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Macrophage mesenchymal migration requires podosome stabilization by Filamin A

Romain Guiet1,2,#

, Christel Vérollet1,2,#

, Isabelle Lamsoul1,2

, Céline Cougoule1,2

, Renaud

Poincloux1,2

, Arnaud Labrousse1,2

, David A. Calderwood3, Michael Glogauer

4, Pierre G.

Lutz1,2

, and Isabelle Maridonneau-Parini1,2,

*

1Centre National de la Recherche Scientifique (CNRS), Institut de Pharmacologie et de Biologie

Structurale (IPBS), Unité Mixte de Recherche 5089, 205 route de Narbonne, Toulouse, France. 2Université de Toulouse; Université Paul Sabatier, UPS, IPBS, Toulouse, France.

3 Department of Pharmacology and Cell Biology and Interdepartmental Program in Vascular Biology and

Therapeutics, Yale University School of Medicine, New Haven, CT, 06520, USA 4 CIHR Group in Matrix Dynamics, University of Toronto, Toronto, Ontario, Canada

* corresponding author : Isabelle.Maridonneau-Parini@ipbs.fr, Phone: 33- (0)5 61 17 54 58,

FAX number: 33- (0)5 61 17 59 94, # RG and CV equally contributed to this work

Running title: Filamin A regulates podosome stability and cell migration

Keywords: Filamin, podosome, Hck, macrophage, 3D migration

Background: Filamin A is an actin-binding

and scaffolding protein. Mutations in the filamin

A gene cause developmental anomalies in

humans.

Results: Filamin A is required for podosome

stabilization, podosome rosette formation,

extracellular matrix degradation and for three-

dimensional mesenchymal migration.

Conclusion: New functions are assigned to

Filamin A.

Significance: Identification of actors

involved in cell migration is crucial for

understanding human developmental disorders.

SUMMARY

Filamin A (FLNa) is a cross-linker of actin

filaments and serves as a scaffold protein

mostly involved in the regulation of actin

polymerisation. It is ubiquitously distributed

and null-mutations have strong consequences

on embryonic development in humans, with

organ defects which suggest deficiencies in

cell migration. We have previously reported

that macrophages, the archetypal migratory

cells, use the protease and podosome-

dependent mesenchymal migration mode in

dense 3D environments, whereas they use the

protease- and podosome-independent

amoeboid mode in more porous matrices.

Since FLNa has been shown to localise to

podosomes, we hypothesized that the defects

seen in patients carrying FLNa mutations

could be related to the capacity of certain cell

types to form podosomes. Using strategies

based on FLNa knock-out, knock-down, and

rescue, we show that FLNa: (i) is involved in

podosome stability and their organization as

rosettes and 3D-podosomes, (ii) regulates the

proteolysis of the matrix mediated by

podosomes in macrophages, (iii) is required

for podosome rosette formation triggered by

Hck and (iv) is necessary for mesenchymal

migration but dispensable for amoeboid

migration. These new functions assigned to

FLNa, particularly its role in mesenchymal

migration, could be directly related to the

defects in cell migration described during the

embryonic development in FLNa-defective

patients.

INTRODUCTION

Filamins are cytoskeletal proteins that

organize actin filaments into networks and link

these networks to cell membranes. Three

isoforms have been identified, Filamin A

(FLNa), the most abundant and widely expressed

isoform localizes to filopodia, lamellipodia,

stress fibers, focal contacts and invadosomes in

osteoclasts and tumour cells (1 ,2 ,3). Filamin B

is associated to stress fibers but does not

normally localize to focal contacts. Filamin C is

primarily expressed in muscle cells (4).

http://www.jbc.org/cgi/doi/10.1074/jbc.M111.307124The latest version is at JBC Papers in Press. Published on February 9, 2012 as Manuscript M111.307124

Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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FLNa is a cross-linker of actin filaments

which forms orthogonal branches, with the actin

networks behaving as weak elastic solids. Such

branches cannot be formed by other cross-linkers

such as alpha-actinin or temporary branching

proteins such as Arp2/3 (5). FLNa is also a

scaffolding protein which binds multiple partners

including membrane receptors, enzymes and

signalling intermediates. Many of these partners

are involved in the regulation of actin

polymerization and FLNa thus participates in

signal transduction related to F-actin

polymerisation and organization. Integrins

interact with FLNa, which mediates a link

between the cytoskeleton and the cell membrane

to control cell adhesion (3). Over 90 binding

partners of FLNa have been identified (4). As a

consequence, mutations in the FLNa gene can

result in a wide range of anomalies which

include cell adhesion and cell migration defects

(4-7). The role of FLNa in cell migration has

been recently emphasized in reviews (4,7),

underlining its essential function for embryonic

development, organ formation and homeostasis.

For example, it emerges that an appropriate level

of FLNa is required for migration of neuron

progenitors during embryonic development (4).

Indeed, in humans, inactivation of the FLNa

gene causes brain malformation and disrupts

directed neuronal migration (4,8). In addition, it

has recently been shown that FLNa is required

for monocyte 2D migration during in vitro

osteoclastogenesis (9). Conversely, cleavage of

FLNa by calpain has also been reported to

facilitate 2D cell migration, suggesting that the

role of FLNa in 2D migration could differ from

one cell type to another (1,7 ,10,11). In vivo, it

has been reported that the metastatic capacity of

FLNa knock-down tumour cells is modified (11).

How FLNa modulates cell migration, especially

in 3D environments, has not been elucidated yet.

In 3D-environments, macrophages use two

distinct migration modes, the amoeboid and

mesenchymal modes, depending on the

architecture of the matrix (12). The protease-

driven mesenchymal migration of macrophages

takes place in dense 3D environments and

involves podosomes (12,13). Macrophages and

monocyte/macrophage-derived cells (dendritic

cells and osteoclasts) constitutively form

podosomes when layered on extracellular matrix

(ECM) proteins, a 2D environment while, in

other cell types, podosomes can form transiently

(14 ,15 ,16). These actin-rich structures are

involved in integrin-mediated cell adhesion and

in proteolytic degradation of the ECM (16).

Interestingly, in cells cultured in 2D, FLNa has

been reported to be associated with podosome

structures in osteoclasts and with invadopodia, a

podosome counterpart, in tumour cells (1

,11,16). Thus, we decided to investigate whether

FLNa regulates the 3D migration process in

macrophages, the archetypal migratory cells, and

whether it is involved in podosome dynamics

and organization.

EXPERIMENTAL PROCEDURES

Antibodies and Reagents

Antibodies against human Filamin A

(hFLNa) clone PM6/317 were obtained from

Chemicon International (Temecula, CA). FLNa

antiserum against mouse Filamin A (mFLNa)

and ASB2 antiserum were described previously

(17 ,18). Rabbit polyclonal anti-Hck Abs (sc-72)

were from Santa Cruz Biotechnology (TEBU-

Bio, France), actin monoclonal Abs, anti-

vinculin Abs were from Sigma-Aldrich (Saint-

Quentin Fallavier, France). Secondary HRP-

conjugated Abs were from Bio-Rad (Hercules,

CA), secondary anti-mouse and anti-rabbit Abs

conjugated to Alexa 488 or Alexa 555 and

TexasRed/Alexa Fluor 488/Alexa Fluor 633 -

coupled phalloidins were from Molecular Probes

(Invitrogen, Cergy Pontoise, France). The

inhibitor of Src kinases SU6656 was purchased

from Sigma, recombinant IFN-γ from

Immunotools (Friesoythe, Germany).

Extracellular matrix proteins: Fibrinogen

(Sigma-Aldrich), Fibronectin (Sigma-Aldrich),

Vitronectin (Fisher scientific, Illkirch France) or

FITC-coupled gelatin (Invitrogen).

Cell culture

Human monocyte-derived macrophages

(MDM). Human monocytes were isolated from

the blood of healthy donors as previously

described (12). The culture medium RPMI 1640

(Invitrogen) containing 10% heat-inactivated

FCS, antibiotics and 20 ng/ml M-CSF

(PeproTech, Rock Hill, NJ) was renewed on the

third day of culture. MDM were used for

experiments at day 7 of differentiation. MDM

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were distributed on glass coverslips coated with

40µg/ml fibrinogen (Fg) as described (13). In

some experiments, 1 hour after plating, SU6656

was added for 30 minutes and cells were fixed in

PFA-sucrose and stained for

immunofluorescence microscopy (see below).

RAW264.7macrophages. RAW264.7

macrophages were cultured in RPMI 1640

containing 10% heat-inactivated FCS,

antibiotics, at 37°C in humidified 5% CO2

atmosphere. RAW264.7 cells were transfected

using the Amaxa electroporation system (19)

with the expression vectors encoding for EGFP,

EGFP-ASB2a-WT or EGFP-ASB2a-LA protein

(20), mouse Hck-shRNA (19), mouse FLNa

(mFLNa) shRNA (18) or human FLNa (hFLNa)

(21). Clones stably expressing shRNA against

FLNa (OpenBiosystem clone # V2HS_131780,

targeting sequence 5'-ggtgatcactgtggacactaa

tagtgaagccacagatgta ttagtgtccacagtgatcacc-3') or

against Hck (5'-ctagttccaaaaa ccgtatgcctcga

ccagataat ctcttgaattatctggtcgaggcatacggcggg-3'

designed and cloned as described in (19)) were

obtained by limiting dilution in parallel to

selection with puromycin and characterized by

western-blot analysis and podosome content.

Among the several clones obtained with similar

phenotypes, one, shown in the result section, was

chosen and used for rescue of mFLNa with

hFLNa. hFLNa-expressing clones were

generated by limiting dilution and selection with

geneticin. Two clones arbitrarily selected were

characterized by western-blot analysis and

podosome content and had similar phenotypes

(only one is shown). RAW264.7 cells were

seeded either on coverslips coated with 10µg/ml

vitronectin for 24 hours or on coverslips coated

with FITC-coupled gelatin and subsequently

coated with vitronectin for matrix degradation.

IFNγ was added to a final concentration of

100U/ml for 24h, cells were fixed using PFA-

Sucrose and stained for immunofluorescence

microscopy (see below).

Fibroblasts. Mouse Embryonic Fibroblasts

(MEF)-3T3 Tet-Off cell clones stably expressing

the constitutively active Hck isoforms, p59Hckca

and p61Hckca

in fusion or not with EGFP

(HckY/F501, Hckca

) have been previously

described (13,22). These cells optimally

expressed Hck after 7 days in doxycycline-free

culture medium. Hck-negative MEF-3T3 Tet-Off

were used as a negative control. MEF3T3

fibroblasts stably expressing p59/p61-Hckca

(-

EGFP) were seeded on glass coverslips and fixed

24 hours later.

NIH3T3 stably expressing FLNa shRNA or

Luciferase shRNA as a control were cultured as

described (18) in the presence of 1mM sodium

pyruvate. NIH-3T3 were seeded on fibronectin

coated coverslips, transfected with the p59/61-

Hckca

-EGFP expression vector using calcium

phosphate (23,24), fixed 12 hours later and

stained for F-actin.

Bone marrow derived macrophages. Mouse

bone marrow derived macrophages (BMDM)

were differentiated for 7 days as described (13).

The culture medium, RPMI 1640 containing

10% heat-inactivated FCS, antibiotics and 20

ng/ml M-CSF (Immunotools) was renewed on

the third day of culture. For immunofluorescence

microscopy experiments, cells were seeded on

fibronectin-coated coverslips. In some

experiments, BMDMs were transduced after 2

days of the differentiation process by adding

mCherry-Lifeact lentiviral vector (106 effective

viral particles for 106 macrophages) as described

(25). At day 7, cells were harvested and

embedded in Matrigel, plated in Lab-tek glass

base chambers and kept in a humidified

atmosphere at 37°C and 5% CO2 for at least 18

h. Cells were then visualized using a Zeiss 710

NLO microscope with a DPPS-laser 561nm

every 10min for 16 hours.

Gelatin-FITC degradation

For the matrix degradation assay, coverslips

were coated with 0.2mg/ml FITC-coupled

gelatin (13). Macrophages were seeded and 24h

later, fixed and stained. Dark areas in FITC

gelatin images were measured using the

threshold command of ImageJ software. The

degradation index was measured as described

previously (13).

3D Migration assay

For migration assays, thick layers of Matrigel

or 2.1 mg/ml Collagen I were polymerized in

Transwell inserts as previously described (12).

Macrophages were starved of serum for at least 2

h, harvested and seeded on top of the matrix at

3.104 cells/Transwell insert. Quantification was

performed as previously described (12). The

percentage of cell migration was obtained as the

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ratio of cells within the matrix to the total

number of counted cells.

Immunofluorescence microscopy

Cells were fixed with 3.7% paraformaldehyde

-150mM Sucrose, permeabilized with 0.1%

TritonX100 (Sigma-Aldrich) and stained with

primary antibodies: anti-hFLNa (1/1000) anti-

mFLNa (1/500) anti-Hck (1/200) and secondary

antibody anti-rabbit or anti-mouse conjugated to

Alexa 488 or Alexa 555 and phalloidin-Texas

Red or phalloidin-Alexa350 or phalloidin-

Alexa633 (1 U/ml). Slides were visualized with

a Leica DM-RB fluorescence microscope or

using a confocal microscope (Leica SP2). Image

stacks were collected using sequential scanning

and a standardized 120 nm z-sampling density.

Images

were processed for brightness and

contrast and filtered for

noise with Adobe

Photoshop, in compliance with the current

ethical

rules. In some experiments, the

fluorescence intensity of FLNa staining was

quantified in epifluorescence images acquired

with a Leica DM-RB microsocope as a function

of the cell size (only spread cells with a surface

above 350 µm2 were considered). For

quantitative analyses of F-actin and FLNa

staining shown in Fig 4A and B, podosomes

were segmented using the auto local threshold

function from Fiji software.

Measurement of podosome life-span.

RAW264.7cells were transfected with the

expression vector encoding for mCherry-Lifeact,

using the Amaxa® electroporation system. Cells

were layered onto vitronectin coated lab-tek

chambers and IFNγ (100U/ml) was added 4

hours later. After 24h, cells were imaged using

an inverted microscope (Leica DMIRB, Leica

Microsystems) equipped with a motorized stage

and an incubator chamber to maintain the

temperature and CO2 concentration constant.

Images were acquired with Metamorph software.

In each experiment, time-lapse images were

acquired every 15 sec in one z-plane over a 15-

30min period for 4–5 representative fields of

view per cell type. Quantification of podosome

life-span was measured manually using ImageJ

software for podosomes appearing and

disappearing during the time-course of the

experiment and results were expressed as the

mean ± SD of more than 50 podosomes from 10-

15 cells from three independent experiments.

Cells were screened visually before

measurement, and polarized cells were not taken

into account.

Western-blot

Proteins were separated with 5-8% SDS-

PAGE gels, proteins transferred on

nitrocellulose membranes and those were stained

with anti-hFLNa (1/10000), anti-mFLNa

(1/5000), anti-Hck (1/1000: Santa Cruz), anti-

actin (1/5000), anti-ASB2 Abs (1/5000), or anti-

phospho tyrosine Abs (4G10, 1/2000) revealed

by secondary horseradish peroxidase-coupled

Abs (1/10000). Signals were visualized with

enhanced chemiluminescence reagents

(Amersham) and quantified using Adobe

Photoshop CS3 software.

Statistical analysis Data are reported as means ± SD. Statistical

comparisons

between two sets of data were

performed with a unilateral Student’s unpaired t

test. Statistical comparisons

between three or

more sets of data were performed with ANOVA,

and a Tukey post-test. Statistical comparisons of

two sets of nominal values were performed with

Fisher’s exact test. Statistical comparisons of

three or more sets of nominal values were

performed with Chi-square test and Bonferonni

correction. *p < 0.05, **p< 0.01, and ***p<

0.001.

In vitro phosphorylation assay

h-FLNa was immunoprecipated as in (20).

Recombinant Hck (WT or KD) was produced in

E.Coli BL21(DE3)pLysS and purifed as

described in (26). h-FLNa was incubated (or not)

with Hck-Wt or Hck-KD in the presence of 1.5

mM ATP, 1.5 mM MgCl2,1.5 mM MnCl2 in

Hepes 100 mM at 30°C for 15 min, before

addition of Laemmli buffer for Western blot

analysis.

RESULTS

1. FLNa is involved in the mesenchymal

but not the amoeboid migration mode in

macrophages.

The migration capacity of bone-marrow

derived macrophages (BMDMs) from

conditional knock-out FLNa mice (9) was

analysed using transwells in which a thick layer

of Matrigel matrix was polymerized (12,13). In

dense, poorly porous matrices such as Matrigel,

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macrophages use the mesenchymal migration

mode (12). It is characterized by an elongated

and protrusive cell shape, and requires proteases,

adhesion proteins, the tyrosine kinase Hck and

formation of 3D podosomes, whilst the Rho-

Kinase (ROCK) is dispensable (12,13,25). As

shown in Fig 1, FLNa-/- BMDMs had a reduced

mesenchymal migration capacity in Matrigel

compared to wt macrophages (Fig. 1A).

In porous matrices such as fibrillar Collagen

I, macrophages migrate using the amoeboid

mode characterized by a rounded cell shape,

dependent on the Rho/ROCK signalling pathway

and independent of proteases and podosomes

(12,25). We observed that in fibrillar Collagen I,

the migration capacity of FLNa-/- BMDMs was

not affected (Fig. 1B).

During mesenchymal migration, and not for

the amoeboid mode, we have reported that 3D

podosomes are observed at the tip of cell

protrusions, where proteolytic degradation of the

matrix is undertaken to create paths for cell

migration (12,25). We thus examined the

formation of 3D podosomes in live FLNa-/-

macrophages embedded into Matrigel and

transduced with mCherry-Lifeact to stain F-actin

(25,27). As shown by video microscopy, the

formation of cell protrusions was strongly

affected when compared to wt BMDMs (Fig. 1C,

Supplemental movies 1 and 2).

To determine whether formation of

podosomes was affected in FLNa-/- BMDMs in

2D environments (layered on coverslips), we

examined podosomes by immunofluorescence

microscopy, in parallel to their capacity to

degrade the ECM, as assayed by gelatin-FITC

degradation. Podosomes can either be spread all

over the ventral face of adherent macrophages,

or limited to specific areas called clusters, or

organized as rosettes (22). Whilst, as expected

from our previous report (13), wt BMDMs

mostly organized their podosomes as rosettes,

FLNa-/- BMDMs had a defect in podosome

rosette formation (Fig. 1D and E) and in matrix

proteolysis (Fig. 1F-I).

Thus, these results show that FLNa-/- mouse

macrophages have a defect in podosomes

rosettes formation, matrix degradation and 3D

mesenchymal migration, which is podosome-

dependent.

2- FLNa is present at podosomes and

podosome rosettes in human macrophages

Having observed a defect of podosomes in

FLNa-/- mouse macrophages, we next examined

whether, in human macrophages, FLNa is

present at podosomes. Human macrophages

derived from blood monocytes (MDM) of

healthy donors, layered on fibrinogen, formed

individual podosomes (Fig. 2A, arrowhead), and

about 25% of the cells spontaneously organized

their podosomes as rosettes (Fig. 2A, arrow).

FLNa was observed at individual podosomes,

forming a ring around the F-actin core (Fig. 2A,

arrowhead), similarly to what has been described

for vinculin, talin and paxillin (16). Moreover,

FLNa accumulated at podosome rosettes where

it co-localized with F-actin (Fig. 2A, arrow and

2D). The αMβ2integrin (CD11b-CD18), the

major leukocyte fibrinogen receptor (28), and

Hck, a Src family tyrosine kinase specifically

expressed in phagocytes (29) which regulates the

organisation of podosomes as rosettes (13), were

also present at podosome rosettes (Fig. 2B-2D),

Thus, in human macrophages FLNa is present

at rings of individual podosomes. Furthermore, it accumulates with, 2-integrins and Hck at

podosome rosettes, suggesting that FLNa could

also play a role in these cell structures in human

macrophages.

3- Filamin A is involved in podosome

stability and podosome rosette formation

As a cross-linker of actin filaments and a

scaffold protein involved in the regulation of

actin polymerisation, FLNa might have a role in

the regulation of podosome stability and

lifespan, and in organization of podosomes as

rosettes.

Thus, different strategies were undertaken to

deplete FLNa: transient expression of ASB2α a

subunit of an E3 ubiquitin ligase complex which

targets FLNa for proteasomal degradation (20),

and stable expression of mouse FLNa shRNA

(18). For this, we used the macrophage cell line

RAW264.7 which is relatively easy to transfect.

When we looked at the localisation of

endogenous FLNa by immunostaining we found

that, like in human MDMs (Fig. 2), it was

present at the podosome ring and accumulated at

podosome rosettes (Fig. S1A), and we also

noticed that the FLNa fluorescence intensity was

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heterogeneous from one cell to another.

Interestingly, we found a positive correlation

between the FLNa fluorescence intensity

calculated in cells of similar size and the rate of

podosome formation and their organisation as

clusters and rosettes (Fig. S1B). A similar

correlation between the intensity of fluorescence

staining with FLNa antibodies and the presence

of podosomes and podosome superstructures was

obtained in human macrophages (data not

shown). In RAW264.7 macrophages expressing

GFP-ASB2α, the expression of FLNa was

decreased and the percentage of cells with

podosome rosettes was reduced compared to

control macrophages expressing GFP or the

ASB2α E3 defective mutant ASB2α-LA (Fig.

S2).

Similarly, in RAW264.7 macrophages stably

transfected to express an shRNA specific for the

mouse FLNa mRNA, we observed that: FLNa

was knocked-down by approximately 60%, the

percentage of cells with podosomes was

diminished, the density of F-actin in clouds (30)

surrounding remnant podosomes was reduced

(Fig. S1C) and podosome rosettes were absent

(Fig. 3A-C). Furthermore, the formation of

rosettes was rescued in those cells by expressing

human FLNa (Fig. 3A-C).

Next, we used time-lapse videomicroscopy to

analyze the role of FLNa on the lifespan of

podosomes in RAW264.7 macrophages

expressing both the anti-FLNa shRNA and the F-

actin binding peptide mCherry-Lifeact (27,31).

As shown in Fig. 3D, the podosome lifespan was

decreased in FLNa-depleted cells, and when

FLNa-depleted RAW264.7 macrophages were

complemented by human FLNa, podosome life-

span was restored.

These results indicate that FLNa plays a

critical role in podosome formation and/or

stability and is required for the organization of

podosomes into rosettes.

4- Hck and FLNa exhibit similar

properties on podosome stability,

organization and functionality.

In osteoclasts, the kinase activity of Src has

been involved in the control of podosome

lifespan (32). In macrophages, the organisation

of podosomes into rosettes has been shown to be

regulated by Hck, and Hck also plays a critical

role in the protease-dependent mesenchymal

migration (13). In addition, the Src family

tyrosine kinase Lck, which is specifically

expressed in lymphocytes, has been shown to

activate the actin cross-linking property of FLNa

(33). So we examined whether the formation of

podosome rosettes induced by Hck involves

FLNa.

We took advantage of a cellular model that

we had previously established to dissect the role

of Hck on podosome rosette formation. It

involves ectopic expression of constitutively

active Hck (Hckca

) in MEF-3T3 Tet-Off

fibroblasts (34). While MEF-3T3 fibroblasts are

unable to form podosomes spontaneously,

fibroblasts expressing Hckca

form podosome

rosettes with the classical “donut” shape

structure, where Hck, F-actin and FLNa

accumulated (Fig. 4A and B, (34)). When

NIH3T3 fibroblasts depleted in FLNa by stable

shRNA expression were transfected with the

Hckca

cDNA construct (18), almost no podosome

rosettes were formed (Fig. 4C-D). These results

indicate that, in the formation of podosome

rosettes, FLNa is either required in the Hck

signalling pathway or an essential component of

these cell structures.

In the next experiments, we investigated the

role of Src kinases in the organization of

podosomes and localization of FLNa. The effect

of SU6656, a broad inhibitor of Src kinases was

examined in human macrophages. We observed

that fewer macrophages formed podosomes and

podosome rosettes (Fig. 5A-B, quantified in C)

as expected from previous results obtained with

PP1, another inhibitor of Src kinases (34). While

FLNa was organized as a duct around the F-actin

core of podosomes in control human

macrophages (Fig 5A, see a’ and a’’), it was

partially removed from the remnant podosomes

in the presence of SU6656 (Fig 5B, see b’ and

b”). Rather, FLNa accumulated with F-actin as

disorganized patches at the cell periphery (Fig.

5B, b). This result suggests that the stability of

podosomes could require a proper FLNa

localization, which is controlled, at least in part,

by Src kinases.

To further examine the role of Hck in the

process of podosome formation, RAW264.7

macrophages in which Hck is specifically down-

regulated via stable transfection of shRNA were

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used (19). The percentage of cells with

podosomes was reduced (21.9+6.1% versus

65.3+13.5%, mean + SD, n=3) and cells formed

fewer podosome rosettes (1.1 + 0.6 versus 10.4 +

3.8%, mean + SD, n=3). Similarly to what had

been observed for FLNa (Fig. 3D), podosomes

also had a shortened life-time in shHck-treated

cells (Fig. 5D), indicating that, like FLNa, Hck is

involved in the stability of podosomes. As above

in the SU6656 experiments, FLNa was partially

removed from the ring of the podosome

remnants (data not shown). When Hck depleted

mouse macrophages were complemented by

human Hck, podosome life-span was restored

(Fig. 5D). In RAW264.7 macrophages

expressing shRNAs against either Hck or FLNa,

the degradation of the extracellular matrix was

found to be reduced (Fig. S3), most likely as a

consequence of the lower number of podosome

rosettes formed in those cells. Thus FLNa and

Hck exhibit similar properties on podosome

organization and function.

Finally, to determine whether Hck and FLNa

also share similar properties in the migration

mode of macrophages, the amoeboid migration

mode of Hck-/- BMDMs was examined. Indeed,

although we have previously reported that Hck-/-

BMDMs are defective in their mesenchymal

migration (13), we had not examined their

amoeboid migration. Thus, as performed in Fig.

1 for FLNa-/- BMDMs, we placed Hck-/- mouse

macrophages into fibrillar collagen I, and no

difference was observed between Hck-/- and wt

cells (23.0 + 5.7 versus 25.2 + 6.9, percentage of

migrating macrophages, n= 5).

Taken together, these results show that FLNa

and Hck i) are two critical components of a

signalling pathway leading to podosome rosette

formation and ECM degradation, ii) are required

for podosome stability and iii) are essential for

the protease-dependent mesenchymal migration

but dispensable for the amoeboid mode.

DISCUSSION

Patients carrying defective genes for FLNa

have a congenital malformation of the human

cerebral cortex called periventricular nodular

heterotopia (35,36). Although the precise

molecular mechanisms involved are not yet

understood, this pathology has been shown to

correlate with a defect in neuronal migration

during brain embryogenesis (7,37-40). The

results of the experiments reported here support

our working hypothesis that FLNa, which is a

well-known cross-linker of 3D actin filament

networks and a scaffolding protein, is involved

in cell migration, and more precisely in the

protease-dependent mesenchymal migration of

macrophages moving in 3D environments. We

also confirm that FLNa is present at podosomes,

which are cell structures involved in

mesenchymal migration via adhesion and matrix

degradation properties, and we have found that

FLNa regulates podosome stability, their

organization as rosettes and in 3D, and the

extracellular matrix degradation activity of

macrophages. All these properties are shared

with Hck.

Adhesion and migration of cells into tissues

are critical processes for organ development,

cell-mediated immunity and wound healing. In

vivo, cell migration takes place mostly in 3D-

environments which can markedly differ

between tissues because of different

composition, porosity, stiffness and viscoelastic

properties. In loose and porous environments

into which cells can glide and squeeze to find

their path in a protease-dispensable manner,

macrophages preferentially use the amoeboid

migration mode (12). In dense and poorly porous

matrices, however, they have to adhere to

proteins of the extracellular matrix via their

integrins and use the protease-dependent

mesenchymal mode to create their own path

(12,25,41,42).

The observation that FLNa-/- macrophages

have a reduced capacity for mesenchymal

migration while they migrate normally using the

amoeboid mode opens the way to the

identification of the molecular mechanisms

involving FLNa in cell migration. Although

FLNa has been described as a regulator of cell

migration in 2D environments (9), this is the first

report showing that FLNa is involved in 3D cell

migration.

In macrophages, proteases involved in matrix

degradation are delivered at podosomes (13,16)

and recently, we have shown that the presence of

podosome rosettes correlates with the

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mesenchymal migration of macrophages in 3D

environments but not with the amoeboid

migration mode (12,13,25 ). Using different

approaches to down-regulate FLNa, we show

that the life-span of podosomes, their

organization as rosettes, the formation of 3D

podosomes when macrophages are embedded

into the matrix and the proteolytic activity on the

extracellular matrix are all regulated by FLNa in

macrophages. These observations could explain

why the mesenchymal migration is specifically

altered in FLNa-/- macrophages. When

podosomes are organized as a rosette they form a

structure related to the osteoclast sealing zone

which exhibits an efficient degradative activity

on the extracellular matrix (13,22). The sealing

zone consists of an array of podosomes

communicating through a dense and

interconnected network of actin filaments

(43,44). Since FLNa is located at the ring of

individual podosomes and not at the core, and

since it regulates the F-actin density in the cloud

(this report), it could be involved in the increased

densification of the actin network

interconnecting podosome cores which occurs

during rosette formation (43). Paxillin and

vinculin were also found at rings and proposed

to cooperate in a force-transfer process to the

actomyosin complex (45). FLNa could be part of

that complex linking integrins to actin to

maintain the podosome structure and the

rosettes’ degrading and protrusive activities

when cells penetrate in a 3D environment. In

fact, in addition to actin filaments, FLNa binds a

large number of other proteins, many of which

such as integrins are key players in cell adhesion

and migration (4).

Similarly, Hck, a phagocyte-specific Src-

family tyrosine kinase, has been shown to be

involved in the formation of podosome rosettes,

in the proteolytic degradation of the matrix and

in mesenchymal migration, but has no apparent

role in the amoeboid migration mode and 2D cell

migration ((13), and this report). In fibroblasts

expressing constitutively active Hck ectopically,

the formation of podosome rosettes occurs

spontaneously, and this was inhibited by

knocking down the expression of FLNa. In

macrophages, podosome rosettes are also poorly

formed when Hck or FLNa are knocked-down,

in which case over-expressing Hck or FLNa can

rescue podosomes. The organization of

podosomes as rosettes triggered by Hck (13,34)

requires Rho, Rac and Cdc42 (34), three proteins

known to interact with FLNa and involved in the

process of actin polymerization (9,46 ).

Interestingly, defective activation of Rac, Cdc42

and Rho in monocytes from the FLNa

conditional knock-out mice used here has been

reported (9). Taking these findings together,

FLNa and Hck could belong to a common

signalling pathway involving Rho GTPases. The

observation that, in vitro, FLNa is

phosphorylated by Hck (Fig. S4) is consistent

with this hypothesis.

The amoeboid movement is a push-and-

squeeze type of migration which helps cells to

find their way into porous matrices (47). In

such cells, which are poorly adhesive since

integrins are not involved, amoeboid migration

is driven by RhoA/ROCK-mediated actomyosin

contractions (47). Although FLNa has been

reported to interact with ROCK (48), we found

no evidence that FLNa is involved in the

amoeboid movement, suggesting that this

property of FLNa is not critical for this

migration mode.

The interaction of FLNa with some of its

effectors is potentially regulated by mechanical

forces (7). In epithelial cells, interaction between

FLNa and β1 integrin forms a mechanosensitive

complex that can sense the tension of the matrix

bi-directionally and, in turn, regulate cellular

contractility/morphogenesis and respond to this

matrix tension (49). The mechanical forces

exerted on macrophages when they infiltrate a

dense matrix could be the mechanism directing

the cells to use mesenchymal rather than

amoeboid migration. In addition, the mechanical

forces exerted at the level of podosomes, where

integrins and FLNa accumulate, could be

contributing to the maintenance of these

structures.

In macrophages and monocyte-derived cells

such as dendritic cells and osteoclasts,

podosomes are constitutive cell structures, but

they can also form transiently in other cell types,

such as smooth muscle cells stimulated with

phorbol esters (14 ,50) or endothelial cells

stimulated with TGFβ (15). Interestingly, during

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the process of vascular repair which implicates

endothelial cell migration, formation of

podosome rosettes also occurs (51), and it has

recently been proposed that podosomes may play

a role during cell movement in embryogenesis

(52). If we consider this hypothesis, it is

conceivable that podosome defects could

account for the malformations of brain, blood

vessels and several other organs observed in

FLNa-null organisms (7,53). Conversely, since

no particular immune disorder has been

described in FLNa-null organisms, the partial

(about 50%) defects in macrophages

mesenchymal migration does not appear

sufficient to initiate immune troubles. In support

of this, Hck-/- mice, which also exhibit partial

defects in mesenchymal macrophage migration

(13), have no apparent immune disorder either.

In contrast to the innate immune response which

involves huge numbers of phagocytes moving

towards infectious sites, cell migration during

embryogenesis generally involves a limited

number of cells moving at a precise time of the

embryonic development. Under these conditions,

migration defects might be more harmful for the

organism.

In conclusion, FLNa and Hck are the only

two proteins described to date as being involved

in mesenchymal migration of macrophages and

not in their amoeboid mode. Both proteins

regulate the stability of podosomes and their

organization as rosettes which are cell structures

involved in mesenchymal movement. In contrast

to Hck which is only expressed in myeloid cells,

FLNa is ubiquitously distributed and null-

mutations have strong consequences on

embryonic development. Our data thus strongly

support the hypothesis that FLNa could be

involved in mesenchymal migration of

embryonic cells, which could in turn explain, at

least in part, the organ defects observed in FLNa

null patients.

Acknowledgements

This study was supported in part by ARC #2010-

120-1733, ARC Equipement # 8505, ANR 2010-

01301. RG was supported by Fondation pour la

Recherche Médicale, CV by Sidaction and DAC

by the NIH (GM068600). We gratefully

acknowledge Clifford A Lowell (UCSF) for

kindly providing Hck-/- mice, Etienne Joly for

critical reading of the manuscript and the TRI

imaging facility.

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FIGURE LEGENDS

Figure 1. FLNa -/- BMDM have decreased abilities to perform 3D mesenchymal migration,

to form podosome rosettes and to degrade Gelatin-FITC.

BMDM from WT or FLNa-/- mice were seeded on thick layers of matrices of Matrigel (A) or

fibrillar collagen I (B), and the percentage of cells migrating into the matrices was quantified

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(mean + SD of four independent experiments). Pictures of WT (a’ and b’) and FLNa-/- cells (a’’

and b’’) migrating respectively in Matrigel (a’ and a’’) and fibrillar collagen I (b’ and b’’) (z is

the depth value of cells in the focal plan marked by an arrow). (C) FLNa-/- BMDM embedded

into Matrigel are defective in forming cell protrusions (see supplemental movie 1 and 2). (scale

bar = 10µm). (D) BMDM from WT or FLNa-/- mice were seeded on fibronectin-coated

coverslips for 16 hours and stained for F-actin. The arrow points to a typical podosome rosette.

(E) Quantification of cells making podosome rosettes in conditions of panel D (arrow) (mean +

SD of four independent experiments). (F) Macrophages seeded on coverslips coated with gelatin-

FITC for 16 hours were then stained for F-actin. The arrow points to a non-degrading cell, the

dotted lines show the degraded areas. (G) Quantification of cells degrading the matrix, in

conditions of panel F (mean + SD of four independent experiments). (H-I) Quantification of the

surface of degraded gelatin-FITC per cell surface, expressed as the mean area degraded per cell

(µm2) in H, and the percentage of degraded area per cell surface in I, in condition of panel F

(mean + SD of four independent experiments, at least 25 cells were quantified per condition).

Figure 2. FLNa is localized at podosomes and accumulates with integrin and Hck at

podosome rosette of human MDMs.

MDMs plated on fibrinogen were stained for microscopy observation for (A) FLNa and F-actin

or (B) CD11b and F-actin, or (C) Hck and F-actin; inserts are magnification of areas depicted by

the white squares. (scale bar = 10µm). (D) Normalized fluorescence intensity profiles along the

white dotted line in panels A, B and C. hFLNa was observed at individual podosomes forming a

ring around the F-actin core (A, arrowhead) and at podosome rosettes (A, arrow), where CD11b

(B, arrow) and Hck (C, arrow) also accumulated.

Figure 3. Macrophages with inhibited FLNa expression have a defect in podosome and

podosome rosettes formation.

(A) RAW264.7 macrophages (Control) or RAW264.7 cells stably expressing shRNA against

mFLNa, and those rescued with stable expression of hFLNa were all seeded on vitronectin-

coated coverslips and treated with -IFN to enhance the formation of podosomes, fixed and

stained for F-actin and FLNa. Cells counted as forming podosomes and podosome rosettes shown

by an arrow presente numerous actin dots (≥5) and rosettes (≥1), respectively (scale bar = 10µm).

(B) Quantification of cells with podosomes or podosome rosettes (mean + SD of three

independent experiments). (C) Western blot against hFLNa, mFLNa, and actin; and

quantification of mFLNa in 3 experiments. (D) RAW264.7 macrophages control or transfected

with mFLNA shRNA alone, or together with a vector expressing hFLNA were transiently

transfected with mCherry-Lifeact to reveal F-actin. The lifespans of podosomes were then

evaluated using time-lapse microscopy, and are plotted for each cell type (mean + SD of three

independent experiments, 5 to 10 podosomes analysed per cell in at least 3 cells per experiment).

Figure 4. The formation of podosome rosettes triggered by expression of Hckca

in

fibroblasts requires FLNa. MEF3T3 fibroblasts and MEF3T3 fibroblasts expressing human

p59Hckca

/p61Hckca

were stained for mFLNa and F-actin and observed by confocal microscopy.

(A) Control cells did not form podosome rosettes while the cells expressing p59 Hckca

/p61Hckca

showed FLNa accumulation at podosome rosettes (arrowheads). (B) MEF3T3 fibroblasts

expressing p59Hckca

/p61Hckca

-GFP and stained for FLNa and F-actin showed accumulation of

Hck and FLNa and F-actin at podosome rosettes (arrowheads). Fluorescence intensity profiles

along the white dotted line are shown. (C) Expression of p59Hckca

/p61Hckca

-GFP in NIH3T3

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stably expressing shRNA against luciferase (control) or against mFLNa were stained for F-actin

(scale bar = 10µm). (D) Quantification of p59Hckca

/p61Hckca

-GFP expressing cells forming

podosome rosettes (mean + SD of three independent experiments).

Figure 5. Src kinase activity is required for podosome formation and for FilaminA

localisation to podosomes.

(A) MDM plated on fibrinogen and (B) treated with SU6656 were stained for hFLNa and F-

actin, before acquisition of confocal micrograph series (z-step=0.1µm) (scale bar = 10µm). (a)’

and (b’) show the average of the F-actin and FLNa fluorescence staining of at least 100

podosomes from control and SU6656 treated cells (scale bar = 1µm). (a”) and (b”) show

fluorescence intensity profiles of the averaged podosomes along the white dotted line in (a’) and

(b’), respectively. (C) Quantification of human MDM with podosomes or podosome rosettes

when seeded on coverslips that were either uncoated or coated with fibrinogen (Fg), and treated

with the Src inhibitor SU6656 (mean + SD of three independent experiments). (D) RAW264.7

macrophages or stably expressing a shRNA against mouse Hck were (or not) transiently

transfected with a human Hck-GFP coding vector (to rescue Hck) and were transiently

transfected with Lifeact-mCherry coding vector to stain F-actin. Podosome lifespans, measured

by time-lapse microscopy, are plotted for each cells type (mean + SD of three independent

experiments, 5 to 10 podosomes analysed per cell in at least 3 cells per experiment).

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Figure 5Guiet et al

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Maridonneau-PariniArnaud Labrousse, David A. Calderwood, Michael Glogauer, Pierre G. Lutz and Isabelle Romain Guiet, Christel Verollet, Isabelle Lamsoul, Celine Cougoule, Renaud Poincloux,Macrophage mesenchymal migration requires podosome stabilization by Filamin A

published online February 9, 2012J. Biol. Chem. 

  10.1074/jbc.M111.307124Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2012/02/09/M111.307124.DC1

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