Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells

Abstract

Interactions between microtubules (MTs) and filamentous actin (f-actin) are involved in directed cell locomotion, but are poorly understood. To test the hypothesis that MTs and f-actin associate with one another and affect each other's organization and dynamics, we performed time-lapse dual-wavelength spinning-disk confocal fluorescent speckle microscopy (FSM) of MTs and f-actin in migrating newt lung epithelial cells. F-actin exhibited four zones of dynamic behavior: rapid retrograde flow in the lamellipodium, slow retrograde flow in the lamellum, anterograde flow in the cell body, and no movement in the convergence zone between the lamellum and cell body. Speckle analysis showed that MTs moved at the same trajectory and velocity as f-actin in the cell body and lamellum, but not in the lamellipodium or convergence zone. MTs grew along f-actin bundles, and quiescent MT ends moved in association with f-actin bundles. These results show that the movement and organization of f-actin has a profound effect on the dynamic organization of MTs in migrating cells, and suggest that MTs and f-actin bind to one another in vivo.

Figure 1.

Organization and dynamics of F-actin in migrating newt lung epithelial cells. (a and b) Z-series of deconvolved optical sections of an Alexa-488 phalloidin-stained cell were used to generate views along the z axis (a, maximum projection) or y axis (b, three-dimensional reconstruction of the boxed area in panel a). Bar, 10 μm. (c and d) Confocal FSM image, taken from a time-lapse series, of a living cell injected with X-rhodamine actin (c) and after fixation and staining with Alexa 488 phalloidin (d). See Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200203022/DC1). F-actin bundles (c and d, arrowheads) prominent in the phalloidin image (c) correspond to linear arrays of speckles in the FSM image (d, boxed bundle shown at 150-s intervals in inset). A bundle in d (starred arrowheads) has no corresponding linear array of speckles in the FSM image (c). Bar, 10 μm; inset, 3 μm. (e–i) Four zones of f-actin dynamics. (e) Single f-actin FSM image from Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200203022/DC1). Bar, 10 μm. (f–i) Kymographs of the four zones of f-actin movement taken along the regions highlighted in (e). F-actin speckles move retrograde in the (f) lamellipodium at 1.6 μm/min, in the (g) lamella at 0.3 μm/min. (h) In the convergence zone, there is no directional movement of f-actin. (i) F-actin moves anterograde in the cell body at 0.4 μ/min. Elapsed time in min:s. Bar, 10 μm. (j) Plot of the actin:fluorescent volume marker ratio along the axis of migration of a cell reveals a steep gradient of actin concentration across the lamellipodium (lp), and relatively constant concentration through the rest of the cell.

Figure 2.

MTs parallel to the leading edge are coupled to the movement of f-actin. (a) Image from Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200203022/DC1) of Cy2 MTs (green) and X-rhodamine f-actin (red). Boxes highlight the regions in the lamellipodium (lp), lamellum (la), convergence zone (cz), and cell body (cb) that were used to construct the kymographs in (b–e). The long axis of the boxes was tilted to match the trajectory of speckles as determined by watching Video 3. Green arrowheads highlight the parallel MTs being analyzed. (b–e) Dual wavelength kymographs of the regions highlighted in panel a. Green and red arrowheads highlight speckles in parallel MTs and the actin meshwork, respectively. Bar, 10 μm.

Figure 3.

MTs perpendicular to the leading edge move at the same rate as the f-actin in the lamellum, but not in the lamellipodium. (a) Single image from the time-lapse series in Video 4 (available at http://www.jcb.org/cgi/content/full/jcb.200203022/DC1) shows an MT (green) oriented perpendicular to the leading edge within the lamellum. (c) Kymograph of the boxed region in (a). Speckles along the MT appear as parallel green streaks in the kymograph as the MT moves retrograde at the exact same rate as the immediately adjacent f-actin speckles (red streaks). (b) Single image from the time-lapse series seen in Video 5 (available at http://www.jcb.org/cgi/content/full/jcb.200203022/DC1) of a perpendicular MT in the lamellipodium. (d) Kymograph of the boxed region in (b). The slope of the green MT streaks in the lamellipodium (lp, green line) are much shallower than the slopes of the red f-actin streaks (red line), indicating f-actin moves faster than the MT at the lamellipodium. In the lamellum (la), the slopes of the red and green streaks match (yellow line), indicating coordinated movement of the MT and f-actin at this region. Bar, 3 μm.

Figure 4.

MTs interact with f-actin bundles. (a and b) MTs align along f-actin bundles in the cell body and grow toward dense f-actin plaques. (a) F-actin bundles (highlighted with red lines) radiate out from an f-actin plaque. (b) Dual wavelength images taken from Video 6 (available at http://www.jcb.org/cgi/content/full/jcb.200203022/DC1). MT ends (green, arrowhead and small arrow) grow toward the f-actin plaque while remaining aligned along f-actin bundles (red). As the end of a MT (arrowhead) moves toward the plaque, a speckle along the MT shaft (large arrow) remains stationary with respect to the f-actin bundle. The figure is oriented with the leading edge toward the top of the figure. Elapsed time in min:s. Bar, 10 μm. (c–e) MTs with nondynamic ends move coordinately with f-actin bundles. (c) Time-lapse series from Video 7 (available at http://www.jcb.org/cgi/content/full/jcb.200203022/DC1) of an MT end (green; small arrowhead) moving coordinately with an f-actin bundle (red, highlighted by white line). A nearby nonbundle actin speckle stays stationary over the same time (large arrow). (d) Plot of X and Y coordinates (increasing time from left to right) of the MT end (c, small arrowhead) and two speckles in the f-actin bundle (c, arrows 1 and 2). The MT and the f-actin speckles have the same trajectories and instantaneous velocities. (e) The distance between the MT end (c, small arrowhead) and a speckle on the MT shaft (c, large arrowhead) remains constant. Absent measurements in the series were slightly out-of-focus frames. The figure is oriented with the leading edge toward the top of the figure. Elapsed time in min:s. Bar, 4 μm.