Monomer Dissociation Research Articles

The Potential Therapeutic Agent Mepacrine Protects Caco-2 Cells against Clostridium perfringens Enterotoxin Action.

Clostridium perfringens enterotoxin (CPE) causes the diarrhea associated with a common bacterial food poisoning and many antibiotic-associated diarrhea cases. The severity of some CPE-mediated disease cases warrants the development of potential therapeutics. A previous study showed that the presence of mepacrine inhibited CPE-induced electrophysiology effects in artificial lipid bilayers lacking CPE receptors. However, that study did not assess whether mepacrine inactivates CPE or, instead, inhibits a step in CPE action. Furthermore, CPE action in host cells is complex, involving the toxin binding to receptors, receptor-bound CPE oligomerizing into a prepore on the membrane surface, and β-hairpins in the CPE prepore inserting into the membrane to form a pore that induces cell death. Therefore, the current study evaluated the ability of mepacrine to protect cells from CPE. This drug was found to reduce CPE-induced cytotoxicity in Caco-2 cells. This protection did not involve mepacrine inactivation of CPE, indicating that mepacrine affects one or more steps in CPE action. Western blotting then demonstrated that mepacrine decreases CPE pore levels in Caco-2 cells. This mepacrine-induced reduction in CPE pore levels did not involve CPE binding inhibition but rather an increase in CPE monomer dissociation due to mepacrine interactions with Caco-2 membranes. In addition, mepacrine was also shown to inhibit CPE pores when already present in Caco-2 cells. These in vitro studies, which identified two mepacrine-sensitive steps in CPE-induced cytotoxicity, add support to further testing of the therapeutic potential of mepacrine against CPE-mediated disease. IMPORTANCEClostridium perfringens enterotoxin (CPE) causes the gastrointestinal (GI) symptoms of a common bacterial food poisoning and several nonfoodborne human GI diseases. A previous study showed that, via an undetermined mechanism, the presence of mepacrine blocks CPE-induced electrophysiologic activity in artificial membranes. The current study now demonstrates that mepacrine also inhibits CPE-induced cytotoxicity in human enterocyte-like Caco-2 cells and that mepacrine does not directly inactivate CPE. Instead, this drug reduces both CPE pore formation and CPE pore activity in Caco-2 cells. These results suggest mepacrine as a therapeutic candidate for treating CPE-mediated GI diseases.

Coupled cluster valence bond theory for open-shell systems with application to very long range strong correlation in a polycarbene dimer.

The Coupled Cluster Valence Bond (CCVB) method, previously presented for closed-shell (CS) systems, is extended to open-shell (OS) systems. The theoretical development is based on embedding the basic OS CCVB wavefunction in a fictitious singlet super-system. This approach reveals that the OS CCVB amplitude equations are quite similar to those of CS CCVB, and thus that OS CCVB requires the same level of computational effort as CS CCVB, which is an inexpensive method. We present qualitatively correct CCVB potential energy curves for all low-lying spin states of P2 and Mn2+. CCVB is successfully applied to the low-lying spin states of some model linear polycarbenes, systems that appear to be a hindrance to standard density functionals. We examine an octa-carbene dimer in a side-by-side orientation, which, in the monomer dissociation limit, exhibits maximal strong correlation over the length of the polycarbene.

Brazilin Inhibits Prostatic Acidic Phosphatase Fibrillogenesis and Decreases its Cytotoxicity.

A 39-amino acid peptide fragment that is derived from prostatic acidic phosphatase (PAP), PAP248-286 , is secreted in large amounts in human semen and forms amyloid fibrils. These fibrils can capture HIV virions and increase the attachment of virions to target cells; as such, they are called a "semen-derived enhancer of virus infection" (SEVI). Therefore, the inhibition of the formation of PAP248-286 amyloid fibrils is of great significance. Herein, we demonstrate that brazilin effectively inhibits PAP248-286 aggregation. The inhibitory effect increases with increasing brazilin concentration. Thioflavin T fluorescence assays and TEM observations confirmed that a few fibrils formed when brazilin was present with PAP248-286 in an equimolar concentration. Circular dichroism spectroscopy indicated that brazilin inhibited the secondary structural transitions from α-helices and random coils into β-sheets. Cytotoxicity assays showed that brazilin significantly decreased the cytotoxicity of the fibrils at 0.01 mmol L-1 . Isothermal titration calorimetry revealed that hydrophobic interactions were the main driving force for the binding of brazilin to the PAP248-286 monomer (dissociation constant, 4.03 μmol L-1 ), and that the binding affinity of brazilin for the fibrils was at least three orders of magnitude lower than that for the monomer. These results indicate that brazilin holds great potential as a small-molecule agent against SEVIs.

Strength durability of gellan gum biopolymer-treated Korean sand with cyclic wetting and drying

Various biological approaches recently have been explored as alternative environmentally-friendly soil improvement strategies in the fields of construction and geotechnical engineering, with the aim of reducing the use of high greenhouse gas emitting construction binders such as cement. Previous studies have shown the effectiveness of microbial biopolymers in soil improvement. However, there are still concerns about the durability and serviceability of biopolymer treated soils, resulting from the biodegradation and hydrolysis behaviors of the biologically produced compounds. In this study, the strength and durability of gellan gum biopolymer treated Jumunjin sand (standard sand of the Republic of Korea) was evaluated under cyclic wetting and drying. The results obtained indicate that the cyclic wetting and drying of gellan gum-treated sands results in a gradual degradation of strength, due to the dissociation of the gellan gum monomers under wetting and imperfect recomposition during re-drying, with an approximately 30% strength reduction over 10 cycles. However, a certain degree of strength recovery and resistance was observed even after numerous cycles, indicating that gellan gum-treated soils can potentially be applied for temporary or medium-term purposes in practical construction.

Direct Visualization and Quantification of the Actin Nucleation andElongation Events in vitro by TIRF Microscopy.

Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing the dynamics of actin filaments at single-filament resolution in vitro. Thanks to the development of various fluorescent probes, we can easily monitor all kinds of events associated with actin dynamics, including nucleation, elongation, bundling, fragmentation and monomer dissociation. Here we present a detailed protocol regarding the visualization and quantification of actin nucleation and filament elongation events by TIRF microscopy in vitro, which is based on the methods previously reported ( Liu et al., 2015 ; Yang et al., 2011 ).

ADF10 shapes the overall organization of apical actin filaments by promoting their turnover and ordering in pollen tubes.

Here, we show that Arabidopsis ADF10 plays an important role in shaping the overall organization of apical actin filaments by promoting their turnover and ordering. ADF10 severs and depolymerizes actin filaments in vitro and is distributed throughout the entire pollen tube. In adf10 mutants, severing and monomer dissociation events for apical actin filaments are reduced, and the apical actin structure extends further toward the tube base than in wild-type tubes. In particular, the percentage of apical actin filaments that form large angles to the tube growth axis is much higher in adf10 pollen tubes, and the actin filaments are more randomly distributed, implying that ADF10 promotes their ordering. Consistent with the role of apical actin filaments in physically restricting the movement of vesicles, the region in which apical vesicles accumulate is enlarged at the tip of adf10 pollen tubes. Both tipward and backward movements of small vesicles are altered within the growth domain of adf10 pollen tubes. Thus, our study suggests that ADF10 shapes the organization of apical actin filaments to regulate vesicle trafficking and pollen tube growth.

Adsorption, Dissociation, and Dehydrogenation of Water Monomer and Water Dimer on the Smallest 3D Aluminum Particle. The O-H Dissociation Barrier Disappears for the Dimer.

We present a detailed mechanistic study on the interaction and reaction of water monomer and water dimer with the smallest 3D aluminum particle (Al6) by employing density functional and explicitly correlated coupled cluster CCSD(T)-F12 theories. Water adsorption, dissociation, and dehydrogenation are considered. For the monomer reaction, where core-valence correlation and an extrapolation to the complete basis set limit is allowed for, our coupled cluster calculations predict the O-H dissociation barrier of about 2 kcal/mol. For the dimer reaction, two distinct reaction paths are identified, initiated by forming separate dimer complexes wherein (H2O)2 adsorbs mainly via the oxygen atom of the donor H2O molecule. The key O-H dissociation transition states of the dimer reaction involve a concerted migration of two H atoms resulting in the dissociation of the donor molecule and formation of the OH-water complex adsorbed on the metal cluster's surface. The most remarkable feature of both dimer reaction energy profiles is the lack of the overall energy barrier for the (rate-determining) O-H dissociation. The hydrogen bond acceptor molecule is suggested to have an extra catalytic effect on the O-H dissociation barrier of the hydrogen bond donor molecule by removing this barrier. A similar effect on the dehydrogenation step is indicated.

Fibril formation from the amyloid-β peptide is governed by a dynamic equilibrium involving association and dissociation of the monomer

Here I review the molecular mechanisms by which water-soluble monomeric amyloid-β (Aβ) peptides are transformed into well-organized supramolecular complexes called amyloid fibrils. The mechanism of amyloid formation is considered theoretically on the basis of experimental results, and the structural and mechanistic similarities of amyloid fibrils to three-dimensional crystals are highlighted. A number of important results from the literature are described. These include the observation that a correct ratio of monomer association and dissociation rate constants is key for formation of well-organized amyloid fibrils. The dynamic nature of the amyloid-β structure is discussed, along with the possibly obligate requirement of the transient formation of a hairpin-like fold prior to its incorporation into amyloid fibrils. Many rounds of monomer association and dissociation events may be present during an apparently silent lag-period. Amongst these association/dissociation events, interaction between the C-terminal regions of the Aβ peptide seems to be more favored. Such association and dissociation events occurring in a "trial-and-error" fashion may be an important requirement for the formation of well-organized amyloid fibrils.

Fragmentation and Coagulation in Supramolecular (Co)polymerization Kinetics.

The self-assembly of molecular building blocks into one-dimensional supramolecular architectures has opened up new frontiers in materials science. Due to the noncovalent interactions between the monomeric units, these architectures are intrinsically dynamic, and understanding their kinetic driving forces is key to rationally programming their morphology and function. To understand the self-assembly dynamics of supramolecular polymerizations (SP), kinetic models based on aggregate growth by sequential monomer association and dissociation have been analyzed. However, fragmentation and coagulation events can also play a role, as evident from studies on peptide self-assembly and the fact that aggregations can be sensitive to mechanical agitations. Here, we analyze how fragmentation and coagulation events influence SP kinetics by theoretical analysis of self-assembling systems of increasing complexity. Our analysis starts with single-component systems in which aggregates are able to grow via an isodesmic or cooperative nucleation–elongation mechanism. Subsequently, equilibration dynamics in cooperative two-component supramolecular copolymerizations are investigated. In the final part, we reveal how aggregate growth in the presence of competing, kinetically controlled pathways is influenced by fragmentation and coagulation reactions and reveal how seed-induced growth can give rise to block copolymers. Our analysis shows how fragmentation and coagulation reactions are able to modulate SP kinetics in ways that are highly system dependent.

Ru55 nanoparticles catalyze the dissociation of H2O monomer and dimer to produce hydrogen: A comparative DFT study

The calculation results show that H2O monomer prefers to adsorb at two top sites (t1, t2) of the icosahedral Ru55 nanoparticle. When two H2O molecules coadsorb on Ru55, they choose to adsorb at the two adjacent vertex and edge sites to form a H2O dimer. The energy barrier for H2O monomer dissociation is 0.43 eV on t1 site and 0.34 eV on t2 site after DFT-D2 correction, indicating the t2 site is more active than the t1 site. The energy barrier for H2O dimer dissociation on t2 site (2H2O→TS1step1HOHH2O) is only 0.11 eV after DFT-D2 correction. During H2O dimer dissociation, the H atom from the H2O locating on the t1 site can automatically transfer to the H2O locating on the more active t2 site. The low energy barriers for the dissociation of H2O monomer and dimer illustrate that Ru55 nanoparticles have a high catalyst activity for H2O dissociation to produce hydrogen.

Acidity of the Poly(acrylamidoxime) Adsorbent in Aqueous Solution: Determination of the Proton Affinity Distribution via Potentiometric Titrations

Poly(acrylamidoxime) fibers are the current state of the art adsorbent for mining uranium from seawater. While the acid dissociation constants, pKa, of characteristic amidoxime and carboxylate ligands have been reported in the literature, the proton affinity distribution of the poly(acrylamidoxime) fiber is yet to be established. Herein, we report the poly(acrylamidoxime) proton affinity distribution between pH 2 and pH 10 via the stable numerical solution of the adsorption integral equation using splines (SAIUS) algorithm. Two peaks in the proton affinity distribution of poly(acrylamidoxime) were observed: the neutral to anionic dissociation of the carboxylate monomer between pH 3.2 and pH 4.4 (pKa ≈ 4.0) and the protonated to neutral dissociation of the acyclic amidoxime monomer between pH 5.6 and pH 6.8 (pKa ≈ 6.1). The acidity constants obtained for the carboxylate and amidoximate monomers vary from the acidity constants of acetic acid and acetamidoxime, respectively. These variations in acidity can b...

Contribution of Electrostatics in the Fibril Stability of a Model Ionic-Complementary Peptide.

In this work we quantified the role of electrostatic interactions in the self-assembly of a model amphiphilic peptide (RADA 16-I) into fibrillar structures by a combination of size exclusion chromatography and molecular simulations. For the peptide under investigation, it is found that a net charge of +0.75 represents the ideal condition to promote the formation of regular amyloid fibrils. Lower net charges favor the formation of amorphous precipitates, while larger net charges destabilize the fibrillar aggregates and promote a reversible dissociation of monomers from the ends of the fibrils. By quantifying the dependence of the equilibrium constant of this reversible reaction on the pH value and the peptide net charge, we show that electrostatic interactions contribute largely to the free energy of fibril formation. The addition of both salt and a charged destabilizer (guanidinium hydrochloride) at moderate concentration (0.3-1 M) shifts the monomer-fibril equilibrium toward the fibrillar state. Whereas the first effect can be explained by charge screening of electrostatic repulsion only, the promotion of fibril formation in the presence of guanidinium hydrochloride is also attributed to modifications of the peptide conformation. The results of this work indicate that the global peptide net charge is a key property that correlates well with the fibril stability, although the peptide conformation and the surface charge distribution also contribute to the aggregation propensity.

Depolymerization of Free-Radical Polymers with Spin Migrations.

The mechanism of depolymerization is one of the most essential issues in chemical engineering and materials science. In this work, we investigate the depolymerization reactions of three typical free-radical poly(alpha-methylstyrene) tetramers by using first-principles density functional theory. The calculated results show that these reactions all need to overcome the energy barriers in the range of 0.58 to 0.77 eV, and that breaking the C-C bond at the chain end leads to the dissociation of alpha-methylstyrene monomers from the polymers. Electronic-structure analysis indicates that the reactions occur easily at the CR3 unsaturated end, and that the frontier molecular orbitals that participate in the reactions are mainly localized at the unsaturated ends. Meanwhile, spin population analysis presents the unique net spin-transfer process in free-radical depolymerization reactions. We hope the current findings can contribute to understanding the free-radical depolymerization mechanism and help guide future experiments.

A realistic description of influence of the magnetic field strength on high gradient magnetic separation

The magnetic field strength is a very important parameter in high gradient magnetic separation (HGMS). It was usually thought that the increase of magnetic field strength will lead to the decrease of the minerals’ susceptibility and the matrices’ surface area available for the buildup of the magnetic particles, which was responsible for the decrease of the mineral recovery in high magnetic field. But the detailed influence of the magnetic field strength on the behavior of the particles of different characteristics in HGMS is still not clear. A very important factor was usually ignored in the process of HGMS, namely the behavior of the low grade intergrowths. In this article, the influence of magnetic field strength on the behavior of the magnetic monomers and intergrowths in HGMS was investigated with the particle buildup model and the particle capture model. The buildup profile of the magnetic monomers increases with the increase of the magnetic field strength despite the decrease of the minerals’ susceptibility. The low grade intergrowths can hardly accumulate on the matrices in a low magnetic field but can largely accumulate on the matrices in a high magnetic field. In a high magnetic field, there exists the competitive accumulation between the low grade intergrowths and the fine monomers. Coarse intergrowths may have larger buildup profile and shorter capture time than the fine monomers and will accumulate preferentially on the matrices. In some circumstances, the competitive accumulation of the particles will lead to the decrease of the recovery in a high magnetic field. A moderate high magnetic field can ensure a high recovery. For a magnetic product of high grade and recovery, high monomer dissociation degree is necessary and the over grinding of the minerals should be avoided, and classifying the minerals to handle the coarse fraction and the fine fraction separately can also be considered.

Top-down mass spectrometry of intact membrane protein complexes reveals oligomeric state and sequence information in a single experiment.

Here we study the intact stoichiometry and top-down fragmentation behavior of three integral membrane proteins which were natively reconstituted into detergent micelles: the mechano-sensitive ion channel of large conductance (MscL), the Kirbac potassium channel and the p7 viroporin from the hepatitis C virus. By releasing the proteins under nondenaturing conditions inside the mass spectrometer, we obtained their oligomeric sizes. Increasing the ion activation (collision energy) causes unfolding and subsequent ejection of a highly charged monomer from the membrane protein complexes. Further increase of the ion activation then causes collision-induced dissociation (CID) of the ejected monomers, with fragments observed which were predominantly found to stem from membrane-embedded regions. These experiments show how in a single experiment, we can probe the relation between higher-order structure and protein sequence, by combining the native MS data with fragmentation obtained from top-down MS.

Structural Basis of pH Dependence of Neoculin, a Sweet Taste-Modifying Protein.

Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness.

Sodium triflate decreases interaggregate repulsion and induces phase separation in cationic micelles.

Dodecyltrimethylammonium triflate (DTATf) micelles possess lower degree of counterion dissociation (α), lower hydration, and higher packing of monomers than other micelles of similar structure. Addition of sodium triflate ([NaTf] > 0.05 M) to DTATf solutions promotes phase separation. This phenomenon is commonly observed in oppositely charged surfactant mixtures, but it is rare for ionic surfactants and relatively simple counterions. While the properties of DTATf have already been reported, the driving forces for the observed phase separation with added salt remain unclear. Thus, we propose an interpretation for the observed phase separation in cationic surfactant solutions. Addition of up to 0.03 M NaTf to micellar DTATf solutions led to a limited increase of the aggregation number, to interface dehydration, and to a progressive decrease in α. The viscosity of DTATf solutions of higher concentration ([DTATf] ≥ 0.06 M) reached a maximum with increasing [NaTf], though the aggregation number slightly increased, and no shape change occurred. We hypothesize that this maximum results from a decrease in interaggregate repulsion, as a consequence of increased ion binding. This reduction in micellar repulsion without simultaneous infinite micellar growth is, probably, the major driving force for phase separation at higher [NaTf].

Mathematical modeling for intracellular transport and binding of HIV-1 Gag proteins

This paper presents a modeling study for the intracellular trafficking and trimerization of the HIV-1 Gag proteins. A set of differential equations including initial and boundary conditions is used to characterize the transport, diffusion, association and dissociation of Gag monomers and trimers for the time period from the initial production of Gag protein monomers to the initial appearance of immature HIV-1 virions near the cell membrane (the time duration Ta). The existence and stability of the steady-state solution of the initial boundary value problems provide a quantitative characterization of the tendency and equilibrium of Gag protein movement. The numerical simulation results further demonstrate Gag trimerization near the cell membrane. Our calculations of Ta are in good agreement with published experimental data. Sensitivity analysis of Ta to the model parameters indicates that the timing of the initial appearance of HIV-1 virions on the cell membrane is affected by the diffusion and transport processes. These results provide important information and insight into the Gag protein transport and binding and HIV-1 virion formation.

Cofilin-1 – Induced Actin Reorganization and Phosphatidylserine Exposure in Platelets

Background: Platelet activation leads to the transbilayer movement of phosphatidylserine (PS) from inner to the outer leaflet of membrane bilayer. Exteriorization of PS promotes platelet procoagulant activity by promoting the assembly of tenase and the prothrombinase complex on platelet membrane. In a previous study, we observed that Rho-associated coiled-coil kinase-1 (ROCK1) deficiency in murine platelets or ROCK inhibition by Y-27632 in human platelets resulted in increase in PS exposure and platelet procoagulant activity. ROCK1 deficient platelets had a marked decrease in phosphorylation of cofilin-1. Cofilin-1 decreases actin filament length by increasing the rate of dissociation of actin monomers and its activity is abolished by phosphorylation. These studies suggested a role for cofilin-1-induced actin reorganization in collagen-induced PS exposure. Cofilin-1 activity is also modulated by its interactions with phosphatidylinositol 4, 5-bisphosphate (PIP2) and a cofilin-1 binding protein, Wdr1, which enhances capacity of cofilin-1 to accelerate depolymerization by capping their barbed ends. Here, we studied the role of cofilin-1 phosphorylation and its interaction with PIP2 and Wdr1 in activation induced PS exposure in platelets.Materials and Methods: We isolated platelet membrane, cytosol and cytoskeleton through differential centrifugation from resting and collagen-stimulated platelets and assessed the relative abundance of cofilin-1 and phosphocofilin-1 by mobility shift in phosphate-affinity polyacrylamide gel electrophoresis. This method allows simultaneous detection of relative proportions of phosphoproteins and its nonphospho counterparts. For cofilin-1 and Wdr-1 distribution, platelets were immobilized on a polylysine-coated cover slip or on a collagen-coated cover slip, fixed, permeabilized and immunostained with appropriate antibody. In addition, PS exposure (FITC lactadherin binding), F-actin (Alexa Fluor 488-phalloidin) and calcium concentration (Fura-2AM fluorescence) were also measured.Results: Immunofluorescence images show that in resting platelets, cofilin-1 is present in distinct patches in the plasma membrane and following activation with collagen, cofilin-1 is redistributed in a discrete granular pattern throughout the cytoplasm. In parallel, we also studied the relative distribution and the phosphorylation status of cofilin-1 in various subcellular fractions of platelets. In resting platelets, cofilin-1 is present in the plasma membrane and in the cytosol but absent in the cytoskeleton. Following activation with collagen, cofilin-1 moves to the cytoskeleton with a concomitant decrease in the plasma membrane fraction. These results suggest that active cofilin-1 is incorporated into reorganizing actin cytoskeleton during platelet activation. In ROCK1-deficient mice, there is increased cofilin-1 in cytoskeletal fraction, which also correlates with increase in PS exposure. Blocking PIP2 hydrolysis by U73122 prevents cofilin-1 translocation and PS exposure. A cell permeable cofilin-1 peptide containing the actin binding site of cofilin-1 (amino acids Trp104 to Met115) at the carboxy terminus (that competitively inhibits cofilin-1 binding to F-actin), blocks cofilin-1 translocation and decreases PS exposure. Platelets from a Wdr1-deficient mice which express less than 2% of wild-type protein in platelets, showed impaired collagen-induced PS exposure despite equivalent increase in intra platelet calcium compared to wild-type platelets.Conclusion: Our results show cofilin-1 trafficking from membranre to cytoskeleton and subsequent actin reorganization precedes PS exposure during platelet activation. In ROCK1-deficient platelets, there is increased cofilin-1 activity due to decreased serine-3 phosphorylation and it is associated with increased cofilin-1 translocation to actin filaments and enhanced PS exposure. In contrast, inhibiting cofilin-1 translocation, either by inhibiting PIP2 hydrolysis or by competitive inhibition by a cell permeable peptide, prevents cofilin-1 trafficking and decreases PS exposure. Furthermore, in Wdr1-deficient mouse platelets, which have diminished cofilin-1 activity, have impaired PS exposure during platelet activation. DisclosuresNo relevant conflicts of interest to declare.

Aip1 Destabilizes Cofilin-Saturated Actin Filaments by Severing and Accelerating Monomer Dissociation from Ends

Depolymerization of actin filaments is vital for the morphogenesis of dynamic cytoskeletal arrays and actin-dependent cell motility. Cofilin is necessary for actin disassembly in cells, and it severs filaments most efficiently at low cofilin to actin ratios, whereas higher concentrations of cofilin suppress severing. However, the cofilin concentration in thymocytes is too high to allow the severing of single-actin filaments. We observed that filaments sever efficiently in thymus cytosol. We identified Aip1 as a critical factor responsible for the severing and destabilization of actin filaments even in the presence of high amounts of cofilin. By fluorescence resonance energy transfer (FRET)-based spectroscopy and single-filament imaging of actin, we show that, besides driving the rapid severing of cofilin-actin filaments, Aip1 also augments the monomer dissociation rate at both the barbed and pointed ends of actin. Our results also demonstrate that Aip1 does not cap the barbed ends of actin filaments, as was previously thought. Our results indicate that Aip1 is a cofilin-dependent actin depolymerization factor and not a barbed-end-capping factor as was previously thought. Aip1 inverts the rules of cofilin-mediated actin disassembly such that increasing ratios of cofilin to actin now result in filament destabilization through faster severing and accelerated monomer loss from barbed and pointed ends. Aip1 therefore offers a potential control point for disassembly mechanisms in cells to switch from a regime of cofilin-saturation and stabilization to one that favors fast disassembly and destabilization.