Self-organized podosomes are dynamic mechanosensors

Abstract

Podosomes are self-organized, dynamic, actin-containing structures that adhere to the extracellular matrix via integrins [1-5]. Yet, it is not clear what regulates podosome dynamics and whether podosomes can function as direct mechanosensors, like focal adhesions [6-9]. We show here that myosin-II proteins form circular structures outside and at the podosome actin ring to regulate podosome dynamics. Inhibiting myosin-II-dependent tension dissipated podosome actin rings before dissipating the myosin-ring structure. As podosome rings changed size or shape, tractions underneath the podosomes were exerted onto the substrate and were abolished when myosin-light-chain activity was inhibited. The magnitudes of tractions were comparable to those generated underneath focal adhesions, and they increased with substrate stiffness. The dynamics of podosomes and of focal adhesions were different. Torsional tractions underneath the podosome rings were generated with rotations of podosome rings in a nonmotile, nonrotating cell, suggesting a unique feature of these circular structures. Stresses applied via integrins at the apical surface directly displaced podosomes near the basal surface. Stress-induced podosome displacements increased nonlinearly with applied stresses. Our results suggest that podosomes are dynamic mechanosensors in which interactions of myosin tension and actin dynamics are crucial for regulating these self-organized structures in living cells.

Figure 1. Myosin tension regulates podosome ring dynamics

Cells were co-transfected with mCherry actin (red) and GFP-MLC (myosin light chain) (green). (A) BHK cells displayed podosome actin rings (Actin, arrow); myosin networks exhibited a circular fibrillar network (MLC, arrow). The overlay revealed a complex circular network of myosin fibers (green) surrounding the actin ring (red) (scale bar 10 µm). (B) For a better visualization, white square in left image of (A) is cropped and enlarged. In the control cell (Cont), the actin ring (left image) is surrounded by the large circular myosin network, and a small faint myosin ring is located in the inner region (middle image). The overlay (right image) shows that the actin ring colocalizes with the small myosin ring in a majority of locations. ML7 (25 µM) induced a very rapid (1 min) and sustained (10 min) disappearance of the actin ring (left images); however, the circular myosin fibers were still present after 1 min and decreased slightly at 10 minutes (middle images). The small myosin ring shrank after 1 min of ML7 myosin (middle image) and a myosin aggregate appeared at the center after 10 min of ML7 (lower middle image). The overlay shows that the stress fibers at the cell periphery were still present despite a relaxation of the cell (Movie S3) and a concurrent disassembly of the podosome actin ring, suggesting that myosin tension is crucial in regulating podosome ring dynamics. (C) The control level of actin fluorescence is normalized by taking the average fluorescence of the cell outside the ring (Non-POD). The fluorescence of the podosome rings was 187±13% of the control fluorescence in the absence of ML7 (POD, (−) ML7), and rapidly decreased to 112± 3.7% after 1 min of ML7 (25µM) and to 107±3% after 10 min of ML7 (*, p<0.01; n=8 different cells; Mean+/− se). (D) The control level of MLC fluorescence is normalized by taking the average fluorescence in the center of the large myosin ring (MLC Center, -ML7). The fluorescence of the large myosin ring surrounding the actin ring was 172±11.6% of the control fluorescence and remained relatively constant after 1 min of ML7 (25µM), and then decreased somewhat to 152±9.1% after 10 min of ML7. Although the level of fluorescence of the large myosin ring did not significantly decrease with ML7 treatment, the level of myosin in the center rapidly increased to 116.5±9.2% after 1 min and to 148±4.3% after 10 min of ML7 (*, p<0.01; n=8 cells; Mean+/− se), likely largely due to the shrinkage of the small myosin ring.

Figure 1. Myosin tension regulates podosome ring dynamics

Cells were co-transfected with mCherry actin (red) and GFP-MLC (myosin light chain) (green). (A) BHK cells displayed podosome actin rings (Actin, arrow); myosin networks exhibited a circular fibrillar network (MLC, arrow). The overlay revealed a complex circular network of myosin fibers (green) surrounding the actin ring (red) (scale bar 10 µm). (B) For a better visualization, white square in left image of (A) is cropped and enlarged. In the control cell (Cont), the actin ring (left image) is surrounded by the large circular myosin network, and a small faint myosin ring is located in the inner region (middle image). The overlay (right image) shows that the actin ring colocalizes with the small myosin ring in a majority of locations. ML7 (25 µM) induced a very rapid (1 min) and sustained (10 min) disappearance of the actin ring (left images); however, the circular myosin fibers were still present after 1 min and decreased slightly at 10 minutes (middle images). The small myosin ring shrank after 1 min of ML7 myosin (middle image) and a myosin aggregate appeared at the center after 10 min of ML7 (lower middle image). The overlay shows that the stress fibers at the cell periphery were still present despite a relaxation of the cell (Movie S3) and a concurrent disassembly of the podosome actin ring, suggesting that myosin tension is crucial in regulating podosome ring dynamics. (C) The control level of actin fluorescence is normalized by taking the average fluorescence of the cell outside the ring (Non-POD). The fluorescence of the podosome rings was 187±13% of the control fluorescence in the absence of ML7 (POD, (−) ML7), and rapidly decreased to 112± 3.7% after 1 min of ML7 (25µM) and to 107±3% after 10 min of ML7 (*, p<0.01; n=8 different cells; Mean+/− se). (D) The control level of MLC fluorescence is normalized by taking the average fluorescence in the center of the large myosin ring (MLC Center, -ML7). The fluorescence of the large myosin ring surrounding the actin ring was 172±11.6% of the control fluorescence and remained relatively constant after 1 min of ML7 (25µM), and then decreased somewhat to 152±9.1% after 10 min of ML7. Although the level of fluorescence of the large myosin ring did not significantly decrease with ML7 treatment, the level of myosin in the center rapidly increased to 116.5±9.2% after 1 min and to 148±4.3% after 10 min of ML7 (*, p<0.01; n=8 cells; Mean+/− se), likely largely due to the shrinkage of the small myosin ring.

Figure 2. Podosome rings exert traction stresses on the matrix when they undergo movements in the cytoplasm

(A) Cells transfected with mCherry actin were seeded on a 6.5 kPa polyacrylamide gel substrate (top left insert: a podosome actin ring, the same as in (B)). (B) The cell displayed an actin ring that expands to the left of the image within two minutes. To better visualize the movement of the ring, timelapse sequences were analyzed with the optical flow method. The dark and blue regions correspond to the location of the podosome ring at time zero and the green and red regions correspond to the location of the podosome ring at 2 min. (C) The podosome ring exerts tractions up to 800 Pa in the direction of the movement of the wave. During the podosome band extension, the peak tractions are exerted at the extremities of the actin band and generated a peak gel deformation up to 0.3 µm (D). (E) A BHK cell, transfected with mCherry actin, was plated on a 3.5 kPa polyacrylamide gel substrate (top left insert: a podosome actin ring, the same as in (F)). Scale bar=20 µm for (A) and (E). (F) Although the size and shape of the podosome ring did not change during a 4-minute period, the optical flow analysis showed that the ring rotated clockwise, with a major actin flow that went from the left of the ring (dark regions) to the upper side of the ring (red regions). (G) The ring rotation exerted torsional tractions on the substrate up to 600 Pa where the actin flow was the highest, resulting a torsional deformation of the substrate up to 0.6 µm (H). (I) The peak tractions during podosome ring movements were computed for cells on different substrate stiffness (n=4 cells each on 2, 3.5, and 6.5 kPa gel respectively). Average peak tractions were 251±26 Pa on 2 kPa gel, 356±109 Pa on 3.5 kPa gel, and 672±100 Pa on 6.5 kPa gel (p<0.01 between 2 kPa and 3.5 kPa gels; p<0.038 between 3.5 kPa and 6.5 kPa gels; Mean+/−se).

Figure 2. Podosome rings exert traction stresses on the matrix when they undergo movements in the cytoplasm

(A) Cells transfected with mCherry actin were seeded on a 6.5 kPa polyacrylamide gel substrate (top left insert: a podosome actin ring, the same as in (B)). (B) The cell displayed an actin ring that expands to the left of the image within two minutes. To better visualize the movement of the ring, timelapse sequences were analyzed with the optical flow method. The dark and blue regions correspond to the location of the podosome ring at time zero and the green and red regions correspond to the location of the podosome ring at 2 min. (C) The podosome ring exerts tractions up to 800 Pa in the direction of the movement of the wave. During the podosome band extension, the peak tractions are exerted at the extremities of the actin band and generated a peak gel deformation up to 0.3 µm (D). (E) A BHK cell, transfected with mCherry actin, was plated on a 3.5 kPa polyacrylamide gel substrate (top left insert: a podosome actin ring, the same as in (F)). Scale bar=20 µm for (A) and (E). (F) Although the size and shape of the podosome ring did not change during a 4-minute period, the optical flow analysis showed that the ring rotated clockwise, with a major actin flow that went from the left of the ring (dark regions) to the upper side of the ring (red regions). (G) The ring rotation exerted torsional tractions on the substrate up to 600 Pa where the actin flow was the highest, resulting a torsional deformation of the substrate up to 0.6 µm (H). (I) The peak tractions during podosome ring movements were computed for cells on different substrate stiffness (n=4 cells each on 2, 3.5, and 6.5 kPa gel respectively). Average peak tractions were 251±26 Pa on 2 kPa gel, 356±109 Pa on 3.5 kPa gel, and 672±100 Pa on 6.5 kPa gel (p<0.01 between 2 kPa and 3.5 kPa gels; p<0.038 between 3.5 kPa and 6.5 kPa gels; Mean+/−se).

Figure 3. Movements and tractions by podosome rings and focal adhesions

(A) BHK cells are co-transfected with m-Cherry actin (left image) and EGFP zyxin (central image) and seeded on collagen-1 coated rigid glass. The zyxin is located in the focal adhesions at the tip of the actin stress fibers, and also colocalizes with the actin in the podosome rings (left image). (B) The focal adhesions and the podosome ring were visualized in EGFP zyxin at a region of interest (white square in (A)) in a timelapse sequence of 8 minutes (left image and central image). The image at the initial time is artificially colored in green, and the image after 8 minutes is artificially colored in red. The overlay of the two images allows the determination of the movement of the podosomes and the focal adhesions (right image). The zyxin located in the focal adhesions before and after 8 minutes completely colocalizes (yellow dots), thus showing little movement of the focal adhesions during this period. However, during the same period, the podosome zyxin ring moved substantially within 8 min (the red band and the green band). (C) A BHK cell was transfected with EGFP-zyxin and seeded on a substrate of 5 kPa. The dynamics of a podosome ring and of focal adhesions were recorded during a 4-minute period. During this period, the focal adhesions exhibit very small movements, while the podosome ring display expansion. (D) A close visualization of the podosome ring is shown at the beginning of the sequence and after 4 min. Analysis of the ring movement with optical flow shows in blue/green (the position of the ring at the beginning of the sequence) and in red (the position at 4 min). The ring exhibited an expansion to the left on its left part, an expansion to the right in the center of the ring, and a formation or reinforcement of podosomes on the right part of the ring. (E) The tractions exerted by the cell are computed during the timelapse sequence of 4 min. A majority of focal adhesions did not exhibit traction changes at all during this period, although a few focal adhesions exhibited traction changes at the cell periphery ranging from 150 to 350 Pa. In contrast, the podosome ring exerted tractions up to 200 Pa, in the same direction of its movements, at sites far from any focal adhesions. Another cell exhibited similar behavior.

Figure 3. Movements and tractions by podosome rings and focal adhesions

(A) BHK cells are co-transfected with m-Cherry actin (left image) and EGFP zyxin (central image) and seeded on collagen-1 coated rigid glass. The zyxin is located in the focal adhesions at the tip of the actin stress fibers, and also colocalizes with the actin in the podosome rings (left image). (B) The focal adhesions and the podosome ring were visualized in EGFP zyxin at a region of interest (white square in (A)) in a timelapse sequence of 8 minutes (left image and central image). The image at the initial time is artificially colored in green, and the image after 8 minutes is artificially colored in red. The overlay of the two images allows the determination of the movement of the podosomes and the focal adhesions (right image). The zyxin located in the focal adhesions before and after 8 minutes completely colocalizes (yellow dots), thus showing little movement of the focal adhesions during this period. However, during the same period, the podosome zyxin ring moved substantially within 8 min (the red band and the green band). (C) A BHK cell was transfected with EGFP-zyxin and seeded on a substrate of 5 kPa. The dynamics of a podosome ring and of focal adhesions were recorded during a 4-minute period. During this period, the focal adhesions exhibit very small movements, while the podosome ring display expansion. (D) A close visualization of the podosome ring is shown at the beginning of the sequence and after 4 min. Analysis of the ring movement with optical flow shows in blue/green (the position of the ring at the beginning of the sequence) and in red (the position at 4 min). The ring exhibited an expansion to the left on its left part, an expansion to the right in the center of the ring, and a formation or reinforcement of podosomes on the right part of the ring. (E) The tractions exerted by the cell are computed during the timelapse sequence of 4 min. A majority of focal adhesions did not exhibit traction changes at all during this period, although a few focal adhesions exhibited traction changes at the cell periphery ranging from 150 to 350 Pa. In contrast, the podosome ring exerted tractions up to 200 Pa, in the same direction of its movements, at sites far from any focal adhesions. Another cell exhibited similar behavior.

Figure 4. Mechanical stresses are transmitted outside-in to the podosomes

(A) An RGD-coated 4.5-µm bead (arrow) was attached to the apical surface of a BHK cell transfected with EGFP-α-actinin. (B) The cell exhibited a large podosome α-actinin ring at the basal surface at the periphery of the cell and a small faint ring inside. (C) Direct displacements of podosome rings were quantified using a sensitive synchronous detection method [38. 39] in response to varying oscillatory stresses (0.8 Hz). Maximum displacements of the podosomes increased from ~30 nm at 8.7 Pa and 17.5 Pa to ~50 nm at 26.2 Pa. Displacements at sites other than the rings are due to stress-induced diaplacements of α-actinin associated with other cytoskeletal structures. The pink dot represents the bead center position. (D) Quantitative analyses of podosome maximum displacements. Average peak displacements were 48±18.7 nm, 53.2±14.6 nm, and 87.4±17.7 nm, respectively with increasing stresses. (*, p<0.01; n = 5 cells, mean ± se) Note that the relationship between the applied stress and the maximum α-actinin displacements was nonlinear.