Nature - USA (2020-06-25)

Article

Actin: optical flow

Angle of actin flow (°)

Number of detections

Angle of cell movement (°)

Number of detections

e

b

Detected spots

Spots inside cell core

Spots direction

Cell direction

Optical flow direction

Cell core

Actin: spots tracking

a

0

20

40

60

80

100

120

0306090

0°

90°

0306090

0

20

40

60

80

100

120

0°

90°

perpendicular

to ridges

parallel to ridges

top view

Lifeact-GFP

primary T cell

0°

90°

f

Cell Speed

Actin Speed

0.0

0.5

1.0

1.5

2.0

Angle of cell movement (°)

Normalized speed (a.u.)

1020 40 50 70 80 1020 4050 7080

0510 15

0.0

0.5

1.0

1.5

2.0

Cell speed (μm/min)

Normalized actin speed (a.u.)

r=- 0. 65

Agarose

side view

800nm

600 nm

800nm

Agarose

Pll-PEG

X (μm)

Y (μm)

0°

90°

0

5

10

15

-5

-10

-15

-20-152-10 -5 0510 15 0

c

d

0-1515-3030-4545-6060-7575-90

5μm

Lifeact-GFP

Extended Data Fig. 7 | Actin-based force transmission in primary T cells.
a, Scheme (top and side view) of cells migrating under agarose on a
non-adhesive, topography-baring substrate. b, Tracks of migrating cells
analysed in c–f. n = 18; mean cell speed = 6.58 μm/min; mean actin retrograde
f low speed = 6.53 μm/min. Starting points of tracks are shifted to the origin.
c, Mean speed of cells in relation to their mean actin retrograde f low speed (in
relation to the substrate). Pearson's rank correlation coefficient r = −0.6484.
A drop in retrograde f low with increasing locomotion speed demonstrates
that, as in the transmembrane clutch paradigm of force transmission, actin
slippage is inversely related to locomotion. d, Snapshots of actin f low analysis
on ridged surfaces of cells expressing Lifeact-GFP. Left, cell core segmented
with Ilastik (pink); the yellow arrow indicates cell direction, the blue arrow
indicates the mean optical f low direction and the green arrow indicates actin
retrograde f low. Middle, optical f low analysis (obtained with a customized
MATLAB code). Right, actin single-spot tracking (TrackMate). Representative
of three independent experiments. e, Angular distribution of actin retrograde
f low and forwards locomotion of cells migrating on nanoridges. Only frames
where optical f low analysis was confirming automatic particle tracking
(deviation of <30°; see d) were subjected to analysis. The histogram on the left

shows predominant retrograde actin f low when aligned along the ridges (90°).

Retrograde actin f low declines towards 0° (perpendicular to the ridges), while

forwards locomotion (right histogram) steadily increases, indicating that

retrograde actin f low couples to topographical barriers and drives

locomotion. n = 422 events of actin f low and cell movements obtained in n = 18

cells from three independent experiments. f, Frame-to-frame speed of cells

migrating on nanoridges increases when forwards locomotion aligns

perpendicular to topographical barriers (90° → 0° ≃ 15% increase). The

orientation-dependent increase of cell speed is paralleled by a steady decrease

of retrograde actin f low (90° → 0° ≃ 55%), indicating that retrograde actin f low

couples to topographical barriers and drives locomotion. Frame-to-frame cell

speed and actin retrograde f low speed were recorded in 18 cells from 3

independent experiments, and pooled to n = 412 (cell speed) and n = 422 (actin

retrograde f low speed) events. The centre shows the mean, and both s.e.m.

(solid lines) and s.d. (thick transparent lines) are shown. Before pooling, cell

speed was normalized to the mean speed of the tracked cell, and actin

retrograde f low speed was normalized to the mean actin retrograde f low

speed.