Drosophila Tropomodulin is required for multiple actin-dependent processes within developing myofibers.

Proper muscle contraction requires the assembly and maintenance of sarcomeres and myofibrils. Although the protein components of myofibrils are generally known, less is known about the mechanisms by which they individually function and together synergize for myofibril assembly and maintenance. For example, it is unclear how the disruption of actin filament (F-actin) regulatory proteins leads to the muscle weakness observed in myopathies. Here, we show that knockdown of Drosophila Tropomodulin (Tmod), results in several myopathy-related phenotypes, including reduction of muscle cell (myofiber) size, increased sarcomere length, disorganization and misorientation of myofibrils, ectopic F-actin accumulation, loss of tension-mediating proteins at the myotendinous junction, and misshaped and internalized nuclei. Our findings support and extend the tension-driven self-organizing myofibrillogenesis model. We show that, like its mammalian counterpart, Drosophila Tmod caps F-actin pointed-ends, and we propose that this activity is crucial for cellular processes in different locations within the myofiber that directly and indirectly contribute to the maintenance of muscle function. Our findings provide significant insights to the role of Tmod in muscle development, maintenance and disease.

Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor.

Arp2/3 complex generates branched actin networks that drive fundamental processes such as cell motility and cytokinesis. The complex comprises seven proteins, including actin-related proteins (Arps) 2 and 3 and five scaffolding proteins (ArpC1-ArpC5) that mediate interactions with a pre-existing (mother) actin filament at the branch junction. Arp2/3 complex exists in two main conformations, inactive with the Arps interacting end-to-end and active with the Arps interacting side-by-side like subunits of the short-pitch helix of the actin filament. Several cofactors drive the transition toward the active state, including ATP binding to the Arps, WASP-family nucleation-promoting factors (NPFs), actin monomers, and binding of Arp2/3 complex to the mother filament. The precise contribution of each cofactor to activation is poorly understood. We report the 3.32-Å resolution cryo-electron microscopy structure of a transition state of Arp2/3 complex activation with bound constitutively dimeric NPF. Arp2/3 complex-binding region of the NPF N-WASP was fused C-terminally to the α and ß subunits of the CapZ heterodimer. One arm of the NPF dimer binds Arp2 and the other binds actin and Arp3. The conformation of the complex is intermediate between those of inactive and active Arp2/3 complex. Arp2, Arp3, and actin also adopt intermediate conformations between monomeric (G-actin) and filamentous (F-actin) states, but only actin hydrolyzes ATP. In solution, the transition complex is kinetically shifted toward the short-pitch conformation and has higher affinity for F-actin than inactive Arp2/3 complex. The results reveal how all the activating cofactors contribute in a coordinated manner toward Arp2/3 complex activation.

A solution to the long-standing problem of actin expression and purification.

Actin is the most abundant protein in the cytoplasm of eukaryotic cells and interacts with hundreds of proteins to perform essential functions, including cell motility and cytokinesis. Numerous diseases are caused by mutations in actin, but studying the biochemistry of actin mutants is difficult without a reliable method to obtain recombinant actin. Moreover, biochemical studies have typically used tissue-purified α-actin, whereas humans express six isoforms that are nearly identical but perform specialized functions and are difficult to obtain in isolation from natural sources. Here, we describe a solution to the problem of actin expression and purification. We obtain high yields of actin isoforms in human Expi293F cells. Experiments along the multistep purification protocol demonstrate the removal of endogenous actin and the functional integrity of recombinant actin isoforms. Proteomics analysis of endogenous vs. recombinant actin isoforms confirms the presence of native posttranslational modifications, including N-terminal acetylation achieved after affinity-tag removal using the actin-specific enzyme Naa80. The method described facilitates studies of actin under fully native conditions to determine differences among isoforms and the effects of disease-causing mutations that occur in all six isoforms.

Single particle cryo-EM analysis of Rickettsia conorii Sca2 reveals a formin-like core.

Spotted fever group Rickettsia undergo actin-based motility inside infected eukaryotic cells using Sca2 (surface cell antigen 2): an âˆ¼ 1800 amino-acid monomeric autotransporter protein that is surface-attached to the bacterium and responsible for the assembly of long unbranched actin tails. Sca2 is the only known functional mimic of eukaryotic formins, yet it shares no sequence similarities to the latter. Using structural and biochemical approaches we have previously shown that Sca2 uses a novel actin assembly mechanism. The first âˆ¼ 400 amino acids fold into helix-loop-helix repeats that form a crescent shape reminiscent of a formin FH2 monomer. Additionally, the N- and C- terminal halves of Sca2 display intramolecular interaction in an end-to-end manner and cooperate for actin assembly, mimicking a formin FH2 dimer. Towards a better structural understanding of this mechanism, we performed single-particle cryo-electron microscopy analysis of Sca2. While high-resolution structural details remain elusive, our model confirms the presence of a formin-like core: Sca2 indeed forms a doughnut shape, similar in diameter to a formin FH2 dimer and can accommodate two actin subunits. Extra electron density, thought to be contributed by the C-terminal repeat domain (CRD), covering one side is also observed. This structural analysis allows us to propose an updated model where nucleation proceeds by encircling two actin subunits, and elongation proceeds either by a formin-like mechanism that necessitates conformational changes in the observed Sca2 model, or via an insertional mechanism akin to that observed in the ParMRC system.

Novel human cell expression method reveals the role and prevalence of posttranslational modification in nonmuscle tropomyosins.

Biochemical studies require large quantities of proteins, which are typically obtained using bacterial overexpression. However, the folding machinery in bacteria is inadequate for expressing many mammalian proteins, which additionally undergo posttranslational modifications (PTMs) that bacteria, yeast, or insect cells cannot perform. Many proteins also require native N- and C-termini and cannot tolerate extra tag amino acids for proper function. Tropomyosin (Tpm), a coiled coil protein that decorates most actin filaments in cells, requires both native N- and C-termini and PTMs, specifically N-terminal acetylation (Nt-acetylation), to polymerize along actin filaments. Here, we describe a new method that combines native protein expression in human cells with an intein-based purification tag that can be precisely removed after purification. Using this method, we expressed several nonmuscle Tpm isoforms (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2) and the muscle isoform Tpm1.1. Proteomics analysis revealed that human-cell-expressed Tpms present various PTMs, including Nt-acetylation, Ser/Thr phosphorylation, Tyr phosphorylation, and Lys acetylation. Depending on the Tpm isoform (humans express up to 40 Tpm isoforms), Nt-acetylation occurs on either the initiator methionine or on the second residue after removal of the initiator methionine. Human-cell-expressed Tpms bind F-actin differently than their Escherichia coli-expressed counterparts, with or without N-terminal extensions intended to mimic Nt-acetylation, and they can form heterodimers in cells and in vitro. The expression method described here reveals previously unknown features of nonmuscle Tpms and can be used in future structural and biochemical studies with Tpms and other proteins, as shown here for α-synuclein.

Molecular mechanisms linking missense ACTG2 mutations to visceral myopathy.

Visceral myopathy is a life-threatening disease characterized by muscle weakness in the bowel, bladder, and uterus. Mutations in smooth muscle γ-actin (ACTG2) are the most common cause of the disease, but the mechanisms by which the mutations alter muscle function are unknown. Here, we examined four prevalent ACTG2 mutations (R40C, R148C, R178C, and R257C) that cause different disease severity and are spread throughout the actin fold. R178C displayed premature degradation, R148C disrupted interactions with actin-binding proteins, R40C inhibited polymerization, and R257C destabilized filaments. Because these mutations are heterozygous, we also analyzed 50/50 mixtures with wild-type (WT) ACTG2. The WT/R40C mixture impaired filament nucleation by leiomodin 1, and WT/R257C produced filaments that were easily fragmented by smooth muscle myosin. Smooth muscle tropomyosin isoform Tpm1.4 partially rescued the defects of R40C and R257C. Cryo-electron microscopy structures of filaments formed by R40C and R257C revealed disrupted intersubunit contacts. The biochemical and structural properties of the mutants correlate with their genotype-specific disease severity.

The LexA-RecA* structure reveals a cryptic lock-and-key mechanism for SOS activation.

The bacterial SOS response plays a key role in adaptation to DNA damage, including genomic stress caused by antibiotics. SOS induction begins when activated RecA*, an oligomeric nucleoprotein filament that forms on single-stranded DNA, binds to and stimulates autoproteolysis of the repressor LexA. Here, we present the structure of the complete Escherichia coli SOS signal complex, constituting full-length LexA bound to RecA*. We uncover an extensive interface unexpectedly including the LexA DNA-binding domain, providing a new molecular rationale for ordered SOS gene induction. We further find that the interface involves three RecA subunits, with a single residue in the central engaged subunit acting as a molecular key, inserting into an allosteric binding pocket to induce LexA cleavage. Given the pro-mutagenic nature of SOS activation, our structural and mechanistic insights provide a foundation for developing new therapeutics to slow the evolution of antibiotic resistance.

Design principles to tailor Hsp104 therapeutics.

The hexameric AAA+ disaggregase, Hsp104, collaborates with Hsp70 and Hsp40 via its autoregulatory middle domain (MD) to solubilize aggregated protein conformers. However, how ATP- or ADP-specific MD configurations regulate Hsp104 hexamers remains poorly understood. Here, we define an ATP-specific network of interprotomer contacts between nucleotide-binding domain 1 (NBD1) and MD helix L1, which tunes Hsp70 collaboration. Manipulating this network can: (a) reduce Hsp70 collaboration without enhancing activity; (b) generate Hsp104 hypomorphs that collaborate selectively with class B Hsp40s; (c) produce Hsp70-independent potentiated variants; or (d) create species barriers between Hsp104 and Hsp70. Conversely, ADP-specific intraprotomer contacts between MD helix L2 and NBD1 restrict activity, and their perturbation frequently potentiates Hsp104. Importantly, adjusting the NBD1:MD helix L1 rheostat via rational design enables finely tuned collaboration with Hsp70 to safely potentiate Hsp104, minimize off-target toxicity, and counteract FUS proteinopathy in human cells. Thus, we establish important design principles to tailor Hsp104 therapeutics.

Conformation of actin subunits at the barbed and pointed ends of F-actin with and without capping proteins.

Advances in cryo-electron microscopy have made possible the determination of structures of the barbed and pointed ends of F-actin, both in the absence and the presence of capping proteins that block subunit exchange. The conformation of the two exposed protomers at the barbed end resembles the "flat" conformation of protomers in the middle of F-actin. The barbed end changes little upon binding of CapZ, which in turn undergoes a major conformational change. At the pointed end, however, protomers have the "twisted" conformation characteristic of G-actin, whereas tropomodulin binding forces a flat conformation upon the second subunit. The structures provide a mechanistic understanding for the asymmetric addition/dissociation of actin subunits at the ends of F-actin and open the way to future studies of other regulators of filament end dynamics.

Structures of the free and capped ends of the actin filament.

The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange, including CapZ at the barbed end and tropomodulin at the pointed end. We describe cryo-electron microscopy structures of the free and capped ends of F-actin. Terminal subunits at the free barbed end adopt a "flat" F-actin conformation. CapZ binds with minor changes to the barbed end but with major changes to itself. By contrast, subunits at the free pointed end adopt a "twisted" monomeric actin (G-actin) conformation. Tropomodulin binding forces the second subunit into an F-actin conformation. The structures reveal how the ends differ from the middle in F-actin and how these differences control subunit addition, dissociation, capping, and interactions with end-binding proteins.

Mechanism of synergistic activation of Arp2/3 complex by cortactin and WASP-family proteins.

Cortactin coactivates Arp2/3 complex synergistically with WASP-family nucleation-promoting factors (NPFs) and stabilizes branched networks by linking Arp2/3 complex to F-actin. It is poorly understood how cortactin performs these functions. We describe the 2.89 Å resolution cryo-EM structure of cortactin's N-terminal domain (Cort1-76) bound to Arp2/3 complex. Cortactin binds Arp2/3 complex through an inverted Acidic domain (D20-V29), which targets the same site on Arp3 as the Acidic domain of NPFs but with opposite polarity. Sequences N- and C-terminal to cortactin's Acidic domain do not increase its affinity for Arp2/3 complex but contribute toward coactivation with NPFs. Coactivation further increases with NPF dimerization and for longer cortactin constructs with stronger binding to F-actin. The results suggest that cortactin contributes to Arp2/3 complex coactivation with NPFs in two ways, by helping recruit the complex to F-actin and by stabilizing the short-pitch (active) conformation, which are both byproducts of cortactin's core function in branch stabilization.

Novel Protein Production Method Combining Native Expression in Human Cells with an Intein-based Affinity Purification and Self-cleavable Tag.

The human proteins used in most biochemical studies are commonly obtained using bacterial expression. Owing to its relative simplicity and low cost, this approach has been extremely successful, but is inadequate for many proteins that require the mammalian folding machinery and posttranslational modifications (PTMs) for function. Moreover, the expressed proteins are typically purified using N- and/or C-terminal affinity tags, which are often left on proteins or leave non-native extra amino acids when removed proteolytically. Many proteins cannot tolerate such extra amino acids for function. Here we describe a protein production method that resolves both these issues. Our method combines expression in human Expi293F cells, which grow in suspension to high density and can process native PTMs, with a chitin-binding domain (CBD)-intein affinity purification and self-cleavable tag, which can be precisely removed after purification. In this protocol, we describe how to clone a target gene into our specifically designed human cell expression vector (pJCX4), and how to efficiently transfect the Expi293F cells and purify the expressed proteins using a chitin affinity resin. Graphic abstract.

Sequential dynein effectors regulate axonal autophagosome motility in a maturation-dependent pathway.

Autophagy is a degradative pathway required to maintain homeostasis. Neuronal autophagosomes form constitutively at the axon terminal and mature via lysosomal fusion during dynein-mediated transport to the soma. How the dynein-autophagosome interaction is regulated is unknown. Here, we identify multiple dynein effectors on autophagosomes as they transit along the axons of primary neurons. In the distal axon, JIP1 initiates autophagosomal transport. Autophagosomes in the mid-axon require HAP1 and Huntingtin. We find that HAP1 is a dynein activator, binding the dynein-dynactin complex via canonical and noncanonical interactions. JIP3 is on most axonal autophagosomes, but specifically regulates the transport of mature autolysosomes. Inhibiting autophagosomal transport disrupts maturation, and inhibiting autophagosomal maturation perturbs the association and function of dynein effectors; thus, maturation and transport are tightly linked. These results reveal a novel maturation-based dynein effector handoff on neuronal autophagosomes that is key to motility, cargo degradation, and the maintenance of axonal health.

Structural insights into assembly and function of the RSC chromatin remodeling complex.

SWI/SNF chromatin remodelers modify the position and spacing of nucleosomes and, in humans, are linked to cancer. To provide insights into the assembly and regulation of this protein family, we focused on a subcomplex of the Saccharomyces cerevisiae RSC comprising its ATPase (Sth1), the essential actin-related proteins (ARPs) Arp7 and Arp9 and the ARP-binding protein Rtt102. Cryo-EM and biochemical analyses of this subcomplex shows that ARP binding induces a helical conformation in the helicase-SANT-associated (HSA) domain of Sth1. Surprisingly, the ARP module is rotated 120° relative to the full RSC about a pivot point previously identified as a regulatory hub in Sth1, suggesting that large conformational changes are part of Sth1 regulation and RSC assembly. We also show that a conserved interaction between Sth1 and the nucleosome acidic patch enhances remodeling. As some cancer-associated mutations dysregulate rather than inactivate SWI/SNF remodelers, our insights into RSC complex regulation advance a mechanistic understanding of chromatin remodeling in disease states.

Tropomodulins Control the Balance between Protrusive and Contractile Structures by Stabilizing Actin-Tropomyosin Filaments.

Eukaryotic cells have diverse protrusive and contractile actin filament structures, which compete with one another for a limited pool of actin monomers. Numerous actin-binding proteins regulate the dynamics of actin structures, including tropomodulins (Tmods), which cap the pointed end of actin filaments. In striated muscles, Tmods prevent actin filaments from overgrowing, whereas in non-muscle cells, their function has remained elusive. Here, we identify two Tmod isoforms, Tmod1 and Tmod3, as key components of contractile stress fibers in non-muscle cells. Individually, Tmod1 and Tmod3 can compensate for one another, but their simultaneous depletion results in disassembly of actin-tropomyosin filaments, loss of force-generating stress fibers, and severe defects in cell morphology. Knockout-rescue experiments reveal that Tmod's interaction with tropomyosin is essential for its role in the stabilization of actin-tropomyosin filaments in cells. Thus, in contrast to their role in muscle myofibrils, in non-muscle cells, Tmods bind actin-tropomyosin filaments to protect them from depolymerizing, not elongating. Furthermore, loss of Tmods shifts the balance from linear actin-tropomyosin filaments to Arp2/3 complex-nucleated branched networks, and this phenotype can be partially rescued by inhibiting the Arp2/3 complex. Collectively, the data reveal that Tmods are essential for the maintenance of contractile actomyosin bundles and that Tmod-dependent capping of actin-tropomyosin filaments is critical for the regulation of actin homeostasis in non-muscle cells.

Mining Disaggregase Sequence Space to Safely Counter TDP-43, FUS, and α-Synuclein Proteotoxicity.

Hsp104 is an AAA+ protein disaggregase, which can be potentiated via diverse mutations in its autoregulatory middle domain (MD) to mitigate toxic misfolding of TDP-43, FUS, and α-synuclein implicated in fatal neurodegenerative disorders. Problematically, potentiated MD variants can exhibit off-target toxicity. Here, we mine disaggregase sequence space to safely enhance Hsp104 activity via single mutations in nucleotide-binding domain 1 (NBD1) or NBD2. Like MD variants, NBD variants counter TDP-43, FUS, and α-synuclein toxicity and exhibit elevated ATPase and disaggregase activity. Unlike MD variants, non-toxic NBD1 and NBD2 variants emerge that rescue TDP-43, FUS, and α-synuclein toxicity. Potentiating substitutions alter NBD1 residues that contact ATP, ATP-binding residues, or the MD. Mutating the NBD2 protomer interface can also safely ameliorate Hsp104. Thus, we disambiguate allosteric regulation of Hsp104 by several tunable structural contacts, which can be engineered to spawn enhanced therapeutic disaggregases with minimal off-target toxicity.

BAR domain proteins-a linkage between cellular membranes, signaling pathways, and the actin cytoskeleton.

Actin filament assembly typically occurs in association with cellular membranes. A large number of proteins sit at the interface between actin networks and membranes, playing diverse roles such as initiation of actin polymerization, modulation of membrane curvature, and signaling. Bin/Amphiphysin/Rvs (BAR) domain proteins have been implicated in all of these functions. The BAR domain family of proteins comprises a diverse group of multi-functional effectors, characterized by their modular architecture. In addition to the membrane-curvature sensing/inducing BAR domain module, which also mediates antiparallel dimerization, most contain auxiliary domains implicated in protein-protein and/or protein-membrane interactions, including SH3, PX, PH, RhoGEF, and RhoGAP domains. The shape of the BAR domain itself varies, resulting in three major subfamilies: the classical crescent-shaped BAR, the more extended and less curved F-BAR, and the inverse curvature I-BAR subfamilies. Most members of this family have been implicated in cellular functions that require dynamic remodeling of the actin cytoskeleton, such as endocytosis, organelle trafficking, cell motility, and T-tubule biogenesis in muscle cells. Here, we review the structure and function of mammalian BAR domain proteins and the many ways in which they are interconnected with the actin cytoskeleton.