Capping Protein (CP) regulates actin dynamics by serving as the major terminator of assembly at the actin filament barbed end (BE). CARMIL antagonizes CP function by reducing CPs affinity for the BE and by uncapping CP-capped filaments, while V-1 (myotrophin) sequesters CP in an inactive complex. Previous work showed that CARMIL can readily retrieve CP from the CP: V-1 complex, thereby converting inactive CP into a version with moderate affinity for the BE. Here we further clarify the mechanism of this exchange reaction, and demonstrate its effect on BE assembly in vitro using solution assays and single filament imaging. We show that the CP: CARMIL complex created by complex exchange slows in a dose-dependent manner the rate of BE assembly, probably by rapidly associating with, and dissociating from, the BE. Moreover, the cellular concentrations of CP, V-1 and CARMIL should allow them to collaborate in a robust CP regulatory cycle in vivo. Finally, we provide evidence that CARMIL is recruited to the plasma membrane, and only at cell edges undergoing active protrusion. Assuming that CARMIL is active only at this location, our data argue that a large pool of inactive CP (CP: V-1) feeds, via CARMIL-driven complex exchange, the formation of weak capping complexes (CP: CARMIL) at the plasma membrane of protruding edges. This mechanism should enhance, relative to unregulated CP, the growth of newly-nucleated actin filaments at the plasma membrane: cytoplasm interface, while maintaining strong suppression of actin assembly elsewhere in the cell. Myotrophin/V-1 is a ubiquitously expressed, 13 kDa ankyrin-repeat protein that binds Capping Protein (CP) 1:1 with an affinity of 20 nM, resulting in a complex that has no affinity for the barbed end. The CP sequestering activity of V-1 may play a major role in buffering the barbed end capping activity of CP in Dictyostelium (Dd), especially given our estimates of the cellular concentrations of CP and V-1 in Dd (1 M and 8 M, respectively). Consistent with biochemical studies of mouse V-1, endogenous Dd CP is pulled down by GST-tagged, wild type Dd V-1 (WT Dd V-1) and is co immuno- precipitated by Flag-tagged, WT Dd V-1. Moreover, these interactions are abrogated when using a version of Dd V-1 (FBM Dd V-1) containing four closely-spaced point mutations that in mouse V-1 greatly attenuate its interaction with CP. Consistent with V-1s ability to inactivate CP, the major cellular terminator of actin assembly, the over expression of WT Dd V-1 results in a significant elevation in total cellular F-actin content. Moreover, this increase scales positively with degree of over expression. As expected, the over expression of FBM Dd V-1 does not alter cellular F-actin levels. The over expression of WT Dd V-1 (but not FBM Dd V-1) also induces the formation of actin-rich, filopodial-like structures, and this effect once again scales positively with the degree of over expression. WT-DdV1 over expression leads not only to a significant increase in the number of filopodia, but also to a significant increase in their length. Moreover, time lapse images of cells co expressing a live-cell reporter for F-actin reveal that the filopodia in WT-DdV1 over-expressing cells are more dynamic than in control cells. Together, these over expression studies suggest that V-1 regulates actin polymerization and filopdial formation in vivo by buffering the level of active CP (where, in the case of V-1 over expression, actin polymerization and filopodia formation are enhanced because more cellular CP in sequestered). These results are consistent with previous studies showing that CP knockdown leads to the explosive formation of filopodia in both B16F1 melanocytes and Dictyostelium, and that V-1 over expression enhances actin polymerization and induces finger-like surface structures in PC12D cells. Efforts to create a Dictyostelium cell line that lacks V-1, which should provide further confirmation of the proteins role in regulating CP in vivo (these KO cells are expected to exhibit a profound decease in cellular F-actin content because the bulk of cellular CP will now be active) are underway.
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