Lamellipodial actin mechanically links myosin activity with adhesion-site formation - PubMed (original) (raw)
Lamellipodial actin mechanically links myosin activity with adhesion-site formation
Grégory Giannone et al. Cell. 2007.
Abstract
Cell motility proceeds by cycles of edge protrusion, adhesion, and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction, and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process.
Figures
Figure 1. The period of retraction depends on lamellipodial actin growth and regeneration during the contractile cycle
(A) DIC kymograph of a MEF spreading on FN (10 µg/ml) showing the generation of periodic waves during periodic contractions. Perfusion of jasplakinolide (200 nM, red line) induces transient cessation of periodic contractions. The cycle resumes by an edge retraction followed by a DIC wave (dashed red lines). Time bar 30 s (t); scale bar 1 µm (d). (B) α-actinin EPI (left) kymograph. Note the periodic network growth (ng) and separation (ns) of α-actinin from the cell tip that created gaps (g) in the α-actinin fluorescence (movie S1B). The dotted lines correspond to the time where pictures 1–6 (right top) were acquired. Rectangles in pictures 1–6 depict the region used to generate the kymograph and intensity profiles (right bottom). The intensity profiles show growth, separation (black arrows), and gap formation (‘g’) in α-actinin network. Left: t = 30 s; d = 1 µm. Right top: scale bar 1 µm. Right bottom: intensity bar (horizontal) arbitrary unit; scale bar (vertical) 1 µm. (C) DIC (left) and α-actinin EPI (middle) kymographs with lines marking the displacement of the cell edge (blue) and the α-actinin network growth (ng), separation (ns), and gap formation (g) (red). The schematic kymograph (right) shows the parameters we quantified that are summarized in table 1. (D) α-actinin EPI kymograph before (up) and after (bottom) perfusion of CB (250 nM). The rate of LP actin growth (slope of white line) is reduced by CB treatment (movie S1D). t = 30 s; d = 2 µm. (E) DIC kymograph. Perfusion of a control solution (up) does not change periodic contractions or DIC waves. Perfusion with 250 nM CB solution (bottom) decreases the period of retraction (movie S1E). t = 30 s; d = 2 µm. (F) Relationship between the retraction period and the actin network growth speed. Error bars show standard deviation. White arrows indicate direction of protrusion.
Figure 2. Lamellipodial actin bending
(A) Micrographs (left) and kymographs (right) of DIC (top), α-actinin TIRF (middle), and merge (bottom). The dashed line depicts the region used to generate the kymographs. (movie S2A). Left: scale bar 2 µm. Right: t = 30 s; d = 2 µm. (B) Identical to (A) except with α-actinin EPI (middle) (movie S2B). (C) Kymographs of DIC (top) and α-actinin TIRF (bottom). Dashed lines mark the start of edge retraction. Note that LP actin bending, visualized in TIRF by the loss in α-actinin-GFP fluorescence, initiates simultaneously with the start of edge retraction. t = 30 s; d = 2 µm. (D) Kymographs of DIC (top) and α-actinin EPI (bottom). Dashed lines mark the end of edge retraction. Note that the gap between LP actin and the edge, visualized by EPI, occurs 5 s after the start of edge retraction simultaneously with the end of the retraction. t = 30 s; d = 2 µm. (E) Schematic of the α-actinin network before (top) and after (bottom) edge retraction showing a side view (first column), imaging by TIRF (second column) and EPI (third column). Green represents α-actinin fluorescence, white outlines the contractile module, grey corresponds to FN, and the blue rectangle corresponds to the glass cover-slip. (F) MEF generating periodic contractions in a medium containing high molecular weight fluorescein dextran imaged with TIRF (top left). Fluorescence intensity profiles (top right) taken along dashed lines 1 and 2 show that the height of the contractile module peaks (red line) at a position corresponding to DIC waves. Schematic representation of the bending contractile module (bottom). Colors correspond to (C) except green represents fluorescein fluorescence. Top left: scale bar 2 µm. Top right: intensity bar (vertical) arbitrary unit; scale bar (horizontal) 1 µm.
Figure 3. Mechanical connections between cell edge and the lamellipodial actin are broken inducing separation and condensation of lamellipodial actin
(A) Cell edge velocity plotted as a function of time and position along the edge of a spreading MEF (movie S3A). The cell edge was detected using computer vision from which edge velocity was calculated (see methods). Lateral propagation of retraction is visible as diagonal yellow lines. (B) Velocity of the rearward movement of two DIC waves plotted as a function of time and position along the cell edge. Note that the front of the waves correspond to edge retractions in (A). For sake of clarity, two of seven detected waves are shown. (C) DIC kymograph from the cell in Figure 3A shows that DIC wave initiates simultaneously with the start of edge retraction (left). In the bottom kymograph the white part of the DIC wave is outline in red. Dashed lines mark the start of edge retraction. Time bar 30 s; scale bar 2 µm. Plot of cell edge velocity during retraction as a function of time relative to the initial detection of the DIC wave peak (red line)(right). The analysis reveals that the DIC wave peak is detected shortly (1.5 s ± 1.5 s, 803 kymographs, 7 waves) after the start of the edge retraction (D) DIC kymograph from the cell in Figure 3A shows the generation of DIC waves and their condensation into persistent structures (red arrows). t = 30 s; d = 2 µm. (E) DIC (top), α-actinin-GFP TIRF (middle), and merge (bottom) micrographs (left column) and kymographs (right column). Dotted lines depict the regions used to generate the kymographs. Note the condensation of α-actinin into bundles (red arrows) (movie S3E). Left: scale bar 2 µm. Right: t = 30 s; d = 2 µm. (F) Identical to (E) except fluorescence is GFP-MLCK. White arrows indicate direction of protrusion
Figure 4. Edge retraction depends on MII activities
(A) DIC kymographs shows that retraction speed is decreased with BBI (50 µM) (top) when compared to controls (middle) while CalA (20 nM) increases retraction speed (bottom) (movie S4A). t = 30 s; d = 2 µm. (B) Bar graphs of periodic contractions retraction speed (left) and duration (middle) and protrusion duration (right) for cells treated with BBI (grey), control (white), and CalA (black). Scatter plot shows an inverse relationship between retraction speed and retraction duration (bottom). Error bars show standard deviation. (C) DIC kymographs show that a MIIB control cell (top) generates stronger retractions compared to a MIIB-KO cell (middle top). MIIB-KO also ruffle extensively (middle bottom) while a MIIB-KO/MIIA-KD cell generates no periodic contractions (bottom). t = 30 s; d = 2 µm. (D) Bar graphs show retraction speed (top left) and duration (top middle) and protrusion duration (top right) for MIIB control cells (black) and MIIB-KO cells (grey) during periodic contractions, while MIIB-KO cells generating extensive ruffling show comparable protrusion speed (white). Bar graphs representing the percentage of cells displaying periodic contractions (black), no periodic contractions (grey) and extensive ruffling (white), for MIIB control cells (bottom left), MIIB-KO cells (bottom middle), and MIIB-KO/MIIA-KD cells (bottom right). Error bars show standard deviation. (movies S4 A–C) Black arrows indicate direction of protrusion.
Figure 5. Periodic contractions initiate adhesion site formation
(A) DIC (top), MLC-EGFP TIRF (middle), and merge (bottom) micrographs (left column) and kymographs (right column). Note that MLC clustered at the back of the contractile module (movie S5A). (B) Same as (A) except with paxillin-GFP TIRF. Note that adhesion sites initiated close to the cell edge, between the cell tip and the previous DIC wave (movie S5B). (C) Laser trap experiment using beads coated with diluted FN7-10 trimers. Beads were positioned on the tip of the protruding cell edge using a laser trap during the generation of periodic contractions. DIC kymographs depicting the movement of the cell edge (upper left, red line) and the movement of a FN-coated bead (upper right, green line). Displacement versus time plot (bottom) showing that the bead is periodically connecting to the actin flow during the retraction (red strips), being moved rearward until the force applied by the laser trap break its connection with actin, pulling back the bead in the trap center. (D) α-actinin-GFP TIRF (top), paxillin-DsRed TIRF (middle), and merge (bottom) micrographs (left column) and kymographs (right column). Note that adhesion sites were present in the area where the LP actin was bending up. Note that α-actinin condensate behind adhesion sites initiated by the previous retraction (movie S5C). (E) Same as (D) but with VASP-GFP TIRF (top), MLC-mRFP TIRF (middle), and merge (bottom). Note the succession of adhesion site rows and MLC aggregates (movie S5D). (F) Thresholding of VASP-GFP (green) and MLC-mRFP (red) fluorescence was performed to quantify the co-localization (yellow) of protein clusters. Both in TIRF (up) and in EPI (bottom). MLC and VASP clusters were excluded (movie S5F). t = 30 s; d = 2 µm. White arrows indicates protrusion direction. Dashed lines indicate the region used to generate kymographs.
Figure 6. Lamellipodial actin is a dense and cohesive network above the LM
(A) Spreading MEF generating periodic contractions. LP actin seen by α-actinin-GFP (left) is preserved after extraction/fixation (right) for EM visualization. (B) DIC image of spreading MEF generating periodic contractions just before (left) and just after (right) extraction. The green contours denote high α-actinin-GFP fluorescence. (C) DIC kymograph generated from the region depicted by the red line in (B). Vertical line indicates detergent extraction. The DIC wave is unchanged by extraction. (D) Blowup of the sub-region in (A) seen in EM (top) and merged with α-actinin fluorescence (green, bottom). (E-F) Higher resolution blowups of the regions depicted in (D) showing that the α-actinin-GFP fluorescence co-localizes with a dense ultrastructure. Green contour in (E) denotes high α-actinin-GFP fluorescence. (G) Visualization of the cytoskeletal ultrastructure (cell from (B,C)) by EM merged with α-actinin fluorescence contours (left) and with α-actininin fluorescence (middle) showing co-localization of α-actinin with the dense actin network visible in detail (right) (stack movie S6). Note the LP actin growth (1), gap in the LP actin (2) and LP actin bundle (3). (H) Stereo anaglyphs (subregions from G, left panel) showing the dense structure corresponding to LP actin atop a more sparse structure (left and right), and LP actin bending during contraction (left).
Figure 7. Schematic representation of lamellipodial actin periodic regeneration
The LP actin (green) is above the LM (gray). Polymerization at the front of the LP actin network causes the back of the network to grow towards the back of the contractile module until it reaches an adhesion site (i) where a MII cluster (blue) forms. MII pulls the LP actin generating high tension on the cell front, causing LP bending, edge retraction, and initiation of new adhesion sites (red) on the ECM (black rectangle) (iia). The LP actin continues to be pulled until it is released from the tip (iii) and edge protrusion restarts. A new LP actin network immediately resumes growth, suggesting that the actin polymerization machinery (yellow) is still present at the cell tip (iv). The released LP actin, still pulled by MII, further condenses into a bundle at the back of the previous adhesion site (v) while the newly growing LP actin reaches the next adhesion site and the cycle begins anew (vi). LP Ruffling (iib) occurred in the case when the total bond energy connecting LP actin to the edge is greater than the bond energy of nascent adhesion sites to the ECM.
References
- Bear JE, Svitkina TM, Krause M, Schafer DA, Loureiro JJ, Strasser GA, Maly IV, Chaga OY, Cooper JA, Borisy GG, et al. Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell. 2002;109:509–521. - PubMed
- Cameron LA, Giardini PA, Soo FS, Theriot JA. Secrets of actin-based motility revealed by a bacterial pathogen. Nat Rev Mol Cell Biol. 2000;1:110–119. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources