Visualization and molecular analysis of actin assembly in living cells

Abstract

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM-kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP-CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

Figure 1

The distributions of GFP–CP (A, C, D, and F) and GFP–Arp3 (B) in living fibroblasts change dramatically over time. Each sequence of images (A–F) shows selected frames from a video sequence demonstrating that the distributions of GFP–CP and GFP–Arp3 change with time. Movies of the complete video sequences for A–F present these data more clearly and are available at www.cooperlab.wustl.edu or from the authors. Arrows in C and D indicate the initial position of a prominent motile spot formed in the lamella (C) or associated with a macropinosome (D). The distribution of GFP (E) is diffuse, as expected. Slight changes in the distribution of GFP over time reflect changes in cell thickness that accompany cellular movements. The sequence in F was obtained using a confocal microscope and shows that the accumulation of GFP–CP at the cell periphery is not due to increased cell thickness at the edge of the cell. Numbers in the lower corner of each image indicate elapsed time in seconds. Bar, 10 μm.

Figure 2

Arp2/3 complex and CP colocalize in punctate structures in the lamella and along the edge of cells. PtK1 cells expressing GFP–CP (left) or GFP–Arp3 (right) were fixed and labeled with antibodies to localize Arp2/3 complex (left) or CP (right), respectively. Green, GFP-tagged proteins; red, antibody labeling. Bottom, merged image of each red-green pair; yellow, regions of overlap of Arp2/3 complex and CP. Bar, 10 μm.

Figure 3

Spots were reduced in number and spot motility was suppressed in migrating cells. Confluent monolayers of PtK1 fibroblasts expressing GFP–Arp3 (A) or GFP–CP (B) were “wounded” to obtain a clear area on the coverslip. Cells migrating into the cleared region were monitored 2–4 h after scratching. Both Arp2/3 and CP were enriched in regions of motile activity at the cell edge. Few motile spots were observed and they were generally restricted to a location at the rear of the extended lamella. Movies of the complete video sequence for each panel are available at www.cooperlab.wustl.edu or from the authors. Bar, 10 μm.

Figure 4

Actin assembly occurs at spots and at the cell periphery. To identify sites of actin assembly, cells were permeabilized with saponin in the presence of rhodamine-labeled G-actin, which indicates sites of actin assembly. Rhodamine-actin (red) was incorporated along the edge of the cell and at GFP–CP spots (green). Yellow, merged image shows regions of overlap of the GFP–CP and rhodamine-actin. Bar, 10 μm.

Figure 5

Actin polymerization is required for formation and movement of spots and for movements at the cell periphery. Fibroblasts expressing GFP–CP were filmed within 2 min after treatment with 1 μM cytochalasin D or with 200 nM latrunculin B. Two images separated by 16 or 12.5 s in each video sequence are shown. The distribution of GFP–CP did not change during this interval. Movies of the complete video sequence, including views of the cell before and after treatment with cytochalasin D or latrunculin B are available at www.cooperlab.wustl.edu or from the authors. Bar, 10 μm.

Figure 6

Actin colocalizes with Arp2/3 complex and CP at the cell periphery and on spots in the lamella. (A and B) Fibroblasts expressing GFP–CP (A, green) or GFP– Arp3 (B, green) were fixed and labeled with anti-actin mAb C4 (red). Yellow, merged images show the colocalization of actin with Arp2/3 complex or CP. (C) Actin filaments, detected with rhodamine-phalloidin are components of spots. Cells expressing GFP–CP (green) were fixed and stained with rhodamine-phalloidin (red). Yellow, merged image shows the colocalization of F-actin with CP. Bar, 10 μm.

Figure 7

Motile spots in the lamella are small, fin-like projections that taper toward their ends. Cells expressing GFP–CP were grown on electron microscopic grids, placed in a flow chamber on the fluorescence microscope to document the movement of spots and the lamella, and then prepared as whole mounts for viewing in the electron microscope. Movies of single cells were obtained before and during fixation for preparing the whole mounts. A shows one frame from the video recording taken using fluorescence microscopy (left) during the addition of fixative. Arrowheads, three regions where GFP–CP spots were actively moving just before fixation. The low quality of the fluorescence image is due to the decreased signal to noise ratio upon addition of the glutaraldehyde-containing fixation solution. Adjacent to the fluorescence view is a stereo pair at low magnification (right) of the same cell viewed using electron microscopy; arrowheads, three structures corresponding to the motile GFP–CP spots. A stereo pair at higher magnification of the structures marked by arrowheads is shown in B. When viewed with the aid of stereo viewers, the stereo pairs show that the fin-like structures project out from the membrane surface. To those observers who view stereo pairs by crossing their eyes, the structures will appear to project away from the viewer. Bars: (A) 2 μm; (B) 1.5 μm.

Figure 8

Small GTPases regulate spot formation and motility at the cell periphery. GFP–Arp3 fibroblasts were microinjected with plasmids for expression of constitutively active (v12) or dominant-negative forms (n17) of rac1 and cdc42. Other cells were microinjected with an active form of RhoA (v14 RhoA) protein (0.5 mg/ml) or with C3 toxin (160–200 μg/ml). GFP–Arp3 was diffusely distributed in cells containing N17rac1, N17cdc42, or C3 toxin. In contrast, motile spots were observed in cells containing V12rac1, V12 cdc42, or V14RhoA. Movies of the complete video sequence for each panel are available at www.cooperlab.wustl.edu or from the authors. Bar, 10 μm.

Figure 9

Expression of PI 5-kinase promotes the movement of spots. Nuclei were injected with DNA for expression of PI 5-kinase and cells were observed 2–3 h after injection. A white asterisk follows one moving spot and a black asterisk in each frame marks the initial position of the spot. Numbers indicate elapsed time in seconds. The complete video sequence for these data are available at www.cooperlab.wustl.edu or from the authors. Bar, 10 μm.

Figure 10

Expression of LIMK-1, which inactivates cofilin, results in decreased motility of spots and of the cell periphery. Nuclei were injected with DNA for expression of LIMK-1 (A) or a mutant form of LIMK-1 lacking a portion of the catalytic kinase domain (B) and cells were observed 2–3 h after injection. Expression of LIMK-1 caused spots to stop moving and, in some cases, to the formation of aggregates of GFP–CP. In contrast, expression of an inactive mutant form of LIMK-1 did not affect spot movement or motility at the cell periphery. Numbers indicate elapsed time in seconds. Movies that show the complete video sequences for these data are available at www.cooperlab.wustl.edu or from the authors. Bar, 10 μm.

Figure 11

Model for signal-induced actin assembly at motile spots and at the leading edge. When Arp2/3 complex is activated, free barbed ends are created near the membrane, which assemble actin (blue). The mechanism for the local activation or recruitment of Arp2/3 complex at sites of assembly is unknown. Small G proteins, phosphoinositides, and an endogenous ActA-like factor may participate in the recruitment or activation of Arp2/3 complex at the membrane. Actin polymerization then proceeds at the new free barbed ends until blocked by CP. The short-lived actin filaments would subsequently disassemble via the action of cofilin. LIM–kinase-1, which inactivates cofilin and is also stimulated by GTP-rac, may regulate the disassembly of actin filaments. Spatial regulation of the processes of actin assembly and disassembly could result in the growth of filaments in the extending lamella and their subsequent disassembly at sites further from the leading edge.