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Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes

Nature Cell Biology volume 18, pages 1253–1259 (2016)Cite this article

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Abstract

Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families1. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.

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Figure 1: Localization of Arp2/3 activation machinery and morphologies of wild-type (wt) and hem1−/− DCs.
Figure 2: Migratory properties of wt and hem1−/− mDCs in vivo and in vitro.
Figure 3: Migratory properties of wt and hem1−/− mDCs in microfluidic devices.
Figure 4: Migratory properties of wt and hem1−/− iDCs.
Figure 5: Lamellipodial position of leukocytes during confined migration.

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Acknowledgements

This work was supported by the German Research Foundation (DFG) Priority Program SP 1464 to T.E.B.S. and M.S., and European Research Council (ERC GA 281556) and Human Frontiers Program grants to M.S. We thank H. Haecker for his generous help with HoxB8 immortalization, J. Renkawitz for critical reading of the manuscript and the Scientific Service Units of IST Austria for excellent technical support.

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  1. Alexander Leithner and Alexander Eichner: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Science and Technology Austria (IST Austria), am Campus 1, 3400 Klosterneuburg, Austria

    Alexander Leithner, Alexander Eichner, Jan Müller, Anne Reversat, Markus Brown, Jan Schwarz, Jack Merrin, Ingrid de Vries, Stefan Wieser, Robert Hauschild & Michael Sixt

  2. Institute for Molecular Biotechnology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030 Vienna, Austria

    Jan Müller, Florian Schur, Jonathan Bayerl & J. Victor Small

  3. Clinical Department of Pathology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria

    Markus Brown & Dontscho Kerjaschki

  4. Institute for Molecular Cell Biology, University of Münster, Schlossplatz 5, 48149 Münster, Germany

    David J. J. de Gorter & Theresia E. B. Stradal

  5. Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany

    Frank P. L. Lai, Klemens Rottner & Theresia E. B. Stradal

  6. Department of Molecular Medicine, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany

    Markus Moser

  7. Division of Molecular Cell Biology, Zoological Institute, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany

    Klemens Rottner

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Contributions

A.L. and A.E. devised and performed most experiments. A.R., J.S., I.d.V. and J.Merrin helped with microfluidic approaches. J.Müller, F.S., J.B. and J.V.S. performed electron tomography. M.B. and D.K. performed intralymphatic injections. S.W. and R.H. helped with quantitative analysis. D.J.J.d.G., F.P.L.L., M.M., K.R. and T.E.B.S. generated hem1-deficient mice. M.S. initiated and supervised the project and wrote the paper with input from all authors.

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Correspondence to Alexander Leithner, Alexander Eichner or Michael Sixt.

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Integrated supplementary information

Supplementary Figure 2 Generation of hem1−/− knock-out mice.

hem1 is located on chromosome 15F3 in the mouse and consists of 31 exons spanning approximately 45 kilobases. (A) Exons four and five were flanked by loxP sites, placing an excisable neomycin resistance cassette into the intron before exon 4. A targeting construct harbouring the neomycin cassette (pthem1-Neo) was used to replace one wt allele in embryonic stem cells (ES line IGD3.21). (B) Genomic Southern hybridization and PCR confirmed the presence of the corresponding alleles in isolated ES cell clones. (C) ES-cell clone #A11 gave rise to chimeric animals with germ line transmission. Resulting mice harbouring the targeted allele were crossed with a mouse expressing flip-recombinase2. Mice carrying the targeted allele and displaying deletion of the neomycin resistance cassette were selected for further backcrossing to C57BL/6J mice until G9. (D) Cre-mediated deletion of the targeted allele was achieved by crossing male hem1 fl/wt animals with female Keratin14-cre mice3 and resulted in animals heterozygously deleted for hem1 (del/wt). (E) Heterozygous mice displayed no phenotype and were crossed to produce mice in which hem1 is deleted on both alleles. For detection of the hem1 targeted allele, primers b and c (for sequences see Supplementary Table 1) were used giving rise to products sized 190 bp for wildtype and 220 bp for the targeted allele, respectively. For detecting hem1 floxed alleles, primers d and e were used to obtain product sizes of 250 bp for wildtype and 350 bp for floxed alleles, respectively. Finally, to detect the deleted allele, primers b, c and e were used, giving rise to a 400 bp product.

Supplementary Figure 3 Morphological and actin nucleation machinery related characterization of wt and hem1−/− i- and mDCs.

(A) Western Blots of wt and hem1−/− i- and mDC lysates for WAVE 1, 2 and 3. WAVE1/3 – GFP transfected HEK cells were used as positive control for antibodies. GAPDH was used as loading control. In case of WAVE2, blot was consecutively stained for WAVE2 and GAPDH. For WAVE1 and 3 equal sample volumes were loaded on two gels, run together and either stained for GAPDH or WAVE1 or 3. (B) Flow cytometry of wt and hem1−/− mDCs for Cd11c, MHCII, CD40, CD80, CD86 and CCR7 levels. (C) DIC images of wt and hem1−/− i- and mDCs. Insets show development over 20 s of regions marked with white rectangles. Scale bars, 10 μm. (D) top: Western Blot of control and WAVE2-/- iDCs lysates for WAVE2. GAPDH was used as loading control. Blot was obtained as described for (A). bottom: brightfield image shows WAVE2-/- iDC. Scale bar, 10 μm. (E) Confocal sections of hem1−/− iDCs expressing eGFP-ArpC5 or eGFP-WASP. Scale bar, 10 μm. (F) Brightfield and TIRF images of wt and hem1−/− iDCs expressing MLC-eGFP. Yellow dotted line marks lamellipodial rim of wt cell. Yellow arrow marks tip of hem1−/− cell. Scale bar, 10 μm. (G) Representative Z projections of fixed and FITC-phalloidin stained wt and hem1−/− i- and mDCs. Scale bar, 10 μm. (H) top: relative phalloidin-FITC fluorescence levels, measured by flow cytometry, of hem1−/− i- and mDCs compared to wt control. Each bar represents one biological replicate. bottom: Western Blots of wt and hem1−/− i- and mDC lysates for total actin. GAPDH was used as loading control. Blot was consecutively stained for Actin and GAPDH (I) Left: Relative frequency of actin filament angles relative to leading edge in wt (blue) and hem1−/− iDCs (green). Right: Automatically tracked actin filaments used to derive data on the left. Black triangles denote position of leading edges. Scale bars, 150 nm. Raw data are shown in Fig. 1e, f.

Supplementary Figure 4 Supporting data for mDC in vivo and in vitro cell migration.

(A) Ratios of wt and hem1−/− mDCs inside/outside of lymphatic vessels 2, 4 and 6 h after application to ear sheets. Blue and green dots, connected with a line represent data from one mouse ear sheet. 2 h: Two-tailed t-test, ∗∗P = 0.0034. n = 12, 4 h: Two-tailed t-test, ∗P = 0.0241. n = 10, 6 h: Two-tailed t-test, ∗∗∗P = 0.0002. All data are pooled from 2 biologically independent experiments. (B) Example z-projections of intravasation endpoint assays 2, 4 and 6 h after application of wt (green) and hem1−/− (magenta) mDCs, quantified in (A). Lymphatic vessels are stained with Lyve-1 (white). Insets: details of regions marked by white squares. Yellow dotted lines mark lymph vessels. (C) Single cell chemotactic indices of wt and hem1−/− mDCs in response to CCL19 gradient, depending on increasing rattail collagen concentrations. Mean (red line) ± s.d. (black bars). Two-tailed t-tests (exponential transformation). 1.1 mg ml−1: n.s. P = 0.9307. n = 82 (wt), n = 80 (hem1−/−). 1.7 mg ml−1: n.s. P = 0.0587. n = 80 (wt), n = 81 (hem1−/−). 2.7 mg ml−1: n.s. P = 0.8518. n = 81 (wt), n = 80 (hem1−/−). Data stem from the same 3 biological replicates as in Fig. 2g. (D) Mean cell area in arbitrary units of wt and hem1−/− mDCs migrating in microfluidic ‘maze’ device. Mean (red line) ± s.d. (black bars). Two-tailed t-test, n.s. P = 0.2564. n = 20 (wt), n = 21 (hem1−/−). Data stem from the same 3 biological replicates as in Fig. 3i.

Supplementary Figure 5 Supporting data for iDC in vitro cell migration and chemokine sensing.

(A) Analysis of the mean square displacement (MSD) over time and Fürth fits (red solid line) of wt and hem1−/− iDCs, migrating in 3D collagen gels in Fig. 4a; blue and green circles, average MSD at given time point. (B) Speed (μm min−1) of hem1−/− iDCs, migrating in 3D Collagen gel, correlated with cell splitting (appearance of second pointed protrusion) as exemplified in brightfield time-lapse series. Mean (green dot) ± s.e.m. (black bars), n = 12. Red dotted line marks time point of splitting events. (C) left: correlation of frame-to-frame speed (μm s−1) and shape factor (1 = completely round, 0 = indefinitely elongated) of wt iDCs, migrating in 3D collagen gel. Red line represents best-fit linear regression. Correlation coefficient = −0.45, ∗∗∗∗P < 0.0001. n = 382 from 34 single cells. right: Two example time-lapse series of wt iDCs in collagen gel. Shape factor in respective frame is shown in yellow. (D) Single cell chemotactic indices of wt and hem1−/− iDCs, migrating in 3D collagen gel in response or absence of CCL3 gradient. Mean (red line) ± s.d. (black bars). CCL3: One-way ANOVA, ∗∗∗∗P < 0.0001. n = 30 (wt), n = 32 (hem1−/−). No chemokine: One-way ANOVA, n.s. n = 30 (wt), n = 32 (hem1−/−). Data are pooled from 3 biological replicates. (E) Average y-displacement (pixel min−1) over time (min) of ctrl (blue) and WAVE2-/- (green) iDCs in response to CCL3 gradient. Mean (dots and triangles) ± s.e.m. (black bars). n = 6 collagen gels pooled from 3 biologically independent experiments. (F) Western blot of wt and hem1−/− iDC lysates from Fig. 4g for total-Akt levels after CCL3 chemokine stimulation. GAPDH was used as loading control. Blot was consecutively stained for total Akt and GAPHD.

Supplementary Figure 6 Schematic representation of migrating wt and WAVE deficient i- and mDCs.

Summary of capabilities of wt and WAVE deficient i- and mDCs regarding (i) general Locomotion, (ii) gradient sensing and (iii) translation of sensing into navigation towards chemokine source. Wt i- and mDCs use their broad lammelipodia to efficiently negotiate complex 3D environments towards the chemokine source. In contrast, the multiple filopodia of WAVE deficient mDCs are inferior in finding optimal paths through complex environments. This slows down cell migration while the basic locomotion and gradient sensing machinery is unaffected. The single, needle shaped protrusion of WAVE deficient iDCs enables high migratory speed but is insufficient to navigate the cell towards chemokine gradients in complex environments.

Supplementary Figure 7 Raw data of Western Blots.

(A) Unprocessed scan of Western Blot for WAVE2 and GAPDH (right), shown in Supplementary Fig. 2A. Left: Marker. (B) Unprocessed scan of Western Blot for WAVE1 (right) and GAPDH (left) shown in Supplementary Fig. 2a. (C) Unprocessed scan of Western Blot for WAVE3 (right) and GAPDH (left) shown in Supplementary Fig. 2A. (D) Unprocessed scan of Western Blot for total Akt (left) and GAPDH (right) shown in Supplementary Fig. 4F. (E) Unprocessed scan of Western Blot for phospho-Akt (left) and GAPDH (right) shown in Fig. 4g. (F) Unprocessed scan of Western Blot for WAVE2 and and GAPDH shown in Supplementary Fig. 2D. (G) Unprocessed scan of Western Blot for Actin and GAPDH shown in Supplementary Fig. 2H.

Supplementary Table 1 Antibodies and Primers.
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Supplementary information

Supplementary Information

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Total internal reflection (TIRF) movies of iDCs migrating on PLL-PEG coated cover slip under agarose in a CCL3 gradient. Cells are expressing eGFP reporters for ArpC5, WASP and Abi1.

ArpC5 and Abi1 localize to the lamellipodial edge in phases of expansion while WASP is absent from the leading edge. Time-laps over 180 s (60 frames, 12 frames s−1). (AVI 2074 kb)

Total internal reflection (TIRF) movies of mDCs migrating on PLL-PEG coated cover slip under agarose in a CCL19 gradient.

Cells are expressing eGFP reporters for Lifeact, ArpC5, WASP and Abi1. ArpC5, WASP and Abi1 localize to the lamellipodial edge in phases of expansion. Time-laps over 180 s (60 frames, 12 frames s−1.) (AVI 3347 kb)

Wt and hem1−/− i- and mDCs in suspension, imaged with differential interference contrast (DIC) microscopy.

hem1−/− iDCs have a pointed, needle-like shape while their wild-type (wt) counter parts display a roundish morphology with veil-like lamellipodia. hem1−/− mDCs round up like their wt counter parts but instead of displaying pronounced lamellipodia they form multiple, finger-like extensions. Time-laps over 1 min (61 frames, 10 frames s−1.) (AVI 1743 kb)

Electron tomography of the lamellipodium of a wt iDC.

The image series shows a z-stack, followed by a close-up, showing branched actin filaments (red circles). Branch junctions are then marked in red, actin filaments radiating from these points are marked in green. (AVI 6725 kb)

Electron tomography of the tip of a hem1−/− iDC.

The image series shows a z-stack, followed by a polarity analysis of actin filaments. Actin filaments are marked in green, red spheres denote barbed filament ends, showing a linear array of parallel filaments. (AVI 4554 kb)

Wt mDCs migrating in 3D collagen gel towards a CCL19 gradient.

Individual cell tracks within the first 60 min are marked by different colors. Time-laps over 180 min (61 frames, 12 frames s−1). The sequence is followed by a close up of an individual wt mDC migrating in a 3D collagen gel towards a CCL19 gradient. Time-laps over 60 s (60 frames, 12 frames s−1). (AVI 256 kb)

hem1−/− mDCs migrating in 3D collagen gel towards a CCL19 gradient.

Individual cell tracks within the first 60 min are marked by different colors. Time-laps over 180 min (61 frames, 12 frames s−1). The sequence is followed by a close up of an individual hem1−/− mDC migrating in a 3D collagen gel towards a CCL19 gradient. Time-laps over 60 s (60 frames, 12 frames s−1). (AVI 6539 kb)

Wt (left) and hem1−/− (right) mDCs migrating in microfluidic ‘maze’ device towards a CCL19 gradient.

Cell bodies are stained with 10 μM TAMRA. Nuclei are stained with NucBlue nuclear dye. Time-laps over 100 min (100 frames, 12 frames s−1). (AVI 16795 kb)

Wt iDCs migrating in 3D collagen gel.

Time-laps over 60 min (61 frames, 12 frames s−1). (AVI 3179 kb)

hem1−/− iDCs migrating in 3D collagen gel.

Time-laps over 120 min (61 frames, 12 frames s−1). The movie also shows examples of the following immature hem1−/− iDC traits: (1) High persistence, (2) Change of direction upon rounding and (3) bifurcations and spiking, followed by a close-up of a migrating hem1−/− DC. (AVI 19060 kb)

Brightfield and total internal reflection (TIRF, red) microscopy movies of a individual Lifeact-eGFP expressing (1) mDC (time-laps over 170 s, 18 frames), (2) T cell (time-laps over 117 s, 40 frames) (3) neutrophil (time-laps over 100 s, 26 frames) and D.rerio keratocyte (time-lapse over 120 s 59 frames).

Cells are migrating confined under a PLL-PEG coated 4 μm micropillar, except D.rerio keratocyte that is migrating on a coverslip. In mDCs, T cells and neutrophils the lamellipodium is rarely visible in the TIRF channel (red). This suggests that it is not involved in force transduction. Instead, the cell body itself seems to be the main driver of locomotion. In contrast, the lamellipodia of keratocytes are in tight contact with the substrate, visualized by a constant TIRF signal in the lamellipodium. (12 frames s−1) (AVI 3093 kb)

Time-lapse spinning disc confocal z-projection (center) and z-reconstructions (oblique top/bottom- and front view) of eGFP-Abi1 expressing mDC, migrating confined under a 3 μm micropillar (time-lapse over 200 s 21 frames).

The lamellipodium, highlighted by a strong eGFP-Abi1 signal, is not in constant contact with the upper or lower substrate. Instead it shows a highly dynamic, fluctuating behavior. (12 frames s−1) (AVI 1019 kb)

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Leithner, A., Eichner, A., Müller, J. et al. Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes. Nat Cell Biol 18, 1253–1259 (2016). https://doi.org/10.1038/ncb3426

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