Podosome rings generate forces that drive saltatory osteoclast migration

Abstract

Podosomes are dynamic, actin-containing adhesion structures that collectively self-organize as rings. In this study, we first show by observing osteoclasts plated on bead-seeded soft substrates that podosome assemblies, such as rings, are involved in tension forces. During the expansion of a podosome ring, substrate displacement is oriented outward, suggesting that podosomal structures push the substrate away. To further elucidate the function of forces generated by podosomes, we analyze osteoclast migration. Determining the centers of mass of the whole cell (G) and of actin (P), we demonstrate that osteoclasts migrate by "jumps" and that the trajectories of G and P are strongly correlated. The velocity of the center of mass as a function of time reveals that osteoclasts rapidly catch up with podosomal structures in a periodic pattern. We conclude that actin dynamics inside the cell are not only correlated with cell migration, but drive it.

FIGURE 1:

Podosomes are involved in maintaining tension in osteoclasts. (A) During adhesion and spreading processes, the podosomes accumulate along the periphery of the cell, in the expanding regions. Several mature osteoclasts expressing GFP-actin are seeded onto a glass coverslip and observed under the microscope using a Nomarski contrast. Insets, fluorescence image of the cell, which is indicated by the white square. Left, 5 min after seeding, the cell is round and actin is scattered in the cytoplasm (inset). Right, 20 min after seeding, the considered cell starts spreading. The fluorescence image (inset) reveals that actin becomes concentrated in podosomes along the periphery of the contact region, where the cell is expanding. (B) Immunofluorescence labeling of adherent osteoclasts spreading on glass: actin stained with phalloidin (green) and vinculin stained with anti-vinculin (red). Top, 10 min after seeding, actin is loosely distributed in the cytoplasm of the osteoclast and vinculin staining confirms the absence of podosomes. Bottom, 25 min after seeding, podosomes form where the cell is expanding. Vinculin marks the periphery (surrounding cloud) of the podosomes. (C) Osteoclasts remain adherent even in the absence of podosomes. At t = 0 s, the cell is spread, in close contact with the substrate (right), and the fluorescence image (left) reveals several rings of podosomes (bright spots). At t = 40 s, the addition of EDTA produces a rapid dissociation of podosomes associated with cell retraction (white arrows).

FIGURE 2:

The podosome ring expansion correlates with the direction of migration. One osteoclast expressing the GFP-actin adherent on glass is imaged under the confocal microscope. The actin organization in the contact area with the substrate is revealed by slightly tilting the reconstructed images along the z-axis. Left, actin, concentrated in the base plane, is organized in two rings, with the largest ring located on the left-hand side. The fluorescence signal from the actin monomers ubiquitously invading the cytoplasm clearly reveals the shape of the cell. Right, 75 min later, the largest ring grows and the smallest disappears. Simultaneously, the cell flattens above the remaining ring, expanding in that region, and also moving to the left (white contour corresponds to the initial shape of the cell shown on the left).

FIGURE 3:

Actin ring structures exert tension forces on the substrate. The dynamics of a live osteoclast, expressing GFP-actin moving on the surface of a soft polyacrylamide gel (stiffness: 0.5 kPa) are shown. The gel containing fluorescent beads (rhodamine, diameter 210 nm) is coated with vitronectin. Images of GFP-actin and of the rhodamine beads were taken every minute. The substrate displacement field is reconstructed by tracking the displacements of the fluorescent beads. (A) The merge of the osteoclast image (GFP-actin) and of the displacement field (arrows indicate the local displacement with respect to the substrate at rest) reveals displacement is significant solely around the podosomal structure (ring). (B) Enlargement of the podosomal structure as delineated by the rectangle in (A). The displacements of the beads clearly reveal that the ring pushes the substrate outward.

FIGURE 4:

Podosome ring expansion and osteoclast migration are correlated. The dynamics of an osteoclast expressing GFP-actin moving on the surface of a polyacrylamide gel (stiffness: 3 kPa) are shown using time-lapse fluorescence microscopy (Supplemental Movie 4). Eight successive images separated by 30 min are displayed. The center of mass of the cell G, the position of the actin structure P, and defined the vector were determined from such images (see Materials and Methods). The points G and P for the first image (t = 0, top, left) are indicated. Bottom, right, successive positions, separated by 5 min, of the center of mass G determined from this image sequence (same scale).

FIGURE 5:

Successive positions of the center of mass G and of actin P in the sample plane (x, y). The time difference between two successive points is 5 min. The black arrow indicates the direction of the motion. The continuous and dashed lines correspond to the trajectory of G and P, respectively, averaged over 10 successive positions. The trajectories of G (full diamonds) and P (open squares) are strongly correlated. The vectors and (gray arrows) are associated to the events A, B, C, and D, and labels 1–5 indicate the minima in the velocity V_G, as defined in Figure 6.

FIGURE 6:

Velocity of the center of mass V_G, distance f, and cell length L as a function of time t. (A) The velocity V_G exhibits peaks, which correspond to a rapid motion of the cell in a given direction (jump). The minima in the velocity V_G are labeled 1–5. (B) The distance f interestingly exhibits the same type of temporal evolution. (C) The cell length L increases slowly when the cell velocity is small, and rapidly decreases during the jumps. Note that the cell jumps ∼10 min after f or L have reached a maximum. Identified here are four events: A, B, C, and D. Successive jumps are separated by ∼2 h.

FIGURE 7:

Temporal cross-correlation (t) between the distance f and the cell velocity V_G. The oscillations of the correlation function confirm the almost periodic character of the jumps, with a period T ≈ 2 h. Inset, enlargement of the central peak. The correlation (t) is maximum for t ≡ t ≈ 10 min, which shows that the cell jumps ∼10 min after f has reached a maximum.

FIGURE 8:

Vectors and associated displacements . The cell center of mass G jumps toward P, as pointed out by the strong correlation between at the maximum (gray dotted arrows) and the corresponding (black arrows). (A) Angle θ_G vs. angle θ_P. The dotted line corresponds to the slope 1, showing that θ_G ≈ θ_P. (B) Jump length vs. maximum distance f. The cell moves over a larger distance when the distance f at the maximum is larger.

FIGURE 9:

Sketch of forces exerted by two growing podosomes. (A) Podosomes in the contact region between an osteoclast and a glass bottom dish were imaged under the confocal microscope. Stacks of pictures were then used to reconstitute a Z-image, rendering the profiles of the podosomes in a vertical plane (t = 0, 22, 44, and 62 s). The sequence shows that the podosomes consists of actin filaments growing rapidly from the cell membrane (thick dotted rectangle on the right) and disappearing from the top (dotted rectangle on the left). (B) We have previously proposed that the actin cores of podosomes are organized in a conical brush (Destaing et al., 2003; Jurdic et al., 2006, Hu et al., 2011). Because actin filaments are cross-linked and growing from the membrane, the actin filaments, due to steric constraints, repel each other in the base plane. Thus two neighboring podosomes tend naturally to repel each other, and generate the negative tension associated with the podosomal structure. Gray arrows, forces exerted by neighboring podosomes on the substrate.