocytosis, cytokinesis and vesicle movement, in addition to locomotion. As a
result, actin is often modulated by several accessory proteins under the
direction of a variety of signaling systems. Nematode sperm, by contrast, are
cells with a specialized function and use their MSP apparatus exclusively for
locomotion. As a consequence, their locomotory machinery is greatly simplified
and enables central motility functions such as vectorial filament assembly and
bundling to be observed in the absence of other cytoskeletal functions. Thus,
these cells provide a valuable adjunct to actin-based systems for
understanding the basic mechanisms of cell crawling. For example, analysis of
Ascaris sperm revealed that cells do not require molecular motor
proteins to crawl (Italiano et al.,
2001; Roberts and Stewart,
2000
). The simplicity of sperm is reflected in the ease with which
leading edge dynamics can be reconstituted in vitro, and we have capitalized
on this feature to identify MPOP as a key component for localized cytoskeletal
assembly. These advantages of nematode sperm as an experimental system should
facilitate identification of the additional components of the signaling system
and thereby help to elucidate the general principles of membrane-cytoskeleton
communication in cell crawling.
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Acknowledgments |
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