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Regulation of focal complex composition and disassembly by the calcium-dependent protease calpain

¹ Department of Pediatrics and Pharmacology, University of Wisconsin, 1300 University Avenue, University of Wisconsin Medical School, Madison, WI 53706, USA
² Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg A-5020, Austria
³ Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, NC 27599, USA

View larger version (43K): [in a new window]   Fig. 1. Effects of calpain inhibition on the cellular morphology of CHO cells plated on fibronectin. (A) Cells were cultured for 12 hours on fibronectin-coated coverslips (10 µg/ml) and co-stained for vinculin (a,c,e) and actin (b,d,f). Cells that expressed calpastatin (c,d) or those treated with ALLN (e,f) demonstrated fewer but larger, more peripheral focal adhesions than controls (a,b). There was also a loss of central actin stress fibers after calpain inhibition. (B) Cells expressing hrEGFP-calpastatin (a,b) displayed similar effects on vinculin and actin distribution to those of control cells (c,d). Insets show GFP-positive cells and arrows show corresponding cells. The cells presented are representative of the phenotype observed from a minimum of three separate experiments. Bars, 10 µm.

View larger version (118K): [in a new window]   Fig. 2. Effects of calpain inhibition on the dynamics of GFP-vinculin (A,B) and GFP/RFP-zyxin (C-F) in CHOK1 cells plated on fibronectin (10 µg/ml). Video sequence of magnified regions of cells co-transfected with GFP-vinculin and control vector (A) or calpastatin (B), co-transfected with GFP-zyxin and control vector (C) or calpastatin (D), or co-transfected with RFP-zyxin and hrEGFP (E) or hrEGFP-calpastatin (F). Frames are from 6 minute intervals (t=0, 6, 12, 18, 24 and 30 minutes). GFP-vinculin and GFP- or RFP-zyxin-containing adhesions in cells that expressed calpastatin (B and D/F, respectively) are larger, more peripheral and stabilized. White arrows show position of adhesive contact sites that were stabilized. In comparison, control cells have smaller more dynamic GFP-vinculin and GFP-zyxin-containing adhesions. Open arrows show the position of representative contacts sites that turnover. Representative images from a minimum of five separate experiments with more than 10 cells viewed per condition (Movies 1-6). Bars, 10 µm.

View larger version (48K): [in a new window]   Fig. 8. Effects of calpain inhibition on RFP-zyxin and GFP—-actinin localization and focal complex disassembly. Video sequences of magnified regions of control cells (a-1) or cells treated with ALLN (m-r). Red shows zyxin and green shows -actinin; areas of co-localization appear as yellow. Images were collected at t=0, 6, 12, 18, 24 and 30 minutes. White arrows show zyxin-containing focal complexes that disperse after -actinin localization. Gray arrows show zyxin-containing focal complexes that translocate to the cell center. Open arrows show stabilized zyxin-containing focal complexes that did not co-localize with -actinin, and did not translocate or disperse in cells treated with ALLN (Movies 9-11). Bars, 20 µm.

View larger version (67K): [in a new window]   Fig. 3. Effects of calpain inhibition on focal adhesion disassociation during nocodozole recovery in cells plated on fibronectin. (A) CAR fibroblasts were transfected with EGFP-zyxin. Cells were pretreated with nocodozole (a) or nocodozole and ALLN (b) and plated in the presence of the same agents on 50 µg/ml fibronectin for 2 hours. Nocodozole was then washed out. Cells were allowed to recover in the presence (b) or absence (a) of ALLN. In control cells contacts were disassembled as cells spread. In cells treated with ALLN, turnover of adhesive contact sites was inhibited. Bars, 10 µm. (B) Quantification of contact site disassembly or formation in control and ALLN-treated cells from four separate experiments.

View larger version (101K): [in a new window]   Fig. 4. Effects of calpain inhibition on focal adhesion targeting of microtubules in cells plated on fibronectin. CAR fibroblasts were stained for paxillin and tubulin. Cells were pretreated with nocodozole and plated on 50 µg/ml fibronectin for 2 hours before nocodozole wash-out. Cells were then allowed to recover in the presence (B) or absence (A) of ALLN and stained for actin (left panel) or paxillin and tubulin (middle and right panel). Top images were fixed 10 minutes after nocodozole wash-out and bottom images were fixed 1.5 hours after washout. Microtubule targeting occurs in the presence (B) or absence (A) of ALLN. Bars, 10 µm.

View larger version (53K): [in a new window]   Fig. 5. Effects of calpain inhibition on -actinin localization and dynamics in CHOK1 cells plated on fibronectin. (A) Representative images of fixed cells treated with (b) and without (a) ALLN, and fixed cells that have been transfected with hrEGFP (c) or hrEGFP-calpastatin (d). Arrows and inserts show GFP-positive cells. (B) Live imaging of cells co-transfected with GFP—-actinin and control vector (a), and cells co-transfected with GFP—-actinin and calpastatin (b). GFP—-actinin was less dynamic and did not localize to focal complexes in cells that expressed calpastatin in comparison to control vector (Movies 7, 8). Bars, 20 µm.

View larger version (49K): [in a new window]   Fig. 6. Effects of calpain inhibition on RFP-zyxin and GFP—-actinin localization in CHOK1 cells. (A) Images from control cells (a-f) or cells treated with ALLN (g-i). Red shows zyxin and green shows -actinin; areas of co-localization appear as yellow. Zyxin showed decreased localization with -actininin in cells treated with ALLN. (B) Quantification of -actinin and zyxin co-localization in control cells or cells treated with ALLN from five separate experiments (P<0.01). Bars, 10 µM.

View larger version (78K): [in a new window]   Fig. 7. Effects of calpain inhibition on the composition of zyxin-containing focal complexes. (A) CHOK1 cells co-transfected with GFP—5-integrin and RFP-zyxin were treated with (d-f) or without (a-c) ALLN and fixed. Zyxin-containing focal complexes contained GFP—5-integrin in both control and calpain-inhibited cells. (B) Cells transfected with zyxin-RFP were treated with (d-f) or without (a-c) ALLN and fixed and stained. Zyxin-containing focal complexes in both control and ALLN-treated cells contained vinculin. Bars, 20 µm.

View larger version (42K): [in a new window]   Fig. 9. Effects of disrupting -actinin localization on focal adhesion dynamics. (A) Still images from video sequences of CHOK1 cells co-transfected with RFP-zyxin and full-length GFP—-actinin (a), -actinin-head—GFP (b) or -actinin-rod—GFP (c) (Movies 12, 13 and 14, respectively) RFP-zyxin-containing focal complexes are more peripheral and less dynamic when -actinin localization to the focal complex is disrupted by -actinin-rod—GFP, similar to calpain inhibition. In contrast, RFP-zyxin in -actinin-head—GFP co-transfected cells exhibits a focal adhesion distribution and dynamics similar to full-length GFP—-actinin (Movies 11-13). Bar, 20 µm. (B) Quantification of focal adhesion dynamics in cells co-transfected with full-length -actinin, -actinin-head—GFP, or -actinin-rod—GFP and RFP-zyxin. Transfection of -actinin-rod—GFP inhibits focal adhesion disassembly when compared with full-length -actinin (P<0.002) and with -actinin-head—GFP (P<0.003). Quantification of focal adhesion dynamics was performed on five cells for each condition.