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Disrupting microtubules with nocodazole (Supplementary Fig. 3B), completely stopped cell migration (Supplementary Movie 2), ruling out an osmotically driven migration mechanism.At low doses (50 n M), latrunculin A did not induce obvious changes in the overall actin cytoskeleton (Supplementary Fig.(f) Representative cell (blue) and nuclear (green) front instantaneous velocity as a function of the nuclear front position in 1.5 μm constrictions.(g) Cell (blue) and nuclear (green) passage time in 20-μm-long constrictions. At 1 μm width, the cell front passed but only a small fraction of the nucleus was engaged in the constriction (Supplementary Fig. Importantly, for all sizes, non-passing DCs engaged in a constriction spent at least as much time in the constriction as passing cells (Supplementary Figs 1H and 2F), showing that non-passage was not due to an early change of direction of the cell induced by the constriction. Blebbi, blebbistatin; Lat A, latrunculin A; MT, microtubules; Noco, nocodazole. Right: percentage of nuclear entry in non-passing cells.
The complexity of three-dimensional (3D) gels also requires cells to restrict the number of their protrusions, to avoid simultaneously engaging multiple paths.
2d), and they initiated nuclear entry into constrictions to the same extent as non-passing control/WT cells (Fig. Arp2/3 inhibition reduced the rate of passage only for constrictions showed a very strong enrichment of actin filaments around the nucleus inside small constrictions (Fig. 6A) and was quantified in live cells, showing that it was temporally restricted to the time during which the nucleus was deformed in the constriction and spatially limited to the constriction (Fig. 6 C–N and Supplementary Movie 4) that required Arp2/3 for efficient passage (Fig. 7A, B and Supplementary Movie 5) ruling out the involvement of adhesion complexes.
Imaging of actin filaments in DCs migrating in dense, 3 mg m L (Fig.
and P-value of all quantifications).(a) Schematic representation of the experimental setup. (c) Top: representative image of an Iaβ-GFP (red)-expressing DC stained with Hoechst (DNA, green), passing through a 2-μm constriction coated with p LL-PEG-Rhodamin (grey levels). Middle: views of the channel cross-section before, along and after the constriction. Bottom: perspective view (45°) of a 3D iso-surface reconstruction. (d) Representative sequential images of a DC stained with Hoechst (green) migrating through a constriction. (e) Percentage of passage through 20-μm-long constrictions. ***P-value Time-lapse recording of migrating cells identified four phases of transmigration (Fig. 2D, E) showed that for intermediate sizes of 1.5 and 2 μm, while a majority of cells could pass through the constriction, nuclear passage induced a significant reduction of cell velocity (41% in 1.5 μm constrictions).
(b) Top: representative image of a field of channels with constrictions. (i) Cell entry, (ii) nuclear entry, (iii) nuclear exit and (iv) cell exit. Numbers above bars represent the number of cells scored. 1d) as follows: (i) cell front entry into the constriction, which did not induce any slowdown; (ii) nuclear engagement and deformation, which led to a strong slowdown in constrictions . Despite that delay, DCs passed through such small constrictions in only and MDA-MB-231 (ref. This suggests that fast immune cells might have a specific mechanism for deforming their nucleus in a shorter time. We conclude that myosin II plays a role in increasing cell speed but is not required in DCs for nuclear deformation through small constrictions.(a) Percentage of passage and (b) passage time in 2-μm-wide constrictions.
The cells’ requirement for Arp2/3 to pass through constrictions can be relieved when nuclear stiffness is decreased by suppressing lamin A/C expression.