Micro-environmental control of cell migration--myosin IIA is required for efficient migration in fibrillar environments through control of cell adhesion dynamics - PubMed (original) (raw)

. 2012 May 1;125(Pt 9):2244-56.

doi: 10.1242/jcs.098806. Epub 2012 Feb 10.

Affiliations

Micro-environmental control of cell migration--myosin IIA is required for efficient migration in fibrillar environments through control of cell adhesion dynamics

Andrew D Doyle et al. J Cell Sci. 2012.

Abstract

Recent evidence suggests that organization of the extracellular matrix (ECM) into aligned fibrils or fibril-like ECM topographies promotes rapid migration in fibroblasts. However, the mechanisms of cell migration that are altered by these changes in micro-environmental topography remain unknown. Here, using 1D fibrillar migration as a model system for oriented fibrillar 3D matrices, we find that fibroblast leading-edge dynamics are enhanced by 1D fibrillar micropatterns and demonstrate a dependence on the spatial positioning of cell adhesions. Although 1D, 2D and 3D matrix adhesions have similar assembly kinetics, both 1D and 3D adhesions are stabilized for prolonged periods, whereas both paxillin and vinculin show slower turnover rates in 1D adhesions. Moreover, actin in 1D adhesions undergoes slower retrograde flow than the actin that is present in 2D lamellipodia. These data suggest an increase in mechanical coupling between adhesions and protrusive machinery. Experimental reduction of contractility resulted in the loss of 1D adhesion structure and stability, with scattered small and unstable adhesions, and an uncoupling of adhesion protein-integrin stability. Genetic ablation of myosin IIA (MIIA) or myosin IIB (MIIB) isoforms revealed that MIIA is required for efficient migration in restricted environments as well as adhesion maturation, whereas MIIB helps to stabilize adhesions beneath the cell body. These data suggest that restricted cell environments, such as 1D patterns, require cellular contraction through MIIA to enhance adhesion stability and coupling to integrins behind the leading edge. This increase in mechanical coupling allows for greater leading-edge protrusion and rapid cell migration.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

Comparison of protrusion efficiency. (A,B) Phase-contrast images of NIH/3T3 fibroblasts migrating on 2D (A) or 1D micropatterned (B) surfaces. Kymograph analysis at the green lines (right panels and insets) shows local protrusion–retraction cycles, with forwards progression of the lamellipodial–lamellar border (red dotted line). Yellow dotted lines indicate protrusion (P) and retraction (R) phases. (C) Analyses of protrusion, retraction and net cellular protrusion per cycle between 1D and 2D conditions. Scale bars: 10 μm unless otherwise indicated. *P<0.05.

Fig. 2.

Fig. 2.

Lamellar movement in 2D migration is associated with local adhesion formation. (A) Phase contrast with narrow (center panel) versus wide (right panel) kymograph analysis of 2D directional migration. The red dotted line indicates the lamellipodial–lamellar border; kymograph analysis is shown at the yellow dotted line. (B) GFP–paxillin expression in the same cell as in A. The narrow kymograph (at the yellow dotted line) shows few adhesions (blue arrowheads) whereas the wide kymograph (green box) demonstrates fluid forwards progression with more adhesions. (C) Lateral separation of adhesion sites causes a reduction in protrusion rate and P–R cycle frequency. The kymographs (right upper panel and enlarged lower panel) show a reduction in frequency. (D) Protrusion rates for 1D, 2D and 2D-spaced micropatterns. (E) Differences in P–R frequency per minute between conditions. *P<0.05. Scale bars: B, 10 μm and C, 3 μm.

Fig. 3.

Fig. 3.

Enhanced adhesion longevity of 1D and 3D matrix adhesions. (A) GFP–paxillin adhesions forming on 2D substrates involve small NAs (magenta triangles) and force-dependent FAs (blue triangles). Yellow triangles show adhesion disassembly of the FAs highlighted earlier. (B) 1D adhesions lack NAs and have a prolonged stability phase. (C) Examples of fluorescence changes over time, which illustrate the assembly and stability phases for NAs (red), FAs (green), 1D fibrillar adhesions (blue, 1DFXs) and 3D matrix adhesions (black, 3DMAs). (D) Assembly rates for NAs, FAs, 1DFXs and 3DMAs. (E) Average length of time of the stability phase for the four different adhesion types. (F) Percentage of adhesions with a stability phase longer than 1000 seconds. (G) GFP–paxillin-expressing NIH/3T3 fibroblast migrating through a 3D CDM. (H) Inset (yellow box in G) illustrates the stability of 3DMAs over an extended time. Scale bars: 10 μm.

Fig. 4.

Fig. 4.

1D fibrillar adhesions show decreased adhesion turnover. (A) Comparison of 2D (upper) and 1D (lower) adhesions during FRAP analyses of GFP–paxillin. Red circles indicate the size of the FRAP region. (B,C) FRAP kinetic analyses of GFP–paxillin. (D) Analysis of GFP–VASP shows no difference in FRAP kinetics. (E) GFP–VASP localization (left) and a corresponding phase-contrast image of a NIH/3T3 fibroblast during 1D migration. (F,G) Photoconversion of mKikGR–vinculin within adhesions on 2D (F) and 1D (G) surfaces and changes over time. The box indicates the areas shown in the kymographs on the right. (H) mKikGR–vinculin average loss of fluorescence after photoconversion for 1D (black circles) and 2D (open circles) vinculin adhesions. (I) _t_1/2 times for 1D and 2D vinculin adhesions. (J) Velocity measurements for vinculin within 1D and 2D adhesions. *P<0.05. Scale bars: E, 10 μm and F, 5 μm.

Fig. 5.

Fig. 5.

Actin retrograde flow in 1D versus 2D conditions. (A) Photoconversion of mKikGR–actin within the lamellipodium of a migrating NIH/3T3 fibroblast showing the rapid kinetics within this region. The red dot indicates the photoconversion site. (B) Pre-conversion images showing 2D lamellipodial actin (2D LA), 2D stress fiber (2D SF) insertion points and the leading edge. The red arrowhead indicates the point of conversion, whereas the white dotted line illustrates the axis of the kymographs shown in C. (C) Kymographs of the photoconverted (red) sites shown in B. (D) mKikGR–actin average loss of fluorescence after photoconversion for 1D (green circles), 2D LA (open circles) and 2D SFs (red circles). (E) _t_1/2 times for 1D, 2D LA and 2D SF actin. (F) Velocity measurements for actin within 1D, 2D FAs and 2D LA. *P<0.05. Scale bars: 5 μm.

Fig. 6.

Fig. 6.

Differential effects of loss of contractility. (A,B) Fibroblasts expressing mApple–paxillin show an altered distribution of adhesions on 2D (A) and 1D (B) surfaces after treatment with 25 μM blebbistatin. Kymographs illustrate the formation of NAs (red arrowheads) at the leading edge on 2D surfaces (A) and random formation on 1D surfaces (B). (C,D) Timelapse series showing AF568-labeled 9EG7 (activated β1 integrin) incorporation into adhesions on 2D surfaces in the control (C) or after treatment with 25 μM blebbistatin (D). Note the rearward movement of integrins in the control (purple arrowheads) and the lack of movement after treatment with blebbistatin (cyan arrowheads). The yellow dotted line indicates the original position of the adhesion. (E,F) Activated β1 integrin incorporation on 1D surfaces in the control (E) or after treatment with blebbistatin (F). Kymographs illustrate that integrins remain stationary and stable in either condition. The white dotted lines show the leading edge; the kymograph is shown at the green dotted line. Scale bars: 5 μm.

Fig. 7.

Fig. 7.

Myosin II isoforms have different roles in cell adhesion and migration under 1D conditions. (A) SiRNA knockdown of MYH9 results in the accumulation of NAs containing GFP–paxillin at the leading edge (green box) and central or posterior FAs (e.g. red box). (B) Time series for the insets in panel A comparing longevity of NAs (upper panel) and FAs (lower panel). (C) Remnants of 1D adhesions at various locations beneath the cell lose GFP–paxillin stability. (D) Time series (6 frames per minute) from the rectangle in panel C illustrates the instability of GFP–paxillin adhesion formation at the leading edge with nascent-like adhesions (red arrowheads). (E) Changes in fluorescence over time at the colored circles in panel C. (F) SiRNA knockdown of MYH10 in fibroblasts expressing GFP–paxillin leads to adhesion turnover distal to the leading edge. GFP TIRF images and DIC are shown. Zero and 14-minute timepoints are presented to illustrate the change in adhesion turnover at different locations. (G) Changes in normalized fluorescence over time at the colored circles in panel F. (H) Ablation of the genes encoding either MIIA or MIIB reveals a differential requirement for MIIA in rapid 1D migration. Scale bars in A, B and C are 5, 2 and 2 μm, respectively. CB (C), cell body. *P<0.05.

Fig. 8.

Fig. 8.

Decreased contractility on 1D patterns results in loss of protrusion efficiency. (A,B) Analysis of protrusion dynamics in the presence of 25 μM blebbistatin on 1D (A) and 2D (B) substrates with the accompanying kymographs (from the yellow dotted line) on the right. The red dotted line in the kymograph indicates the lamellipodial–lamellar border. (C) Comparison of P–R frequency for both control and blebbistatin-treated cells. (D) Forwards movement of the lamella (red dotted line in A and B) for both control and blebbistatin-treated cells. *P<0.05 compared with dimensional control.

References

    1. Burnette D. T., Manley S., Sengupta P., Sougrat R., Davidson M. W., Kachar B., Lippincott-Schwartz J. (2011). A role for actin arcs in the leading-edge advance of migrating cells. Nat. Cell Biol. 13, 371-382 -PMC -PubMed
    1. Choi C. K., Vicente-Manzanares M., Zareno J., Whitmore L. A., Mogilner A., Horwitz A. R. (2008). Actin and alpha-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner. Nat. Cell Biol. 10, 1039-1050 -PMC -PubMed
    1. Cukierman E., Pankov R., Stevens D. R., Yamada K. M. (2001). Taking cell-matrix adhesions to the third dimension. Science 294, 1708-1712 -PubMed
    1. Doyle A. D. (2009). Generation of micropatterned substrates using micro photopatterning. Curr. Protoc. Cell Biol. Chapter 10, Unit 10 15 -PMC -PubMed
    1. Doyle A. D., Wang F. W., Matsumoto K., Yamada K. M. (2009). One-dimensional topography underlies three-dimensional fibrillar cell migration. J. Cell Biol. 184, 481-490 -PMC -PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources