Nature | Vol 582 | 25 June 2020 | 583between two surfaces, T cell locomotion depends on integrin-mediated
force transmission^6 ,^8 ,^9 ,^12 (see also Supplementary Discussion). This find-
ing was in contrast to our collagen gel data and to the in vivo finding
that T cells retain substantial migratory capacity following integrin
depletion^8 ,^9. We reasoned that the major difference between confine-
ment within a lymph node or within a fibrillar matrix and confinement
between two planar surfaces is geometrical complexity. Hence, we
introduced arrays of variably spaced pillars that intersected the two
surfaces (Fig. 2a), thereby mimicking the geometry of a fibrillar gel
while keeping the surface chemistry unchanged^13. Between the pil-
lars, the migratory capacity of talin-KO cells was restored (Fig. 2b–d,
Extended Data Fig. 2d, e, Supplementary Video 4). While translocation
velocities of wild-type cells were not affected by increased pillar spac-
ings, the performance of talin-KO cells dropped in wider-spaced pillars,
suggesting that tight contact with curved surfaces enables propulsion
in the absence of integrins (Supplementary Video 4). We next turned to
channels in which cells are fully enclosed by surfaces (Fig. 2e). Within
smooth-walled 5 × 5-μm channels, control cells migrated persistently,
while talin-KO cells were immobile (Fig. 2e, f, Supplementary Video 5).
We next introduced surface texture as a serrated pattern with a 6-μm
period and found that the migration of talin-KO cells was restored
(Fig. 2g, h, Extended Data Fig. 2f, Supplementary Video 5). In channels
that contained both smooth and serrated sectors, wild-type cells trans-
ited freely between the sectors. Following cation depletion with EDTA,
which inactivates integrins (and several other cell-surface adhesion
molecules), cells within smooth areas slowed down or stalled, while
cells within serrated sectors kept migrating until they encountered
a smooth area (Fig. 2i, j, Supplementary Video 6). The same principle
was conserved when we tested dendritic cells and neutrophil granulo-
cytes, two other types of amoeboid leukocytes. Notably, EDTA-treatedaefbAdhesive cells (N mm–2)ControlTalin KO Spreading area per cell (μm )ControlTalin KO2D 2D c 3DFc-ICAM1d 2.5DLifeact-GFPControlTalin KOControlTalin KO05101520Velocity (μm min–1)ControlTalin KOCellActin****
****CellActinLifeact-GFP
00:00
02:00Lifeact-GFP
00:00
02:00g5 μm3D view
****0100200
150250505 μm
Side viewMSD (μm2 )(^00204060)
10,000
20,000
30,000
40,000
50,000
Time (min)
MSD (
μm
2 )
Time (min)
0
5,000
10,000
15,000
20,000
0204060
0
10
20
30
40
50 Control
Talin KO
Control
Talin KO
Fig. 1 | Adhesive and migratory properties of talin-KO T cells. a, The number
of cells adhering to the 2D surface. Data are mean ± s.e.m. of four independent
experiments; *P = 0.00076, paired t-test. b, Surface area (μm^2 ) of cells
spreading on Fc-ICAM1-coated glass. Control (n = 139) and talin-KO (n = 81) cells
of three independent experiments are shown. Data are mean ± s.d.;
P < 0.0001, Mann–Whitney U-test. c, Mean square displacement (MSD) of
control (n = 51, in black) and talin-KO (n = 52, in red) cells migrating in 3D
collagen gel from three independent experiments. Data are mean ± s.e.m.;
P = 0.2243, Mann–Whitney U-test. d, MSD of control (n = 290, in black) and
t alin-KO (n = 284, in red) cells migrating in a 5-μm confiner from four
independent experiments. Data are mean ± s.e.m.; P < 0.0001, Mann–Whitney
U-test. e, Snapshots of TIRF microscopy of a control cell (left) and a talin-KO cell
(right) expressing Lifeact-GFP under 5-μm confinement. Representative of
three independent experiments. The green arrowheads mark the uropod and
the orange arrowheads indicate the cell front. TIRF images in cyan are at
t = 0 min and in red at t = 2 min. The yellow dashed line is used for the kymograph
in f. Scale bars, 5 μm. Time is shown in min:s. f, Kymographs of a control cell and
talin-KO cell under 5-μm confinement as shown in e. Cell velocity is indicated by
the green dashed line, and actin retrograde f low is shown by the cyan dashed
line. Horizontal scale bar, 5 μm; vertical scale bar, 1 min. g, Retrograde f low
velocities for control (n = 42) and talin-KO (n = 36) cells from three independent
experiments. P < 0.0001, Mann–Whitney U-test.
Cell speed (
μm min
–1)
0
5
(^10) *
0
5
10
15
(^20)
0
5
10
15
(^20)
Cell speed(μ
m min
–1)
EDTA– + – + – +
2.5D Serratedchannel Smoothchannel
3D view
Side view
5 μ
m
2.5D + pillars
5 μ
m
Side view
3D view
34 μμmm
Pillar spacing 56 μμmm
2.5D
Serrated channel
Smooth channel
0
50
100
150
(^200) ***
Talin KO
60:00
Control^5
μm
44:00
Control
Talin KO
6 μm
Serrated Smooth
5 μ
m
60:00
60:00
ControlTalin KO
0
5
10
(^15) ****
Cell velocity(μ
m min
–1)
Cell velocity(μ
m min
–1)
0
5
10
20
SerratedSmooth
NS
15
25
SerratedSmooth
ControlTalin KO
3 μ
m
4 μ
m
5 μ
m
6 μ
m
Control
∞
3 μ
m
4 μ
m
5 μ
m
6 μ
m
3 μ
m
4 μ
m
5 μ
m
6 μ
m
3 μ
m
4 μ
m
5 μ
m
6 μ
m
Talin KO
(^0) ∞
5
10
15
20
0
5
10
15
20
2.5D (no pillar)∞
EDTA (%)
afterbefore
Cell speed
NS
Displacement (
μm)
a
c
ij
f
g
e
h
b
d
0
100
200
300
400
500 ****
0
100
200
300
400
500
Control
∞
Talin KO
∞
y^ (
Control μm)
–1000
100
Talin KO
6 μm
–100 0100
–1000
100
5 μm
–100 0100
4 μm
–100 0100
3 μm
–100 0100
x (μm)
Fig. 2 | Migration of T cells in textured conf inement. a, Experimental setup
for b–d. b, Cell trajectories analysed in c and d during 1 h. The black crosses
indicate the start of the individual tracks. c, d, Cell speed (c), mean ± s.d.;
P = 0.0021, otherwise not significant (P > 0.9999); and displacement (d),
mean ± s.d.; P = 0.0089 and P < 0.0001, otherwise not significant, Kruskal–
Wallis test followed by post hoc Dunn’s test. n = 25, 20, 75 and 61 for control cells
in respective 3-μm, 4-μm, 5-μm and 6-μm zones. Data are from three
independent experiments. n = 64, 115, 124 and 94 for talin-KO cells in respective
3-μm, 4-μm, 5-μm and 6-μm zones. Data are from six independent experiments.
e, f, Migration in smooth channels. Data are from four independent
experiments. In e, 5 × 5-μm smooth channels are shown. Top, scheme of the
channel. Representative snapshots at t = 44 min of Lifeact-GFP-expressing
control cells (middle) and at t = 60 min talin-KO cells (bottom) are displayed.
Tracks are shown in yellow. Scale bars, 20 μm. In f, the cell velocity is shown.
n = 99 for control cells, n = 24 for talin-KO cells. Data are mean ± s.d.;
P < 0.0001, Mann–Whitney U-test. g, h, Migration in serrated channels. Data
are from three (control) and four (KO) independent experiments. In g, the
serrated topography with a 6-μm period in a 5 × 5-μm channel is shown. Top,
scheme of the channel. A representative snapshot at t = 60 min of the
Lifeact-GFP control cell (middle) and the talin-KO cell (bottom) is displayed.
Tracks are shown in yellow. Scale bars, 20 μm. In h, the cell velocity in channels
is shown. n = 59 (control serrated), 25 (control smooth), 8 (talin-KO serrated)
and 5 (talin-KO smooth). Data are mean ± s.d.; *P = 0.0009, Kruskal–Wallis test
followed by post hoc Dunn’s test. i, j, Cell speed before and after the addition of
10 mM EDTA. Data are from three independent experiments. 2.5D: n = 73 cells;
serrated channel: n = 23; and smooth channel: n = 27. In i, the cell speed before
(−) and after (+) the addition of EDTA in respective devices is shown.
**P < 0.0001 and *P = 0.0167, Wilcoxon matched-pairs signed-rank test.
In j, the change in cell speed after versus before the addition of EDTA is shown.
Data are mean ± s.d.; not significant (NS; P > 0.9999), *P = 0.0004 and
**P < 0.0001, Kruskal–Wallis test followed by post hoc Dunn’s tests.