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The ability of cells to generate forces through actin filament turnover was an early adaptation in evolution. While much is known about how actin filaments grow, mechanisms of their disassembly are incompletely understood. The best-characterized actin disassembly factors are the cofilin family proteins, which increase cytoskeletal dynamics by severing actin filaments. However, the mechanism by which severed actin filaments are recycled back to monomeric form has remained enigmatic. We report that cyclase-associated-protein (CAP) works in synergy with cofilin to accelerate actin filament depolymerization by nearly 100-fold. Structural work uncovers the molecular mechanism by which CAP interacts with actin filament pointed end to destabilize the interface between terminal actin subunits, and subsequently recycles the newly-depolymerized actin monomer for the next round of filament assembly. These findings establish CAP as a molecular machine promoting rapid actin filament depolymerization and monomer recycling, and explain why CAP is critical for actin-dependent processes in all eukaryotes. The cofilin family proteins are actin disassembly factors but the disassembly mechanism is poorly understood. Here authors show that cyclase-associated-protein (CAP) works in synergy with cofilin to accelerate actin filament depolymerization by nearly 100-fold and reveal how CAP destabilizes the interface between terminal actin subunits.

Actin polymerization powers key cellular processes, including motility, morphogenesis, and endocytosis. The actin turnover cycle depends critically on “re-charging” of ADP-actin monomers with ATP, but whether this reaction requires dedicated proteins in cells, and the underlying mechanism, have remained elusive. Here we report that nucleotide exchange catalyzed by the ubiquitous cytoskeletal regulator cyclase-associated protein (CAP) is critical for actin-based processes in vivo. We determine the structure of the CAP–actin complex, which reveals that nucleotide exchange occurs in a compact, sandwich-like complex formed between the dimeric actin-binding domain of CAP and two ADP-actin monomers. In the crystal structure, the C-terminal tail of CAP associates with the nucleotide-sensing region of actin, and this interaction is required for rapid re-charging of actin by both yeast and mammalian CAPs. These data uncover the conserved structural basis and biological role of protein-catalyzed re-charging of actin monomers. Depolymerized ADP-actin monomers must be recharged with ATP for new rounds of filament assembly. Here the authors show that cyclase-associated protein (CAP) catalyzes actin nucleotide exchange in vivo and their CAP–actin complex structure reveals the molecular mechanism of CAP-mediated actin nucleotide exchange.

Focal adhesions (FAs) connect inner workings of cell to the extracellular matrix to control cell adhesion, migration and mechanosensing. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton. By using super-resolution iPALM microscopy, we identify two additional nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments, beneath the previously identified α-actinin cross-linked actin filaments, appear critical for adhesion maturation and controlled cell motility, whereas the adjacent Tpm3.2-actin filament layer beneath seems to facilitate adhesion disassembly. Mechanistically, Tpm3.2 stabilizes ACF-7/MACF1 and KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Tpm3.2 depletion leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during migration. Thus, FAs are composed of distinct actin filament layers, and each may have specific roles in coupling adhesions to the cytoskeleton, or in controlling adhesion dynamics. Focal adhesions are dynamic structures that link the cell to the extracellular matrix. Here, the authors report that focal adhesions contain tropomyosin-decorated actin filaments, and show evidence that suggests specific functions in adhesion dynamics and cell migration.

Membrane-integral pyrophosphatases (M-PPases) are crucial for the survival of plants, bacteria, and protozoan parasites. They couple pyrophosphate hydrolysis or synthesis to Na + or H + pumping. The 2.6-angstrom structure of Thermotoga maritima M-PPase in the resting state reveals a previously unknown solution for ion pumping. The hydrolytic center, 20 angstroms above the membrane, is coupled to the gate formed by the conserved Asp 243 , Glu 246 , and Lys 707 by an unusual \"coupling funnel\" of six α helices. Comparison with our 4.0-angstrom resolution structure of the product complex suggests that helix 12 slides down upon substrate binding to open the gate by a simple binding-change mechanism. Below the gate, four helices form the exit channel. Superimposing helices 3 to 6, 9 to 12, and 13 to 16 suggests that M-PPases arose through gene triplication.

Contractile actomyosin bundles are key force-producing and mechanosensing elements in muscle and non-muscle tissues. Whereas the organization of muscle myofibrils and mechanism regulating their contractility are relatively well-established, the principles by which myosin-II activity and force-balance are regulated in non-muscle cells have remained elusive. We show that Caldesmon, an important component of smooth muscle and non-muscle cell actomyosin bundles, is an elongated protein that functions as a dynamic cross-linker between myosin-II and tropomyosin-actin filaments. Depletion of Caldesmon results in aberrant lateral movement of myosin-II filaments along actin bundles, leading to irregular myosin distribution within stress fibers. This manifests as defects in stress fiber network organization and contractility, and accompanied problems in cell morphogenesis, migration, invasion, and mechanosensing. These results identify Caldesmon as critical factor that ensures regular myosin-II spacing within non-muscle cell actomyosin bundles, and reveal how stress fiber networks are controlled through dynamic cross-linking of tropomyosin-actin and myosin filaments. In this study the authors report that Caldesmon controls force-balance and architecture of stress fibers through dynamic cross-linking of actin and myosin filaments. Caldesmon depletion led to consequent problems in cell morphogenesis, motility and mechanosensing.

In this paper, we suggest a two-player differential game model of transboundary pollution that accounts for time-dependent environmental absorption efficiency, which allows the biosphere to switch from a carbon sink to a source. We investigate the impact of negative externalities resulting from a transboundary pollution non-cooperative game wherein countries are dynamically involved. Based on a linear-quadratic specification for the instantaneous revenue function, we assess differences related to both transient path and steady state between cooperative solution, open-loop and Markov perfect Nash equilibria (MPNE). Regarding the methodological contribution of the paper, we suggest a particular structure of the conjectured value function to solve MPNE problems with multiplicative interaction between state variables in one state equation, so that third-order terms that arise in the Hamilton–Jacobi–Bellman equation are made negligible. Using a collocation procedure, we confirm the validity of the particular structure of the conjectured value function. The results suggest unexpected contrasts in terms of pollution control and environmental absorption efficiency management: (i) in the long run, an open-loop Nash equilibrium (OLNE) allows equivalent emissions to the social optimum but requires greater restoration efforts; (ii) although an MPNE is likely to end up with lower emissions and greater restoration efforts than an OLNE, it has a much greater chance of falling in the emergency area; (iii) the absence of cooperation and or precommitment becomes more costly as the initial absorption efficiency decreases; (iv) more heavily discounted MPNE strategies are less robust than OLNE to prevent irreversible switching of the biosphere from a carbon sink to a source.

Immune evasion facilitates survival of Borrelia , leading to infections like relapsing fever and Lyme disease. Important mechanism for complement evasion is acquisition of the main host complement inhibitor, factor H (FH). By determining the 2.2 Å crystal structure of Factor H binding protein A (FhbA) from Borrelia hermsii in complex with FH domains 19–20, combined with extensive mutagenesis, we identified the structural mechanism by which B . hermsii utilizes FhbA in immune evasion. Moreover, structure-guided sequence database analysis identified a new family of FhbA-related immune evasion molecules from Lyme disease and relapsing fever Borrelia . Conserved FH-binding mechanism within the FhbA-family was verified by analysis of a novel FH-binding protein from B . duttonii . By sequence analysis, we were able to group FH-binding proteins of Borrelia into four distinct phyletic types and identified novel putative FH-binding proteins. The conserved FH-binding mechanism of the FhbA-related proteins could aid in developing new approaches to inhibit virulence and complement resistance in Borrelia .

Actin polymerization generates forces for cellular processes throughout the eukaryotic kingdom, but our understanding of the ‘ancient’ actin turnover machineries is limited. We show that, despite > 1 billion years of evolution, pathogenic Leishmania major parasite and mammalian actins share the same overall fold and co-polymerize with each other. Interestingly, Leishmania harbors a simple actin-regulatory machinery that lacks cofilin ‘cofactors’, which accelerate filament disassembly in higher eukaryotes. By applying single-filament biochemistry we discovered that, compared to mammalian proteins, Leishmania actin filaments depolymerize more rapidly from both ends, and are severed > 100-fold more efficiently by cofilin. Our high-resolution cryo-EM structures of Leishmania ADP-, ADP-Pi- and cofilin-actin filaments identify specific features at actin subunit interfaces and cofilin-actin interactions that explain the unusually rapid dynamics of parasite actin filaments. Our findings reveal how divergent parasites achieve rapid actin dynamics using a remarkably simple set of actin-binding proteins, and elucidate evolution of the actin cytoskeleton. The authors report here the structure-function analysis of highly divergent actin from Leishmania parasite. The study reveals remarkably rapid dynamics of parasite actin as well as the underlying molecular basis, thus providing insight into evolution of the actin cytoskeleton.

Contractile actomyosin bundles, stress fibers, contribute to morphogenesis, migration, and mechanosensing of non-muscle cells. In addition to actin and non-muscle myosin II (NMII), stress fibers contain a large array of proteins that control their assembly, turnover, and contractility. Calponin-3 (Cnn3) is an actin-binding protein that associates with stress fibers. However, whether Cnn3 promotes stress fiber assembly, or serves as either a positive or negative regulator of their contractility has remained obscure. Here, we applied U2OS osteosarcoma cells as a model system to study the function of Cnn3. We show that Cnn3 localizes to both NMII-containing contractile ventral stress fibers and transverse arcs, as well as to non-contractile dorsal stress fibers that do not contain NMII. Fluorescence-recovery-after-photobleaching experiments revealed that Cnn3 is a dynamic component of stress fibers. Importantly, CRISPR/Cas9 knockout and RNAi knockdown studies demonstrated that Cnn3 is not essential for stress fiber assembly. However, Cnn3 depletion resulted in increased and uncoordinated contractility of stress fibers that often led to breakage of individual actomyosin bundles within the stress fiber network. Collectively these results provide evidence that Cnn3 is dispensable for the assembly of actomyosin bundles, but that it is required for controlling proper contractility of the stress fiber network.

Several types of network and non-network licenses control users’ access to software. The literature on efficient selection of software licenses has mostly addressed queuing models intended to estimate the probability of denial of access for a user. Under that approach, however, the lower one sets the rejection probability, the greater the likelihood that licenses will be underused. We follow the literature in describing demand for software access with an Erlang queuing model and present a general integer program for optimizing mixed-license software portfolios under constraints on two types of risk: access rejection and license underutilization. The program, in which the number of licenses of each type is a decision variable, minimizes the total licensing cost while satisfying constraints on access denial and license underutilization. We evaluate and illustrate the ability of an efficient enumerative algorithm to solve the common problem of selecting an appropriate mix of floating and network named user licenses. Our case-study analysis indicates the potential for reducing the cost of licensing while also limiting problems associated with lack of access and underutilization.