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Rapid leukocyte migration by integrin-independent flowing and squeezing

Abstract

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 protrusive 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 expansion, 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.

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Figure 1: Migration of integrin-deficient dendritic cells into lymph nodes.
Figure 2: Integrin-independent interstitial leukocyte migration in vivo.
Figure 3: Integrin-independent leukocyte migration in 3D networks in vitro.
Figure 4: Myosin II-dependent nuclear squeezing at the cell rear.
Figure 5: Myosin II-independent protrusive migration of dendritic cells.

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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.Fö. performed intravital microscopy in lymph nodes. B.L.B. and M.K. generated the integrin αv mouse. S.J.M. and D.R.C. generated the talin1 mouse. R.Fä. generated the integrin β1 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.

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Correspondence to Michael Sixt.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-9 with Legends, Supplementary Table and Legends to Supplementary Movies 1-14. (PDF 5406 kb)

Supplementary Movie 1

The file contains Supplementary Movie 1 which shows integrin-independent migration of dendritic cells within lymph nodes of living mice. 33 h after subcutaneous injection of a 1:1 mixture of differentially labelled dendritic cells (DCs) into hind footpads, migrating DCs in the popliteal lymph node were visualized with intravital, two-photon microscopy. This representative movie shows directed migration of both wild-type (WT) (CFSE, green) and integrin-/- (Itg-/-) (TAMRA, red) DCs from the subcapsular sinus (left) through the interfollicular area towards the T cell cortex (right). Quantification of velocity, directional persistence and mean square displacement did not show any significant differences between WT and Itg-/- cells (Fig. 2e, f, data not shown). (MOV 7490 kb)

Supplementary Movie 2

The file contains Supplementary Movie 2 which shows migration of dendritic cells into lymphatic vessels in dermal ear explants (“crawl-in”). Interstitial migration of exogenously added TAMRA-labelled dendritic cells (DCs) (red in left and right panel) within dermal ear explants was visualized with wide-field fluorescent microscopy. Native ear dermis was pre-stained with LYVE-1 antibody to detect lymphatic vessels (green, in left panel). DCs approached the lymphatics in a directed manner and subsequently entered the lumen of the lymph vessel. Time-lapse over 80 min (160 frames, 30 frames/s). (MOV 3129 kb)

Supplementary Movie 3

The file contains Supplementary Movie 3 which shows dendritic cells migrating independent of integrins within the dermis and into lymphatic vessels. Interstitial migration of TAMRA-labelled dendritic cells (DCs, red) into lymphatics of dermal explants was visualized with wide-field fluorescence microscopy. Native ear dermis was pre-stained with LYVE-1 antibody to detect lymphatic vessels (green). Migration was recorded over 42 min (42 frames, 15 frames/s). Wild-type (WT) (left), integrin-/- (Itg-/-) (middle) and talin1-/- (Tln1-/-) (right) enter the lumen of lymphatic vessels (overlay) and did not show any difference in migration speed (Fig. 2b-c, Supplementary Fig. 5). Scale bar: 50 µm. (MOV 5255 kb)

Supplementary Movie 4

The file contains Supplementary Movie 4 which shows integrin-dependent spreading of dendritic cells on 2D in vitro surfaces. Spreading of LPS-matured dendritic cells (DCs) on a 2D plastic surface with immobilized chemokine was monitored over 43 min (86 frames, 15 frames/s). While wild-type (WT) DCs (left) show spreading and migration, integrin-/- (Itg-/-) (middle) and talin1-/- (Tln1-/-) (right) do not adhere and migrate on the 2D substrate. Bright-field microscopy, 20x objective. (MOV 4864 kb)

Supplementary Movie 5

The file contains Supplementary Movie 5 which shows integrin-independent migration of dendritic cells in 3D in vitro matrices. Dendritic cells (DCs) within 3D collagen matrices were polarized and attracted by a diffusive chemotactic gradient (CCL19 on top). Wild-type (WT) DCs (top) and integrin-/- (Itg-/-) DCs (bottom) both migrated towards the CCL19 source without any significant differences in speed and directionality (Fig. 3c). Right boxes illustrate directional migration as animated chemotaxis plot graphs. Collagen gel: 1.6 mg ml-1; time-lapse over 4 h (60 frames, 10 frames/s), 10x objective. (MOV 9597 kb)

Supplementary Movie 6

The file contains Supplementary Movie 6 which shows rearward-pulling of a fibroblast in a 3D in vitro matrix. Transduction of retrograde pulling forces to the extracellular matrix visualized as retrograde fibre dislocation at the leading edge of a migrating fibroblast; collagen gel: 1.6 mg ml-1, DIC microscopy, time-lapse over 48 min (24 frames, 30 frames/s). (MOV 742 kb)

Supplementary Movie 7

The file contains Supplementary Movie 7 which shows alternating phases of rear end contraction when leukocytes migrate in 3D matrices. Directed migration of a dendritic cell towards CCL19 (left) in a 3D collagen gel. Two phases of migration can be distinguished: cytoplasmic streaming and contraction at the trailing edge while the branched cell front stagnates (1), the protruding leading edge drags the static, non-streaming cell body behind (2), followed again by cytoplasmic streaming and contraction at the trailing edge (similar to 1). These phases do not depend on integrins and can be also observed for other leukocytes (see also Supplementary Videos 10 and 11). Rearward pulling of collagen fibers can not be observed. Double-headed arrows monitor the length of the leading (yellow) and the trailing edge (light blue); collagen gel: 1.6 mg ml-1; DIC microscopy, time-lapse over 13 min 30 s (81 frames, 10 frames/s; total number of frames: 252). (MOV 11937 kb)

Supplementary Movie 8

The file contains Supplementary Movie 8 which shows myosin light chain localisation at the cell rear during leukocyte migration. Directed migration of a wild-type dendritic cell (DC) towards CCL19 (left). DCs were transfected with myosin light chain (MLC)-GFP to visualize the site of contraction within the cell. The MLC-GFP localization at the rear in phases of contraction is independent of the presence of integrins (Fig. 4a, Supplementary Fig. 8a). Top: DIC microscopy, middle: fluorescence intensity of MLC-GFP, bottom: 3D intensity profile of MLC-GFP; collagen gel: 1.6 mg ml-1, DIC and fluorescence microscopy, time-lapse over 3 min 50 s (46 frames, 10 frames/s). (MOV 5779 kb)

Supplementary Movie 9

The file contains Supplementary Movie 9 which shows that 3D leukocyte migration requires rear end contraction. Upper row: Directed migration of wild-type dendritic cells (DCs) towards CCL19 (top). Inhibition of contraction by 50 µM blebbistatin (myosin II inhibitor, middle) and 30 µM Y27632 (ROCK inhibitor, right) leads to significant speed reduction compared to untreated DCs (left) (see also Fig. 4b); collagen gel: 1.6 mg ml-1, time-lapse over 4 h (4 min/frame), 10x objective. Lower row: Inhibition of contraction (50 µM blebbistatin, middle; 30 µM Y27632, left) leads to immobile cell rears, while the fronts still protrude forward. Few cells still migrate towards CCL19 (top) when contraction is blocked; collagen gel: 1.6 mg ml-1, time-lapse over 1 h 25 min (75 frames, 1 min/frame), 20x objective. (MOV 10892 kb)

Supplementary Movie 10

The file contains Supplementary Movie 10 which shows that leukocyte rear end contraction squeezes the nucleus through narrow spaces in 3D matrices. Directed migration of dendritic cells (DCs) in collagen gels towards CCL19, nuclei are stained green. Untreated wild-type (WT) (top, left) and integrin-/- (Itg-/-) (top, right) DCs deform the nucleus during migration, whereas inhibition of contraction (50 µM blebbistatin) leads in WT (bottom, left) and Itg-/- (bottom, right) to an immobile cell rear with a nucleus that is “left behind” and a cell front that protrudes towards the chemotactic source; collagen gel: 1.6 mg ml-1, DIC and fluorescence microscopy, time-lapse over 12 min (60 frames, 15 frames/s). (MOV 5589 kb)

Supplementary Movie 11

The file contains Supplementary Movie 11 which shows granulocyte nuclear squeezing by rear contraction in 3D matrices. Directed migration of wild-type (WT) bone marrow granulocytes towards C5a (applied on top). Left column: Similar to dendritic cells (DCs) (Supplementary Video 9), inhibition of contraction by treatment of cells with 50 µM blebbistatin (myosin II inhibitor, bottom) leads to significant speed reduction compared to untreated cells (top) (see also Supplementary Fig. 8e); collagen gel: 3 mg ml-1, time-lapse over 15 min (30 s/frame), 10x objective. Middle column: Boxes illustrate directional migration as animated chemotaxis plot graphs. Right column: Directed migration of WT bone marrow granulocytes towards C5a, nuclei are stained green. Untreated granulocytes (top) deform the nucleus during migration, whereas the nucleus of 50 µM blebbistatin-treated granulocytes (bottom) is “dragged” (see also Supplementary Fig. 8c, d); collagen gel: 5 mg ml-1, DIC and fluorescence microscopy, time-lapse over 7 min 24 s (76 frames, 15 frames/s). (MOV 9666 kb)

Supplementary Movie 12

The file contains Supplementary Movie 12 which shows that basal leukocyte locomotion (without contraction) is characterized by dragging of the nucleus and more resting phases. Residual migration without contraction (50 µM blebbistatin): Dendritic cells either drag the nucleus behind with elongated appearance and low speed (cell 1) or appear morphologically normal with high speed (cell 2); time-lapse over 5 h 25 min (325 frames, 15 frames/s), 20x objective. Characteristically, residual migration leads to more resting phases (cell 3) when the cell rear gets stuck while the cell front still protrudes forward (see also Supplementary Fig. 9), time-lapse over 1 h 12 min (132 frames, 15 frames/s), 20x objective, collagen gel: 0.75 mg ml-1. (MOV 1497 kb)

Supplementary Movie 13

The file contains Supplementary Movie 13 which shows rescue of basal leukocyte locomotion (without nuclear squeezing) in 3D matrices of low external resistance. Residual migration without contraction (50 µM blebbistatin) depends on the external resistance of the collagen network. While dendritic cell (DC) migration is severely impaired in dense 3D collagen gels (3 mg ml-1, top) due to lack of rear end contraction, myosin II-independent DC migration can be rescued in sparse gels (1.6 mg ml-1, bottom). Time-lapse over 4 h (60 frames, 10 frames/s), 10x objective. (MOV 9984 kb)

Supplementary Movie 14

The file contains Supplementary Movie 14 which shows that anterior actin expansion is essential for leukocyte migration in 3D matrices. Directed migration of wild-type dendritic cells towards CCL19 in the presence of different concentrations of latrunculin B: While 500 nM latrunculin B (right) leads to immobility and rounding up, cells treated with 100 nM latrunculin B (middle) migrate with reduced speed compared to control (no inhibitor, right). Upper row: collagen gel: 1.6 mg ml-1, time-lapse over 1 h 30 min (2 min/frame), 10x objective. Lower row: collagen gel: 1.6 mg ml-1, DIC and fluorescence microscopy, time-lapse over 11 min (90 frames, 10 frames/s). (MOV 8460 kb)

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Lämmermann, T., Bader, B., Monkley, S. et al. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 453, 51–55 (2008). https://doi.org/10.1038/nature06887

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