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

Area of Science:

• Cell Biology
• Biophysics
• Mechanobiology

Background:

• Cell migration relies on dynamic adhesions for force exertion and sensing.
• Calpain proteases, including calpain 1 and calpain 2, are implicated in focal adhesion regulation.
• The small regulatory subunit, Capn4, is essential for calpain heterodimer function.

Purpose of the Study:

• To investigate the role of the calpain regulatory subunit (Capn4) in cellular traction force production and mechanosensing during fibroblast migration.
• To compare the functions of Capn4 with the catalytic subunits of calpain 1 (Capn1) and calpain 2 (Capn2).

Main Methods:

• Culturing Capn4-deficient and rescued fibroblasts on flexible polyacrylamide substrates.
• Measuring traction forces exerted by cells.
• Applying mechanical stimuli to assess cellular responses.
• Utilizing siRNA to knockdown Capn1 and Capn2 expression in fibroblasts.

Main Results:

• Capn4-deficient cells exhibited ~75% lower total force output, with forces being more random and less dynamic compared to rescued cells.
• Capn4 deficiency led to reduced cell adhesion and impaired responses to mechanical stimulation.
• Fibroblasts lacking calpain 1 or calpain 2 showed defects in mechanosensing but not in force production or adhesion.
• Capn4-deficient cells displayed aberrant stress fibers with fewer vinculin-containing adhesions.

Conclusions:

• The calpain small subunit (Capn4) is critical for mechanical force generation and mechanosensing in migrating fibroblasts.
• Capn4 may have functions independent of or secondary to its role as a regulatory subunit for calpains 1 and 2.
• Understanding Capn4's role provides insights into the molecular mechanisms of cell migration and mechanobiology.