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First published online April 24, 2006
doi: 10.1242/10.1242/jcs.02838

Journal of Cell Science 119, 1914-1925 (2006)
Published by The Company of Biologists 2006
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Spatiotemporal dynamics of actin-rich adhesion microdomains: influence of substrate flexibility

Olivier Collin1, Philippe Tracqui1,*, Angélique Stephanou1, Yves Usson2, Jocelyne Clément-Lacroix1 and Emmanuelle Planus1,*,{ddagger}

1 Equipe DynaCell, Laboratoire TIMC-IMAG, CNRS UMR5525, Institut de l'Ingénierie et de l'Information de Santé-Faculté de Médecine, 38706 La Tronche CEDEX, France
2 Equipe RFMQ, Laboratoire TIMC-IMAG, CNRS UMR5525, Institut de l'Ingénierie et de l'Information de Santé-Faculté de Médecine, 38706 La Tronche CEDEX, France


Figure 1
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  		Fig. 1. Actin cytoskeleton structure and podosome rosette appearance of spread 3T3 fibroblasts depend on substrate flexibility. 3T3 GFP-actin transfected fibroblasts observed by fluorescence microscopy. A 3T3 fibroblast spread on rigid substrate (glass) exhibits a larger number of actin stress fibres (A) than a fibroblast on very flexible substrate (0.08% bis-acrylamide) (B). The cell appears more spherical and loses most of its actin stress fibres on 0.08% gel. However 3T3 fibroblasts can exhibit podosome rosettes, i.e. large ring-shaped bands made of an actin cloud surrounding dense actin dots on both rigid (C) and flexible substrates (D) (0.2% gel), although the frequency of appearance of the rosettes is higher for rigid substrates than for flexible ones. Bars, 10 µm.

 

Figure 2
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  		Fig. 2. Podosome rosettes are adherent structures. (A) Observation of 3T3 fibroblasts by total internal fluorescence microscopy show that podosomes are dense actin dots. (B) Confocal interference reflection microscopy (CIRM) revealed that podosomes are structures in close contact with the substrate and are therefore involved in cell adhesion. (C) Rosette in 3T3 fibroblast viewed by fluorescence. This ring-shaped structure is composed of a large number of podosomes, all adherent to the substrate (D), as observed with CIRM. Furthermore, they can coexist with other adherent structures such as focal contacts. Bars, 10 µm.

 

Figure 3
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  		Fig. 3. The distance between neighbouring podosomes depends on the substrate flexibility. (A) Total internal fluorescence microscopy of a podosome cluster. Podosomes are hardly visible over the dense actin cloud. (B) Image post-processing by a top-hat filter reveals the location of podosomes. Bars, 10 µm. (C) Computation of the mean distance between nearest podosomes for 16 pairs of podosomes per cell over full time-lapse sequences and for each substrate rigidity (n=96 for 0.08% gel; n=112 for 0.2% gel; and n=144 for glass). Data are expressed as mean ± s.d.

 

Figure 4
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  		Fig. 4. Individual podosome lifespan depends on the substrate flexibility. (A) 3T3 fibroblast observed by fluorescence microscopy exhibiting an actin cluster. (B-E) Movement of the cluster over 3 minutes revealed an apparent motion induced by appearance and disappearance of non-moving podosomes (circled). (F) The lifespan of podosomes calculated by averaging the lifespan of about ten podosomes per cell, and for each substrate type (n=68 for 0.08% gel; n=63 for 0.2% gel; and n=99 for glass). Data are expressed as mean ± s.d. Bars, 20 µm (A); 10 µm (B-E).

 

Figure 5
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  		Fig. 5. Podosomes are more stable and more individualised on rigid substrates. Podosome rosette observed by confocal microscopy for actin GFP in a 3T3 cell seeded on rigid (A) and flexible substrate (B). The 3D reconstruction was made by measuring fluorescence density. High-density regions were coloured in red whereas weaker densities were coloured in green. The rosette on the rigid substrate is composed of individual actin dots covered by a weak actin cloud. (B) When seeded on flexible substrate, the rosette appears more blurred and actin dots are very difficult to distinguish from the denser actin cloud. Bars, 10 µm. (C) Line scan of the fluorescence profile of FITC beads when spread on polyacrylamide gels, allowing the computation of the width at half height of the main peak. (D) Mean width for glass and flexible substrates illustrates the optical diffusion caused by the gel (n=20).

 

Figure 6
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  		Fig. 6. FRAP analysis of the rosette. (A) The rosette before photo bleaching. A region of interest (ROI) is boxed in the rosette band. (B) After photobleaching the recovery of fluorescence in the ROI was measured. Bars, 10 µm. (C) Experimental data with an exponential recovery. The identified characteristic time is 17 seconds.

 

Figure 7
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  		Fig. 7. Spatial dynamics of the rosettes depends on substrate rigidity. Simultaneous evolution with time of the expansion/contraction rate E (blue curve) and of the rotation rate {omega} (grey curve). (B) Simultaneous evolution of the orthogonal shear rate S1 (green curve) and of the oblique shear rate S2 (grey curve). (C) Velocity field superimposed to the rosette image at time t=120 seconds, which corresponds to the maximum expansion rate (1); (D) Velocity field superimposed to the rosette image at time t=255 seconds which corresponds to the maximum contraction rate (2). This rosette was observed on a rigid substratum (glass). (E) Mean rate of expansion and contraction averaged over several rosettes and for each value of substrate rigidity computed by n=8 for 0.08% gel; n=7 for 0.2% gel; and n=16 for glass. Data are expressed as mean ± s.d.

 

Figure 8
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  		Fig. 8. Motion of the rosettes is associated with the protrusion of the cell membrane. (A-F) 3T3 cell exhibiting growth and shrinkage of a rosette over 5 minutes with time intervals of 1 minute. The movement of the rosette along the cell periphery allows protrusion and retraction of the membrane as it grows and shrinks. Bar, 10 µm.

 

Figure 9
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  		Fig. 9. Podosome rosettes can be a periodically emerging structure. (A,C,E) Diversity of the profiles exhibited by expansion/contraction rates E(t). (A) Contraction profile without rosette expansion (on flexible substratum). (C) alternating symmetrical expansion and contraction phases with decreasing signal amplitude (on rigid substratum). (E) Alternating asymmetrical expansion and contraction phases with decreasing signal amplitude (on rigid substratum). (B,D,F) Associated Fourier transforms of the E(t) time series in A-C, respectively.

 

Figure 10
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  		Fig. 10. Effect of drug treatment on podosome dynamics. 3T3 fibroblasts spread on glass exhibit a podosome rosette visualised by GFP actin fluorescence. (A-D) Soon after treatment, cyto D inhibits actin wave propagation (B, arrow). Five minutes after treatment, the actin cytoskeleton disrupts, inhibiting new podosome formation. Podosomes previously formed stay still for several minutes in their position during actin disruption (C,D, arrows). Depolymerising the microtubule cytoskeleton using nocodazole has no effect on rosette propagation (E,F). Myosin inhibition with 2,3-BDM (G,H) or blebbistatin (I,J) completely inhibits podosome rosettes (arrows). Bars, 10 µm.

 

Figure 11
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  		Fig. 11. Schematic representation of patterns of podosome groups adherent on rigid (A) or flexible (B) substrates. The zone of inhibition (zi) preventing the formation of new podosomes in a close vicinity of an existing podosome increases with the substrate flexibility. The reduction of contractile forces induced by the attachment to flexible substrate (B') compared with rigid substrate (A') should cause a cell-substrate immature adhesive interface at the podosome level and then reduce its lifespan and regulate minimum distance (d) between podosomes.

 

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© The Company of Biologists Ltd 2006