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doi: 10.1242/10.1242/jcs.00147

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Spatial regulation of actin dynamics: a tropomyosin-free, actin-rich compartment at the leading edge

Vera DesMarais1,*, Ilia Ichetovkin1, John Condeelis1 and Sarah E. Hitchcock-DeGregori2

1 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
2 Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA



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  		Fig. 5. Quantification of the relative locations of tropomyosin, F-actin, and the Arp2/3 complex at the leading edge. Cells analyzed were double-labeled for tropomyosin (LC24) and F-actin (rhodamine-phalloidin, A,B,C,J,K), tropomyosin ({alpha}f9d) and barbed ends (biotin-labeled G-actin, D,E,F), and tropomyosin (LC24) and the Arp2/3 complex (anti-p34, G,H,I) as in Figs 2,3,4. For panels A-I, cells were either unstimulated (A,D,G), or stimulated with EGF for 50 seconds (B,E,H) or 3 minutes (C,F,I). The fluorescence intensity (arbitrary units) is the mean of the cell perimeter in regions of the lamellipodium for 0.22 µm wide steps (see Materials and Methods). Quantification is shown within 1 µm of the cell membrane. After EGF stimulation, the F-actin, barbed ends and the Arp2/3 complex distribution peak within 0.5 µm of the membrane, while tropomyosin increases deeper in the lamellipodium. In panel J, quantification of F-actin is shown up to 4 µm beyond the cell membrane without EGF stimulation, 50 seconds and 3 minutes after EGF stimulation. Regions corresponding to the leading edge and the base of the lamellipodium are indicated. In panel K, quantification of tropomyosin (LC24) is shown up to 4 µm beyond the cell membrane without EGF stimulation, 50 seconds and 3 minutes after EGF stimulation. Error bars indicate standard error, with n=15 (A,B,C,J,K) and n=25 (D,E,F,G,H,I).

 


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  		Fig. 1. Immunoblots of extracts of unstimulated MTLn3 cells with antibodies against tropomyosin. The positions of TM2 and TM5a standards in relation to the bands of the blots are indicated. The {alpha}f9d antibody recognizes long and short tropomyosin isoforms encoded by the {alpha}TM and ßTM genes. These include, from top to bottom, TM1 and TM6 (not distinguishable on this blot), TM2, TM3, and minor amounts of short isoforms. The TM311 antibody shows the presence of at least three high molecular weight isoforms: TM1/6, TM2 and TM3. The CGß6 antibody identifies the two faster migrating major forms seen in the {alpha}f9d and TM311 blots as TM2 and TM3. The blots using LC24 and CG3 recognize the major short tropomyosins expressed in MTLn3 cells as TM4 and one or more products of the {gamma}TM gene.

 


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  		Fig. 2. Tropomyosin is absent from regions of the leading edge that contain a dense F-actin network. Cells were either unstimulated (A-D,M), or stimulated with EGF for 50 seconds (E-H) or 3 minutes (I-L,O). Phalloidin-stained F-actin is shown in panels A, E, and I and tropomyosin (antibody LC24) in panels B, F, and J. Double-labeling overlays are shown (C,G,K; green, F-actin; red, tropomyosin), as well as phase contrast images (D,H,L). Panel M shows double-labeling of unstimulated cells and panel O shows double labeling of cells stimulated with EGF for 3 minutes. In panels M and O, the white square indicates an area that is shown at higher magnification in the panels below (panel N and P, from left to right: double labeling, F-actin, tropomyosin). The size of the square is 6 µm by 6 µm. The scale bar in panel L indicates 10 µm and applies to panels A-L. The scale bar in panel M also indicates 10 µm and applies to panels M and O. Arrows indicate regions at the leading edge that show F-actin staining but no tropomyosin. Arrowheads indicate labeling of actin stress fibers by both rhodamine-phalloidin and LC24 (tropomyosin). The diffuse, non-stress fiber actin network in the base of the lamellipodium is indicated by an asterisk and contains both F-actin and tropomyosin.

 


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  		Fig. 3. Tropomyosin is not associated with the free barbed ends of growing actin filaments at the leading edge in resting and EGF-stimulated MTLn3 cells. Cells were either unstimulated (A-E), or stimulated with EGF for 50 seconds (F-J) or 3 minutes (K-T). Cells were double-labeled for barbed ends (A,F,K,P) and tropomyosin ({alpha}f9d,B,G,L; CG3, Q). Double-labeling overlays are shown (C,H,M,R; red, tropomyosin; green, barbed ends), as well as phase-contrast images (E,J,O,T). The white square in the overlay images (C,H,M,R) indicates an area that is shown at higher magnification directly below (D,I,N,S); from left to right: double labeling, barbed ends, tropomyosin. The size of the square is 7.5 µm by 7.5 µm. Bar, 10 µm. Arrows indicate regions at the leading edge with barbed ends, demonstrating the absence of tropomyosin. Arrowheads indicate regions of overlap between tropomyosin and barbed ends within stress fibers.

 


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  		Fig. 4. Tropomyosin is absent from regions of the leading edge containing the Arp2/3 complex. Cells were either unstimulated (A-D), or stimulated with EGF for 50 seconds (E-H) or 3 minutes (I-P), and double-labeled for the Arp2/3 complex (anti-p34; A,E,I,M) and tropomyosin (LC24, B,F,J; IV15, N). Double-labeling overlays are shown in C, G, K and O (red, tropomyosin; green, the Arp2/3 complex). Phase contrast images are present in D, H, L and P. Bar, 10 µm. Arrows indicate regions at the leading edge where the Arp2/3 complex is localized demonstrating the absence of tropomyosin.

 


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  		Fig. 6. Tropomyosin inhibits F-actin severing by cofilin. Rhodamine-biotin-labeled actin filaments were polymerized and attached to the surface of the perfusion chamber with anti-biotin antibody. Chambers were perfused with buffer or recombinant rat TM5a. All chambers were washed with cofilin storage buffer, and the first set of images was taken (time 0). Cofilin in the same buffer was perfused and 1 minute later another set of images was taken of the same field. Filaments saturated with TM5a did not show any severing while control filaments were severed by cofilin (arrowheads). While control fields show an increase in the number of filaments, pre-treatment of filaments with TM5a leads to the complete inhibition of this effect (right panel; a. u., arbitrary units).

 


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  		Fig. 7. Model of the actin compartments defined by tropomyosin in the cell. Three compartments are shown. Green, the dynamic leading edge compartment of the lamellipodium contains short actin filaments that are severed by cofilin. Here the Arp2/3 complex induces branch formation on newly polymerized actin filaments to give highly branched actin filaments (see top box). Tropomyosin is absent. Red: the second compartment is the base of the lamellipodium. It contains longer, unbranched actin filaments as well as tropomyosin bound to actin, free cofilin and free Arp2/3 complex. Tropomyosin stabilizes actin filaments and protects them from cofilin severing and Arp2/3 complex-induced branch formation (see bottom box). White: the third compartment is the main body of the cell that contains stress fibers stabilized by tropomyosin, as in the red compartment, and other actin binding proteins. The main body of the cell also contains free cofilin and Arp 2/3 complex.

 

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