Mooren Olivia L, McConnell Patrick, DeBrecht James D, Jaysingh Anshuman, Cooper John A
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO.
bioRxiv. 2024 May 13:2024.05.13.593916. doi: 10.1101/2024.05.13.593916.
Actin polymerization is often associated with membrane proteins containing capping-protein-interacting (CPI) motifs, such as CARMIL, CD2AP, and WASHCAP/Fam21. CPI motifs bind directly to actin capping protein (CP), and this interaction weakens the binding of CP to barbed ends of actin filaments, lessening the ability of CP to functionally cap those ends. The protein V-1 / myotrophin binds to the F-actin binding site on CP and sterically blocks CP from binding barbed ends. CPI-motif proteins also weaken the binding between V-1 and CP, which decreases the inhibitory effects of V-1, thereby freeing CP to cap barbed ends. Here, we address the question of whether CPI-motif proteins on a surface analogous to a membrane lead to net activation or inhibition of actin assembly nucleated by Arp2/3 complex. Using reconstitution with purified components, we discovered that CARMIL at the surface promotes and enhances actin assembly, countering the inhibitory effects of V-1 and thus activating CP. The reconstitution involves the presence of an Arp2/3 activator on the surface, along with Arp2/3 complex, V-1, CP, profilin and actin monomers in solution, recreating key features of cell physiology.
肌动蛋白聚合作用通常与含有帽蛋白相互作用（CPI）基序的膜蛋白相关，如CARMIL、CD2AP和WASHCAP/Fam21。CPI基序直接与肌动蛋白帽蛋白（CP）结合，这种相互作用会削弱CP与肌动蛋白丝末端的结合，降低CP对这些末端进行功能性封端的能力。蛋白V-1/肌养蛋白结合到CP上的F-肌动蛋白结合位点，并在空间上阻止CP结合肌动蛋白丝末端。含有CPI基序的蛋白也会削弱V-1与CP之间的结合，从而降低V-1的抑制作用，进而使CP能够封端肌动蛋白丝末端。在此，我们探讨了在类似于膜的表面上，含有CPI基序的蛋白是否会导致由Arp2/3复合物引发的肌动蛋白组装的净激活或抑制这一问题。通过使用纯化成分进行重组，我们发现表面的CARMIL能促进和增强肌动蛋白组装，抵消V-1的抑制作用，从而激活CP。重组过程涉及表面存在Arp2/3激活剂，以及溶液中的Arp2/3复合物、V-1、CP、肌动蛋白单体结合蛋白和肌动蛋白单体，重现了细胞生理学的关键特征。
