ARTICLES
Rapid leukocyte migration by integrin-
independent flowing and squeezing
Tim La
¨
mmermann
1
, Bernhard L. Bader
3
, Susan J. Monkley
4
, Tim Worbs
5
, Roland Wedlich-So
¨
ldner
2
, Karin Hirsch
1
,
Markus Keller
3
, Reinhold Fo
¨
rster
5
, David R. Critchley
4
, Reinhard Fa
¨
ssler
1
& Michael Sixt
1
All metazoan cells carry transmembrane receptors of the integrin family, which couple the contractile force of the
actomyosin cytoskeleton to the extracellular environment. In agreement with this principle, rapidly migrating leukocytes use
integrin-mediated adhesion when moving over two-dimensional surfaces. As migration on two-dimensional substrates
naturally overemphasizes the role of adhesion, the contribution of integrins during three-dimensional movement of
leukocytes within tissues has remained controversial. We studied the interplay between adhesive, contractile and protrus ive
forces during interstitial leukocyte chemotaxis in vivo and in vitro. We ablated all integrin heterodimers from murine
leukocytes, and show here that functional integrins do not contribute to migration in three-dimensional environments.
Instead, these cells migrate by the sole force of actin-network expan sion, which promotes protrusive flowing of the leading
edge. Myosin II-dependent contraction is only required on passage through narrow gaps, where a squeezing contraction of
the trailing edge propels the rigid nucleus.
The current model of metazoan cell migration is frequently described
as a multistep cycle: F-actin polymerization at the cell front pushes
out a membrane protrusion that subsequently becomes anchored to
an extracellular substrate by transmembrane receptors of the integrin
family. Integrins are dynamically coupled to the cytoskeleton and
transduce the internal force that is generated when myosin II con-
tracts the actin network. Contraction imposes retrograde pulling
forces on the integrins, which in turn facilitates forward locomotion
of the cell body
1–3
. The mammalian integrin family consists of 24
different functional heterodimers with individual binding specifici-
ties for cellular and extracellular ligands
4
. The integrin repertoire of
each cell type defines which substrate it can use for this ‘haptokinetic’
(adhesion driven) mode of migration. Such intimate linkage between
substrate-specific adhesion and migration restricts the migrating
cells to preformed pathways, and thereby creates the determinism
that is essential for many of the precise cell trafficking and positioning
processes underlying compartmentalization and patterning during
development and regeneration. As all mammalian cells with the
exception of erythrocytes carry integrins on their surface, it is rea-
sonable to assume that haptokinesis is a universal phenomenon
4,5
.
Leukocytes are outstanding cells, as they are scattered throughout
the body and have the potential to infiltrate any type of tissue. Rather
than following exact routes and being restricted to specific compart-
ments, leukocytes frequently undergo stochastic swarming with single
cell migration velocities that are up to 100 times faster than mesench-
ymal and epithelial cell types
6,7
. What makes leukocytes so quick and
flexible? In contrast to slow cells, migrating leukocytes undergo fre-
quent shape changes and were therefore morphologically described as
‘‘amoeboid’’
8
. How these shape changes relate to actual locomotion is
poorly investigated, and it is currently unknown if amoeboid migra-
tion represents a specialized strategy or just an accelerated variant of
the above introduced migration paradigm that is only well established
for slow cells. We therefore studied the inter-dependency of adhesion,
protrusion and contraction in inflammatory cells. We demonstrate
that integrin-mediated adhesion is only necessary to overcome tissue
barriers like the endothelial layer, while interstitial migration is auto-
nomous from the molecular composition of the extracellular environ-
ment. Such adhesion-independent migration is driven by protrusive
flowing of the anterior actin network of the cell and supported by
squeezing actomyosin contractions of the trailing edge to propel the
rigid nucleus through narrow spaces.
Dendritic cells migrate without integrins
As a model system to study interstitial leukocyte migration in vivo,we
focused on dendritic cells (DCs), phagocytes that reside within peri-
pheral tissues such as skin. They become activated upon wounding or
infection, sample antigens and quickly migrate via the afferent lymph-
atic vessels into the draining lymph node where they act as antigen
presenting cells
9
. DC migration is primarily guided by the two chemo-
kines CCL19 and CCL21; these are expressed in lymphatic endothe-
lium and the lymph nodes’ T-cell area, and bind the CC-chemokine
receptor 7 (CCR7), which is upregulated on DCs upon activation
10
.
Flow cytometric analysis of DCs generated from mouse bone-
marrow-derived stem cells revealed expression of the b
1
, b
2
, b
7
and
a
v
integrin families (Fig. 1a). To investigate the role integrins play in
DC migration in vivo, we used a combinatorial mouse genetics
approach to delete all integrin heterodimers from the surface of
DCs, thereby generating phenotypically normal pan-integrin-
deficient (integrin
2/2
) DCs (Fig. 1a, see Supplementary Material,
Methods, and Supplementary Figs 1, 2, 4c). We co-injected 1:1 mix-
tures of differentially labelled integrin
2/2
and wild-type DCs into the
dermis of mouse footpads and quantified their arrival in T-cell areas
of the draining lymph nodes. Surprisingly, wild-type and integrin
2/2
DCs arrived and localized in the T-cell area in an indistinguishable
manner (Fig. 1b, c, Supplementary Fig. 3), whereas CCR7
2/2
DCs,
which cannot interpret the directional information, failed to enter
the lymph node (Fig. 1c) as previously described
10
. We next targeted
integrin functionality indirectly by deleting the talin1 gene in DCs
1
Department of Molecular Medicine,
2
Junior Research Group Cellular Dynamics and Cell Patterning, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
3
Department
of Nutritional Medicine, Technische Universita
¨
tMu¨nchen, Munich, 85350 Freising, Germany.
4
Department of Biochemistry, University of Leicester, Leicester LE1 9HN, UK.
5
Institute
of Immunology, Hannover Medical School, 30625 Hannover, Germany.
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(Supplementary Fig. 4a). The interaction of talin1 with integrin
cytoplasmic domains is required for integrin activation, ligand bind-
ing and coupling of F-actin to adhesion sites
11
. Accordingly, talin1
gene ablation in DCs did not affect the levels of integrin expression,
but completely abolished their ability to bind ligands (Supple-
mentary Fig. 4b–d). Again, arrival of talin1
2/2
DCs in lymph nodes
did not significantly differ from wild-type DCs (Fig. 1c).
To allow for a detailed comparison of the cellular dynamics of
wild-type and integrin
2/2
DCs within the different physiological
environments of their migration route, we employed two in situ
imaging approaches (Fig. 2). Representing the starting point of DC
migration, the interstitial space of the dermis is dominated by fibrillar
arrays of collagen bundles
12
. Using a newly developed set-up of ex vivo
live cell imaging within explanted ear dermis, we could directly visu-
alize DCs moving towards and entering the afferent lymphatic vessels
by wide-field microscopy (Fig. 2b, c, Supplementary Fig. 5). Single
cell tracking revealed no difference in the migratory behaviour of
wild-type, integrin
2/2
and talin1
2/2
DCs (Fig. 2c, d).
The lymph node constitutes a fundamentallydifferent environment
for cellular migration, as it contains almost no freely accessible extra-
cellular matrix molecules but is densely packed with lymphocytes and
stroma cells
13,14
. To analyse the dynamics of intranodal DC migration
from the subcapsular sinus towards the T-cell area, we employed a
set-up of intravital two-photon microscopy of the popliteal lymph
nodes
15
. Evaluating a range of motility parameters, including cell
velocity, directional persistence, mean square displacement, motility
coefficients and morphology, we found that the movement behaviour
of wild-type and integrin
2/2
DCs was indistinguishable (Fig. 2e, f,
Supplementary Videos 1–3, data not shown).
2D but not 3D migration is integrin-dependent
On their way into the lymph node, DCs do not cross significant tissue
barriers, such as continuous endothelial or epithelial linings
12
.As
these processes have been shown to be integrin-dependent
16
, we also
tested for extravasation of integrin
2/2
leukocytes from the blood
stream into the inflamed dermis, and found that it was indeed abo-
lished (Fig. 2g, Supplementary Fig. 6). This finding corroborates the
extravasation model in which integrin-mediated tight immobiliza-
tion of leukocytes to the luminal endothelial surface is necessary to
counteract the shear forces imposed by the blood flow
16
. In line with
this in vivo finding, we found that integrin
2/2
DCs were entirely
unable to adhere to and migrate on two-dimensional (2D) substrates.
Gravity was obviously not sufficient to confine the non-adherent cells
to planar surfaces and allow the transduction of traction forces
b
a
1.0
0.5
0.0
48 h
WT DC
Itg
–/–
DC
Laminin
CCR7
–/–
Itg
–/–
DC
WT
DC
β
1
β
2
β
3
β
7
α
4
α
5
α
L
α
M
α
v
α
X
Itg
–/–
Tln1
–/–
32 h 48 h 48 h32 h
c
Ratio of –/– DC in
T cortex
B
B
T
B
Figure 1
|
Migration of integrin-deficient dendritic cells into lymph nodes.
a
, Flow cytometric analysis of integrin subunits of wild-type (WT) and
integrin-deficient (Itg
2/2
) dendritic cells (DCs) (integrin, unshaded; isotype
control, grey-shaded).
b, c, In vivo migration of DCs after subcutaneous
injection into hind footpads of WT mice. At the time points indicated,
arrival of differentially labelled DCs in the lymph node was analysed.
b, Localization of DCs after 48 h. Composite of six separate images of a single
lymph node. Scale bar, 200 mm; B, B-cell follicle; T, T-cell cortex.
c, Quantitative histology. Dotted line at 0.5, 50% of DCs that migrated into
the lymph node are knockout DCs; Tln1, talin1. Red dots indicate single
experiments (1 lymph node); box shows median, 25%, 75% percentile;
whiskers show minimum, maximum.
T
B
B
E
LN
LV
BV
D
LYVE-1
DC
Speed (µm min
–1
)
F
2,303
= 2.1, P = 0.13
WT DC
Itg
–/–
DC
t
129
= 1.35,
P = 0.18
Granulocytes
per mm
2
*
*
H
2
= 13.3, P = 0.001
Interstitial migration within skin and entry into lymphatics
Migration within lymph node
Extravasation from blood vessel
a
g
c
WTItg
–/–
Tln1
–/–
b
d
2
4
6
8
Tln1
–/–
0.76
±0.13
Itg
–/–
0.76
±0.17
WT
0.79
±0.14
DP:
0
0
2,000
4,000
6,000
8,000
Tln1
–/–
Itg
–/–
WT
e
2
4
6
8
Speed
(µm min
–1
)
f
0.41
±0.15
Itg
–/–
0.41
±0.18
WT
DP:
0
DCLYVE-1 LYVE-1/DC
Figure 2
|
Integrin-independent
interstitial leukocyte migration
in vivo.a
, Scheme of the dendritic
cell (DC) migration path from the
skin via lymphatic vessels to the
lymph node; B, B-cell follicle; BV,
blood vessel; D, dermis; E, epidermis;
LN, lymph node; LV, lymphatic
vessel; T, T-cell cortex.
b–d, Migration of DCs within the
dermis of ear explants.
b, Tracks of
DCs (red) entering LYVE-1
1
LVs
(green).
c,Confocalimagesofwhole
mount ear explants 2 h after addition
of DCs (red). DCs locate within the
lumen of LVs; all scale bars, 50 mm.
d, Speed and directional persistence
(DP, mean 6 s.d.) of single cells (dots)
for wild-type (WT), integrin
2/2
(Itg
2/2
)andtalin1
2/2
(Tln1
2/2
)
DCs. Red line, mean.
e, f, Intravital
two-photon microscopy.
e,3D
tracking of WT (green, blue line) and
Itg
2/2
(red, yellow line) DCs
migrating in the interfollicular areas.
f, Quantification of the average speed
and DP (mean 6 s.d.) of tracked
single cells (dots). Red line, mean.
g, Quantification of granulocytes
that extravasated from blood into ear
dermis (n 5 6 for each mouse line).
Red line, median. *P , 0.05 (post hoc).
ARTICLES NATURE
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(Fig. 3a, b, Supplementary Fig. 4d, Supplementary Video 4). We
conclude that extravasation requires integrin-mediated adhesion.
To better mimic the interstitial microenvironment, we established
chemotaxis assays within artificial three-dimensional (3D) matrices
of fibrin (a ligand for b
2
and b
3
integrins) and collagen I (a ligand for
several members of the b
1
integrin subfamily). In this system, DCs
persistently migrated along soluble gradients of CCL19 and showed
amoeboid morphology and velocities that were comparable to our
(and previously published
17
) in vivo observations. In agreement with
our in vivo data and in contrast to 2D migration, integrin
2/2
and
talin1
2/2
DCs migrated with speed and directional persistence that
were indistinguishable from wild-type cells (Fig. 3c, Supplementary
Fig. 7a, Supplementary Video 5). We also observed integrin-
independent migration for chemotaxing B cells and granulocytes,
suggesting a broader prevalence of this functional principle (Fig. 3d,
Supplementary Fig. 7b).
Functional dissociation of front and back
To mechanistically understand how cells can move in the absence of
integrin-mediated traction forces, we compared the integrin-
dependent movement pattern of fibroblasts with that of leukocytes
and found a striking difference. Leading edge protrusions of
fibroblasts translocated collagen fibres towards the cell body, indi-
cating the presence of rearward pulling forces and demonstrating
that protrusion, adhesion and contraction are tightly coupled in this
cell type (Supplementary Video 6)
18,19
. In contrast, chemotaxing DCs
protruded without signs of anterior pulling forces while the trailing
edge displayed an irregularly alternating contractile pattern. Con-
traction was characterized by shrinkage of the cell rear with con-
comitant forward-streaming of cytoplasmic matter (Supplementary
Video 7). When we dynamically visualized activated myosin II, the
motor system mediating contraction
1,2,20
, by time-lapse imaging of
DCs expressing a myosin light chain–GFP fusion protein, we found
accumulation at the cell rear during contractile phases (Fig. 4a,
Supplementary Fig. 8a, Supplementary Video 8). During non-
contractile phases, the trailing edge remained motionless and
appeared to be passively dragged by the protruding cell front
(Supplementary Video 7). These observations suggest that in
contrast to slow moving cells, protrusion and contraction are
spatio-temporally dissociated in leukocytes.
Receptor-mediated force transduction can only support forward
movement if retrograde (contractile) but not anterograde (protrus-
ive) forces are coupled to the environment
2,3
. To address how
actomyosin contraction functionally contributes to leukocyte loco-
motion, we pharmacologically inhibited myosin II or its upstream-
activator, Rho kinase. Irrespective of the presence of integrin
function, either treatment severely reduced migration speeds of
DCs, granulocytes and B cells (Fig. 4b, Supplementary Fig. 8e, data
not shown). Nevertheless, the chemotactic gradient was still able to
polarize the cell population. The leading edges of cells remained
dynamic and protruded with normal speed towards the chemokine
source. However, the cells were unable to move their trailing edges
ICAM-1
No. of adherent cells
Speed (µm min
–1
)
ICAM-1
H
2
=12.3
P<0.001
H
2
=16.3
P<0.001
F
2,9
=2.8, P=0.12
t
64
=0.6,
P=0.53
t
65
=1.6,
P=0.11
*
*
*
*
FN
WT
0
400
800
2D 3D
a
bc d
2
0
DP:
4
8
10
6
Speed (µm min
–1
)
4
0
12
16
8
Itg
–/–
Tln1
–/–
WT
WT
0.81
± 0.09
0.78
± 0.08
0.76
± 0.1
Itg
–/–
Tln1
–/–
WT
Itg
–/–
Tln1
–/–
Itg
–/–
WT
Granulocyte B blast
Itg
–/–
WT Itg
–/–
Tln1
–/–
Figure 3
|
Integrin-independent leukocyte migration in 3D networks
in vitro.a
, 2D adhesion versus 3D migration of DCs. b, 2D adhesion on
ICAM-1 and fibronectin (FN) 1 h after LPS stimulation. Top, quantification
of the number of adherent cells (ICAM-1, n 5 6; FN, n 5 8 for each
experiment). Bars, median values with interquartile range, *P , 0.05 (post
hoc). Bottom, morphology of wild-type (WT), integrin
2/2
(Itg
2/2
) and
talin1
2/2
(Tln1
2/2
) DCs. c, d, Velocities of chemotaxing leukocytes in 3D
collagen matrices.
c, Top, DCs (4 experiments per group); d, granulocytes, B
cells. Dots, single cells; red line, mean.
c, Bottom, single cell tracks of
chemotaxing DCs; values indicate mean DP 6 s.d.
CCL19
CCL19
CCL19 CCL19 CCL19 CCL19
– Bleb
H
2
= 184.2, P < 0.001
T = 378
P < 0.001
T = 300
P < 0.001
*
*
R
e
ar
Front
R
e
ar
Front
Bleb
Speed (µm min
–1
)
Blebbistatin
WTItg
–/–
Blebbistatin
Y27632 Y27632
8
6
4
2
0
Speed (µm min
–1
)
6
4
2
0
a
b
d
c
0:30 0:35 0:45
Y27632
Bleb
0:00
0:00
0:00 1:00 3:10 5:00 6:40 8:10 10:00
0:00 1:00 3:10 5:00 6:40 8:10 10:00
1:50 3:00 5:00 6:40 8:10 10:00
3:00 5:00 6:40 8:10 10:001:50
0:55 1:05 1:10
MLC–GFP
intensity
Figure 4
|
Myosin II-dependent nuclear squeezing at the cell rear.
a
, Sequence of a myosin light chain (MLC)–GFP expressing wild-type (WT)
dendritic cell (DC) migrating towards CCL19 in a 3D collagen gel; upper
row, differential interference contrast (DIC) microscopy; lower row,
MLC–GFP intensity profiles (encoded in pseudo-colours with red
representing highest levels). Time in min:s.
b, Left, velocities of single DCs
(dots; red line, median), and right, DC morphology (coloured red, DIC
microscopy), chemotaxing towards CCL19 in 3D collagen gels upon
pharmacological inhibition.
c, Speed of cell bodies (rear) versus cell
protrusions (front) of DCs in the presence of inhibitor; red line, median.
Bleb, blebbistatin; *P , 0.05 (post hoc).
d, e, Time-lapse sequence of a WT
(
d) and integrin
2/2
(e) DC either with (lower row) or without (upper row)
blebbistatin; DIC microscopy, nuclei (green). Time in min:s; scale bars, 5 mm
(
a), 10 mm(b, d, e).
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(Fig. 4b–e, Supplementary Fig. 8b–d, Supplementary Video 9). This
functional dissociation between front and back caused up to 30-fold
cell elongation, and demonstrates that the leading edge migrates
autonomously and without a need for receptor-mediated coupling
of contractile forces to the extracellular matrix.
Trailing edge contraction propels the nucleus
How does trailing edge contraction contribute to cell body loco-
motion? In fibroblasts and leukocytes moving on 2D substrates,
actomyosin contraction at the back is required to disassemble recep-
tor binding-sites and subsequently retract the membrane
2,21,22
.
Hence, blocking contraction causes membrane tethers at the trailing
edge. Consistent with an adhesion-independent migration mode, we
could not observe membrane tethers in myosin II-inhibited leuko-
cytes migrating in 3D gels. We considered that the elongated pheno-
type with its rounded back was caused by the inability to move an
internal resistance through narrow gaps within the gel. As the nucleus
is the least elastic cellular compartment
23
, we visualized DNA within
chemotaxing DCs and granulocytes. In untreated cells, we observed
continuous shape changes of nuclei, indicating deformation forces,
while upon myosin II inhibition, spherical nuclei were immobilized
at the rear ends of the cells (Figs 4d, e, Supplementary Fig. 8b–d,
Supplementary Videos 10, 11).
Protrusion drives basal locomotion
Although ‘locked’ nuclei caused migration arrest in most cells, a few
still showed locomotion. This residual migration was often charac-
terized by cell body elongation and dragging of the nucleus (Fig. 5a,
Supplementary Video 12). Such non-contractile movement was in
line with our previous observations showing motile phases in the
absence of trailing edge contractions. We hypothesized that such
myosin II-independent migration might occur in areas of the col-
lagen gel where external resistance was low due to increased spacing
of the collagen fibres. To establish the relationship between internal
contractile force and external resistance, we analysed DC chemotaxis
in collagen gels of varying fibre spacing (Fig. 5b). In all collagen
densities, myosin II-inhibited DCs were slower than untreated cells
but importantly, they ‘caught up’ at lowest gel densities (Fig. 5b,
Supplementary Video 13). A comparison of instantaneous velocities
further showed that myosin II inhibition did not prevent DCs from
reaching the same peak values as untreated cells (Supplementary
Fig. 9a–c).
We conclude that, in contrast to the ‘blebbing’ model of amoeboid
cell migration
24
, cortical actin network contraction does not mediate
locomotion itself but rather facilitates a protrusive mode of migra-
tion in confined environments where protrusion alone is unable to
act against counter-forces. So in constricted areas, the cell overcomes
internal and external resistance (rigidity of the nucleus and fibre
density) by contraction (myosin II). Contractile force deforms the
resistance and facilitates a purely protrusive mode of migration.
To challenge the concept of protrusive movement, we treated che-
motaxing DCs with latrunculin B, which interferes with actin poly-
merization by depleting the pool of available functional G-actin
monomers
25
. We found that this treatment reduced cell migration
velocity in a dose-dependent manner (Fig. 5c). At intermediate con-
centrations, the trailing edge remained contractile and the nucleus
was ‘pushed’ to the cell front (Fig. 5d, Supplementary Video 14).
Consistent with a model where protrusion determines the speed of
cell migration while contraction is only activated to overcome
external resistances, the speed reduction of latrunculin B-treated cells
was independent of gel density (Fig. 5b, c).
Discussion
We show that leukocytes migrate in the absence of specific adhesive
interactions with the extracellular environment. This subversion of
the metazoan principle makes them autonomous from the tissue
context, and allows them to quickly and flexibly navigate through
any organ without adaptations to alternating extracellular ligands.
This strategy is in stark contrast to the haptokinetic migration prin-
ciple that leads along preformed pathways and therefore promotes
deterministic positioning. Adhesion independency better suits stoch-
astic movement or chemotaxis where cells randomly migrate or fol-
low soluble cues. Astonishingly, the protrusive flow of F-actin
appears sufficient to drive rapid leukocyte locomotion in environ-
ments with large pore-size. This resembles the locomotion principle
of nematode sperm cells that is entirely driven by treadmilling poly-
mers of major sperm protein
26
. Only in narrow areas do leukocytes
activate the contractile module to squeeze and propel the internal
resistance of the nucleus in a manner resembling neuronal
nucleokinesis
27
. This ‘flowing and squeezing’ migration model
fulfils a central requirement for immune cell movement: the pericel-
lular environment is transiently deformed but never digested
28
or
CCL19
F
2,147
= 18.9, P < 0.001
H
2
= 53.6, P < 0.001
H
2
= 3.1, P < 0.22
ab
c
d
Blebbistatin
Blebbistatin 100 nM Lat B 100 nM Lat B
Speed (µm min
–1
)
Speed (µm min
–1
)CCL19
00
718
14 34
25 45
33 49
38 58
46 61
51
6
4
2
0
0:00 1:50 2:40 3:50 5:40 6:20 7:40
6
4
2
0
6
4
2
0
6
4
2
0
0 100
H
2
= 120.1, P < 0.001
*
*
*
*
*
*
ns
0.75 1.5
Collagen (mg ml
–1
)
3
*
*
Latrunculin B (nM)
500
64
CCL19
Figure 5
|
Myosin II-independent protrusive migration of dendritic cells.
a
, Two types of residual migration of wild-type (WT) dendritic cells (DCs)
treated with blebbistatin in a standard 3D collagen gel. DCs either drag the
nucleus behind with elongated appearance and low speed (right column) or
appear morphologically normal with high speed (left column). Time in min.
b, Top panel, confocal reflection microscopy of 3D collagen networks of
different densities. Lower panels, velocities of single chemotaxing WT DCs
(dots) in collagen gels with varying densities. Upper graph, no inhibitor; red
line, mean. Middle graph, blebbistatin; red line, median. Bottom graph,
100 nM latrunculin B (partial actin depolymerization); red line, median.
c, Velocity of single WT DCs (dots) in the presence of differentconcentrations
of latrunculin B (1.6 mg ml
21
collagen); red line,median. In b and c, *P , 0.05
(post hoc).
d, Time-lapse sequence of a 100 nM latrunculin B-treated WT DC;
DIC microscopy, nuclei(green). Time, min:s. Blue boxes(in
b and c) highlight
experimental conditions that were used for follow-up experiments indicated
by the blue arrow. Scale bars: 10 mm(
a, d), 50 mm(b).
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otherwise permanently remodelled which avoids ‘collateral damage’,
caused by infiltrating cells.
The fact that leukocytes are able to move autonomously generates
a new level of regulatory possibilities. Because surface bound chemo-
kines and other immobilized extracellular signals do trigger integrin
affinity
16
(unlike soluble chemokines), leukocyte integrins should no
longer be viewed as force transducers during locomotion but as
switchable immobilizing anchors that stop, slow down or confine
high intrinsic motility to specifically assigned surfaces
29,30
. The role
of integrins is therefore mostly to mediate retention, invasion, cell–
cell communication and cell–cell adhesion
31
.
METHODS SUMMARY
Generation of integrin-deficient leukocytes. Integrins and talin1 were targeted
by generating mice with the genotype a
v
flox/flox
(Supplementary Fig. 1), b
1
flox/flox
(ref. 32), b
2
2/2
(ref. 33), b
7
2/2
(ref. 34), Mx1Cre
1/2
(ref. 35) and talin1
flox/flox
(ref. 36), Mx1Cre
1/2
, respectively. Cre expression in the haematopoietic system
was induced by intraperitoneal injection of 250 mg Poly (I)?Poly (C) (Amersham
Biosciences). 10–14 d after knockout induction DCs were generated from bone
marrow suspension and matured with lipopolysaccharide (LPS). The DC culture
was depleted for granulocytes and remaining integrin-positive contaminants by
magnetic sorting (Miltenyi Biotech). DCs used for migration assays were .99%
enriched for b
1
and a
v
integrin knockout cells.
In situ live cell imaging. For dermal ex vivo microscopy, mouse ears were
mechanically separated in dorsal and ventral halves, fluorescently stained with
LYVE-1 antibody and immobilized with the epidermal side down. The dermis
was overlaid with fluorescently labelled DCs and time lapse movies were
recorded with an automated Leica MZ 16 FA stereomicroscope (Visitron
Systems).
In vitro 3D chemotaxis assays. Cells were suspended in PureCol (INAMED) and
cast in custom built migration chambers (standard collagen concentration:
1.6 mg ml
21
). After polymerization, gels were overlaid with culture medium
containing the chemoattractant (CCL19, C5a and CXCL13 for DCs, granulo-
cytes and B cells, respectively) and subsequently imaged using wide-field fluor-
escence video microscopes with differential interference contrast (Zeiss). For
nucleus visualization, SYTO-dyes (Invitrogen) were used. For visualization of
myosin light chain, DCs were nucleofected using Amaxa technology. Inhibitors
were titrated and used 50 mm blebbistatin (Sigma) and 30 mm Y27632
(Calbiochem).
Statistical analysis. t-tests and analysis of variance (ANOVA) were performed
after data were confirmed to fulfil the criteria of normal distribution and equal
variance, otherwise Kruskal–Wallis tests or Mann–Whitney U-tests were
applied. If overall ANOVA or Kruskal–Wallis tests were significant, we per-
formed a post hoc test. Analyses were performed with Sigma Stat 2.03. For
further statistical details, see Supplementary Table.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 22 November 2007; accepted 6 March 2008.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank S. Cremer for help with statistical analysis, Z. Werb
and P. Friedl for critical reading of the manuscript, and M. Bauer for technical
support. This work was financed by the German Research Foundation (DFG), the
Austrian Science Foundation (FWF) and the Max Planck Society. Work in D.R .C.’s
laboratory was supported by the Wellcome Trust.
Author Contributions T.L. and M.S. designed and performed the experiments and
analysed the data. M.S. wrote the paper. T.W. and R.Fo
¨
. performed intravital
microscopy in lymph nodes. B.L.B. and M.K. generated the integrin a
v
mouse. S.J.M.
and D.R.C. generated the talin1 mouse. R.Fa
¨.
generated the integrin b
1
and the
quadruple integrin knockout mouse and provided general support. K.H. assisted
with experiments. R.W.S. contributed to data analysis and experimental design.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. Correspondence and requests for materials should be
addressed to M.S. (sixt@biochem.mpg.de).
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