Quantitative variations of ADF/cofilin’s multiple actions on actin filaments with pH

ABSTRACTActin Depolymerizing Factor (ADF)/cofilin is the main protein family promoting the disassembly of actin filaments, which is essential for numerous cellular functions. ADF/cofilin proteins disassemble actin filaments through different reactions, as they bind to their sides, sever them, and promote the depolymerization of the resulting ADF/cofilin-saturated filaments. Moreover, the efficiency of ADF/cofilin is known to be very sensitive to pH. ADF/cofilin thus illustrates two challenges in actin biochemistry: separating the different regulatory actions of a single protein, and characterizing them as a function of specific biochemical conditions. Here, we investigate the different reactions of ADF/cofilin on actin filaments, over four different values of pH ranging from pH 6.6 to pH 7.8, using single filament microfluidics techniques. We show that lowering pH reduces the effective filament severing rate by increasing the rate at which filaments become saturated by ADF/cofilin, thereby reducing the number of ADF/cofilin domain boundaries, where severing can occur. The severing rate per domain boundary, however, remains unchanged at different pH values. The ADF/cofilin-decorated filaments (refered to as “cofilactin” filaments) depolymerize from both ends. We show here that, at physiological pH (pH 7.0 to 7.4), the pointed end depolymerization of cofilactin filaments is barely faster than that of bare filaments. In contrast, cofilactin barbed ends undergo an “unstoppable” depolymerization (depolymerizing for minutes despite the presence of free actin monomers and capping protein in solution), throughout our range of pH. We thus show that, at physiological pH, the main contribution of ADF/cofilin to filament depolymerization is at the barbed end.A number of key cellular processes rely on the proper assembly and disassembly of actin filament networks 1. The central regulator of actin disassembly is the ADF/cofilin protein family 2, 3, which comprises three isoforms in mammals: cofilin-1 (cof1, found in nearly all cell types), cofilin-2 (cof2, found primarily in muscles) and Actin Depolymerization Factor (ADF, found mostly in neurons and epithelial cells). We refer to them collectively as “ADF/cofilin”.Over the years, the combined efforts of several labs have led to the following understanding of actin filament disassembly by ADF/cofilin. Molecules of ADF/cofilin bind stoechiometrically 4, 5 to the sides of actin filaments, with a strong preference for ADP-actin subunits 6–10. Though ADF/cofilin molecules do not contact each other 11, they bind in a cooperative manner, leading to the formation of ADF/cofilin domains on the filaments 5, 7, 9, 12, 13. Compared to bare F-actin, the filament portions decorated by ADF/cofilin (refered to as “cofilactin”) are more flexible 14, 15 and exhibit a shorter right-handed helical pitch, with a different subunit conformation 11, 16–19. Thermal fluctuations are then enough to sever actin filaments at (or near) domain boundaries8, 9, 13, 20, 21. Cofilactin filaments do not sever, but depolymerize from both ends 13 thereby renewing the actin monomer pool.ADF/cofilin thus disassembles actin filaments through the combination of different actions. As such, it vividly illustrates a current challenge in actin biochemistry: identifying and quantifying the multiple reactions involving a single protein. This is a very difficult task for bulk solution assays, where a large number of reactions take place simultaneously, and single-filament techniques have played a key role in deciphering ADF/cofilin’s actions 9, 13, 20, 22–24. In particular, the microfluidics-based method that we have developed over the past years, is a powerful tool for such investigations 25. It has recently allowed us to quantify the kinetics of the aforementioned reactions, and to discover that ADF/cofilin-saturated filament (cofilactin) barbed ends can hardly stop depolymerizing, even when ATP-G-actin and capping protein are present in solution 13.In addition, ADF/cofilin is very sensitive to pH 4, 5, 26–29. In cells, pH can be a key regulatory factor 30. It can vary between compartments, between cell types, and be specifically modulated. We can consider that a typical cytoplasmic pH would be comprised between 7.0 and 7.4. Recently, we have quantified the different reactions involving ADF/cofilin at pH 7.8 13, leaving open the question of how these reaction rates are indivdually affected by pH variations. For instance, it has been reported that ADF/cofilin is a more potent filament disassembler at higher pH values 4, 5, 26–29 but the actual impact of pH on the rate constants of individual reactions has yet to be characterized. Moreover, whether the unstoppable barbed end depolymerization that we have recently discovered for ADF/cofilin-saturated filaments at pH 7.8 13 remains significant at lower, more physiological pH values is an open question.Here, we investigate how the different contributions of ADF/cofilin (using unlabeled ADF, unlabeled cof1 and eGFP-cof1) to actin filament disassembly depend on pH, which we varied from 6.6 to 7.8. We first present the methods which we have used to do so, based on the observation of individual filaments, using microfluidics (Fig. 1). We measured cofilin’s abitility to decorate actin filament by binding to its sides (Fig. 2), and the rate at which individual cofilin domains severed actin filaments (Fig. 3). We next quantified the kinetic parameters of filament ends, for bare and ADF/cofilin-saturated (cofilactin) filaments (Fig. 4), and we specifically quantified the extent to which the barbed ends of cofilactin filaments are in a state which can hardly stop depolymerizing (Fig. 5). We finally summarize our results (Fig. 6).