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Regular Article
PLATELETS AND THROMBOPOIESIS
Regulation of actin polymerization by tropomodulin-3 controls
megakaryocyte actin organization and platelet biogenesis
Zhenhua Sui,1 Roberta B. Nowak,1 Chad Sanada,2 Stephanie Halene,3 Diane S. Krause,2 and Velia M. Fowler1
1
3
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA; 2Department of Laboratory Medicine, Yale Stem Cell Center, and
Division of Hematology, Department of Internal Medicine, Yale School of Medicine, New Haven, CT
The actin cytoskeleton is important for platelet biogenesis. Tropomodulin-3 (Tmod3), the
only Tmod isoform detected in platelets and megakaryocytes (MKs), caps actin filament
(F-actin) pointed ends and binds tropomyosins (TMs), regulating actin polymerization
• Tmod3-null embryos have
and stability. To determine the function of Tmod3 in platelet biogenesis, we studied
macrothrombocytopenia due
Tmod32/2 embryos, which are embryonic lethal by E18.5. Tmod32/2 embryos often show
to impaired MK cytoplasmic
hemorrhaging
at E14.5 with fewer and larger platelets, indicating impaired platelet
morphogenesis with defective
biogenesis. MK numbers are moderately increased in Tmod32/2 fetal livers, with only
proplatelet formation.
a slight increase in the 8N population, suggesting that MK differentiation is not signif• F-actin polymerization and
icantly affected. However, Tmod32/2 MKs fail to develop a normal demarcation membrane
organization are disrupted in
system (DMS), and cytoplasmic organelle distribution is abnormal. Moreover, cultured
Tmod3-null MKs and in their
Tmod32/2 MKs exhibit impaired proplatelet formation with a wide range of proplatelet bud
proplatelet buds.
sizes, including abnormally large proplatelet buds containing incorrect numbers of von
Willebrand factor-positive granules. Tmod32/2 MKs exhibit F-actin disturbances, and
Tmod32/2 MKs spreading on collagen fail to polymerize F-actin into actomyosin contractile bundles. Tmod3 associates with TM4 and
the F-actin cytoskeleton in wild-type MKs, and confocal microscopy reveals that Tmod3, TM4, and F-actin partially colocalize near the
membrane of proplatelet buds. In contrast, the abnormally large proplatelets from Tmod32/2 MKs show increased F-actin and
redistribution of F-actin and TM4 from the cortex to the cytoplasm, but normal microtubule coil organization. We conclude that
F-actin capping by Tmod3 regulates F-actin organization in mouse fetal liver-derived MKs, thereby controlling MK cytoplasmic morphogenesis, including DMS formation and organelle distribution, as well as proplatelet formation and sizing. (Blood.
2015;126(4):520-530)
Key Points
Introduction
Blood platelets arise from megakaryocyte (MK) precursors.1 During
maturation, MKs become polyploid and grow up to 50 to 100 mm in
size, forming a well-organized demarcation membrane system (DMS)
(also termed invaginated membrane system) in their cytoplasm, defining the cytoplasmic domains of platelet territories, and providing
the membrane reservoir for release into proplatelet protrusions.2,3
Released proplatelets are then processed into mature platelets in the
circulation.4,5 Platelet biogenesis relies on cytoskeletal rearrangements
of both microtubules and actin filament (F-actin) networks.2,3,6,7
Microtubule assembly and dynein-mediated microtubule sliding are
important for proplatelet elongation and organelle trafficking into
proplatelet extensions,2,6,8,9 whereas F-actin plays a role in numbers
and branching of proplatelet protrusions,1 and their morphology,
size, and release from the proplatelet extensions.2,3,7 Analyses of human
mutations and gene-targeted mice have identified several actin
cytoskeletal components involved in this process,7 including nonmuscle myosin IIA (NMIIA) heavy chain (MYH9),10-13 actin
depolymerizing factor (ADF)/n-cofilin,14 filamin A (FLNA),15,16
Wiskott-Aldrich syndrome protein (WASp),17 Rho GTPases,18,19 the
formin DIAPH1 (mDia1),20 and a-actinin1.21,22 Human mutations
and mouse knockouts for these actin cytoskeletal factors often result
in thrombocytopenias with abnormally sized and poorly functioning
platelets, as well as aberrant MKs characterized by perturbations in
F-actin organization and impaired formation of proplatelet extensions.
However, MK numbers and polyploidy are relatively unaffected by
mutations in these cytoskeletal proteins.11,15,18
In addition to defects in F-actin organization, a common feature
underlying the MK phenotypes of some of these mutants is an inability to elaborate the DMS that defines the cytoplasmic territories destined to become platelets and supplies the extensive membrane
reservoir for proplatelet formation.23 This is particularly evident
in Myh92/2 (NMIIA-null) mouse MKs, which have an uneven
organelle distribution and fragmentary DMS with defective F-actin
organization.10,11 Patel-Hett et al have demonstrated that the
DMS is supported by a spectrin-actin cytoskeleton,24 similar to
platelet membranes.25 Transduction of mouse MKs with a spectrin
Submitted September 15, 2014; accepted May 4, 2015. Prepublished online
as Blood First Edition paper, May 11, 2015; DOI 10.1182/blood-2014-09601484.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
Z.S. and R.B.N. contributed equally to this study.
The online version of this article contains a data supplement.
520
© 2015 by The American Society of Hematology
BLOOD, 23 JULY 2015 x VOLUME 126, NUMBER 4
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BLOOD, 23 JULY 2015 x VOLUME 126, NUMBER 4
tetramer-disrupting construct inhibits formation of the DMS, impairs
formation of proplatelet extensions, and leads to loss of the characteristic barbell shape of newly formed proplatelets.24 Actomyosin
contraction and spectrin-actin networks both rely upon F-actin stability, yet other studies have demonstrated that platelet biogenesis
depends upon F-actin disassembly and turnover promoted by ADF/
n-cofilin.14 To reconcile these apparently contradictory roles for
F-actin dynamics, we hypothesize that a balance between F-actin
stability and turnover may be critical for platelet biogenesis, with
F-actin stabilizing proteins in MKs counteracting F-actin disassemblypromoting proteins such as ADF/n-cofilin.
Tropomodulins (Tmods) play key roles in stabilizing F-actin
networks by binding tropomyosins (TMs) and capping the pointed
ends of TM-associated F-actin.26 TMs bind along the length of F-actin
and inhibit disassembly by ADF/cofilin or severing by gelsolin, and
TM-mediated F-actin stabilization is enhanced by Tmods.27-33
Tmods are components of membrane-associated TM–F-actin networks, including the spectrin-actin network of the erythrocyte membrane (Tmod1), ocular lens fiber cell membranes (Tmod1), polarized
epithelial cell plasma membranes (Tmod3), and the sarcoplasmic
reticulum membranes of skeletal muscle (Tmod3), where Tmodmediated stabilization of TM–F-actin is critical for membrane
morphology, mechanics, and physiology.34-37 Tmod3 is also
involved in endothelial cell migration,38 erythroblast enucleation
and erythroblast-macrophage adhesion in erythropoiesis,39 and
insulin-stimulated GLUT4 trafficking in adipocytes.40
In this study, we show that Tmod3 is the only Tmod in platelets
and MKs and confirm that TM4 (Tpm4.2, encoded by the Tpm4/dTm
gene41) is the predominant TM in MKs and platelets.23,42 We
investigated a role for Tmod3 in platelet formation by studying
Tmod32/2 mice, which are embryonic lethal at E14.5 to E18.5 with
anemia due to impaired fetal liver erythropoiesis.39 Tmod32/2
embryos also display hemorrhages, which may be partly due to
reduced platelet numbers and enlarged platelets in the embryonic
circulation. Tmod32/2 fetal liver MKs have reduced proplatelet
formation in vitro, but no significant changes in MK ploidy and
a moderate increase in MK numbers. However, Tmod32/2 MK
cytoplasmic morphogenesis is impaired, with insufficient DMS
formation and aberrant organelle distribution, and many proplatelet
buds from cultured MKs are abnormally large with incorrect numbers
of von Willebrand factor (VWF) negative and positive granules.
Tmod3 associates with TM4 and the actin cytoskeleton in MKs,
and absence of Tmod3 leads to defective F-actin polymerization and
organization in MKs and proplatelet buds. Deletion of Tmod3 may
disrupt F-actin in MKs via perturbation of TM4–F-actin interactions,
resulting in F-actin instability and reorganization, leading to defective DMS formation and organelle distribution, thereby impairing proplatelet formation, morphology, and organelle content.
Materials and methods
Mice
Tmod32/2 embryos were as described.39 All experiments were performed according to the National Institutes of Health animal care guidelines, as approved by
The Scripps Research Institute’s Institutional Animal Care and Use Committee.
X-gal and hematoxylin and eosin (H&E) staining
E14.5 embryos were subjected to whole-mount X-gal staining as described.39
Embryos were then fixed, paraffin-embedded, and sectioned for H&E staining.
TROPOMODULIN-3 CONTROLS PLATELET FORMATION AND SIZE
521
In vitro culture of MKs
Dissociated cells from E13.5 to E14.5 mouse fetal livers were cultured in
Dulbecco’s Modified Eagle’s medium (Gibco Life Technologies) with 5 ng/mL
recombinant murine thrombopoietin (mTPO, Life Technologies), 10% fetal
bovine serum (HyClone, Thermo Scientific), penicillin, and streptomycin.43
MKs were collected using a 2-step bovine serum albumin (BSA) density
gradient on day 3 and incubated in Dulbecco’s Modified Eagle’s medium with
mTPO for an additional 24 hours until proplatelet formation.43
Immunofluorescence and confocal microscopy
Whole mouse embryos were processed for cryosectioning and labeled for antibody as described.39 For imaging of fetal liver cytospins, E14.5 fetal livers were
dissociated into Dulbecco’s phosphate-buffered saline (PBS), suspended cells
were fixed in 4% paraformaldehyde overnight at 4°C, permeabilized, blocked,
and labeled with antibodies in suspension, and then spun onto slides.39 For
staining of proplatelet-forming MKs from in vitro cultures, cells were fixed
in 4% paraformaldehyde for at least 30 minutes at room temperature and
then centrifuged onto poly-L-lysine coated slides.43 Cells or tissue preparations
were permeabilized in 0.3% Triton X-100 in PBS and blocked overnight at
4°C in 2% BSA with 1% goat serum in PBS 1 0.1% Triton X-100. Slides
were incubated with primary antibodies (see supplemental Table 1 on the
Blood Web site) overnight at 4°C, followed by secondary antibodies
(supplemental Table 1) for 2 hours at room temperature, and mounted in
ProLong Gold antifade reagent (Life Technologies). Images were acquired
using a Zeiss LSM 780 confocal laser scanning fluorescence microscope, and
analyzed using Volocity 6.3 (PerkinElmer) or ImageJ software as indicated in
figure legends.
Transmission electron microscopy (TEM)
Peripheral blood from E14.5 mouse embryos was collected by allowing
embryos to bleed out into Dulbecco’s PBS with 5 mM EDTA and added to
K3EDTA containing tubes (Becton Dickinson). Blood or dissociated fetal
liver cells were fixed overnight in ice-cold 4% paraformaldehyde and 1.5%
glutaraldehyde in 0.1 M sodium cacodylate at pH 7.2. Cells or tissues were
processed for embedding and thin sectioning as described.44 Sections were
examined on a Philips CM100 electron microscope (FEI). Images were
collected using a MegaView III CCD camera (Olympus Soft Imaging
Solutions GmbH).
Ploidy analysis
Dissociated E14.5 fetal liver cells were cultured in StemSpan Serum-Free
Expansion Medium (Stemcell Technologies) supplemented with 30% BSA,
insulin, and transferrin (Stemcell Technologies) and 50 ng/mL mTPO
(ConnStem) for 4 days at 37°C. Cultured cells were harvested for flow
cytometry as described (supplemental Figure 2).45
Western blotting
Mouse blood was collected via cardiac puncture of 3-month-old male mice
directly into K3EDTA Vacutainer tubes (Becton Dickinson). Human peripheral blood from healthy donors was collected by venipuncture into K3EDTA
according to an Institutional Review Board–approved human subjects blood
collection protocol (11-5773). Red blood cells and leukocytes were removed by
centrifugation at 200g for 10 minutes, followed by collection of platelets from
the plasma by centrifugation at 1000g for 5 minutes. MKs were obtained from
in vitro cultured mouse fetal liver cells on day 3 as described above. Western
blotting was performed as described.39 Primary and secondary antibodies are
shown in supplemental Table 1.
Co-immunoprecipitation (co-IP)
Cultured MKs were lysed at 4°C in modified radioimmunoprecipitation assay
buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% sodium dodecyl
sulfate, and 1% NP40) supplemented with protease inhibitor cocktail (1:100,
Sigma-Aldrich), centrifuged for 10 minutes at 4°C in an Eppendorf microcentrifuge to remove insoluble material, and affinity-purified rabbit anti-Tmod3 (1 mg),
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BLOOD, 23 JULY 2015 x VOLUME 126, NUMBER 4
Figure 1. Tmod3 is expressed in platelets and MKs.
(A) Western blotting of Tmod expression in human and
mouse platelets. (B) Western blotting of Tmods in MKs
cultured from mouse fetal liver (E13.5); 10 ng purified
recombinant Tmod1 and Tmod3 proteins were used
as controls (A-B). (C) LacZ staining of sections from
Tmod31/1 (left) and Tmod31/2 (right) E14.5 embryos.
Embryos were dissected and stained with X-gal and
H&E. Yellow arrows indicate large MKs with bright blue
staining. Bar, 40 mm. Images were acquired with a 320
objective (N.A. 0.5) using a Zeiss Axioskop microscope
and a Zeiss AxioCam ICc3 color camera. (D) Immunofluorescence staining for b-galactosidase (green)
and CD41 (red) in cryosections of Tmod31 /1 and
Tmod31 /2 E14.5 embryos. Bar, 20 mm. Images were
acquired using a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a 360 oil-immersion
objective (N.A. 1.4).
rabbit anti-TM4, or pre-immune control rabbit IgG (1 mg) was added.
After incubation for 1 hour, mMACS Protein A MicroBeads (Miltenyi Biotec)
was added, incubated overnight at 4°C, run through MACS separation columns
(Miltenyi Biotec), and antibody-protein complexes were eluted with sodium
dodecyl sulfate-gel sample buffer. Western blotting of co-IP products and input
whole-cell lysate were then performed, with images acquired with an Odyssey
Imager (LI-COR Biosciences).46
Statistical analysis
Data are presented as mean 6 SEM for small sample sizes (,10) and
mean 6 SD for large sample sizes (.30). A 2-tailed unpaired Student
t test was used for data analysis. P , .05 was considered statistically
significant. Prism Version 5 software (GraphPad) and Microsoft Excel
were used for statistical analysis.
Results
Tmod3 is present in platelets and MKs
Tmod3 is the only Tmod isoform in the human platelet proteome,
with an estimated 11 900 copies per platelet, considerably less
abundant than cofilin (CFL1 1 CFL2) at 337 200 copies, or profilin
(PFN1) at 504 000 copies per platelet.42 Western blotting confirms
that Tmod3 is present in both human and mouse platelets, whereas no
Tmod1 is detected (Figure 1A). Similar results are obtained using in
vitro cultured MKs from E13.5 mouse fetal livers (Figure 1B). LacZ
staining of fetal livers from Tmod31/2 mice39 also reveals Tmod3
expression in large multinucleated MKs (Figure 1C), along with fainter
blue staining in other cells, as previously shown.39 Immunostaining for
CD41 and b-gal confirmed abundant b-gal in CD411, multinucleated
MKs (Figure 1D). Indeed, Tmod3 expression appears to be higher in
MKs in comparison with other fetal liver cell types. Based on the
restricted expression of Tmod2 and Tmod4 in neurons and skeletal
muscle, respectively,26 Tmod3 is the only Tmod in platelets and fetal
liver MKs.
Tmod32/2 embryos have hemorrhages and fewer platelets with
abnormal morphology
We previously noticed small hemorrhages in ;75% of Tmod32/2
embryos at E14.5 (Figure 2A).39 As Tmod3 is expressed in endothelial cells38,39 and platelets (Figure 1), hemorrhages may be due to
endothelial barrier and/or platelet dysfunction. We focused on the
platelet phenotype, because Tmod32/2 embryos had fewer circulating
platelets (;50% of wild-type [WT]) (Figure 2B,C), with larger and
more variable sizes (Figure 2B-E). Tmod32/2 platelets were also more
irregular in shape and often displayed small protrusions, unlike
WT platelets (Figure 2D). TEM revealed more vesicular structures
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BLOOD, 23 JULY 2015 x VOLUME 126, NUMBER 4
TROPOMODULIN-3 CONTROLS PLATELET FORMATION AND SIZE
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Figure 2. Tmod32/2 embryos show hemorrhages
with abnormal platelets. (A) Representative images
of whole Tmod31/1 (left) and Tmod32/2 (right) embryos at E14.5. Bar, 1 mm. (B) Immunofluorescence
staining for CD41 (gray) and Hoechst (blue) in
cryosections of blood vessels of Tmod31/1 (left)
and Tmod32/2 (right) embryos, revealing circulating
CD411 platelets in situ. Bar, 10 mm. Images are single
optical sections acquired using a Zeiss LSM 780
confocal laser scanning fluorescence microscope
with a 3100 oil-immersion objective (N.A. 1.4), zoom
1. (C) Relative numbers of platelets in peripheral blood
of Tmod31/1 and Tmod32/2 E14.5 embryos. Tmod31/1,
63 6 20 (n 5 4 fields from 2 Tmod31/1 embryos);
Tmod32/2, 30 6 7 (n 5 5 fields from 2 Tmod32/2
embryos). *P , .05. (D) Representative images of
Tmod31/1 (top) and Tmod32/2 (bottom) platelets
stained with CD41. Bar, 4 mm. Images are compressed
Z stacks of optical sections acquired using a Zeiss
LSM 780 confocal laser scanning fluorescence microscope with a 3100 oil-immersion objective (N.A. 1.4),
zoom 3. (E) Platelet sizes in peripheral blood of
Tmod31/1 and Tmod32/2 embryos at E14.5, measured from images as in (B). Platelet diameters were
determined from line scans across platelets using
Volocity 6.3 software. Tmod32/2 average platelet
diameters were ;31.5 greater than Tmod31/1 platelets. Tmod31/1, 1.73 6 0.46 mm (n 5 114); Tmod32/2,
2.21 6 0.76 mm (n 5 98). ***P , .001. (F) Representative TEM images of Tmod31/1 and Tmod32/2
platelets (left and middle panels; Bar, 1 mm), with high
magnification views of circumferential microtubule
rings (red arrows, right panel; Bar, 200 nm). Average
WT platelet diameter in TEM images is ;3 mm, similar
to previous studies,10 but larger than measurements
from fluorescence optical sections (B), which include
measurements across the short and long axes of the
asymmetric platelets (E).
with clear lumens in some Tmod32/2 platelets (Figure 2F), and confocal microscopy of GPIba revealed internal staining in contrast to
typical membrane staining of WT platelets (supplemental Figure 1).
This could be due to partial activation of Tmod32/2 platelets leading
to GPIba internalization47 or impaired membrane biogenesis (see
below in Figures 4 and 5). However, no changes were evident in the
morphology of the circumferential microtubule coils of Tmod32/2
platelets (Figure 2F, red arrows). (Tmod31/2 embryos and platelets
were similar to WT, due to increased Tmod3 expression in heterozygotes.39) Therefore, Tmod32/2 embryos exhibit decreased numbers of platelets with variable sizes and abnormal morphologies,
resembling a macrothrombocytopenia.
MK numbers are increased but ploidy is not significantly
different in the absence of Tmod3
To investigate whether reduced platelet numbers in Tmod32/2 embryos
are due to decreased MK number and/or impaired differentiation, we
quantified CD411 MKs in fetal liver cryosections. WT fetal livers
contained sparsely distributed bright CD411 MKs, as expected,
whereas Tmod32/2 fetal livers contained ;1.5-fold more CD411 MKs
(Figure 3A-B). This may reflect increased MK progenitors, based on
increased colony-forming unit (CFU)-MKs in Tmod32/2 fetal livers
(Figure 3C). We then examined the ploidy of isolated fetal liver
MKs differentiated in vitro with mTPO (Figure 3D and supplemental
Figure 2). As expected, the average ploidy of WT fetal liver MKs
was close to ;8N, which is lower than that of bone marrow MKs
from adults (Figure 3D-E).48-50 However, the relative numbers
of Tmod32/2 MKs with different ploidy were not significantly
different, except for a small increase in 8N MKs (from 32% to 40%)
(Figure 3D). Furthermore, there was no change in average ploidy
(Figure 3E). Thus, alterations in MK numbers and ploidy are
unlikely to account for decreased numbers of circulating platelets in
Tmod32/2 embryos. Increased MKs and progenitors in Tmod32/2
fetal livers may reflect a compensatory response to the deficit in
platelet production by Tmod3-deficient MKs (see below).
Tmod32/2 MKs exhibit abnormal DMS formation and
organelle distributions
We next investigated whether the platelet deficits in Tmod32/2
embryos were due to abnormalities in MK cytoplasmic morphogenesis. TEM of mature MKs in WT fetal liver sections reveals
an extensive DMS surrounding electron-dense granules within
cytoplasmic platelet territories (Figure 4A, left). In contrast,
Tmod32/2 MKs displayed two types of morphology, with about
half resembling immature MKs containing large clumps of vesicles
resembling a pre-DMS structure51 (Figure 4A, middle), and the
other half containing relatively normal, elongated DMS tubules
surrounding platelet territories (Figure 4A, right). In this latter
type of Tmod32/2 MK, the DMS often enclosed larger cytoplasmic
regions containing fewer electron-dense granules (Figure 4A,
right).
To further investigate DMS morphology and organelle distribution in MKs, we immunostained the DMS with anti-GPIba51,52
and mitochrondria with anti-TOM20, and imaged isolated MKs by
confocal fluorescence microscopy (Figure 4B). Although all largesized Tmod31/1 or Tmod31/2 MKs with multilobular nuclei contained
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Proplatelet formation is impaired in Tmod32/2 MKs
To test the ability of Tmod32/2 MKs to produce proplatelets, we
cultured fetal liver MKs in mTPO and observed the morphologies
and numbers of MKs forming proplatelets.43 Proplatelet buds formed
from cultured Tmod32/2 MKs contained microtubule rings but were
more variable in size and often much larger than WT proplatelets
(Figure 5A). Furthermore, Tmod32/2 fetal livers contained about half
as many proplatelet-forming MKs as WT fetal livers (Figure 5B),
indicating that the proplatelet-forming ability of Tmod32/2 MKs is
impaired. For the subset of Tmod32/2 MKs that did form proplatelets,
proplatelet buds exhibited an approximately threefold increase in
average area (Figure 5C), consistent with larger platelet sizes in embryonic blood (Figure 2E). The Tmod32/2 MKs that do not form
proplatelets may correspond to MKs with clumped DMS vesicles
(Figure 4A, middle, and supplemental Figure 3).
Proplatelet buds formed from cultured Tmod32/2 MKs had defective organelle composition, based on reduced staining for VWF,
a marker of a-granules (Figure 5D-E). In proplatelet buds formed from
Tmod31/1 MKs, the number of VWF1 puncta is linearly related to
proplatelet bud area (Figure 5F), as shown previously.10 However, in
the absence of Tmod3, there are 2 populations of proplatelet buds: one
containing VWF1 puncta in proportion to bud area, similar to WT, and
another with abnormally large proplatelet buds, containing variable
numbers of VWF1 puncta with no relationship to size (Figure 5G).
The Tmod32/2 proplatelet buds with VWF1 content that scales with
size may originate from regions of MK cytoplasm with relatively
normal DMS tubule arrangements, whereas the abnormally large
Tmod32/2 proplatelet buds with variable VWF1 content may originate
from DMS-poor regions of MK cytoplasm with uneven organelle distributions (Figure 4).
Figure 3. MK numbers are increased, whereas ploidy is not significantly changed
in the absence of Tmod3. (A) Immunofluorescence staining for CD41 (green) and
Hoechst (blue) on cryosections of Tmod31/1 (top) and Tmod32/2 (bottom) fetal
livers from E14.5 embryos. Images are single optical sections acquired using a
Zeiss LSM 780 confocal laser scanning fluorescence microscope with a 320 oilimmersion objective (N.A. 1.4). Bar, 100 mm. (B) Numbers of MKs per field (n 5 46
fields from controls; 2 Tmod31/1 and 2 Tmod31/2 fetal livers, and n 5 42 fields
from 2 Tmod32/2 fetal livers). **P , .05. (C) Numbers of CFU-MKs per 1 3 105
fetal liver cells from E15.5 embryos (n 5 14 fields from 4 Tmod31/1 fetal livers,
n 5 4 fields from 1 Tmod32/2 fetal liver) (left). Representative image of a WT
CFU-MK colony (right). Tmod32/2 CFU-MK colonies were similar in appearance
(not shown). Bar, 200 mm. ***P , .001 (D) Percentages of MKs with different ploidy
(Tmod31/1, n 5 5; Tmod31/2, n 5 3; Tmod32/2, n 5 2 fetal livers). *P , .05. (E)
Average MK ploidy from Tmod31/1, Tmod31/2, and Tmod32/2 fetal livers.
DMS membrane tubules organized into one or more rows of territories
around the cell circumference, DMS tubule organization in Tmod32/2
MKs was variable, forming a narrow band of territories around the
circumference of some MKs (Figure 4B), but only a few territories
in others (Figure 4C). Strikingly, mitochondria were clearly visible
within each platelet territory in Tmod31/2 MKs, but in Tmod32/2 MKs
only some territories contained mitochondria, and many mitochondria
collected in cytoplasmic aggregates near the nucleus (Figure 4B-C).
Further, in yet other Tmod32/2 MKs, consistent with the TEM images (Figure 4A, middle), the DMS appeared as one or two large
clumps of vesicles, resembling pre–DMS-like structures.51 Nonetheless, these Tmod32/2 MKs were large with similar multilobular nuclear morphology as Tmod31/2 MKs in which the DMS had formed
tubules surrounding territories (supplemental Figure 3). Thus, although
Tmod32/2 MKs achieve high nuclear ploidy similar to Tmod31/1 and
Tmod31/2 (Figure 3C-D), cytoplasmic morphogenesis is impaired, with
abnormal DMS formation that does not create normal platelet territories with appropriate distributions of organelles.
F-actin polymerization is defective in Tmod32/2 MKs
To investigate whether defective F-actin polymerization underlies
abnormalities in Tmod32/2 MKs and impaired proplatelet formation, we phalloidin-stained isolated fetal liver MKs. In control MKs,
F-actin is associated with GPIba-labeled DMS tubules and plasma
membrane, with little cytoplasmic staining (Figure 4B-C). In contrast, in Tmod32/2 MKs, brightly stained cytoplasmic aggregates
of F-actin are often present (Figure 4B, red arrows), in addition to
F-actin at the DMS tubules and plasma membrane (Figure 4B-C).
In the most abnormal Tmod32/2 MKs with no DMS-defined platelet territories, F-actin was present in the clumps of pre–DMS-like
vesicles (supplemental Figure 3). Additionally, WT MKs extend
protrusions and pseudopods with membrane-associated F-actin,
whereas Tmod32/2 MKs form rudimentary protrusions, with cytoplasmic rather than membrane-associated F-actin (supplemental
Figure 5).
To directly determine whether F-actin polymerization might
be defective in Tmod32/2 MKs, we exploited the ability of MKs
to assemble F-actin bundles upon spreading on collagen-coated
coverslips. As expected, WT fetal liver-derived MKs assembled
robust F-actin bundles as well as podosomes on their ventral surfaces
(Figure 6A).11,17 Tmod3-stained puncta were often observed associated with the F-actin bundles, suggesting the F-actin pointed ends
are capped (Figure 6A, bottom panels, arrowheads). NMIIA also
assembles along the F-actin bundles of WT MKs (Figure 6B),
appearing in a striated pattern indicative of their contractile
function.53 In contrast, in Tmod32/2 MKs spreading on collagen,
F-actin polymerization with NMIIA into bundles is severely
impaired (Figure 6C). Instead, F-actin forms amorphous aggregates
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TROPOMODULIN-3 CONTROLS PLATELET FORMATION AND SIZE
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Figure 4. Tmod32/2 MKs show abnormal ultrastructure with incomplete DMS formation, abnormal
organelle distribution, and defects in F-actin. (A)
Representative TEM images of Tmod31/1 and Tmod32/2
MKs at lower (upper panels) and higher (lower panels)
magnification. All Tmod31/1 MKs showed DMS tubules
surrounding platelet territories with abundant electrondense a-granules (left). In contrast, about half of
Tmod32/2 MKs contained large clumps of vesicles
(middle), whereas others appeared with some DMS
tubules surrounding platelet territories, which were
often larger and with fewer electron-dense granules
(right). Bar, 5 mm (upper panels) and 1 mm (lower
panels). (B-C) Immunofluorescence staining of GPIba,
F-actin, mitochondria (TOM20), and nuclei (Hoechst)
in isolated Tmod31/2 and Tmod32/2 fetal liver MKs.
Grayscale images are shown for each stain, and the
merge shows GPIba (green), mitochondria (red), and
nuclei (blue). In Tmod31/2 MKs, GPIba and F-actin are
associated with DMS membranes surrounding platelet
territories that contain mitochondria. Tmod31/2 MK
morphology is indistinguishable from Tmod31/1 (data
not shown). In Tmod32/2 MKs, GPIba-labeled DMS
membranes form fewer territories, which are deficient
in mitochondria, F-actin accumulates in abnormal foci
(B, red arrows), and mitochondria aggregate in the
cytoplasm and around the nucleus (C, white arrowheads). Images are single optical sections selected
from Z stacks acquired using a Zeiss LSM 780 confocal
laser scanning fluorescence microscope with a 3100 oilimmersion objective (N.A. 1.4) at zoom 2 (B) or zoom 5
(C). Asterisk in (C), nucleus. Bars, 8 mm (B); 2 mm (C).
near the periphery of the spreading MKs, and NMIIA does not
assemble (Figure 6C). Thus, Tmod3 regulates F-actin polymerization
in fetal liver-derived MKs.
F-actin and TM4 organization are perturbed in Tmod32/2
proplatelet buds
Tmod3 binds TMs to cap and stabilize F-actin pointed ends,33 with
TM4 as the predominant TM in platelets.42,54-56 Indeed, TM4 is
detected in whole-cell lysates of in vitro cultured MKs from mouse
fetal livers (supplemental Figure 4A-B), whereas long, muscle-type
TMs are not detected (data not shown). Co-IP shows that MK Tmod3
and TM4 are associated with actin and associated cytoskeletal proteins (NMIIA, FLNA, and b2-spectrin) (supplemental Figure 4A-B).
Confocal fluorescence microscopy of proplatelets further reveals that
Tmod3 and TM4 partially colocalize with F-actin and are enriched
at the cortex of proplatelet buds (supplemental Figure 4C-F), similar
to cortical enrichment of F-actin in circulating embryonic platelets
(supplemental Figure 1).
We hypothesized that absence of Tmod3 leading to disruption
of Tmod3–TM4-actin associations might alter F-actin abundance
and/or organization in Tmod32/2 proplatelet buds. Tmod32/2
proplatelet buds exhibited highly variable F-actin levels, with
some similar to WT and others with up to approximately four- to
fivefold more F-actin (Figure 7A-B). Moreover, similar to VWF1
puncta (Figure 5G), normal-sized Tmod32/2 proplatelet buds had
relatively normal F-actin levels, whereas larger buds had highly
variable levels of F-actin (Figure 7C).
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SUI et al
Next, we evaluated F-actin and TM4 distributions in proplatelet
buds. Tmod31/1 proplatelet buds exhibited cortical enrichment of
F-actin and TM4 (Figure 7D,G, supplemental Figure 4D,F, and
supplemental Figure 6). However, in Tmod32/2 proplatelet buds,
the relative F-actin and TM4 intensity in the cytoplasm was increased
as compared with the cortex, indicating TM4 and F-actin redistribution
(Figure 7D,G and supplemental Figure 6). Quantification showed
an approximate threefold increase for F-actin in the cytoplasmic
vs cortical intensity ratio per proplatelet bud (Figure 7E), and an
approximate twofold increase for TM4 (Figure 7H). Notably, the
increased cytoplasm/cortex ratios of F-actin and TM4 were more
pronounced in the abnormally large Tmod32/2 proplatelet buds
(Figure 7F,I). Thus, the deletion of Tmod3 disrupts TM4–F-actin
cytoskeletal organization, with more pronounced effects in abnormally large proplatelet buds.
Abnormal F-actin organization was also evident in Tmod32/2
circulating embryonic platelets, where foci and F-actin aggregates
were present in the platelet interior, in contrast to Tmod31/1 embryonic
platelets where F-actin concentrates at the cortex were present
(supplemental Figure 1). We then examined F-actin organization in
embryonic platelets spreading on collagen-coated coverslips. WT
platelets formed peripheral lamellipodia with F-actin at the leading
edge, as well as an internal F-actin ring and central concentration
(Figure 7J). In contrast, Tmod32/2 platelets formed lamellipodia at
their periphery, but not the internal F-actin ring, and, in others, F-actin
formed irregular bundles or partial rings (Figure 7J). Thus, Tmod3
regulates F-actin organization in proplatelets from cultured MKs and
in embryonic circulating platelets.
Discussion
Figure 5. Proplaletet formation by Tmod32/2 MKs in culture is impaired, and
Tmod32/2 proplatelets have abnormal levels of VWF1 granules. (A) Immunofluorescence staining of a-tubulin in proplatelets produced by MKs cultured in vitro
from Tmod31/1 and Tmod32/2 fetal livers reveals uniformly small sizes for Tmod31/1,
but highly variable sizes for Tmod32/2 proplatelets, including some very large ones.
Arrowhead and arrow (lower panel) are examples of a large and small Tmod32/2
proplatelet, respectively. Bar, 10 mm. (B) Percentages of MKs cultured in vitro that
form proplatelets. ***P , .001. (C) Sizes of proplatelet buds produced by cultured
MKs. Tmod31/1, 5.23 6 2.39 mm2 (n 5 96); Tmod32/2, 15.48 6 9.31 mm2 (n 5 98).
***P , .001. Proplatelet sizes (areas, mm2) were measured from a-tubulin–stained
proplatelets using Volocity 6.3 software to circle their perimeter. (D) Immunofluorescence staining of VWF (green) and a-tubulin (red) in proplatelet buds from
cultured MKs. Bar, 5 mm. (E) Total VWF per proplatelet based on the ratio of VWF
fluorescence intensity vs a-tubulin intensity. Tmod31/1, 0.80 6 0.49 (n 5 46);
Tmod32/2, 0.45 6 0.27 (n 5 48). ***P , .001. (F-G) Number of VWF1stained puncta per proplatelet bud with respect to area (solid grey line for Tmod31/1,
solid black line for Tmod32/2). The number of VWF1 granules per proplatelet bud was
linearly dependent on proplatelet area for Tmod31/1 but not Tmod32/2 MKs (R2 5
0.6017 vs R2 5 0.2112, respectively). However, the subset of Tmod32/2 proplatelet
We identified a new feature of Tmod32/2 mouse embryos: hemorrhaging with macrothrombocytopenia. Tmod3 is the only Tmod in
mouse fetal liver MKs and platelets, and is important for cytoplasmic
morphogenesis of MKs. Tmod32/2 MKs display an immature or incomplete DMS, which forms insufficient tubules for platelet territories
and the membrane reservoir required for proplatelet protrusion, leading
to impaired ability to produce proplatelets. Tmod32/2 MKs may
contain pre–DMS-like vesicular clumps,51 due to delay or inability to
remodel the DMS, or contain reduced DMS tubules creating fewer
platelet territories with irregular organelle distributions. Tmod32/2
MKs produce 2 varieties of proplatelet buds: ones within a normal
size range (;2 to 20 mm2),10 and others that are abnormally large (up to
;60 mm2). In normal-sized WT and Tmod32/2 proplatelet buds,
VWF1 granule content scales with proplatelet bud size, suggesting that
Tmod3 regulation of F-actin is not required for organelle transport
down proplatelet extensions, which depends on microtubule-dependent
transport and sliding processes.8,57 However, F-actin polymerization is aberrant in Tmod32/2 MKs, and increased F-actin and
redistribution of F-actin and TM4 from the cortex to the cytoplasm
Figure 5 (continued) buds with areas smaller than or equal to the area of the largest
Tmod31/1 proplatelet bud (demarcated by blue dotted line), revealed a stronger
linear correlation (R2 5 0.4945, dashed black line). Thus, in Tmod32/2 proplatelets,
the overall weakness of the linear relationship of the number of VWF1 granules per
proplatelet bud as a function of proplatelet area is driven primarily by the unusually
large, aberrant proplatelet buds. Images are single optical sections acquired using
a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a 3100 oilimmersion objective (N.A. 1.4) at zoom 0.6 (A) or zoom 2 (D).
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TROPOMODULIN-3 CONTROLS PLATELET FORMATION AND SIZE
527
Figure 6. Tmod3 regulates F-actin polymerization
and organization in MKs. (A) Tmod3 is associated
with F-actin bundles that assemble in MKs spread on
collagen. Immunofluorescence staining of F-actin (red)
and Tmod3 (green) in WT MKs spread on collagen for
30 minutes. F-actin polymerizes into bundles and foci
on the ventral surface of the MKs. Boxed regions
shown at high magnification (lower panels) reveal
Tmod3 puncta associated with the F-actin bundles
(yellow arrrowheads). Bars, 10 mm. (B-C) Immunofluorescence staining of F-actin (red) and NMIIA (green)
in Tmod31/1 and Tmod32/2 MKs spread on collagen.
In WT MKs, F-actin assembles into parallel bundles, as
well as intensely stained foci on the ventral surface,
termed podosomes.17 NMIIA is associated with the
F-actin bundles (but not the podosomes) in a striated
pattern in WT MKs, whereas both F-actin and NMIIA
form amorphous aggregates near the cell periphery
in Tmod3 2 /2 MKs. Boxed regions shown at higher
magnification (lower sets of panels in [B-C]). Scale
bars, 10 mm. Images are single optical sections from
the ventral surface of MKs, acquired using a Zeiss LSM
780 confocal laser scanning fluorescence microscope
with a 363 oil-immersion objective (N.A. 1.4) at zoom 1
(top panels) and zoom 3 (boxed regions).
is only observed in abnormally large Tmod32/2 proplatelet buds.
Therefore, normal-size Tmod32/2 proplatelet buds may originate
from regions of MK cytoplasm containing DMS tubules, whereas
abnormally large proplatelet buds may originate from DMS-poor
regions of cytoplasm with irregular distributions of organelles and
F-actin aggregates.
How might Tmod3 control MK DMS formation, organelle distribution, and proplatelet formation? The DMS is associated with
F-actin (Figure 4B-C)23 in a spectrin-actin network,24 and spectrin
tetramer formation and F-actin assembly are known to influence
DMS formation and platelet biogenesis.14,23,24,58,59 Spectrin is
also implicated in intracellular vesicular transport,60-64 suggesting
Tmod3 may influence DMS and organelle distributions in MKs via
stabilization of spectrin-actin networks associated with intracellular
membranes. A precedent for direct association of Tmod3 with an
intracellular membrane receptor is that of Tmod3 with sAnk1.5,
a transmembrane protein in the sarcoplasmic reticulum of skeletal
muscle.36 Alternatively, Tmod3 may associate with MK and proplatelet
membranes indirectly via F-actin’s links to spectrin networks or to
FLNA, which binds GP1ba.7,25
Several lines of evidence in our study support the idea that
impaired DMS formation and aberrant organelle distributions in
Tmod32/2 MKs are caused by a disrupted F-actin cytoskeleton.
Tmod32/2 MKs display aberrant cytoplasmic accumulations of
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528
SUI et al
BLOOD, 23 JULY 2015 x VOLUME 126, NUMBER 4
Figure 7. Tmod32/2 proplatelets have defects in F-actin and TM4 organization, and circulating Tmod32/2 platelets fail to organize an F-actin ring after spreading
on collagen. (A) Immunofluorescence staining of F-actin (red) and a-tubulin (green) in proplatelet-forming Tmod31/1 (top) and Tmod32/2 (bottom) fetal liver MKs. Bar,
20 mm. (B) Relative level of F-actin vs a-tubulin per proplatelet bud, calculated from fluorescence intensity ratios as in Figure 5. Tmod31/1: 0.82 6 0.44 (n 5 51); Tmod32/2: 1.72 6 1.0
(n 5 45). ***P , .001. (C) F-actin/tubulin intensity per proplatelet bud with respect to area. (D) High magnification images and representative line scans of a-tubulin (green)
and F-actin (red) fluorescence in Tmod31/1 and Tmod32/2 proplatelets. Bars, 5 mm. (E) F-actin cytoplasm vs cortex intensity per proplatelet bud. Tmod31/1 : 0.55 6 0.17
(n 5 50); Tmod32/2 : 1.47 6 0.74 (n 5 42). ***P , .001. (F) F-actin cytoplasm vs cortex intensity per proplatelet bud with respect to area. (G) High magnification
images and representative line scans of TM4 (green), a-tubulin (blue), and F-actin (red) fluorescence in Tmod31/1 and Tmod32/2 proplatelets. Bars, 5 mm. (H) TM4
cytoplasm vs cortex intensity per proplatelet bud. Tmod31/1: 0.75 6 0.17 (n 5 60); Tmod32/2: 1.17 6 0.24 (n 5 60). (I) TM4 cytoplasm vs cortex intensity per proplatelet bud with
respect to area. (J) Rhodamine-phalloidin staining for F-actin in Tmod31/1 (left) and Tmod32/2 (right) embryonic platelets spread on collagen for 30 minutes. Bar, 2 mm. Images are
single optical sections acquired using a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a 1003 oil-immersion objective (N.A. 1.4) at zoom 1 (A) or
zoom 3 (D,G,J). Fluorescence intensities of a-tubulin and F-actin in proplatelets (B-C) were determined using ImageJ software, with areas of proplatetets determined from
a-tubulin staining as described in Figure 5. Ratios of cytoplasm to cortex staining intensities for F-actin and TM4 (E-F,H-I) were determined from intensities within small ;0.5 mm
diameter circles placed over the edge or middle of the proplatelet, using ImageJ software. Line scans of TM4 and F-actin signals across proplatelets were performed also with ImageJ
software (D,G). Blue dotted lines indicate maximum x- and y-coordinate values of Tmod31/1 data.
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TROPOMODULIN-3 CONTROLS PLATELET FORMATION AND SIZE
F-actin (Figure 4B, supplemental Figures 3 and 5), and fail to
polymerize F-actin during spreading on collagen (Figure 6C).
Tmod3 is present in these F-actin bundles in WT MKs (Figure 6A),
consistent with Tmod3 capping and promotion of F-actin assembly
in the MK actin cytoskeleton. In vitro, Tmod3’s F-actin–capping
activity is enhanced by Tmod3 binding TM, which binds along
F-actin and stabilizes filaments.26,30,33 TM4 (TPM4 gene) is the
primary TM in platelets42,54-56 and MKs (Figure 1 and supplemental Figure 4), and Tmod3 associates with TM4 and the
actin cytoskeleton, which includes FLNA, b2-spectrin, and
NMIIA (supplemental Figure 4). Since Tmod3 binds directly to
TMs,30,33 including TM4, 65 it is likely that Tmod3 caps TM4coated F-actin linked to FLNA, b2-spectrin, and/or NMIIA in MKs
and proplatelets.
Loss of Tmod3 leads to F-actin instability,30,33,34 yet overall,
more F-actin is observed in large Tmod32/2 proplatelet buds
(Figure 7B,C), and F-actin aggregates are observed in Tmod32/2
MK cytoplasm (Figure 4B and supplemental Figure 5). What
might explain this? Increased actin monomer levels resulting from
disassembly of Tmod3-capped F-actin would be available for
polymerization nucleated by other actin assembly factors present in
MKs and proplatelets, such as WASp17,58 or mDia1.20 In other cell
types, assembly of diverse F-actin structures are promoted by actin
nucleation factors that compete for a limited pool of globular actin;
thus when one assembly factor and pathway is eliminated, the others
promote excessive F-actin assembly into alternative cytoskeletal
structures.66,67
The actin cytoskeletal proteins that play important roles in MK
platelet biogenesis (FLNA, ADF/n-cofilin, Rac/Cdc42, RhoA, and
WASp) are also required for platelet spreading, adhesion, and granule
secretion to form clots.7,68,69 Indeed, Tmod3-null embryonic platelets spreading on collagen display defective F-actin polymerization
and reorganization (Figure 7J). Although Tmod3-null platelets form
lamellipodia, an actin–polymerization-dependent process, they do not
form the internal F-actin rings observed during organelle centralization.47,68 Moreover, abnormal numbers of VWF1 granules in
Tmod32/2 proplatelets (Figure 5), and fewer granules and more empty
vesicular structures observed by TEM in circulating platelets in
529
Tmod32/2 embryos (Figure 2) support the idea that Tmod32/2 platelet
function is likely defective. Future studies with a MK-restricted Tmod3
deletion in adult mice will be required to define the precise role of
Tmod3 in platelet function in thrombosis and hemostasis.
Acknowledgments
The authors thank Malcolm Wood in the The Scripps Research
Institute Core Microscopy Facility for TEM of MKs and platelets,
Zaverio Ruggeri and Alessandro Zarpellon for anti-GPIba antibodies and discussions, and David Gokhin for help with editing.
This study was supported by grants from the National Institutes
of Health, National Heart, Lung, and Blood Institute (R01-HL083464)
(V.M.F), and National Institute of Diabetes and Digestive and Kidney
Diseases (R01 DK094934 and R01 DK086267) (D.S.K.), and funding
from the State of Connecticut Stem Cell Research Fund (D.S.K).
Authorship
Contribution: Z.S. analyzed protein expression, performed MK
cultures, confocal microscopy and quantitative image analyses, prepared figures, and wrote portions of the manuscript; R.B.N. bred
mice, performed embryo analysis, histology and confocal microscopy, and prepared the figures; C.S. and S.H. performed fetal liver
dissections for flow cytometry, analyzed data, and prepared the figures; D.S.K. provided intellectual input, experimental design, data
analysis, and read the manuscript; and V.M.F. conceived the project,
designed experiments, interpreted data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Velia M. Fowler, Department of Cell and
Molecular Biology, CB163, The Scripps Research Institute, 10550 N.
Torrey Pines Rd, La Jolla, CA 92037; e-mail: [email protected].
References
1. Italiano JE Jr, Lecine P, Shivdasani RA, Hartwig
JH. Blood platelets are assembled principally at
the ends of proplatelet processes produced by
differentiated megakaryocytes. J Cell Biol. 1999;
147(6):1299-1312.
2. Hartwig JH, Italiano JE Jr. Cytoskeletal
mechanisms for platelet production. Blood
Cells Mol Dis. 2006;36(2):99-103.
3. Machlus KR, Italiano JE Jr. The incredible
journey: from megakaryocyte development to
platelet formation. J Cell Biol. 2013;201(6):
785-796.
4. Junt T, Schulze H, Chen Z, et al. Dynamic
visualization of thrombopoiesis within bone
marrow. Science. 2007;317(5845):1767-1770.
5. Thon JN, Montalvo A, Patel-Hett S, et al.
Cytoskeletal mechanics of proplatelet maturation
and platelet release. J Cell Biol. 2010;191(4):
861-874.
6. Patel SR, Hartwig JH, Italiano JE Jr. The
biogenesis of platelets from megakaryocyte
proplatelets. J Clin Invest. 2005;115(12):
3348-3354.
7. Thon JN, Italiano JE Jr. Does size matter in
platelet production? Blood. 2012;120(8):
1552-1561.
8. Bender M, Thon JN, Ehrlicher AJ, et al.
Microtubule sliding drives proplatelet elongation
and is dependent on cytoplasmic dynein.
Blood. 2015;125(5):860-868.
14. Bender M, Eckly A, Hartwig JH, et al. ADF/
n-cofilin-dependent actin turnover determines
platelet formation and sizing. Blood. 2010;
116(10):1767-1775.
9. Patel SR, Richardson JL, Schulze H, et al.
Differential roles of microtubule assembly
and sliding in proplatelet formation by
megakaryocytes. Blood. 2005;106(13):
4076-4085.
15. Jurak Begonja A, Hoffmeister KM, Hartwig JH,
Falet H. FlnA-null megakaryocytes prematurely
release large and fragile platelets that circulate
poorly. Blood. 2011;118(8):2285-2295.
10. Pertuy F, Eckly A, Weber J, et al. Myosin IIA
is critical for organelle distribution and F-actin
organization in megakaryocytes and platelets.
Blood. 2014;123(8):1261-1269.
11. Eckly A, Strassel C, Freund M, et al. Abnormal
megakaryocyte morphology and proplatelet
formation in mice with megakaryocyte-restricted
MYH9 inactivation. Blood. 2009;113(14):
3182-3189.
12. Zhang Y, Conti MA, Malide D, et al. Mouse
models of MYH9-related disease: mutations in
nonmuscle myosin II-A. Blood. 2012;119(1):
238-250.
13. Eckly A, Rinckel JY, Laeuffer P, et al. Proplatelet
formation deficit and megakaryocyte death
contribute to thrombocytopenia in Myh9 knockout
mice. J Thromb Haemost. 2010;8(10):2243-2251.
16. Nurden P, Debili N, Coupry I, et al.
Thrombocytopenia resulting from mutations in
filamin A can be expressed as an isolated
syndrome. Blood. 2011;118(22):5928-5937.
17. Sabri S, Foudi A, Boukour S, et al. Deficiency in
the Wiskott-Aldrich protein induces premature
proplatelet formation and platelet production in the
bone marrow compartment. Blood. 2006;108(1):
134-140.
18. Pleines I, Dütting S, Cherpokova D, et al.
Defective tubulin organization and proplatelet
formation in murine megakaryocytes lacking Rac1
and Cdc42. Blood. 2013;122(18):3178-3187.
19. Suzuki A, Shin JW, Wang Y, et al. RhoA is
essential for maintaining normal megakaryocyte
ploidy and platelet generation. PLoS ONE. 2013;
8(7):e69315.
20. Pan J, Lordier L, Meyran D, et al. The formin
DIAPH1 (mDia1) regulates megakaryocyte
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
530
BLOOD, 23 JULY 2015 x VOLUME 126, NUMBER 4
SUI et al
proplatelet formation by remodeling the actin and
microtubule cytoskeletons. Blood. 2014;124(26):
3967-3977.
21. Kunishima S, Okuno Y, Yoshida K, et al.
ACTN1 mutations cause congenital
macrothrombocytopenia. Am J Hum
Genet. 2013;92(3):431-438.
22. Guéguen P, Rouault K, Chen JM, et al. A
missense mutation in the alpha-actinin 1 gene
(ACTN1) is the cause of autosomal dominant
macrothrombocytopenia in a large French family.
PLoS ONE. 2013;8(9):e74728.
23. Schulze H, Korpal M, Hurov J, et al.
Characterization of the megakaryocyte
demarcation membrane system and its role in
thrombopoiesis. Blood. 2006;107(10):3868-3875.
24. Patel-Hett S, Wang H, Begonja AJ, et al. The
spectrin-based membrane skeleton stabilizes
mouse megakaryocyte membrane systems and is
essential for proplatelet and platelet formation.
Blood. 2011;118(6):1641-1652.
25. Hartwig JH, DeSisto M. The cytoskeleton of the
resting human blood platelet: structure of the
membrane skeleton and its attachment to actin
filaments. J Cell Biol. 1991;112(3):407-425.
26. Yamashiro S, Gokhin DS, Kimura S, Nowak RB,
Fowler VM. Tropomodulins: pointed-end capping
proteins that regulate actin filament architecture in
diverse cell types. Cytoskeleton (Hoboken). 2012;
69(6):337-370.
27. Weber A, Pennise CR, Babcock GG, Fowler VM.
Tropomodulin caps the pointed ends of actin
filaments. J Cell Biol. 1994;127(6 pt 1):1627-1635.
28. Weber A, Pennise CR, Fowler VM. Tropomodulin
increases the critical concentration of barbed
end-capped actin filaments by converting ADP.P
(i)-actin to ADP-actin at all pointed filament ends.
J Biol Chem. 1999;274(49):34637-34645.
29. Yamashiro S, Cox EA, Baillie DL, Hardin JD,
Ono S. Sarcomeric actin organization is
synergistically promoted by tropomodulin,
ADF/cofilin, AIP1 and profilin in C. elegans.
J Cell Sci. 2008;121(pt 23):3867-3877.
30. Lewis RA, Yamashiro S, Gokhin DS, Fowler VM.
Functional effects of mutations in the
tropomyosin-binding sites of tropomodulin1 and
tropomodulin3. Cytoskeleton (Hoboken). 2014;
71(7):395-411.
31. Bernstein BW, Bamburg JR. Tropomyosin binding
to F-actin protects the F-actin from disassembly
by brain actin-depolymerizing factor (ADF).
Cell Motil. 1982;2(1):1-8.
32. Burgess DR, Broschat KO, Hayden JM.
Tropomyosin distinguishes between the two actinbinding sites of villin and affects actin-binding
properties of other brush border proteins. J Cell
Biol. 1987;104(1):29-40.
33. Yamashiro S, Gokhin DS, Sui Z, Bergeron SE,
Rubenstein PA, Fowler VM. Differential actinregulatory activities of tropomodulin1 and
tropomodulin3 with diverse tropomyosin and actin
isoforms. J Biol Chem. 2014;289(17):
11616-11629.
34. Weber KL, Fischer RS, Fowler VM. Tmod3
regulates polarized epithelial cell morphology.
J Cell Sci. 2007;120(pt 20):3625-3632.
35. Moyer JD, Nowak RB, Kim NE, et al.
Tropomodulin 1-null mice have a mild spherocytic
elliptocytosis with appearance of tropomodulin 3
in red blood cells and disruption of the membrane
skeleton. Blood. 2010;116(14):2590-2599.
36. Gokhin DS, Fowler VM. Cytoplasmic gammaactin and tropomodulin isoforms link to the
sarcoplasmic reticulum in skeletal muscle fibers.
J Cell Biol. 2011;194(1):105-120.
37. Nowak RB, Fischer RS, Zoltoski RK, Kuszak JR,
Fowler VM. Tropomodulin1 is required for
membrane skeleton organization and hexagonal
geometry of fiber cells in the mouse lens. J Cell
Biol. 2009;186(6):915-928.
38. Fischer RS, Fritz-Six KL, Fowler VM. Pointed-end
capping by tropomodulin3 negatively regulates
endothelial cell motility. J Cell Biol. 2003;161(2):
371-380.
39. Sui Z, Nowak RB, Bacconi A, et al.
Tropomodulin3-null mice are embryonic lethal
with anemia due to impaired erythroid terminal
differentiation in the fetal liver. Blood. 2014;
123(5):758-767.
40. Lim CY, Bi X, Wu D, et al. Tropomodulin3 is
a novel Akt2 effector regulating insulin-stimulated
GLUT4 exocytosis through cortical actin
remodeling. Nat Commun. 2015;6:5951.
41. Geeves MA, Hitchcock-DeGregori SE, Gunning
PW. A systematic nomenclature for mammalian
tropomyosin isoforms. J Muscle Res Cell Motil.
2015;36(2):147-153.
42. Burkhart JM, Vaudel M, Gambaryan S, et al. The
first comprehensive and quantitative analysis of
human platelet protein composition allows the
comparative analysis of structural and functional
pathways. Blood. 2012;120(15):e73-e82.
43. Thon JN, Italiano JE. Visualization and
manipulation of the platelet and megakaryocyte
cytoskeleton. Methods Mol Biol. 2012;788:
109-125.
44. Gokhin DS, Lewis RA, McKeown CR, et al.
Tropomodulin isoforms regulate thin filament
pointed-end capping and skeletal muscle
physiology. J Cell Biol. 2010;189(1):95-109.
45. Smith EC, Thon JN, Devine MT, et al. MKL1
and MKL2 play redundant and crucial roles in
megakaryocyte maturation and platelet formation.
Blood. 2012;120(11):2317-2329.
46. Gokhin DS, Tierney MT, Sui Z, Sacco A, Fowler
VM. Calpain-mediated proteolysis of tropomodulin
isoforms leads to thin filament elongation in
dystrophic skeletal muscle. Mol Biol Cell. 2014;
25(6):852-865.
47. Kovacsovics TJ, Hartwig JH. Thrombin-induced
GPIb-IX centralization on the platelet surface
requires actin assembly and myosin II activation.
Blood. 1996;87(2):618-629.
48. de Alarcon PA, Graeve JL. Analysis of
megakaryocyte ploidy in fetal bone marrow
biopsies using a new adaptation of the feulgen
technique to measure DNA content and estimate
megakaryocyte ploidy from biopsy specimens.
Pediatr Res. 1996;39(1):166-170.
49. Izumi T, Kawakami M, Enzan H, Ohkita T. The
size of megakaryocytes in human fetal, infantile
and adult hematopoiesis. Hiroshima J Med Sci.
1983;32(3):257-260.
50. Zimmet J, Ravid K. Polyploidy: occurrence in
nature, mechanisms, and significance for the
megakaryocyte-platelet system. Exp Hematol.
2000;28(1):3-16.
51. Eckly A, Heijnen H, Pertuy F, et al. Biogenesis of
the demarcation membrane system (DMS) in
megakaryocytes. Blood. 2014;123(6):921-930.
52. Poujol C, Ware J, Nieswandt B, Nurden AT,
Nurden P. Absence of GPIbalpha is responsible
for aberrant membrane development during
megakaryocyte maturation: ultrastructural study
using a transgenic model. Exp Hematol. 2002;
30(4):352-360.
53. Chen Y, Boukour S, Milloud R, et al. The
abnormal proplatelet formation in MYH9-related
macrothrombocytopenia results from an
increased actomyosin contractility and is rescued
by myosin IIA inhibition. J Thromb Haemost.
2013;11(12):2163-2175.
54. MacLeod AR, Talbot K, Smillie LB, Houlker C.
Characterization of a cDNA defining a gene
family encoding TM30p1, a human fibroblast
tropomyosin. J Mol Biol. 1987;194(1):1-10.
55. Lewis WG, Cote GP, Mak AS, Smillie LB. Amino
acid sequence of equine platelet tropomyosin.
Correlation with interaction properties. FEBS Lett.
1983;156(2):269-273.
56. Lees-Miller JP, Yan A, Helfman DM. Structure
and complete nucleotide sequence of the gene
encoding rat fibroblast tropomyosin 4. J Mol Biol.
1990;213(3):399-405.
57. Richardson JL, Shivdasani RA, Boers C, Hartwig JH,
Italiano JE Jr. Mechanisms of organelle transport and
capture along proplatelets during platelet production.
Blood. 2005;106(13):4066-4075.
58. Chen Y, Aardema J, Kale S, et al. Loss of the
F-BAR protein CIP4 reduces platelet production
by impairing membrane-cytoskeleton remodeling.
Blood. 2013;122(10):1695-1706.
59. Machlus KR, Thon JN, Italiano JE Jr. Interpreting
the developmental dance of the megakaryocyte:
a review of the cellular and molecular processes
mediating platelet formation. Br J Haematol. 2014;
165(2):227-236.
60. Hegmann TE, Lin JL, Lin JJ. Probing the role of
nonmuscle tropomyosin isoforms in intracellular
granule movement by microinjection of
monoclonal antibodies. J Cell Biol. 1989;109(3):
1141-1152.
61. Percival JM, Hughes JA, Brown DL, et al.
Targeting of a tropomyosin isoform to short
microfilaments associated with the Golgi complex.
Mol Biol Cell. 2004;15(1):268-280.
62. Levine J, Willard M. Fodrin: axonally transported
polypeptides associated with the internal
periphery of many cells. J Cell Biol. 1981;90(3):
631-642.
63. He M, Tseng WC, Bennett V. A single divergent
exon inhibits ankyrin-B association with the
plasma membrane. J Biol Chem. 2013;288(21):
14769-14779.
64. Muresan V, Stankewich MC, Steffen W, Morrow JS,
Holzbaur EL, Schnapp BJ. Dynactin-dependent,
dynein-driven vesicle transport in the absence of
membrane proteins: a role for spectrin and acidic
phospholipids. Mol Cell. 2001;7(1):173-183.
65. Uversky VN, Shah SP, Gritsyna Y,
Hitchcock-DeGregori SE, Kostyukova AS.
Systematic analysis of tropomodulin/tropomyosin
interactions uncovers fine-tuned binding
specificity of intrinsically disordered proteins.
J Mol Recognit. 2011;24(4):647-655.
66. Burke TA, Christensen JR, Barone E, Suarez C,
Sirotkin V, Kovar DR. Homeostatic actin
cytoskeleton networks are regulated by assembly
factor competition for monomers. Curr Biol. 2014;
24(5):579-585.
67. Rotty JD, Wu C, Haynes EM, et al. Profilin-1
serves as a gatekeeper for actin assembly by
Arp2/3-dependent and -independent pathways.
Dev Cell. 2015;32(1):54-67.
68. Bearer EL, Prakash JM, Li Z. Actin dynamics
in platelets. Int Rev Cytol. 2002;217:137-182.
69. Thon JN, Italiano JE. Platelets: production,
morphology and ultrastructure. Handb Exp
Pharmacol. 2012;(210):3-22.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2015 126: 520-530
doi:10.1182/blood-2014-09-601484 originally published
online May 11, 2015
Regulation of actin polymerization by tropomodulin-3 controls
megakaryocyte actin organization and platelet biogenesis
Zhenhua Sui, Roberta B. Nowak, Chad Sanada, Stephanie Halene, Diane S. Krause and Velia M.
Fowler
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