A role for actin arcs in the leading-edge advance of migrating cells

Abstract

Epithelial cell migration requires coordination of two actin modules at the leading edge: one in the lamellipodium and one in the lamella. How the two modules connect mechanistically to regulate directed edge motion is not understood. Using live-cell imaging and photoactivation approaches, we demonstrate that the actin network of the lamellipodium evolves spatio-temporally into the lamella. This occurs during the retraction phase of edge motion, when myosin II redistributes to the lamellipodial actin and condenses it into an actin arc parallel to the edge. The new actin arc moves rearward, slowing down at focal adhesions in the lamella. We propose that net edge extension occurs by nascent focal adhesions advancing the site at which new actin arcs slow down and form the base of the next protrusion event. The actin arc thereby serves as a structural element underlying the temporal and spatial connection between the lamellipodium and the lamella during directed cell motion.

Figure 1

Retrograde actin flow rates change several times over a single edge protrusion/retraction cycle. a) Electron micrograph of a rotary-shadowed cell after live-cell extraction. b) Actin-tdEOS speckle image of an entire PtK1 cell with a corresponding FSM flow map and a higher magnification of the leading edge flow. Vector colors reflect flow speed (color bar), and arrows reflect direction. Scale bars in (a) and (b), 1 μm and 10 μm respectively. c) Edge motion rates relative to retrograde actin flow. Schematic of how the speckle flow data was binned and resulting rearward speckle flow kymograph showing the change in retrograde flow rates during protrusion (open arrows) and retraction (closed arrows) of the leading edge. Each data bin was 5 μm across and 1 μm high. d) Schematic shows how the edge protrusion and retrograde flow data was binned across the leading edge. Bins for edge protrusion were set at 500 nm parallel to the edge. Bins for retrograde flow were set at 1 μm parallel to the edge and 3 μm into the cell. e) Edge protrusion/retraction velocity and rearward actin velocity maps of the same cell used for the kymograph in (c). f) Edge position, edge velocity, and rearward actin flow of the region denoted by the dotted lines in (e) plotted over time. Stars in edge velocity and rearward actin flow graphs denote retractions and arrowheads denote protrusions corresponding to increases in rearward actin flow. Arrowheads denote slowing rearward actin flow immediately after edge retraction.

Figure 2

Differential actin filament turnover during protrusion and retraction. a) Montage of unconverted actin-tdEOS (green) at the edge and actin-tdEOS photo-converted in the lamellipodium during edge protrusion. b) Montage of unconverted and converted actin-tdEOS molecules during edge traction. Yellow line denotes region of photo-conversion and arrowheads denote actin bundles formation. c) Quantification of fluorescence loss of converted actin-tdEOS molecules in the lamellipodium during edge protrusion (green line) or retraction (red line). d) Still frames showing the actin bundle formed after retraction is arc shaped. e) Montage showing unconverted and converted channels before, 0 minutes, and 6 minutes after photo-conversion of actin-tdEOS molecules incorporated into actin arcs in the lamella (box). f) Time montage showing the recovery of fluorescence from the unconverted channel and loss of fluorescence from the converted channel of actin-tdEOS in the lamella. Scale bar, 5 μm.

Figure 3

Actin arc dynamics at the leading edge. a) Three frames of a time-lapse recording of actin-mRFP showing the cytoskeletal organization at the leading edge during the transition from protrusion to retraction and back to protrusion. Star denotes actin network in the lamellipodium and arrow denotes actin arcs in the lamella. Yellow arrowheads show actin filaments associated with focal adhesions. Red arrowheads show a newly forming actin arc. b) Time-lapse montage of the box in (a) showing the formation of an actin arc (red arrowheads) between protrusion events (white lines). Green arrowhead shows the actin arc formed during previous retraction event. Yellow arrowheads show the removal of actin arcs. c) Kymograph of the line in (a) showing multiple protrusion and retraction events over 1 hr. Red arrows denote the first frame an actin arc was observable and yellow dotted line shows lamellar advance. d) Actin-mGFP and zyxin-mCherry montage showing an actin arc can form (white arrowheads) before coming in contact with focal adhesions (yellow arrow). e) Protrusion/retraction map showing edge velocity (shown by color bar) changes over time across the edge of the cell in (a). F) Plot of the edge velocity from the dotted line in (e). g) Fourier transforms of the velocity profiles represented by the colored lines in (e). h) Distribution of the period of the protrusion/retraction cycle among 41 cells. Scale bar, 10 μm. The change in intensity in (b) from frame 10 to 11 is due to focusing.

Figure 4

Myosin II activity condenses the lamellipodium into an actin arc. a) Organization of actin-mRFP, Myosin IIA-GFP, and overlay during edge protrusion. Notice myosin II localizes with older actin arcs in the lamella. b) Time-lapse montages of actin-mRFP, myosin IIA-GFP and an overlay showing the co-localization of myosin II with newly forming actin arcs. Arrowheads show co-translocation of myosin IIA and the newly formed actin arc. c) Kymograph showing myosin IIA dynamics over three protrusion/retraction cycles. Arrowheads denote the appearance and arrows denote the movement of myosin IIA. d) Actin-mRFP before and after 25 μm blebbistatin. Kymograph shows the protrusion retraction cycle of the edge before and after blebbistatin. Note that the structure and movement of the actin arcs is diminished in the presence of blebbistatin. e) Protrusion/retraction map showing edge motion before and after blebbistatin addition (arrow). Edge retractions denoted by arrowheads. Scale bars, 10 μm.

Figure 5

Oscillatory edge motion and net edge extension. a) Edge velocity map along the edge and a single region (bottom graph) along the edge from a crawling cell. b) Edge position map of the same cell as in (a). Edge position map was created by color-coded the lowest edge position as blue and the highest as red as in the color bar. This allows for relative edge position along the same regions as in the velocity map in (a) to be graphically displayed over time. Bottom graph in (b) shows the relative edge position at one point along the edge. Red dotted line and green dotted line graphically show the protrusion amplitude and retraction amplitude of one protrusion respectively. c) Protrusion amplitudes plotted against edge oscillation frequencies for individual cells. Correlation coefficient: −0.5129 (confidence interval: −0.7087, −0.2437). d) Migration rate plotted edge oscillation frequencies for individual cells. Correlation coefficient: 0.1966 (confidence interval: −0.1182, 0.4755). e) Migration rate plotted against protrusion amplitudes for individual cells. Correlation coefficient: 0.2650. (confidence interval: −0.0464, 0.5295). f) Migration rate plotted against the ratio of protrusion and retraction amplitudes in individual cells. Correlation coefficient: 0.4254 (confidence interval: 0.1355, 0.6482). g-h) Edge position and rearward flow velocity plotted as in Fig. 1(f) for a cell that demonstrates net edge growth (g) and a cell that does not (h). Note the pattern of rearward actin flow is similar in both cells. Pearson’s correlation coefficient was used to quantify the correlation and the 95% confidence interval for each pair was computed by using the Fisher transformation.

Figure 6

Differential slippage of focal adhesions in crawling vs. non-crawling cells correlates with new actin arc movement. a) Overlay of actin-mGFP (blue) and zyxin-mCherry either at 0 minutes (magenta) or after 14 minutes (green). Arrows in zyxin-mCherry overlay show focal adhesions that move during this time period. b) Focal adhesion movement compared to preexisting adhesions. New focal adhesions during protrusion are labeled purple and the same adhesions after the next edge retraction are labeled green in two cells with different migrating rates (fast cell- 0.25 μm/min; slow cell- 0.04 μm/min). The preexisting adhesion in each cell is diplayed in yellow. Total distance of the new adhesion from the previous adhesion during protrusion is shown by magenta double-headed arrows and the net distance after new adhesion slippage during edge retraction is shown by the green double-headed arrows. c) Quantification net distance between new adhesions and preexisting adhesions for the fast cell in (b) (n= 38 adhesions) and a slow cell in (b) (n= 29 adhesions). This distance was calculated for each new adhesion after the first and second edge retraction event for which they are associated. d) Quanitification of net adhesion advance from focal adhesion labeled with vincullin. Average distance of a population of 8 cells (n= 132 adhesions), and for the fastest (0.31 μm/min; n= 17 adhesions) and slowest (0.01 μm/min; n= 39 adhesions) cell in the population are displayed. e) Time-lapse montage of actin-mGFP and Zyxin-mCherry in a crawling cell. White arrow denotes the base of the first retraction to protrusion transition point and the yellow arrow denotes the second. White arrowhead shows the pre-existing focal adhesion. Yellow arrowhead shows a nascent adhesion appearing and maturing (growing larger). f) Similar time-lapse montage as in (e) in a cell that did not exhibit net forward movement of its edge. White arrowhead denotes a preexisting adhesion. Yellow and green arrowheads denote nascent focal adhesion formation and maturation. Note that adhesion move rearward (lines) during edge retraction. Red and green brackets show the net movement of the base of the protrusion retraction cycle and focal adhesion advance. g) Kymograph from a cell that increases its rate of migration. There is no advance of the base protrusion after two protrusion/retraction cycles (arrows) but there is advance after the third cycle (arrow). Yellow and green lines denote rapid and slow actin arc translocation respectively. Error bars represent standard error of the mean. Scale bar, 10 μm.

Figure 7

The lamella moves forward through actin arc treadmilling. a) Edge velocity map, edge position map and kymograph showing dynamics and advance of the leading edge. Dotted yellow lines denote advance of the lamella. b-c) The lamella advances by treadmilling of actin arcs: b) Actin-mGFP and vincullin-mCherry in a cell before and after net edge extension. Stars denote the lamellipodium and brackets denote the lamella. White arrowheads show focal adhesions at the boundary between the lamellipodium and lamella. c) Actin montage of box in (b) shows actin arcs are removed from the back of the lamella (arrowheads in actin montage) while new focal adhesion assembly (arrowheads in vincullin montage) leads to edge advance. Scale bar, 10 μm. The change in intensity in 5C from frame 5 to 6 is due to focusing.

Figure 8

Model of the structural dynamics of the actin cytoskeleton underlying edge motion. Leading edge advance is broken down into discrete steps. Step (1) shows the base of a previous retraction where a newly created actin arc is coupled to a focal adhesion. Hypothetically, the new lamellipodial protrusion could push off the arc to drive the membrane forward. During protrusion (2), actin filament polymerization occurs behind the plasma membrane and depolymerization occurs a few microns away from the edge. Actin filaments treadmill through the lamellipodium during protrusion, and nascent adhesions form. At the peak of protrusion (2) myosin II filaments form in the lamellipodium and a local network contraction (similar to that proposed for keratocyte cell body translocation) occurs which drives actin arc formation and edge retraction (3). In cells that show net advance, the new actin arc slows at the nascent adhesion (4) due to most likely to strong coupling between the arc, adhesion, and growth substrate. The base of the retraction in (4) is shifted forward compared to (1). As a consequence, the start of the new protrusion in (5) is also shifted forward and the edge protrudes farther than in (2). In cells that do not show net advance, the actin arc and adhesion slip rearward during edge retraction (arrow from (3) to (1)). This indicates that there is still strong coupling between the actin arc and the adhesion, and also indicates a weak coupling between the adhesion and the growth substrate. Actin arc addition to the front of the lamella is balanced by actin arc removal at the back of the lamella (5). Lamellipodial and arc actin filaments are yellow. Focal adhesions and associated actin filaments are green. Myosin II filaments are red. Relative actin rearward flow rates are represented by blue arrows.