F-actin binding is essential for coronin 1B function in vivo.
Coronins are conserved F-actin binding proteins that have been implicated in a variety of processes including fibroblast migration, phagocytosis, and chemotaxis. Recent data from our lab indicate that coronin 1B coordinates Arp2/3-dependent actin filament nucleation and cofilin-mediated filament turnover at the leading edge of migrating fibroblasts. Analysis of coronin function has been hampered by the lack of a clear understanding of how coronin interacts with F-actin. Here, we identify a surface-exposed conserved arginine residue at position 30 (R30), which is crucial for coronin 1B binding to F-actin both in vitro and in vivo. Using actin co-sedimentation, we demonstrate that coronin 1B binds with high affinity to ATP/ADP-P(i)-F-actin (170 nM) and with 47-fold lower affinity to ADP-F-actin (8 microM). In contrast to a previous study, we find no evidence for enhanced cofilin binding to F-actin in the presence of either coronin 1B or coronin 1A. Instead, we find that coronin 1B protects actin filaments from cofilin-induced depolymerization. Consistent with an important role for interactions between coronin 1B and F-actin in vivo, an R30D coronin mutant that does not bind F-actin localizes inefficiently to the leading edge. Furthermore, our analysis indicates that F-actin binding is absolutely required for coronin 1B to exert its effects on whole-cell motility and lamellipodial dynamics.
Cai
Liang
L
Lineberger Comprehensive Cancer Center, Department of Cell & Developmental Biology, University of North Carolina at Chapel Hill, North Carolina 27599-7295, USA.
Makhov
Alexander M
AM
Bear
James E
JE
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D004331:Drosophila melanogaster; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008854:Microscopy, Electron; D008958:Models, Molecular; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011554:Pseudopodia / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats
D000199:Actins; D008840:Microfilament Proteins; C072061:coronin proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine