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Investigations into the biosynthetic pathways of three families of actin-targeting macrolides lead to insights into their convergent or combinatorial evolution, along with the identification of the first free-living bacterial source of macroalga-derived luminaolides. Actin-targeting macrolides comprise a large, structurally diverse group of cytotoxins isolated from remarkably dissimilar micro- and macroorganisms. In spite of their disparate origins and structures, many of these compounds bind actin at the same site and exhibit structural relationships reminiscent of modular, combinatorial drug libraries. Here we investigate biosynthesis and evolution of three compound groups: misakinolides, scytophycin-type compounds and luminaolides. For misakinolides from the sponge Theonella swinhoei WA, our data suggest production by an uncultivated 'Entotheonella' symbiont, further supporting the relevance of these bacteria as sources of bioactive polyketides and peptides in sponges. Insights into misakinolide biosynthesis permitted targeted genome mining for other members, providing a cyanobacterial luminaolide producer as the first cultivated source for this dimeric compound family. The data indicate that this polyketide family is bacteria-derived and that the unusual macrolide diversity is the result of combinatorial pathway modularity for some compounds and of convergent evolution for others.

Actin and spectrin play important roles in neurons, but their organization in axons and dendrites remains unclear. We used stochastic optical reconstruction microscopy to study the organization of actin, spectrin, and associated proteins in neurons. Actin formed ringlike structures that wrapped around the circumference of axons and were evenly spaced along axonal shafts with a periodicity of ∼180 to 190 nanometers. This periodic structure was not observed in dendrites, which instead contained long actin filaments running along dendritic shafts. Adducin, an actin-capping protein, colocalized with the actin rings. Spectrin exhibited periodic structures alternating with those of actin and adducin, and the distance between adjacent actin-adducin rings was comparable to the length of a spectrin tetramer. Sodium channels in axons were distributed in a periodic pattern coordinated with the underlying actin-spectrin—based cytoskeleton.

New Arp2/3 complex inhibitors The actin cytoskeleton is dynamic and plays a critical role in several cell biological processes such as cell adhesion, migration and endocytosis. In addition, several pathogens exploit this system to invade and survive within host cells. The Arp2/3 complex nucleates actin filaments and has a unique property to form branched actin networks. In this study, Nolen et al . describe the generation of two types of small molecules that bind to different sites on the Arp2/3 complex and inhibit its actin nucleating activity in vitro and in vivo . These inhibitors provide a powerful approach to study Arp2/3 complex-mediated actin reorganization events in living cells. The actin cytoskeleton plays a critical role in cell biological processes such as cell adhesion, migration and endocytosis. Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movement. Two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments are now described; these inhibitors provide a powerful approach for studying the Arp2/3 complex in living cells. Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movements 1 . However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones; this is because of the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-0993548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying the Arp2/3 complex in living cells.

Force generation by actin assembly shapes cellular membranes. An experimentally constrained multiscale model shows that a minimal branched actin network is sufficient to internalize endocytic pits against membrane tension. Around 200 activated Arp2/3 complexes are required for robust internalization. A newly developed molecule-counting method determined that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Simulations predict that actin self-organizes into a radial branched array with growing ends oriented toward the base of the pit. Long actin filaments bend between attachment sites in the coat and the base of the pit. Elastic energy stored in bent filaments, whose presence was confirmed by cryo-electron tomography, contributes to endocytic internalization. Elevated membrane tension directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, spatially constrained actin filament assembly utilizes an adaptive mechanism enabling endocytosis under varying physical constraints. The outer membrane of a cell is a tight but elastic barrier that controls what enters or leaves the cell. Large molecules typically cannot cross this membrane unaided. Instead, to enter the cell, they must be packaged into a pocket of the membrane that is then pulled inside. This process, called endocytosis, shuttles material into a cell hundreds of times a minute. Endocytosis relies on molecular machines that assemble and disassemble at the membrane as required. One component, a protein called actin, self-assembles near the membrane into long filaments with many repeated subunits. These filaments grow against the membrane, pulling it inwards. But it was not clear how actin filaments organize in such a way that allows them to pull on the membrane with enough force – and without a template to follow. Akamatsu et al. set about identifying how actin operates during endocytosis by using computer simulations that were informed by measurements made in living cells. The simulations included information about the location of actin and other essential molecules, along with the details of how these molecules work individually and together. Akamatsu et al. also developed a method to count the numbers of molecules of a key protein at individual sites of endocytosis. High-resolution imaging was then used to create 3D pictures of actin and endocytosis in action in human cells grown in the laboratory. The analysis showed the way actin filaments arrange themselves depends on the starting positions of a few key molecules that connect to actin. Imaging confirmed that, like a pole-vaulting pole, the flexible actin filaments bend to store energy and then release it to pull the membrane inwards during endocytosis. Finally, the simulations predicted that the collection of filaments adapts its shape and size in response to the resistance of the elastic membrane. This makes the system opportunistic and adaptable to the unpredictable environment within cells.

Actin filament assembly and disassembly are vital for cell functions. MICAL Redox enzymes are important post-translational effectors of actin that stereo-specifically oxidize actin’s M44 and M47 residues to induce cellular F-actin disassembly. Here we show that Mical-oxidized (Mox) actin can undergo extremely fast (84 subunits/s) disassembly, which depends on F-actin’s nucleotide-bound state. Using near-atomic resolution cryoEM reconstruction and single filament TIRF microscopy we identify two dynamic and structural states of Mox-actin. Modeling actin’s D-loop region based on our 3.9 Å cryoEM reconstruction suggests that oxidation by Mical reorients the side chain of M44 and induces a new intermolecular interaction of actin residue M47 (M47-O-T351). Site-directed mutagenesis reveals that this interaction promotes Mox-actin instability. Moreover, we find that Mical oxidation of actin allows for cofilin-mediated severing even in the presence of inorganic phosphate. Thus, in conjunction with cofilin, Mical oxidation of actin promotes F-actin disassembly independent of the nucleotide-bound state. MICAL Redox enzymes post-translationally modify F-actin to promote its cellular destabilization. Here, the authors present a 3.9Å cryoEM structure of Mical-oxidized F-actin, showing its nucleotide-state dependent dynamic instability and susceptibility to cofilin-induced severing in the presence of inorganic phosphate.

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro . Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties. In vitro models of actin organization show the formation of vortices, asters and stars. Here Fritzsche et al . show that such actin structures form in living cells in a manner dependent on the Arp2/3 complex but not myosin, and such structures influence membrane architecture but not cortex elasticity.

The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state 1 – 3 . It remains unclear how F-actin hydrolyses ATP and subsequently undergoes subtle conformational rearrangements that ultimately lead to filament depolymerization by actin-binding proteins. Here we present cryo-electron microscopy structures of F-actin in all nucleotide states, polymerized in the presence of Mg 2+ or Ca 2+ at approximately 2.2 Å resolution. The structures show that actin polymerization induces the relocation of water molecules in the nucleotide-binding pocket, activating one of them for the nucleophilic attack of ATP. Unexpectedly, the back door for the subsequent release of inorganic phosphate (P i ) is closed in all structures, indicating that P i release occurs transiently. The small changes in the nucleotide-binding pocket after ATP hydrolysis and P i release are sensed by a key amino acid, amplified and transmitted to the filament periphery. Furthermore, differences in the positions of water molecules in the nucleotide-binding pocket explain why Ca 2+ -actin shows slower polymerization rates than Mg 2+ -actin. Our work elucidates the solvent-driven rearrangements that govern actin filament assembly and aging and lays the foundation for the rational design of drugs and small molecules for imaging and therapeutic applications. Cryo-electron microscopy structures of skeletal F-actin show solvent-driven rearrangements governing actin filament assembly and aging with potential application in design of drugs and small molecules for imaging and therapy.

ATP-hydrolysis-coupled actin polymerization is a fundamental mechanism of cellular force generation 1 – 3 . In turn, force 4 , 5 and actin filament (F-actin) nucleotide state 6 regulate actin dynamics by tuning F-actin’s engagement of actin-binding proteins through mechanisms that are unclear. Here we show that the nucleotide state of actin modulates F-actin structural transitions evoked by bending forces. Cryo-electron microscopy structures of ADP–F-actin and ADP-P i –F-actin with sufficient resolution to visualize bound solvent reveal intersubunit interfaces bridged by water molecules that could mediate filament lattice flexibility. Despite extensive ordered solvent differences in the nucleotide cleft, these structures feature nearly identical lattices and essentially indistinguishable protein backbone conformations that are unlikely to be discriminable by actin-binding proteins. We next introduce a machine-learning-enabled pipeline for reconstructing bent filaments, enabling us to visualize both continuous structural variability and side-chain-level detail. Bent F-actin structures reveal rearrangements at intersubunit interfaces characterized by substantial alterations of helical twist and deformations in individual protomers, transitions that are distinct in ADP–F-actin and ADP-P i –F-actin. This suggests that phosphate rigidifies actin subunits to alter the bending structural landscape of F-actin. As bending forces evoke nucleotide-state dependent conformational transitions of sufficient magnitude to be detected by actin-binding proteins, we propose that actin nucleotide state can serve as a co-regulator of F-actin mechanical regulation. The nucleotide state of actin modulates F-actin structural transitions evoked by bending forces.

We used cryo-electron microscopy (cryo-EM) to reconstruct actin filaments with bound AMPPNP (β,γ-imidoadenosine 5′-triphosphate, an ATP analog, resolution 3.1 Å), ADP-Pi (ADP with inorganic phosphate, resolution 3.1 Å), or ADP (resolution 3.6 Å). Subunits in the three filaments have similar backbone conformations, so assembly rather than ATP hydrolysis or phosphate dissociation is responsible for their flattened conformation in filaments. Polymerization increases the rate of ATP hydrolysis by changing the positions of the side chains of Q137 and H161 in the active site. Flattening during assembly also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up multiple favorable interactions with the DNase-I binding loop in subdomain 2 of the adjacent subunit. Subunits at the barbed end of the filament are likely to be in this favorable conformation, while monomers are not. This difference explains why filaments grow faster at the barbed end than the pointed end. When phosphate dissociates from ADP-Pi-actin through a backdoor channel, the conformation of the C terminus changes so it distorts the DNase binding loop, which allows cofilin binding, and a network of interactions among S14, H73, G74, N111, R177, and G158 rearranges to open the phosphate release site.

Improper lengths of actin-thin filaments are associated with altered contractile activity and lethal myopathies. Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance; however, its role is under dispute. Using nuclear magnetic resonance data and molecular dynamics simulations, we generated the first atomic structural model of the binding interface between the tropomyosin-binding site of cardiac leiomodin and the N-terminus of striated muscle tropomyosin. Our structural data indicate that the leiomodin/tropomyosin complex only forms at the pointed end of thin filaments, where the tropomyosin N-terminus is not blocked by an adjacent tropomyosin protomer. This discovery provides evidence supporting the debated mechanism where leiomodin and tropomodulin regulate thin filament lengths by competing for thin filament binding. Data from experiments performed in cardiomyocytes provide additional support for the competition model; specifically, expression of a leiomodin mutant that is unable to interact with tropomyosin fails to displace tropomodulin at thin filament pointed ends and fails to elongate thin filaments. Together with previous structural and biochemical data, we now propose a molecular mechanism of actin polymerization at the pointed end in the presence of bound leiomodin. In the proposed model, the N-terminal actin-binding site of leiomodin can act as a \"swinging gate\" allowing limited actin polymerization, thus making leiomodin a leaky pointed-end cap. Results presented in this work answer long-standing questions about the role of leiomodin in thin filament length regulation and maintenance.