Undyala Vishnu V, Dembo Micah, Cembrola Katherine, Perrin Benjamin J, Huttenlocher Anna, Elce John S, Greer Peter A, Wang Yu-Li, Beningo Karen A
Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA.
J Cell Sci. 2008 Nov 1;121(Pt 21):3581-8. doi: 10.1242/jcs.036152. Epub 2008 Oct 7.
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.
细胞迁移涉及细胞与底物黏附的动态形成和释放，而机械力的施加和检测就在此过程中发生。钙蛋白酶家族的钙依赖性蛋白酶成员被认为在这些过程中起核心作用，可能是通过调节黏着斑动力学来实现的。普遍存在的钙蛋白酶，即钙蛋白酶1（μ-钙蛋白酶）和钙蛋白酶2（m-钙蛋白酶），是异二聚体，分别由Capn1和Capn2基因编码的大催化亚基以及Capn4基因编码的小调节亚基组成。我们研究了钙蛋白酶调节性小亚基在细胞迁移过程中产生牵引力和机械传感方面的作用。将Capn4基因缺陷或拯救后的细胞接种在柔性聚丙烯酰胺底物上，用于检测牵引力和施加机械刺激。Capn4基因缺陷细胞的总力输出比拯救后的细胞低约75%，并且与拯救后的细胞相比，Capn4基因缺陷细胞中的力分布更随机，动态性更低。此外，Capn4基因缺陷细胞的黏附性比野生型细胞弱，并且它们也无法通过推或拉柔性底物，或通过与细胞外基质的背侧受体结合来对机械刺激做出反应。令人惊讶的是，分别通过siRNA介导敲低Capn1或Capn2而使钙蛋白酶1或钙蛋白酶2缺陷的成纤维细胞，虽然也无法对机械刺激做出反应，但在力的产生或黏附方面并未表现出相同的缺陷。有趣的是，与Capn1和Capn2敲低细胞相比，Capn4基因缺陷细胞中的应力纤维异常，并且含有共定位的含纽蛋白黏附物也更少。总之，这些结果表明钙蛋白酶小亚基在成纤维细胞迁移过程中产生机械力和介导机械传感方面起重要作用。此外，Capn4基因产物可能执行与其作为钙蛋白酶1和钙蛋白酶2的调节亚基的作用继发或独立的功能。
