TY - JOUR
T1 - Reconstitution of Arp2/3-nucleated actin assembly with proteins CP, V-1, and CARMIL
AU - Mooren, Olivia L.
AU - McConnell, Patrick
AU - DeBrecht, James D.
AU - Jaysingh, Anshuman
AU - Cooper, John A.
N1 - Publisher Copyright:
© 2024 Elsevier Inc.
PY - 2024/11/18
Y1 - 2024/11/18
N2 - Actin polymerization is often associated with membrane proteins containing capping-protein-interacting (CPI) motifs, such as capping protein, Arp2/3, myosin I linker (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.
AB - Actin polymerization is often associated with membrane proteins containing capping-protein-interacting (CPI) motifs, such as capping protein, Arp2/3, myosin I linker (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.
KW - CARMIL
KW - CPI-motif proteins
KW - myotrophin
KW - protein conformation
KW - V-1
KW - WASHCAP
UR - http://www.scopus.com/inward/record.url?scp=85207812299&partnerID=8YFLogxK
U2 - 10.1016/j.cub.2024.09.051
DO - 10.1016/j.cub.2024.09.051
M3 - Article
C2 - 39437783
AN - SCOPUS:85207812299
SN - 0960-9822
VL - 34
SP - 5173-5186.e4
JO - Current Biology
JF - Current Biology
IS - 22
ER -