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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2128-2129
CORRESPONDENCE
Dynamic Association of Moesin With the Membrane Skeleton of
Thrombin-Activated Platelets
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LETTER |
To The Editor:
In the June 15, 1998 issue of Blood, Serrador et
al1 report on redistribution of ezrin and moesin to the
uropod of polarized T lymphoblasts, suggesting their role in
establishing cell-cell contacts. The ERM (ezrin-radixin-moesin) family
proteins colocalize in nonhematopoietic cells with actin filaments in
surface projections, microvilli, microspikes, filopodia, ruffles, etc,
where they function in regulated linkage of plasma membrane proteins
with actin in the cytoskeleton.2,3 Cells contain soluble
pools of ERM monomers, which are dormant due to intramolecular
association of their N- and C-terminal regions.4
Phosphorylation and other activation reactions conformationally unmask
binding sites and promote assembly of target-associated oligomeric ERM
structures.2,3 Although ezrin is the most broadly expressed
ERM protein, moesin is quantitatively dominant in
leukocytes5 and is the only ERM protein in
platelets.6
When smooth surfaced circulating platelets are stimulated to
participate in hemostasis, they undergo rapid cytoskeletal
rearrangements, developing filopodia and ruffling lamellae. To better
understand the role of ERM proteins in blood cells, we used
established approaches to determine moesin localization in resting and
thrombin-activated platelets.
Immunofluorescent microscopy showed moesin localized at the
periphery of resting platelets with the central cytoplasmic cores essentially unstained (Fig 1A, first
panel). One minute after thrombin addition, moesin became localized
in newly formed filopodial and lamellipodial projections, and
double staining showed filamentous actin also localized in platelet
projections (Fig 1A). However, moesin appears to colocalize with only a
subset of actin filaments, because scoring of micrographs indicated
that the majority (65%, n = 400) of activated platelets (1 minute)
had submembranous moesin staining with hollow-appearing unstained
centers, whereas 95% had actin staining of the entire cytoplasm. This
distinction between moesin and actin filament localization, shown here
for suspension-activated platelets, was not noted in a study of
glass-adherent platelets.6
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| Fig 1.
Moesin localization in resting and thrombin-treated
platelets. (A) Resting platelets (0 minute) and platelets treated in
suspension with thrombin for 1 to 5 minutes were cytocentrifuged,
fixed, permeabilized, and double-stained with rabbit antimoesin (top)
and rhodamine phalloidin (bottom). (B) Subcellular fractionation
showing moesin distribution. Stirred resting and thrombin-treated
platelets were lysed with Triton X-100 and fractionated9
into low-speed pellet (cytoskeletal fraction), high-speed pellet
(membrane skeleton fraction), and supernatant (soluble fraction).
Immunoblots of fractions from 107 platelets show GPIIb
(PMI-1 MoAb) and moesin (clone 38 MoAb; Transduction Labs,
Lexington, KY). The Coomassie blue stained gels include, on the left,
total lysate of resting platelets with major proteins identified. (C)
Time course of moesin incorporation into the membrane skeleton
(quantitation of immunoblots; mean ± SEM; n = 4 to 6).
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To explore the association of moesin with actin-based structures, we
used selective Triton X-100 solubilization and differential sedimentation to generate operationally defined subcellular platelet fractions.7 Resting and thrombin-treated platelets were
lysed and separated into the cytoskeleton fraction, membrane skeleton fraction, and soluble fraction. Thrombin initiates rapid rearrangements including peripheral actin filament assembly and cross-linking, causing
actin binding protein (ABP), talin, myosin,  -actinin, and actin to
become incorporated into the cytoskeleton fraction7,8 (Fig
1B, lower left). Several membrane skeletal proteins, including a
subfraction of GPIIb/IIIa, also redistribute to the cytoskeletal fraction9 (Fig 1B, GPIIb blot). In contrast, moesin,
which was found exclusively in the soluble fraction in resting
platelets, redistributed to the membrane skeleton fraction, which is
known to contain short actin filaments, vinculin, spectrin, and
ABP9 (Fig 1B, moesin blot).
Quantitation showed that moesin redistribution to the membrane skeleton
fraction was rapid, increasing dramatically in the first minute after
thrombin addition, reaching maximal levels (18% of moesin molecules
incorporated) after 2 to 3 minutes, and then decreasing (Fig 1C). Thus,
incorporation of moesin molecules into the membrane skeleton coincides
with early activation events that include formation of filopodial
extensions and onset of platelet aggregation (not shown).
Although their tissue distributions differ, the ERM proteins are
approximately 70% identical in sequence, structurally similar, and
considered functionally equivalent.2,3 A recent study of
permeabilized cells demonstrated an absolute requirement for moesin (or
ezrin or radixin) for actin filament assembly mediated by Rho family
GTPases.10 Another response of platelet moesin to thrombin
stimulation involves its phosphorylation, which occurs on threonine-558
near an actin binding site.6 Moesin phosphorylation is a
rapid response, maximal in the first minute and then declining toward
basal levels.6 Because known ERM activation pathways intersect at the stage of conformational unmasking of soluble dormant
monomers,2,3 the timing of these events suggests that
the observed dynamic association of moesin with the membrane cytoskeleton and its relocation to transient filopodia and lamellipodia are downstream reactions of moesin phosphorylation.
These findings strongly suggest that moesin is involved in platelet
cytoarchitectural rearrangements, filopodia and lamellipodia formation,
which are important for the transformation of nonadhesive platelets to
the adhesive hemostatically active state. It was suggested for
polarized lymphocytes that moesin, through its linkage with the
cytoskeleton, promotes cell:cell adhesion by redistributing linked
surface membrane adhesion molecules to the uropod.1 Similarly, platelet moesin, by agonist-induced relocation to filopodial extensions, might redistribute and concentrate linked surface receptors, thus contributing to platelet aggregation.
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ACKNOWLEDGMENT |
The authors thank Dr Mark Ginsberg (Scripps Research Institute, La
Jolla, CA) for PMI-1 monoclonal antibody and Drs John Hartwig and Fred
S. Rosen for advice. This work was supported by National Institutes of
Health Grants No. AI39574 and GM36652.
Anna Shcherbina
Dianne M. Kenney
The Center for Blood Research
Harvard
Medical School
Boston, MA
Anthony Bretscher
Section on
Biochemistry, Molecular and Cell Biology
Cornell University
Ithaca, NY
Eileen Remold-O'Donnell
The Center for Blood
Research
Harvard Medical School
Boston, MA
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REFERENCES |
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CD43 interacts with moesin and ezrin and regulates its redistribution to the uropods of T lymphocytes at the cell-cell contacts.
Blood
91:4632, 1998[Abstract/Free Full Text]
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Bretscher A, Reczek D, Berryman M:
Ezrin: A protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures.
J Cell Sci
110:3011, 1997[Abstract]
3.
Tsukita S, Yonemura S, Tsukita S:
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Curr Opin Cell Biol
9:70, 1997[Medline]
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Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.
Mol Biol Cell
6:1061, 1995[Abstract]
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Shcherbina A, Bretscher A, Kenney DM, Remold-O'Donnell E:
Moesin, the major ERM protein of lymphocytes and platelets, differs from ezrin in its insensitivity to calpain.
FEBS Lett
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6.
Nakamura F, Amieva MR, Furthmayr H:
Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets.
J Biol Chem
270:31377, 1995[Abstract/Free Full Text]
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Fox JEB:
The platelet cytoskeleton.
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Hartwig JH, Kung S, Kovacsovics T, Janmey PA, Cantley LC, Stossel TP, Toker A:
D3 Phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate.
J Biol Chem
271:32986, 1996[Abstract/Free Full Text]
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Fox JEB, Lipfert L, Clark EA, Reynolds CC, Austin CD, Brugge JS:
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Mackay DJG, Esch F, Furthmayr H, Hall A:
Rho- and Rac-dependent assembly of focal adhesion complexes and actin filaments in permeabilized fibroblasts: An essential role for ezrin/radixin/moesin proteins.
J Cell Biol
138:927, 1997[Abstract/Free Full Text]
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