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First published online 29 June 2004
doi: 10.1242/jcs.01216

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Caldesmon mutant defective in Ca²⁺-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility

Department of Biological Sciences, University of Iowa, 340 Biology Building East, Iowa City, IA 52242-1324, USA

View larger version (9K): [in a new window]   Fig. 1. Constructs of human fibroblast caldesmon (CaD) and their corresponding expressing cell lines used in this study. The C-terminal fragment (CaD39) consists of amino acids 244 to 538. Ca2+/CaM-binding domains are labeled as gray boxes and their amino acid sequences are listed. The italic underlined letters represent the replacement of tryptophan residues with alanine in the Ca2+/CaM-binding-site-defective mutant (CaD39-AB). The expression levels of various constructs relative to endogenous CHO CaD in each stable cell lines are indicated by the number of + signs in parentheses.

View larger version (11K): [in a new window]   Fig. 2. Binding of CaD39 and CaD39-AB to CaM. The binding of either CaD39 (filled circles) or CaD39-AB (open circles) to CaM was measured by a modified ELISA method (Lin et al., 1991). Wells in microtiter plate were coated with 100 µl of 5 µg ml–1 bovine brain CaM at 4°C overnight. Bound Ca2+-CaM was incubated with increasing amounts (0-60 pmole) of CaD39 or CaD39-AB, subsequently with monoclonal antibody C21 against CaD, and then with horseradish-peroxidase-conjugated anti-mouse IgG. After a 30-minute incubation with substrate (ABTS), the color intensity (O.D.) was determined by an ELISA reader at 405 nm. Averages from three independent experiments were plotted in the figure, with error bars representing the standard deviation. Mutant CaD39-AB almost abolished its binding to Ca2+-CaM, whereas wild-type CaD39 binds to Ca2+-CaM in a saturable fashion.

View larger version (24K): [in a new window]   Fig. 3. Actin binding of CaD39 or CaD39-AB. Recombinant protein CaD39 at 2.01 µM, 4.02 µM, 4.47 µM, 4.92 µM (A) or CaD39-AB at 2.68 µM, 5.37 µM, 5.82 µM, 7.76 µM (B) was incubated with 9.3 µM F-actin in 10 mM imidazole buffer (pH 7.0) containing 30 mM KCl, 0.1 mM EGTA and 1 mM DTT. After incubation and subsequent centrifugation, both supernatants and pellets were adjusted to equivalent volumes and analysed by SDS-PAGE. `S' and `P' represent the supernatant (free) and pellet (bound) fraction, respectively, of CaD in each reaction. The binding data from three independent experiments were quantified using Scion Image software (C), which showed no significant difference of actin-binding ability between CaD39 and CaD39-AB.

View larger version (51K): [in a new window]   Fig. 4. Both CaD39 and CaD39-AB potentiate the actin binding of tropomyosin. Using the same buffer conditions as in Fig. 3, 9.3 µM F-actin and 3.6 µM hTM3 were incubated alone (1S and 1P), with 0.5 µM CaD39 (2S and 2P) or with 0.5 µM CaD39-AB (3S and 3P). After incubation and subsequent centrifugation, both supernatants (S) and pellets (P) were dissolved and analysed by SDS-PAGE. Like wild-type CaD39, mutant CaD39-AB protein potentiated tropomyosin binding to actin filaments.

View larger version (31K): [in a new window]   Fig. 5. Inhibition of actin/tropomyosin-activated HMM ATPase by CaD39 and CaD39-AB. Rates of HMM ATPase were measured at 29°C in 20 mM imidazole buffer (pH 7.0), 2 mM MgCl2, 50 mM KCl, 0.1 mM EGTA and 1 mM DTT. 12 µM F-actin, 2.5 µM hTM5 and 0.04 µM HMM were incubated in the absence or presence of 2.8 µM CaD39 or CaD39-AB. The reversal of CaD inhibition was measured in the presence of 2.5 µM CaM and 2 mM CaCl2. The actin/tropomyosin-activated HMM ATPase activity (4.58 nmole phosphate released per minute per mg of HMM) obtained under this assay condition was used as 100%. Abbreviations: A, actin; TM, tropomyosin.

View larger version (72K): [in a new window]   Fig. 6. Stable clones expressing CaD39-AB. Total proteins were prepared from five independent stable cell lines (C35, C42, m4-2, m5-8 and m4-65) and a non-expressing cell line C3. Equivalent amounts of protein extract were resolved using SDS-PAGE and the portion of the Coomassie-Blue-stained gel was shown (B) to indicate the equal loading in the gel. The expression levels of CaD39-AB in the stable clones were analysed by western blot (A).

View larger version (150K): [in a new window]   Fig. 7. Distribution of CaD39-AB in overexpressing clones. Cultured cells from different clones were stained with C21 antibody (B,E,H) and phalloidin (C,F,I). (A-C) Control cells from non-expressing clone C3. (D-F) 39C15 cells overexpressing wild-type CaD39. (G-I) m4-65 cells overexpressing CaD39-AB. When used at 1000-fold dilution, C21 antibody could not detect the endogenous CHO CaD (B) and only the localization of overexpressed exogenous proteins could be visualized (E,H). Although wild-type CaD39 incorporated into stress fibers (E), mutant CaD39-AB diffused throughout the cells and was relatively concentrated at many small ruffle regions (H). In addition to cell shape changes, the stress fiber arrays were disrupted in CaD39-AB-overexpressing cells (I). Scale bar, 10 µm.

View larger version (83K): [in a new window]   Fig. 8. Actin bundles isolated from CaD39-AB-expressing cells contain much less force-expressed protein than that from CaD39-expressing cells. Lane 1, 39C15; lane 2, m5-8; lane 3, m4-65. (A) Western blot analysis of total protein extract from 39C15, m5-8 and m4-65 using C21 anti-CaD antibody. (B) SDS-PAGE analysis of tropomyosin-enriched actin bundles isolated from interphase cells of 39C15, m5-8 and m4-65. (C-E) Western blot analyses of isolated tropomyosin-enriched actin bundles with C21 antibody (C,E) and with CG3 anti-TM5 antibody (D). Despite the comparable CaD expression levels, the portion of the force-expressed protein associated with actin bundles was much less in both m5-8 and m4-65 cells than that in 39C15 cells. After calculating the differences in the initial cell numbers used for the actin bundle isolation and in the volumes loaded in the gel, the yield of actin bundles isolated from m4-65 cells was 2.2 times lower than that for 39C15 cells.

View larger version (114K): [in a new window]   Fig. 9. Effects of CaD39-AB expression on stress fibers and focal adhesions. (A-D) Control C3 cells. (E-H) 39C15 cells expressing CaD39. (I-L) m4-65 cells expressing CaD39-AB. Double staining was performed with phalloidin (A,E,I) and anti-vinculin monoclonal antibody (B,F,J). (C,G,K) Merged images of A and B, E and F, and I and J, respectively. Representative areas in C, G and K are highlighted in boxes and enlarged in D, H and L, respectively. Scale bars, 15 µm (C,G,K), 3 µm (D,H,L).

View larger version (13K): [in a new window]   Fig. 10. Effects of CaD39-AB on cell growth. 4x104 39C15 (filled circles), m5-8 (open circle) and m4-65 (filled triangles) cells were plated on 60-mm dishes. Cells from each plate were trypsinized and counted every 24 hours. Cell numbers were plotted against culturing time. Calculated doubling times for 39C15, m5-8 and m4-65 were 16 hours, 21 hours and 24 hours, respectively. The expression level of mutant CaD39-AB protein was inversely proportional to growth rate.

View larger version (15K): [in a new window]   Fig. 11. Centroid tracks of control C3 (A) and mutant m4-65 cells (B) during wound healing. The interval between adjacent centroid is 5 minutes. Persistent, rapid movement in C3 cells was reflected by a linear track and well-separated centroids with time (A). By contrast, little translocation with frequent direction change in mutant m4-65 cells was represented by a zigzag track and clusters of centroids with time (B). Scale bar, 10 µm.

View larger version (107K): [in a new window]   Fig. 12. Phase-contrast images and outlines of migrating control C3 cells during wound healing. Derived from original video taping, the phase-contrast images (A) of migrating cells starts 2.5 hours after wounding and continues for 12.5 minutes at intervals of 2.5 minutes. (B) The outlines of a representative cell (A, arrow) in which membrane extension zones are filled with red. A single membrane sheet covering nearly the entire leading edge was actively extending and retracting during the 12.5-minute period. Scale bar, 10 µm.

View larger version (111K): [in a new window]   Fig. 13. Phase-contrast images and outlines of migrating mutant m5-8 cells during wound healing. Derived from original video taping, the phase-contrast images (A) of migrating cells starts 2.5 hours after wounding and continues for 12.5 minutes at intervals of 2.5 minutes. (B) The outlines of a representative cell (A, arrow) in which membrane extension zones are filled with red. Small, discrete membrane extensions were observed in CaD39-AB-expressing cells. Scale bar, 10 µm.

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