LILAC: enhanced actin imaging with an optogenetic Lifeact.

Lifeact is a popular peptide-based label of actin filaments in live cells. We have designed an improved Lifeact variant, LILAC, that binds to actin in light using the LOV2 protein. Light control allows the user to modulate actin labeling, enabling image analysis that leverages modulation for an enhanced view of F-actin dynamics in cells. Furthermore, the tool reduces actin perturbations and cell sickness caused by Lifeact overexpression.

4-Hydroxyacetophenone modulates the actomyosin cytoskeleton to reduce metastasis.

Metastases are the cause of the vast majority of cancer deaths. In the metastatic process, cells migrate to the vasculature, intravasate, extravasate, and establish metastatic colonies. This pattern of spread requires the cancer cells to change shape and to navigate tissue barriers. Approaches that block this mechanical program represent new therapeutic avenues. We show that 4-hydroxyacetophenone (4-HAP) inhibits colon cancer cell adhesion, invasion, and migration in vitro and reduces the metastatic burden in an in vivo model of colon cancer metastasis to the liver. Treatment with 4-HAP activates nonmuscle myosin-2C (NM2C) (MYH14) to alter actin organization, inhibiting the mechanical program of metastasis. We identify NM2C as a specific therapeutic target. Pharmacological control of myosin isoforms is a promising approach to address metastatic disease, one that may be readily combined with other therapeutic strategies.

Snapshots of a molecular swivel in action.

Members of the serine family of site-specific recombinases exchange DNA strands via 180° rotation about a central protein-protein interface. Modeling of this process has been hampered by the lack of structures in more than one rotational state for any individual serine recombinase. Here we report crystal structures of the catalytic domains of four constitutively active mutants of the serine recombinase Sin, providing snapshots of rotational states not previously visualized for Sin, including two seen in the same crystal. Normal mode analysis predicted that each tetramer's lowest frequency mode (i.e. most accessible large-scale motion) mimics rotation: two protomers rotate as a pair with respect to the other two. Our analyses also suggest that rotation is not a rigid body movement around a single symmetry axis but instead uses multiple pivot points and entails internal motions within each subunit.

How Actin Tracks Affect Myosin Motors.

Cellular organization through cytoskeletal trafficking is a process of fundamental importance. Highly specialized systems evolved that enable motors to identify and select the optimal tracks for motility. In this chapter, we examine the profound effect of actin filament networks on myosin motility patterns. We argue that the myosin classes have adaptations that allow them to detect local structural and chemical cues on actin. These cues are often arranged in a coherent manner on actin filament networks, allowing for directed transport over long distances. We identify a number of potentially important cues, ranging from the biochemical states of actin subunits all the way to multi-filament networks and bundles.

Investigations of human myosin VI targeting using optogenetically controlled cargo loading.

Myosins play countless critical roles in the cell, each requiring it to be activated at a specific location and time. To control myosin VI with this specificity, we created an optogenetic tool for activating myosin VI by fusing the light-sensitive Avena sativa phototropin1 LOV2 domain to a peptide from Dab2 (LOVDab), a myosin VI cargo protein. Our approach harnesses the native targeting and activation mechanism of myosin VI, allowing direct inferences on myosin VI function. LOVDab robustly recruits human full-length myosin VI to various organelles in vivo and hinders peroxisome motion in a light-controllable manner. LOVDab also activates myosin VI in an in vitro gliding filament assay. Our data suggest that protein and lipid cargoes cooperate to activate myosin VI, allowing myosin VI to integrate Ca2+, lipid, and protein cargo signals in the cell to deploy in a site-specific manner.

Pharmacological activation of myosin II paralogs to correct cell mechanics defects.

Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior.

Competition between Coiled-Coil Structures and the Impact on Myosin-10 Bundle Selection.

Coiled-coil fusions are a useful approach to enforce dimerization in protein engineering. However, the final structures of coiled-coil fusion proteins have received relatively little attention. Here, we determine the structural outcome of adjacent parallel and antiparallel coiled coils. The targets are coiled coils that stabilize myosin-10 in single-molecule biophysical studies. We reveal the solution structure of a short, antiparallel, myosin-10 coiled-coil fused to the parallel GCN4-p1 coiled coil. Surprisingly, this structure is a continuous, antiparallel coiled coil where GCN4-p1 pairs with myosin-10 rather than itself. We also show that longer myosin-10 segments in these parallel/antiparallel fusions are dynamic and do not fold cooperatively. Our data resolve conflicting results on myosin-10 selection of actin filament bundles, demonstrating the importance of understanding coiled-coil orientation and stability.

Myosin light chain kinase steady-state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates.

Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat /Km ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. SIGNIFICANCE OF THE STUDY: Phosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.

The development and application of a quantitative peptide microarray based approach to protein interaction domain specificity space.

Protein interaction domain (PID) linear peptide motif interactions direct diverse cellular processes in a specific and coordinated fashion. PID specificity, or the interaction selectivity derived from affinity preferences between possible PID-peptide pairs is the basis of this ability. Here, we develop an integrated experimental and computational cellulose peptide conjugate microarray (CPCMA) based approach for the high throughput analysis of PID specificity that provides unprecedented quantitative resolution and reproducibility. As a test system, we quantify the specificity preferences of four Src Homology 2 domains and 124 physiological phosphopeptides to produce a novel quantitative interactome. The quantitative data set covers a broad affinity range, is highly precise, and agrees well with orthogonal biophysical validation, in vivo interactions, and peptide library trained algorithm predictions. In contrast to preceding approaches, the CPCMAs proved capable of confidently assigning interactions into affinity categories, resolving the subtle affinity contributions of residue correlations, and yielded predictive peptide motif affinity matrices. Unique CPCMA enabled modes of systems level analysis reveal a physiological interactome with expected node degree value decreasing as a function of affinity, resulting in minimal high affinity binding overlap between domains; uncover that Src Homology 2 domains bind ligands with a similar average affinity yet strikingly different levels of promiscuity and binding dynamic range; and parse with unprecedented quantitative resolution contextual factors directing specificity. The CPCMA platform promises broad application within the fields of PID specificity, synthetic biology, specificity focused drug design, and network biology.

Pushed to the edge: hundreds of Myosin 10s pack into filopodia and could cause traffic jams on actin.

Myosin 10 (Myo10) is a vertebrate-specific motor protein well known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the numbers of Myo10 in filopodia. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. Here we combined SDS-PAGE analysis with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it tends to be enriched at opposite ends of the cell. Hundreds of Myo10 are found in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane deformations in addition to the numbers of Myo10 required for filopodia initiation. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation.

Actin filament bundling by fimbrin is important for endocytosis, cytokinesis, and polarization in fission yeast.

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.

HDAC3-dependent reversible lysine acetylation of cardiac myosin heavy chain isoforms modulates their enzymatic and motor activity.

Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, PCAF, associate with cardiac sarcomeres, and a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study, we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to the A band of sarcomeres and was capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and ß-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the K(m) for the actin-activated ATPase activity of both α- and ß-MHC isoforms. By an in vitro motility assay, we found that lysine acetylation increased the actin sliding velocity of α-myosin by 20% and ß-myosin by 36%, compared to their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli, independent of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.

Unconventional myosin traffic in cells reveals a selective actin cytoskeleton.

Eukaryotic cells have a self-organizing cytoskeleton where motors transport cargoes along cytoskeletal tracks. To understand the sorting process, we developed a system to observe single-molecule motility in a cellular context. We followed myosin classes V, VI, and X on triton-extracted actin cytoskeletons from Drosophila S2, mammalian COS-7, and mammalian U2OS cells. We find that these cells vary considerably in their global traffic patterns. The S2 and U2OS cells have regions of actin that either enhance or inhibit specific myosin classes. U2OS cells allow for 1 motor class, myosin VI, to move along stress fiber bundles, while motility of myosin V and X are suppressed. Myosin X motors are recruited to filopodia and the lamellar edge in S2 cells, whereas myosin VI motility is excluded from the same regions. Furthermore, we also see different velocities of myosin V motors in central regions of S2 cells, suggesting regional control of motor motility by the actin cytoskeleton. We also find unexpected features of the actin cytoskeletal network, including a population of reversed filaments with the barbed-end toward the cell center. This myosin motor regulation demonstrates that native actin cytoskeletons are more than just a collection of filaments.

An engineered substance P variant for receptor-mediated delivery of synthetic antibodies into tumor cells.

We have developed and tested a robust delivery method for the transport of proteins to the cytoplasm of mammalian cells without compromising the integrity of the cell membrane. This receptor-mediated delivery (RMD) technology utilizes a variant of substance P (SP), a neuropeptide that is rapidly internalized upon interaction with the neurokinin-1 receptor (NK1R). Cargos in the form of synthetic antibody fragments (sABs) were conjugated to the engineered SP variant (SPv) and efficiently internalized by NK1R-expressing cells. The sABs used here were generated to bind specific conformational forms of actin. The internalized proteins appear to escape the endosome and retain their binding activity within the cells as demonstrated by co-localization with the actin cytoskeleton. Further, since the NK1R is over-expressed in many cancers, SPv-mediated delivery provides a highly specific method for therapeutic utilization of affinity reagents targeting intracellular processes in diseased tissue.

Structured post-IQ domain governs selectivity of myosin X for fascin-actin bundles.

Without guidance cues, cytoskeletal motors would traffic components to the wrong destination with disastrous consequences for the cell. Recently, we identified a motor protein, myosin X, that identifies bundled actin filaments for transport. These bundles direct myosin X to a unique destination, the tips of cellular filopodia. Because the structural and kinetic features that drive bundle selection are unknown, we employed a domain-swapping approach with the nonselective myosin V to identify the selectivity module of myosin X. We found a surprising role of the myosin X tail region (post-IQ) in supporting long runs on bundles. Moreover, the myosin X head is adapted for initiating processive runs on bundles. We found that the tail is structured and biases the orientation of the two myosin X heads because a targeted insertion that introduces flexibility in the tail abolishes selectivity. Together, these results suggest how myosin motors may manage to read cellular addresses.

Actin cross-link assembly and disassembly mechanics for alpha-Actinin and fascin.

Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and alpha-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. alpha-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo.

Unconventional processive mechanics of non-muscle myosin IIB.

Proper tension maintenance in the cytoskeleton is essential for regulated cell polarity, cell motility, and division. Non-muscle myosin IIB (NMIIB) generates tension along actin filaments in many cell types, including neuronal, cardiac, and smooth muscle cells. Using a three-bead optical trapping assay, we recorded NMIIB interactions with actin filaments to determine if a NMIIB dimer cycles along an actin filament in a processive manner. Our results show that NMIIB is the first myosin II to exhibit evidence of processive stepping behavior. Analysis of these data reveals a forward displacement of 5.4 nm and, surprisingly, frequent backward steps of -5.9 nm. Processive stepping along the long pitch helix of actin may provide a mechanism for disassembly of fascin-actin bundles. Forward steps and detachment are weakly force-dependent at all forces, consistent with rate-limiting and force-dependent ADP release. However, backward steps are nearly force-independent. Our data support a model in which NMIIB can readily move in both directions at stall, which may be important for a general regulator of cytoskeleton tension.

The dual mode of action of bistramide A entails severing of filamentous actin and covalent protein modification.

This study provides comprehensive characterization of the mode of action of bistramide A and identifies structural requirements of bistramide-based compounds that are responsible for severing actin filaments and inhibiting growth of cancer cells in vitro and in vivo. We rationally designed and assembled a series of structural analogs of the natural product, including a fluorescently labeled conjugate. We used TIRF microscopy to directly observe actin filament severing by this series of small molecules, which established that the combination of the spiroketal and the amide subunits was sufficient to enable rapid actin filament disassembly in vitro. In addition, we demonstrated that the enone subunit of bistramide A is responsible for covalent modification of the protein in vitro and in A549 cells, resulting in further increase in the cytotoxicity of the natural product. Our results demonstrate that bistramide A elicits its potent antiproliferative activity by a dual mechanism of action, which entails both severing of actin filaments and covalent sequestration of monomeric actin in the cell.

A myosin motor that selects bundled actin for motility.

Eukaryotic cells organize their contents through trafficking along cytoskeletal filaments. The leading edge of a typical metazoan cytoskeleton consists of a dense and complex arrangement of cortical actin. A dendritic mesh is found across the broad lamellopodium, with long parallel bundles at microspikes and filopodia. It is currently unclear whether and how myosin motors identify the few actin filaments that lead to the correct destination, when presented with many similar alternatives within the cortex. Here we show that myosin X, an actin-based motor that concentrates at the distal tips of filopodia, selects the fascin-actin bundle at the filopodial core for motility. Myosin X moves individual actin filaments poorly in vitro, often supercoiling actin into plectonemes. However, single myosin X motors move robustly and processively along fascin-actin bundles. This selection requires only parallel, closely spaced filaments, as myosin X is also processive on artificial actin bundles formed by molecular crowding. Myosin X filopodial localization is perturbed in fascin-depleted HeLa cells, demonstrating that fascin bundles also direct motility in vivo. Our results demonstrate that myosin X recognizes the local structural arrangement of filaments in long bundles, providing a mechanism for sorting cargo to distant target sites.

The stepping pattern of myosin X is adapted for processive motility on bundled actin.

Myosin X is a molecular motor that is adapted to select bundled actin filaments over single actin filaments for processive motility. Its unique form of motility suggests that myosin X's stepping mechanism takes advantage of the arrangement of actin filaments and the additional target binding sites found within a bundle. Here we use fluorescence imaging with one-nanometer accuracy to show that myosin X takes steps of ∼18 nm along a fascin-actin bundle. This step-size is well short of the 36-nm step-size observed in myosin V and myosin VI that corresponds to the actin pseudohelical repeat distance. Myosin X is able to walk along bundles with this step-size if it straddles two actin filaments, but would be quickly forced to spiral into the constrained interior of the bundle if it were to use only a single actin filament. We also demonstrate that myosin X takes many sideways steps as it walks along a bundle, suggesting that it can switch actin filament pairs within the bundle as it walks. Sideways steps to the left or the right occur on bundles with equal frequency, suggesting a degree of lateral flexibility such that the motor's working stroke does not bias it to the left or to the right. On single actin filaments, we find a broad mixture of 10-20-nm steps, which again falls short of the 36-nm actin repeat. Moreover, the motor leans to the right as it walks along single filaments, which may require myosin X to adopt strained configurations. As a control, we also tracked myosin V stepping along actin filaments and fascin-actin bundles. We find that myosin V follows a narrower path on both structures, walking primarily along one surface of an actin filament and following a single filament within a bundle while occasionally switching to neighboring filaments. Together, these results delineate some of the structural features of the motor and the track that allow myosin X to recognize actin filament bundles.