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Regular Article
PLATELETS AND THROMBOPOIESIS
Megakaryocytes assemble podosomes that degrade matrix and protrude
through basement membrane
Hannah Schachtner,1 Simon D. J. Calaminus,1 Amy Sinclair,2 James Monypenny,3 Michael P. Blundell,4 Catherine Leon,5
Tessa L. Holyoake,2 Adrian J. Thrasher,4 Alison M. Michie,2 Milica Vukovic,2 Christian Gachet,5 Gareth E. Jones,3
Steven G. Thomas,6 Steve P. Watson,6 and Laura M. Machesky1
1
University of Glasgow College of Medical, Veterinary and Life Sciences and Beatson Institute for Cancer Research, Bearsden, Glasgow, UK; 2Paul O’Gorman
Leukaemia Research Centre, Institute of Cancer Sciences, University of Glasgow, Gartnavel General Hospital, Glasgow, UK; 3King’s College London, Randall
Division, Guy’s Campus, London, UK; 4Molecular Immunology Unit, Wolfson Centre for Gene Therapy of Childhood Diseases and Centre for Immunodeficiency,
University College London Institute of Child Health, London, UK; 5UMR_S949 INSERM-Université de Strasbourg, Etablissement Français du Sang-Alsace,
Strasbourg, France; and 6University of Birmingham, Edgbaston, Birmingham, UK
Megakaryocytes give rise to platelets via extension of proplatelet arms, which are
released through the vascular sinusoids into the bloodstream. Megakaryocytes and
• Murine and human
their precursors undergo varying interactions with the extracellular environment in the
bone marrow during their maturation and positioning in the vascular niche. We
megakaryocytes assemble
demonstrate that podosomes are abundant in primary murine megakaryocytes
podosomes.
adherent on multiple extracellular matrix substrates, including native basement
• Megakaryocyte podosomes
membrane. Megakaryocyte podosome lifetime and density, but not podosome size,
remodel matrix.
are dependent on the type of matrix, with podosome lifetime dramatically increased on
collagen fibers compared with fibrinogen. Podosome stability and dynamics depend on actin cytoskeletal dynamics but not matrix
metalloproteases. However, podosomes degrade matrix and appear to be important for megakaryocytes to extend protrusions
across a native basement membrane. We thus demonstrate for the first time a fundamental requirement for podosomes in
megakaryocyte process extension across a basement membrane, and our results suggest that podosomes may have a role in
proplatelet arm extension or penetration of basement membrane. (Blood. 2013;121(13):2542-2552)
Key Points
Introduction
Megakaryocytes (Mks) maintain thrombopoiesis by releasing
cytoplasmic fragments called proplatelets into the blood.1 Prior to
proplatelet formation, Mks undergo initial differentiation in the
osteoblastic niche, a hypoxic compartment dominated by extracellular matrix (ECM) proteins collagen I and fibronectin. At some
point in the maturation process, cells migrate to the vascular niche,
where they send out long projections (proplatelets) across the
basement membrane of a sinusoidal vessel. Platelets form as the
long proplatelet arms encounter the shear forces of the bloodstream, with a proportion of platelet formation occurring within the
bloodstream.2,3 The vascular niche is dominated by the presence of
collagen IV, laminin, and fibrinogen, and by endothelial cells.4,5
The interaction of the Mk with its niche environment via integrins
and other surface molecules is essential to Mk function.6
Both Mk migration and proplatelet budding require the dynamic
rearrangement of the actin cytoskeleton. Primary initiators of actin
assembly include the Arp2/3 complex and its regulators, the
Wiskott-Aldrich syndrome protein (WASp) family proteins.
Podosomes are highly dynamic dot-like matrix contacts formed
by Mks7 as well as other myeloid-derived cells such as dendritic
cells, macrophages, and osteoclasts.8,9 Podosomes consist of an
F-actin-rich core with an integrin-associated ring structure. Typical
core proteins include Arp2/3 complex, WASp, and cortactin
whereas integrins, vinculin, talin, paxillin,10 and myosin IIA11
localize to the ring structure. Under some conditions, podosomes
form superstructures called rosettes, where clusters of podosomes
assume a ring shape.12 Podosomes are dynamic, with a lifetime of
up to 12 minutes, which is shorter than related structures like
invadopodia (tens of minutes to more than 10 hours) and focal
adhesions (up to 1 hour).13 However, these structures are thought
to be important in mechanosensory processes and thus can be
longer-lived on more rigid substrates.14,15 Indeed, not only the size
and the shape of podosome rosettes but also the lifetime of
individual podosomes is dependent on stiffness of the underlying
substratum and myosin-IIA-regulated tension.15 In addition to their
role in adhesion, podosomes drive the degradation of ECM
proteins via matrix metalloproteases (MMPs).16,17
Sabri et al (2006)7 first observed podosomes in Mks and found
that WASp2/2 Mks were unable to form podosomes. WASp2/2 Mks
prematurely formed proplatelets in the bone marrow, contributing
to thrombocytopenia. They speculated that premature formation of
platelets could be due to a loss of the normal inhibition of proplatelet
Submitted July 25, 2012; accepted December 21, 2012. Prepublished online
as Blood First Edition paper, January 10, 2013; DOI 10.1182/blood-2012-07443457.
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.
The online version of this article contains a data supplement.
There is an Inside Blood commentary on this article in this issue.
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© 2013 by The American Society of Hematology
BLOOD, 28 MARCH 2013 x VOLUME 121, NUMBER 13
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BLOOD, 28 MARCH 2013 x VOLUME 121, NUMBER 13
budding by podosome assembly on collagen I, which might prohibit
release of platelets prematurely before the vascular niche is reached.7
Here we provide a detailed analysis of Mk podosome structures
and dynamics and we demonstrate the ability of Mk podosomes to
degrade ECM. In contrast to Sabri et al,7 we find that Mks form
podosomes on multiple ECM substrates, including fibrinogen. Mks
form classical podosomes with F-actin cores driven by actin
polymerization via the Arp2/3 complex and WASp and surrounded
by rings of vinculin. We find that the underlying substrate can
affect podosome numbers, density, and lifetime. We demonstrate
podosome-driven degradation of fibrinogen, and we find that
protease activity is essential for the assembly of protrusive actin
structures crossing a native basement membrane. We propose that
podosomes may play a pivotal role in Mk motility and remodelling
of ECM, including activities such as extension of proplatelet arms
across the basement membrane of a sinusoidal vessel.
Materials and methods
MEGAKARYOCYTE PODOSOMES REMODEL MATRIX
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Imaging of live and fixed megakaryocytes
Immunofluorescence of fixed cells was performed by standard methods and
is described in supplemental Methods.
For Mk lifetime experiments, glass-bottom dishes (MatTek, Ashland,
MA) were incubated with 100 mg/mL fibrinogen or 100 mg/mL Horm
collagen at 4°C overnight. Dishes were blocked with heat-inactivated 0.5%
bovine serum albumin for 1 hour. Lifeact-GFP cells were seeded in serumfree medium containing 250 ng/mL stromal-derived factor (SDF) 1a and
directly imaged at 37°C and 5% CO2 supply or in the presence of HEPES
(N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid) buffer to stabilize
the pH. Images were taken every 10 or 20 seconds to decrease phototoxic
side effects. A Nikon A1R confocal microscope (Tokyo, Japan) or a Nikon
TIRF (Tokyo, Japan) microscope was used with a 603/1.4 NA or a 1003/
1.4 NA oil objective. Images were recorded using a 4-channel PMT
detector or a Evolve 512 EMCCD camera (Photometrics, Tuscon, AZ) for
20 minutes to 4 hours. ImageJ 1.46i was used for podosome lifetime
calculation. Podosomes (at least 10 per cell) forming during the length of
the movie were defined as actin-rich dots and analyzed manually.
For fibrinogen and basement membrane degradation assays, we adapted
similar assays that had been previously used to analyze cancer cells or
fibroblasts (eg, Hotari et al24) and the details are in supplemental Methods.
Animals and reagents
Statistical analysis
Animal experiments were done according to UK Home Office Regulations.
Prior to isolation of Mks or native peritoneal basement membranes, mice
were sacrificed by an approved Schedule I method. Control mice from
C57BL/6 or mixed background were used as appropriate. The Lifeact mice
and WASp2/2 mice have a C57BL/6 background and were described
before.18,19 MYH92/2 mice were previously described.20 All antibodies
and reagents are described in supplemental Methods.
Isolation of bone marrow-derived Mks
Mks were isolated from the bone marrow from the hind legs of mice .6 weeks
old. Both tibiae and femurs were flushed followed by the depletion of immune
cells with magnetic beads as described before.21 After 7 days of differentiation,
mature Mks were purified with a 1.5%/3% bovine serum albumin gradient for
45 minutes and constituted .60% of the enriched cell population as reported
previously.22
Isolation of human megakaryocytes from cord blood
Human cord blood samples were obtained after informed written consent in
accordance with the Declaration of Helsinki from the mothers according to
the Scottish National Blood Transfusion Service. Mononuclear cell fractions
were isolated from whole blood with density gradient separation using
Histopaque-1077 (Sigma-Aldrich, St. Louis, MO). Cells were enriched for
CD34 using magnetic bead labeling and separation (Miltenyi Biotec Inc.,
Auburn, CA) according to the manufacturer’s instructions. Purity of enriched
populations was examined using flow cytometry for CD34 expression
(CD34-APC; BD Biosciences, Oxford, UK) and all samples were >80%
CD341 . Cells were seeded at approximately 1 3 105/mL in serum-free
medium supplemented with StemSpan CC220, a cytokine cocktail
containing recombinant human interleukin 6, interleukin 9, stem cell factor,
and thrombopoietin (Stem Cell Technologies, Grenoble, France), cultured
for 7 days, and then reseeded in fresh medium for a further 4 to 7 days. Mks
were isolated as described above for murine Mks.
Lentiviral transfection of megakaryocytes
Lentiviral constructs for WASp tagged with green fluorescent protein (GFP)
have been previously described.23 Lentivirus particles were produced in
HEK293T cells and virus titer was determined with Lenti-X™ qRT-PCR
titration kit from Clontech. Mks were incubated on day 2 with a multiplicity
of infection value of 30 for 24 hours until day 3. Cells were supplemented
with fresh full Dulbecco’s modified Eagle medium containing 100 ng/mL
stem cell factor and 50 ng/mL thrombopoietin during the following 3 days
(days 4-6) prior to experiments.
Statistical analysis was done by standard methods with advice from Dr
Gabriela Kalna (see supplemental Methods).
Results
Megakaryocytes form podosomes on multiple ECM surfaces
Murine Mks were spread on Horm collagen (predominantly collagen
I), and stained for Arp2/3 complex, revealing F-actin-rich dot-like
structures (Figure 1A). Actin puncta were verified to be podosomes
through colocalization of F-actin and WASp (Figure 1B) and the
formation of a vinculin ring structure around the F-actin-rich core
(Figure 1C red square). Interestingly, some of the spread Mks
displayed elongated structures showing aligned podosome assembly along the collagen fibers (Figure 1B arrow). These structures
are also apparent in Figure 1A, but together with actin stress fibers,
which appear as straighter F-actin-rich linear bundles. WASp is widely
known to be essential for podosome formation in hematopoietic
cells,9,25,26 including one report where Mks were studied.7 We
confirmed that WASp2/2 Mks did not form podosomes when
spread on collagen (Figure 1D), demonstrating an absolute
requirement for WASp to initiate podosome formation.
To examine podosome formation on other bone marrow-relevant
matrix molecules, Mks were spread on fibrinogen.27 In comparison
with collagen I, Mks spread significantly less well on fibrinogen
but had a higher number of podosomes per unit area (Figure 1E
and Figure 2A-B). The podosomes were similar in size to those on
collagen and contained WASp (Figure 2A), the Arp2/3 complex
(not shown), and vinculin (Figure 2B red square). Podosomes
formed on fibronectin (Figure 2C) and on glass (Figure 2D) with
no added matrix when cells were spread in serum-containing
medium. Although WASp2/2 Mks did not have podosomes, they
spread out similarly to control cells on both collagen and fibrinogen
(supplemental Data File 1). Primary human Mks differentiated
from cord blood also formed podosomes (supplemental Data File 2).
These were not as prominent as the murine Mk podosomes, but had
clear vinculin ring structures and actin cores (supplemental Data File
2). Because murine Mks were more readily obtainable than human
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SCHACHTNER et al
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Figure 1. Mks form abundant podosomes on Horm
collagen and fibrinogen matrix. Mks were spread on
100 mg/mL Horm collagen-coated surfaces for 3 hours
and then fixed and stained for F-actin and (A) the Arp2/3
complex, (B) WASp, and (C) vinculin. (D) Mks spread
on collagen from WASp2/2 mice were stained for Factin and vinculin. The right column of pictures shows
the merge of F-actin (red, phalloidin) and WASp, Arp2/3
complex, or vinculin (green). Red squares indicate
magnified area of the cell. Red arrows in panel B
indicate podosomes along what appears to be a collagen fiber. (E) Table shows the average plus or minus
the standard error of the mean (SEM) for the spread Mk
surface area in mm2, the mean number of podosomes
formed, podosomes formed per mm2, and the size of
podosomes for Mks spread for 3 hours on 100 mg/mL
Horm collagen or 100 mg/mL fibrinogen-coated surfaces
fixed and stained for F-actin and WASp. The podosome
size was determined by measuring at least 100
podosomes of 5 different cells per experiment. Data
are representative of 3 experiments. Asterisk indicates
significant difference between collagen and fibrinogen
data with a P value , .05. Pictures were taken with
a confocal microscope using a 603 objective. Scale
bars represent 10 mm.
Mks, we performed all of our subsequent experiments with murine
cells. We thus conclude that Mks can make podosomes on multiple
types of substrate that resemble podosomes in myeloid cell types and
that podosome formation is not a prerequisite for cell spreading.
Podosome formation requires the activity of the Arp2/3 complex
Because the Arp2/3 complex strongly localized to Mk podosomes
and had been previously described in podosomes of dendritic cells
and macrophages,28,29 we examined the effect of inhibiting the
Arp2/3 complex on Mk podosomes. Addition of 20 mM CK666, a
small-molecule inhibitor of the Arp2/3 complex,29,30 inhibited
spreading of Mks on collagen by over 50% and reduced the total
number of podosomes to less than 20% of controls (Figure 3;
supplemental Data File 3A-B). The net effect of CK666 on
podosomes was similar whether it was added before (Figure 3) or
after the first 2.5 hours of Mk spreading (supplemental Data
File 4). Thus, it is likely that the Arp2/3 complex plays a
fundamental role in Mk podosome formation and maintenance.
The few residual podosomes that did form in the presence of
CK666 contained filamentous actin and showed no significant
difference in size in comparison with the dimethylsulfoxide
(DMSO) control. These could simply reflect incomplete inhibition
of Arp2/3-mediated actin assembly (data not shown).
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BLOOD, 28 MARCH 2013 x VOLUME 121, NUMBER 13
MEGAKARYOCYTE PODOSOMES REMODEL MATRIX
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Figure 2. Mks form podosomes on different substrata. Cells were spread for 3 hours on 100 mg/mL
fibrinogen or fibronectin-coated surfaces or on glass,
and then fixed and stained for F-actin (red, phalloidin)
and WASp (A, C, D) or vinculin (B) (green). The right
panel of pictures shows the merge of both channels.
Mks were spread for 3 hours on 100 mg/mL Horm
collagen-coated surface 6250 ng/mL SDF. Cells were
analyzed for (E) the number of podosomes, (F) cell
surface area, and (G) podosomes per mm2. Scale bars
represent 10 mm. Red square indicates enlarged area
to highlight podosomes. Data are representative of at
least 3 experiments.
Myosin IIA has also been implicated in force transduction in
association with actin filaments in podosomes, although it is not
essential for their formation.14,31 Interestingly, nonmuscle myosin
IIA is the only isoform present in Mks and platelets.32 Inhibiting
myosin II prior to cell spreading caused a slight increase in the
number of podosomes per area (Figure 3C,E-G; supplemental Data
File 3C). This might have been due to slightly reduced spreading,
although the differences were not statistically significant. To
explore a spreading-related effect, we showed that blebbistatin had
no significant effect on surface area or the number and size of
podosomes in Mks spread first for 2.5 hours and then treated for 30
minutes with inhibitor (supplemental Data File 4). This indicates
that myosin II does not play a significant role in Mk podosome
assembly. Furthermore, no effect of myosin II inhibition on podosome
size could be detected on collagen or fibrinogen (data not shown).
These results were confirmed with myosin-IIA-deficient Mks, which
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SCHACHTNER et al
BLOOD, 28 MARCH 2013 x VOLUME 121, NUMBER 13
Figure 3. Mk podosome formation requires the activity of the Arp2/3 complex. Cells were spread for 3 hours on 100 mg/ml Horm collagen-coated surfaces fixed and stained
for F-actin (green) and WASp (red). The right panel of pictures shows the merge of both channels. The cells were treated with different inhibitors: (A) 0.1% DMSO was used as
control for (B) 20 mM CK666, (C) 10 mM blebbistatin, (D) 5 mM GM6001. Scale bars represent 10 mm. Quantification of the effect of the inhibitors on (E) the number of podosomes,
(F) cell surface area, and (G) podosomes per mm2 were determined on Horm collagen (red columns) or fibrinogen (black columns). Statistical analysis was done using a 1-way
analysis of variance. Asterisk indicates significant difference from the control value with a P value , .05. Error bars indicate 6SEM. Data are representative of 3 experiments.
did not show any difference in the number of podosomes, the spread
cell-surface area, or the podosome size compared with litter-matched
controls spread on fibrinogen (supplemental Figure 5A-C).
Endothelial cells, macrophages, and dendritic cells all produce
podosomes capable of degrading ECM.9,33,34 Cancer cells assemble
invadopodia, which resemble podosomes and are dependent on
MMPs for their assembly.35 Treatment of Mks with a broadspectrum MMP inhibitor, GM6001, had no effect on podosome
formation or podosome size in Mks on collagen or fibrinogen
(Figure 3D-G; supplemental Data File 3D). We confirmed that the
GM6001 was able to inhibit invadopodia formation in MDA-MB231 cells coplated with Mks on a fibrinogen surface (not shown).
Thus, GM6001-sensitive MMPs are not required for podosome
assembly or maintenance in Mks.
The lifetime of podosomes is dependent on the
underlying substratum
Podosomes are highly dynamic structures with a lifetime between
2 and 12 minutes in macrophages, dendritic cells, and osteoclasts
spreading on glass, fibronectin, or collagen I.11,14,25,34,36 In the
present study, we have measured the lifetime of podosomes in Mks
isolated from Lifeact-GFP mice18 using real-time total internal
reflection fluorescence (TIRF) or confocal microscopy. Podosomes are highly dynamic structures, undergoing rapid formation
and dissolution as illustrated on a fibrinogen surface (Figure 4A;
supplemental Movie 1). The mean lifetime of podosomes on
fibrinogen was 5 6 0.3 minutes (Figure 4B), with no podosome
lasting for longer than 15 minutes (Figure 4C black columns). In
comparison, podosomes had a significantly longer half-life on
collagen I of over 16 6 1.1 minutes, with more than 30% lasting beyond
20 minutes (Figure 4C red columns). Furthermore, the podosomes
sometimes assembled in a linear arrangement along the collagen
fibers (Figure 4A bottom panel; supplemental Movie 2). Longlasting podosomes were also observed in GFP-WASp-expressing
Mks spreading on collagen (supplemental Movie 3). These results
suggest that Mks sense the matrix environment and assemble podosomes along collagen fibers and that this influences their lifetime.
Podosome-driven degradation of fibrinogen is MMP and
myosin II dependent
One of the key features of podosomes is the proteolytic degradation
of ECM proteins. Podosome-driven degradation of ECM proteins
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MEGAKARYOCYTE PODOSOMES REMODEL MATRIX
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Figure 4. Mk podosomes are longer-lived on
collagen. (A) Lifeact-GFP cells were spread on 100
mg/mL fibrinogen (top images) or 100 mg/mL Horm
collagen-coated surfaces (bottom images) and were
imaged in real time with a TIRF microscope for 20
minutes (fibrinogen) and 2 hours (Horm collagen) using
a 1003 objective. Pictures were taken every 10
seconds (fibrinogen) or 20 seconds (Horm collagen).
Red squares show enlarged single podosomes. (B) The
average lifetime 6SEM was determined by measuring
100 podosomes in total of 10 different cells. Data were
collected of 4 independent experiments. (C) Cumulative
frequency analysis of podosome lifetime was completed
in 2-minute intervals. A Student t test was used for the
statistical analysis. Asterisk indicates significant difference with a P value , .05. Error bars indicate 6SEM.
See also supplemental Movie 1 (podosomes on
fibrinogen), supplemental Movie 2 (podosomes on
collagen), and supplemental Movie 3 (WASp in podosomes on collagen).
like collagen or fibronectin is well established in cell types such as
macrophages or dendritic cells25,37 but not in Mks. To investigate
whether Mk podosomes can remodel matrix, Mks were spread
on Alexa 488-labeled fibrinogen. Small holes (red arrows) in the
fibrinogen matrix were indicative of podosome-mediated degradation (Figure 5A). Approximately 40% of Mks showed evidence of
degradation (Figure 5E-F) and this was reduced in the presence of
the MMP inhibitor GM6001 and the myosin IIA inhibitor blebbistatin
by more than 50% (Figure 5B-C). MYH92/2 Mks, which displayed
similar numbers of podosomes (supplemental Data File 5A-C),
also showed a similar reduction in numbers of cells degrading
fibrinogen matrix and showed a significant decrease in the amount
of fibrinogen degradation (Figure 5D-F). Furthermore, the extent of
matrix degradation was severely inhibited, most notably in the
presence of GM6001, where the degradation was reduced by over
80% (Figure 5E-F). Comparison of the matrix-degrading and nondegrading Mks indicated that there was a significant increase in
nuclear size (indicating a more mature Mk) associated with those
Mks degrading the fibrinogen matrix (supplemental Figure 6).
Thus, mature Mks used both MMPs and myosin IIA to orchestrate
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Figure 5. Fibrinogen degradation depends on MMPs
and myosin IIA activity. Mks were spread on 100 mg/mL
488-labeled fibrinogen-coated surfaces (green) for
3 hours and stained for F-actin (red) in the presence
of (A) 0.1% DMSO as control for (B) 5 mM GM6001 and
(C) 10 mM blebbistatin. (D) MYH92/2 Mks were spread
on 488-labeled fibrinogen-coated surfaces. Pictures
show from left to right the merge of F-actin and fibrinogen, with red squares indicating magnified areas of
F-actin and fibrinogen and dotted lines marking the
outline of the cell. (E) The percentage of Mks that were
degrading fibrinogen was analyzed and (F) the percentages of total degradation were measured and normalized. Black columns represent inhibitor-treated samples,
and white columns represent control and MYH92/2 Mks.
Images were taken with a confocal microscope using a
603 objective. A total of 20 cells were analyzed per
experiment for n 5 6 experiments. Scale bars represent
10 mm. Statistical analysis was done with a Student
t test. Asterisk represents significant difference with a
P value , .05. Error bars indicate 6SEM. Blebb.,
blebbistatin; KO, knockout.
fibrinogen remodelling via their podosomes. Of note, we were
unable to visualize degradation of collagen matrix, most likely
due to technical reasons, because the fibers were thick and not flat
along the coverglass.
The formation of protrusive actin-rich structures by Mks on
a native basement membrane is dependent on
podosome formation
For Mks to release platelets into the bloodstream, they need to form
protrusions known as proplatelets that can cross the basement
membrane in the blood vessel wall.2,3 To study the potential role of
podosomes in extension of processes across basement membrane,
we isolated peritoneal basement membranes and monitored Mks
for spreading and podosome formation. Mks spread on native basement
membrane to a similar extent as they did on Horm collagen (eg,
1586 6 60 mm2, compared with 1116 6 91.7 mm2 on collagen;
Figure 1 and Figure 6). The presence of podosomes was confirmed
by immunofluorescence microscopy corresponding to the podosome
markers, cortactin and vinculin, in combination with F-actin
(Figure 6A-B). The cortactin antibody showed clear staining in
F-actin-rich dot-like structures in the cell, whereas vinculin demonstrated clear ring structure formation around the actin puncta
(Figure 6B). Thus, Mks form similar podosomes on a native
basement membrane to those formed on a 2-dimensional fibrinogen or
collagen spread surface.
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Figure 6. Mks form podosomes and protrusive
actin-rich structures on a native basement membrane. Cells were spread for 3 hours on a native
basement membrane and then fixed and stained for
F-actin (red) and (A) cortactin or (B) vinculin (green).
Based on the F-actin staining, the cell area of 20 cells
per experiment was analyzed, resulting in an average
cell size of 1586 6 60 mm2. Data are representative of
at least 3 experiments. The right column of images
shows the merge of both channels. Scale bars represent 10 mm. (C-E) Real-time imaging of Lifeact-GFP
Mks (green) was done for 5 hours, with pictures taken
every 15 minutes, and the Mks were preincubated
on the membrane for 2 hours. The membrane was
prestained for collagen IV (red). Every time point shows
the XY perspective and the XYZ perspective (shown in
the red box). Images represent the maximum intensity
projection of the taken z-stacks. Red arrows indicate
actin-rich protrusion crossing the membrane. Axis graph
shows direction of the XYZ perspectives. Images were
taken with a confocal microscope using a 403 objective. Scale bar represents 20 mm. See also supplemental Movie 4.
To investigate if Mks were able to form protrusions that crossed
a native basement membrane, Lifeact-GFP Mks were spread on the
membrane and imaged in real time. The spreading Mks formed
clear actin-rich dots as shown by the images in the xy-plane in
Figure 6C-E. Furthermore, confocal imaging in the Z-plane (red
boxed images) revealed that over time actin protrusions were
formed which were able to cross the membrane (Figure 6C-E red
arrows; supplemental Movie 4). In addition to the formation of
actin protrusions through the membrane, analysis of the intensity of
the membrane outside the cell and underneath the cell as monitored
by staining for collagen type IV indicated a reduction in the
intensity of staining underneath the cell. This therefore revealed
degradation of collagen type IV in the peritoneal basement
membrane by the Mks (data not shown). Therefore, Mks were
able to degrade the ECM and thereby extend protrusions across the
membrane.
Mk protrusion across basement membrane depends on
metalloprotease activity
To investigate the relevance of podosome assembly in the process
of crossing a native basement membrane, Mks were treated with
different inhibitors during spreading on the basement membrane.
Control Mks spread fully on the membrane and formed actin-rich
podosomes. A Z-section through an Mk on basement membrane
reveals actin-containing structures (red) interacting with and
crossing through the membrane (green) (Figure 7A red box and
yellow arrowhead). Around 25% of the control cells formed actin
elongations, which were able to invade the membrane (Figure 7A,C).
The Arp2/3 complex inhibitor CK666 significantly decreased both
the podosome formation and the percentage of protrusions that
penetrated the membrane by almost 90% relative to the control
(Figure 7B,C). We observed similar results with WASp2/2 Mks
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Figure 7. Mks use podosomes to cross a native
basement membrane. Mks were spread for 3 hours on
a native basement membrane fixed and stained for Factin (red) and collagen IV (green). The right column of
pictures shows the merge of both channels. Representative images are shown for (A) 0.1% DMSO used as
control and (B) 20 mM CK666. Pictures show slice of
taken z-stack from each treatment and the corresponding cross section, XYZ perspective. (C) The percentage
of actin protrusions crossing the basement membrane
was analyzed for different inhibitors; 20 mM CK666, 5
mM GM6001, 10 mM blebbistatin, 5 mM GM6001/10 mM
blebbistatin, and WASp2/2 Mks. Yellow arrowhead indicates actin-rich protrusion crossing the membrane.
Red line represents position of cross section. The cross
sections of at least 10 different cells were analyzed per
experiment. Data are representative of at least 3 independent experiments. Images were taken with a
confocal microscope using a 403 objective. Scale bars
represent 20 mm. Statistical analysis was done by
performing a 1-way analysis of variance. Asterisk indicates significant difference with a P value , .05. Error
bars indicate 6SEM.
consistent with podosomes having a role in formation of protrusions
across the basement membrane (Figure 7C). Furthermore, the
inhibition of MMPs with 5 mM GM6001 induced a 50% reduction of actin-based protrusions across the basement membrane
(Figure 7C). In contrast, inhibition of myosin IIA had no effect on
the formation of protrusions across the membrane either in the
absence or presence of MMP inhibition (supplemental Figure 7).
Therefore, protrusion formation across native basement membranes is dependent on podosome formation and MMP activity but
not on myosin IIA.
Discussion
Podosomes are so named because they can be thought of as cellular
“foot processes,” regulating adhesion, migration, and matrix
interactions of a variety of cell types. Typically, podosomes are
found in highly motile monocytic lineage cells such as dendritic
cells, osteoclasts, and macrophages, where they are thought to be
important for mediating the interactions between such cells and the
ECM. We describe here the first detailed characterization of Mk
podosomes, and we propose that they are important modulators of
Mk interactions with matrix and confer matrix-degrading activities.
Our study has important implications for understanding how Mks
can cross ECM and basement membranes and may contribute to our
understanding of how they deposit platelets into the bloodstream.
We extend the initial observations of Sabri et al (2006), who
first noticed that Mks assemble podosomes on collagen, and we
show that Mks make classical podosomes with vinculin rings
around a central actin core.38,39 However, we now show that Mks
also assemble podosomes on all matrices that we tested, including
native mouse peritoneal basement membranes. Although podosomes were ubiquitous, the number of podosomes per unit area
depended on the underlying matrix and was variable. For example,
Mks formed significantly more podosomes per unit area on
fibrinogen than on Horm collagen. A thin layer of fibrinogen likely
forms an inflexible monolayer on glass (compare Alexa 488fibrinogen continuity in Figure 5) in contrast to more flexible and
3-dimensional collagen fibers. Stiffer substrates promote integrin
clustering, suggesting a potential link between substrate elasticity
and responsive adhesion assembly.40 Furthermore, when macrophages were placed on different micropatterned substrata, podosome formation was actively encouraged on fibrinogen, whereas
gelatin and a thin layer of collagen IV on glass were suppressive to
podosome formation.31 Thus, different integrins engaged by each
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 28 MARCH 2013 x VOLUME 121, NUMBER 13
matrix might affect podosome formation, with fibrinogen favoring
b3 integrins and b3 integrin being the most abundant b subunit in
Mks, whereas collagen recruits b1 integrins, which are less abundant
in platelets41 and likely Mks. Interestingly, podosome size and
shape did not appear to vary on the different matrices or following
inhibitor treatments. Thus, podosomes form with some consistent
stoichiometry and arrangement of proteins and may be more of
a continuous network than individual structures, as indicated also
by superresolution microscopy.20
Although Mks can make podosomes on multiple types of matrix,
additional factors such as SDF, which plays an important role in
Mk maturation and migration, could regulate their formation.22
Indeed, Mks formed more podosomes in the presence of SDF-1a,
while spreading was unaffected (Figure 2). This indicates that
SDF-1a induces podosome formation and may contribute to Mk
migration induced by a chemotactic gradient of SDF-1a.
The spatial localization of podosomes is dependent on the
matrix, because in contrast to uniform thin fibrinogen, where
podosomes were more or less uniformly distributed, Mks formed
podosomes along collagen fibers in a linear fashion. Not only did
matrix affect the position and density of podosomes, but it also
affected their lifetime quite dramatically. Labernadie et al31 found
that macrophage podosomes either avoided or collected on top of
various micropatterns in matrix, but to our knowledge this is the
first report of podosomes preferentially aligning along collagen
fibers. Similar structures were recently described in fibroblasts as
linear invadosomes, which formed along collagen I fibers.42 The
distribution of fibrinogen and collagen in bone marrow is very
diverse; collagen is present in the entire bone marrow,43 whereas
fibrinogen is only located at the vascular sinusoid.27 This could
have profound affects on the function of podosomes. For example,
linear podosome structures could encourage not just adhesion but
also guidance clues for protrusion, migration, as well as degradation
hotspots within Mks.
It is still a subject of debate whether invadopodia and podosomes
are distinct or the same structures.12,39,44 Our data demonstrate the
fundamental requirement for both actin polymerization and the
WASp-Arp2/3 complex pathway for podosome formation and
maintenance. We thus show that Mks form podosomes by similar
mechanisms as other cell types.29,45-47 Interestingly, the inhibition
of MMP or myosin IIA or loss of the myosin II heavy chain MYH9
had no significant effect on podosome formation. This provides
further evidence that podosomes are distinct from invadopodia,
which are dependent on MMP activity, or focal adhesions, which
require myosin IIA activity.13,31 We suggest that protease dependence might be one way to distinguish between invadopodia
and podosomes, with the main architecture of podosomes being
insensitive to protease inhibition, whereas invadopodia do not
assemble in the presence of MMP inhibitors.13,35
Despite their MMP-independent formation, podosomes are
associated with degradation of ECM.12 Our data clearly demonstrate that Mk podosomes can degrade ECM proteins, and that on
fibrinogen this degradation is MMP and myosin IIA dependent.
Our results agree with other reports showing MMP and myosin IIA
involvement in podosome-driven degradation.11,16 Unfortunately,
we could not reliably detect podosome driven proteolysis of
collagen, because Mks seeded on a fluorescein-isothiocyanatelabeled gelatin substratum were not able to attach in a sufficient
number to allow quantification (data not shown). In addition, Alexa
488-labeled Horm collagen did not form a monolayer, which made
the quantification of degradation technically unreliable. However,
it is likely that Mks do possess collagenase activity, because it was
MEGAKARYOCYTE PODOSOMES REMODEL MATRIX
2551
shown by Lane et al48 that they express MMP-9, and our serial
analysis of gene expression database identified expression of
MMP-14, MMP-24, MMP-25, and MMP-9.48
For the first time in Mks, we observed abundant formation of
podosomes and actin-rich protrusions crossing a native basement
membrane. Furthermore, the crossing of these actin protrusions
was MMP dependent, implying the need for Mk-driven matrix
degradation. Strikingly, the loss of podosomes caused by inhibition
of the Arp2/3 complex or the deficiency in WASp resulted in a
significant reduction of the percentage of protrusions across the
basement membrane. Interestingly myosin IIA inhibition indicated
that myosin-mediated contractility played no role in protrusion
formation, suggesting that manipulation of the matrix through
contraction forces was not required.
Our data clearly link podosomes, degradation of ECM proteins,
and protrusions across a basement membrane. This link implies
that podosomes could contribute to effective delivery of platelets
into the bloodstream during proplatelet formation. As such, it is
tempting to hypothesize that the lack of podosomes in WiskottAldrich syndrome patients49 could contribute to the thrombocytopenia associated with this disorder and the accumulation of platelets
in the bone marrow of WASp2/2 mice.7 Our study illuminates the
role of podosomes in Mks and extends the knowledge of their
dynamics related to the underlying substrate. Further investigations
are needed to understand these complex structures and their role in
migration, adhesion, and proplatelet formation.
Acknowledgments
The authors thank David Strachan for help in design of ImageJ
plugins for image analysis, Margaret O’Prey and Kurt Anderson
and the Beatson Imaging facility for technical help, Gabriela Kalna
for help and advice with statistical analysis, and the Glasgow
Experimental Cancer Medicine Centre for human samples.
This work was funded by the British Heart Foundation (FS/09/
034/27756) (H.S., L.M.M., S.G.T., and S.P.W.), by a core grant
from Cancer Research UK (L.M.M.), by the Wellcome Trust and
GOSHCC (A.J.T.), by Cancer Research UK programme grant
C11074/A11008 (T.L.H.), and Cell sorting grant KKL501 (T.L.H.).
Authorship
Contribution: H.S. performed most of the experiments, wrote the
paper, and analyzed the data; S.C. performed some of the
experiments and wrote the paper; A.S., A.M., and M.V. performed
some of the experiments; J.M., C.L., and C.G. contributed vital
reagents; M.B. contributed vital reagents and performed some of
the experiments; A.J.T., T.L.H., and G.E.J. contributed vital
reagents and contributed to manuscript writing and study
conception; S.G.T. and S.P.W. contributed to manuscript writing,
study conception, and design; and L.M.M. conceived the study and
wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Laura M. Machesky, The Beatson Institute for
Cancer Research, Glasgow University College of Medical Veterinary and Life Sciences, Garscube Estate, Switchback Rd, Glasgow,
G61 1BD, UK; e-mail: [email protected].
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2552
BLOOD, 28 MARCH 2013 x VOLUME 121, NUMBER 13
SCHACHTNER et al
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2013 121: 2542-2552
doi:10.1182/blood-2012-07-443457 originally published
online January 10, 2013
Megakaryocytes assemble podosomes that degrade matrix and protrude
through basement membrane
Hannah Schachtner, Simon D. J. Calaminus, Amy Sinclair, James Monypenny, Michael P. Blundell,
Catherine Leon, Tessa L. Holyoake, Adrian J. Thrasher, Alison M. Michie, Milica Vukovic, Christian
Gachet, Gareth E. Jones, Steven G. Thomas, Steve P. Watson and Laura M. Machesky
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