Hao Bingqing, Beningo Karen A
bioRxiv. 2023 Mar 9:2023.03.07.531592. doi: 10.1101/2023.03.07.531592.
Cell migration is a fundamental process pertaining to many critical physiological events. The ability to form and release adhesion structures is necessary for cell migration. The Calpain family of cysteine proteases are known to target adhesion proteins as their substrates and modulate adhesion dynamics. The two best studied Calpains, Calpain 1 and Calpain 2 form catalytically active holoenzymes through heterodimerization with a common non-catalytic regulatory small subunit known as Calpain 4. In previous studies, we determined that calpains are important in the production of traction forces and in the sensing of localized mechanical stimulation from the external environment. We found that perturbation of either Calpain 1 or 2 had no effect on the generation of traction forces. However, traction forces were weak when Calpain 4 was silenced. On the other hand, silencing of Calpain 1, 2, or 4 resulted in deficient sensing of external mechanical stimuli. These results together suggest that Calpain 4 functions independent of the catalytic large subunits in the generation of traction forces but functions together with either catalytic subunit in sensing external mechanical stimuli. The small subunit Calpain 4 contains 268 a.a. and is composed of 2 domains, the N-terminal domain V and C-terminal domain VI. Domain VI is a calmodulinlike domain containing five consecutive EF-hand motifs, of which the fifth one heterodimerizes with a large subunit. Moreover, domain V contains the common sequence GTAMRILGGVI that suggests cell membrane interactions. Given these attributes of domain V and VI of Calpain 4, we speculated that an individual domain might provide the functional properties for either traction or sensing. Therefore, each domain was cloned and expressed individually in cells and assayed for traction and sensing. Results revealed that over-expression of domain V was sufficient to rescue the traction forces defect in cells while overexpression of domain VI did not rescue the traction force. Consistent with our hypothesis, overexpression of domain VI rescued the sensing defect in cells while overexpression of domain V had no effect. These results suggest that individual domains of Calpain 4 do indeed function independently to regulate either traction force or the sensing of external stimuli. We speculate that membrane association of Calpain 4 is required for the regulation of traction force and its association with a catalytic subunit is necessary for mechanosensing.
细胞迁移是一个与许多关键生理事件相关的基本过程。形成和释放黏附结构的能力是细胞迁移所必需的。已知半胱氨酸蛋白酶钙蛋白酶家族以黏附蛋白为底物，并调节黏附动力学。研究得最透彻的两种钙蛋白酶，钙蛋白酶1和钙蛋白酶2，通过与一种常见的非催化调节小亚基（称为钙蛋白酶4）异源二聚化形成具有催化活性的全酶。在先前的研究中，我们确定钙蛋白酶在产生牵引力以及感知来自外部环境的局部机械刺激方面很重要。我们发现，干扰钙蛋白酶1或2对牵引力的产生没有影响。然而，当钙蛋白酶4沉默时，牵引力较弱。另一方面，沉默钙蛋白酶1、2或4会导致对外界机械刺激的感知不足。这些结果共同表明，钙蛋白酶4在产生牵引力方面独立于催化大亚基发挥作用，但在感知外部机械刺激方面与任一催化亚基共同发挥作用。小亚基钙蛋白酶4包含268个氨基酸，由2个结构域组成，即N端结构域V和C端结构域VI。结构域VI是一个类钙调蛋白结构域，包含五个连续的EF手基序，其中第五个与大亚基异源二聚化。此外，结构域V包含常见序列GTAMRILGGVI，提示与细胞膜相互作用。鉴于钙蛋白酶4的结构域V和VI的这些特性，我们推测单个结构域可能为牵引力或感知提供功能特性。因此，每个结构域都被单独克隆并在细胞中表达，并检测其牵引力和感知能力。结果显示，结构域V的过表达足以挽救细胞中的牵引力缺陷，而结构域VI的过表达不能挽救牵引力缺陷。与我们的假设一致，结构域VI的过表达挽救了细胞中的感知缺陷，而结构域V的过表达没有影响。这些结果表明，钙蛋白酶4的单个结构域确实独立发挥作用，以调节牵引力或对外界刺激的感知。我们推测，钙蛋白酶4与膜的结合对于调节牵引力是必需的，而其与催化亚基的结合对于机械传感是必要的。
