The calpain small subunit regulates cell-substrate mechanical interactions during fibroblast migration

Abstract

Cell migration involves the dynamic formation and release of cell-substrate adhesions, where the exertion and detection of mechanical forces take place. Members of the calpain family of calcium-dependent proteases are believed to have a central role in these processes, possibly through the regulation of focal adhesion dynamics. The ubiquitous calpains, calpain 1 (mu-calpain) and calpain 2 (m-calpain), are heterodimers consisting of large catalytic subunits encoded by the Capn1 and Capn2 genes, respectively, and the small regulatory subunit encoded by Capn4. We have examined the role of the calpain regulatory small subunit in traction force production and mechanosensing during cell migration. Capn4-deficient or rescued cells were plated on flexible polyacrylamide substrates, for both the detection of traction forces and the application of mechanical stimuli. The total force output of Capn4-deficient cells was approximately 75% lower than that of rescued cells and the forces were more randomly distributed and less dynamic in Capn4-deficient cells than in rescued cells. Furthermore, Capn4-deficient cells were less adhesive than wild-type cells and they also failed to respond to mechanical stimulations by pushing or pulling the flexible substrate, or by engaging dorsal receptors to the extracellular matrix. Surprisingly, fibroblasts deficient in calpain 1 or calpain 2 upon siRNA-mediated knockdown of Capn1 or Capn2, respectively, did not show the same defects in force production or adhesion, although they also failed to respond to mechanical stimulation. Interestingly, stress fibers were aberrant and also contained fewer colocalised vinculin-containing adhesions in Capn4-deficient cells than Capn1- and Capn2-knockdown cells. Together, these results suggest that the calpain small subunit plays an important role in the production of mechanical forces and in mediating mechanosensing during fibroblast migration. Furthermore, the Capn4 gene product might perform functions secondary to, or independent of, its role as a regulatory subunit for calpain 1 and calpain 2.

Fig. 1

Inhibition of traction forces in Capn4^−/− fibroblasts. (A,B) Vector plots of traction stress indicate magnitude and direction of traction stresses exerted by cells on the substrate, for Capn4^−/− cells stably re-expressing the small subunit (A) and Capn4^−/− cells (B). (C) Bar graph shows average integrated traction forces magnitude from each type of the cells. Capn4^−/− cells show a significant reduction in traction forces compared with wild-type or rescued cells (Student's t-test P=0.0003).

Fig. 2

Defects in the dynamics of traction forces and energy output for Capn4^−/− fibroblasts. (A) Color rendering of the magnitude of traction stress at 6, 12, 20 and 28 minutes in rescued Capn4^−/− fibroblasts and in Capn4^−/− cells shows weak, scattered traction forces for Capn4^−/− fibroblasts, whereas control cells exert strong forces at the leading and trailing edges. Color scale represents a minimum magnitude of 1×10³ dynes/cm² and a maximum of 2.9×10⁵ dynes/cm². (B) Plots of average integrated traction forces against time show a much higher degree of dynamics for rescued Capn4^−/− cells than Capn4^−/− fibroblasts. (C) Dynamics of traction forces measured by taking a ratio between average integrated traction forces and the s.d. measured over a period of 45 minutes. Rescued Capn4^−/− fibroblasts have a much smaller value than Capn4^−/− cells (four cells, each recorded over 45 minutes), indicating a larger fluctuation in forces. The difference in the energy stored in the substrate is even more striking (Student's t-test P=0.00001), with a much lower output for Capn4^−/− fibroblasts than rescued cells (n=13 and n=23, respectively).

Fig. 3

Generation of normal traction forces by cells defective in calpain 1 or calpain 2. (A) Average integrated traction forces generated by MEF cells treated with siRNA for either Capn4, Capn1 or Capn2 or cells overexpressing calpastatin. Neither silencing of calpain 1 or calpain 2, nor overexpression of calpastatin, causes a detectable reduction in traction forces. Data are compiled from measurements of a minimum of 15 cells for each cell type. Although wild-type MEF cells transfected with siRNA against Capn4 show a significant reduction in the magnitude of traction stress (Student's t-test P=0.0001) (n=19). (B,C) Immunofluorescence of the calpain small subunit in MEFs transfected with control RNA (B), or with siRNA against Capn4 (C) shows a striking reduction in the amount of small subunit upon siRNA-mediated gene silencing. (D) RT-PCR demonstrates an 88% reduction in the amount of Capn4 mRNAs. (E) Level of calpastatin protein expressed in wild-type MEF cells transfected with GFP plasmid alone and cells in which calpastatin-GFP has been overexpressed.

Fig. 4

Involvement of calpain 1, calpain 2 and the calpain small subunit in cellular responses to mechanical forces. Images of Capn4^−/− cells or rescued cells are recorded at 0 minutes, prior to micromanipulation, and at 20, 40, and 60 minutes after pushing on the substrate with a blunted microneedle against the direction of cell migration. Thin arrows indicate the direction of cell migration and thick arrowheads show the direction of pushing. Scale bar: 10 μm. The chart indicates a response (+) or a failure to respond (−) to stimulation under each of the cellular conditions.

Fig. 5

Failure of cells defective in calpain to respond to the engagement of dorsal integrins. Capn4^−/− (B,D) or rescued (A,C) fibroblasts were cultured on the surface (A,B), or within two layers (C,D), of fibronectin-coated polyacrylamide substrates. Only rescued cells respond to the engagement of dorsal integrins by adopting an elongated shape. Scale bar: 10 μm. The response is quantified by taking the ratio of the length to the width of cells (E). In addition, similar results are obtained with siRNA-induced gene silencing using NIH3T3 cells. Each bar represents mean ± s.e.m. of 25 cells from three experiments.

Fig. 6

Capn4^−/− fibroblasts are defective in adhesiveness and adhesion: stress fiber linkage. (A) Rescued Capn4^−/− fibroblasts, Capn1- and Capn2-knockdown and calpastatin-overexpressing MEF cells, but not Capn4^−/− cells, adhere strongly to the substrate in a centrifugation assay. Each bar represents mean ± s.e.m. results from three separate experiments, expressed as a percentage of control as defined by wild-type fibroblasts. Actin and vinculin immunofluorescence of representative control fibroblasts (B,C) and Capn4^−/− fibroblasts (D,E) show the abnormal organization of these structures in the knockout cells. Scale bar: 10 μm. (F) Colocalization analysis of actin and vinculin indicates a decreased association of prominent actin fibers with vinculin-containing adhesions in the Capn4^−/− fibroblasts compared with the control and Capn1- and Capn2-silenced fibroblasts, as well as cells overexpressing calpastatin. The analysis involves counting the total number of vinculin-containing adhesions and those associated with actin stress fibers in the anterior region (n=15 cells).