The cryo-EM 3D image reconstruction of isolated Lethocerus indicus Z-discs.

The Z-disk of striated muscle defines the ends of the sarcomere, which repeats many times within the muscle fiber. Here we report application of cryoelectron tomography and subtomogram averaging to Z-disks isolated from the flight muscles of the large waterbug Lethocerus indicus. We use high salt solutions to remove the myosin containing filaments and use gelsolin to remove the actin filaments of the A- and I-bands leaving only the thin filaments within the Z-disk which were then frozen for cryoelectron microscopy. The Lethocerus Z-disk structure is similar in many ways to the previously studied Z-disk of the honeybee Apis mellifera. At the corners of the unit cell are positioned trimers of paired antiparallel F-actins defining a large solvent channel, whereas at the trigonal positions are positioned F-actin trimers converging slowly towards their (+) ends defining a small solvent channel through the Z-disk. These near parallel F-actins terminate at different Z-heights within the Z-disk. The two types of solvent channel in Lethocerus are similar in size compared to those of Apis which are very different in size. Two types of α-actinin crosslinks were observed between oppositely oriented actin filaments. In one of these, the α-actinin long axis is almost parallel to the F-actins it crosslinks. In the other, the α-actinins are at a small but distinctive angle with respect to the crosslinked actin filaments. The utility of isolated Z-disks for structure determination is discussed.

Double-headed binding of myosin II to F-actin shows the effect of strain on head structure.

Force production in muscle is achieved through the interaction of myosin and actin. Strong binding states in active muscle are associated with Mg·ADP bound to the active site; release of Mg·ADP allows rebinding of ATP and dissociation from actin. Thus, Mg·ADP binding is positioned for adaptation as a force sensor. Mechanical loads on the lever arm can affect the ability of myosin to release Mg·ADP but exactly how this is done is poorly defined. Here we use F-actin decorated with double-headed smooth muscle myosin fragments in the presence of Mg·ADP to visualize the effect of internally supplied tension on the paired lever arms using cryoEM. The interaction of the paired heads with two adjacent actin subunits is predicted to place one lever arm under positive and the other under negative strain. The converter domain is believed to be the most flexible domain within myosin head. Our results, instead, point to the segment of heavy chain between the essential and regulatory light chains as the location of the largest structural change. Moreover, our results suggest no large changes in the myosin coiled coil tail as the locus of strain relief when both heads bind F-actin. The method would be adaptable to double-headed members of the myosin family. We anticipate that the study of actin-myosin interaction using double-headed fragments enables visualization of domains that are typically noisy in decoration with single-headed fragments.

Taxonomic Delineation of the Old World Species Stomphastis thraustica (Lepidoptera: Gracillariidae) Feeding on Jatropha gossypiifolia (Euphorbiaceae) that Was Collected in the New World and Imported as a Biocontrol Agent to Australia.

We provide the identification and species delineation of this biocontrol agent as Stomphastis thraustica (Meyrick in Trans Ent Soc Lond 80(1):107-120, 1908) belonging to the family Gracillariidae. We clarify the distribution pattern of S. thraustica, its host plant preferences, and present taxonomic and molecular diagnoses based on original morphological and genetic data as well as data retrieved from historic literature and genetic databases. Following our own collecting efforts in three continents Africa, South America, and Australia as well as our study of historic museum collection material, we present many new distribution records of S. thraustica for countries and territories in the world including the new discovery of this species in the Neotropical region and we report its introduction in Australia as a biocontrol agent. Using mitogenomic and COI gene data, we clarified that the closest relative of S. thraustica is Stomphastis sp. that occurs in Madagascar and Australia and feeds on the same host plant as S. thraustica - Jatropha gossypiifolia L. (Euphorbiaceae). The molecular sequence divergence in the mitochondrial DNA barcode fragment between these two closely related species S. thraustica and Stomphastis sp. is over 5.7% supporting that they are different species.

Exploration of RNA-Seq data to identify a potential pathogen of the leaf-mining moth, Stomphastis thraustica (Meyrick, 1908) (Lepidoptera: Gracillariidae).

The leaf-mining moth, Stomphastis thraustica (Meyrick, 1908) was imported to Australia as a potential biological control agent of an exotic weed, bellyache bush (Jatropha gossypiifolia), from Peru. The insect colony has been maintained in the quarantine facility for over eight years but recently, significant mortality was observed in the culture. The larvae demonstrated swollen intersegments with a fragile integument. The infected larvae are cloudy muted green or yellowish whereas a healthy late instar larva is a vivid green. They slowly dehydrate and eventually die, at which point the larval body becomes rubbery and turns to black. We used next generation sequencing to identify the cause of mortality in the insects. Total RNA was extracted from 20 larvae in two cohorts, one with and one without apparent symptoms of disease, for deep sequencing on NovaSeq platform after eukaryote ribosomal RNA depletion. We identified several non-insect sequences belonging to viruses, bacteria, and fungi, but none of those showed significant abundance or enrichment in the infected dataset. The sequences related to a unicellular yeast, Saccharomyces cerevisiae, and they were among the highly expressed non-insect contigs; more than 5% of reads in both libraries mapped to the genome of this opportunistic microorganism.

Structure of the Flight Muscle Thick Filament from the Bumble Bee, Bombus ignitus, at 6 Å Resolution.

Four insect orders have flight muscles that are both asynchronous and indirect; they are asynchronous in that the wingbeat frequency is decoupled from the frequency of nervous stimulation and indirect in that the muscles attach to the thoracic exoskeleton instead of directly to the wing. Flight muscle thick filaments from two orders, Hemiptera and Diptera, have been imaged at a subnanometer resolution, both of which revealed a myosin tail arrangement referred to as "curved molecular crystalline layers". Here, we report a thick filament structure from the indirect flight muscles of a third insect order, Hymenoptera, the Asian bumble bee Bombus ignitus. The myosin tails are in general agreement with previous determinations from Lethocerus indicus and Drosophila melanogaster. The Skip 2 region has the same unusual structure as found in Lethocerus indicus thick filaments, an α-helix discontinuity is also seen at Skip 4, but the orientation of the Skip 1 region on the surface of the backbone is less angled with respect to the filament axis than in the other two species. The heads are disordered as in Drosophila, but we observe no non-myosin proteins on the backbone surface that might prohibit the ordering of myosin heads onto the thick filament backbone. There are strong structural similarities among the three species in their non-myosin proteins within the backbone that suggest how one previously unassigned density in Lethocerus might be assigned. Overall, the structure conforms to the previously observed pattern of high similarity in the myosin tail arrangement, but differences in the non-myosin proteins.

Tau induces formation of α-synuclein filaments with distinct molecular conformations.

Recent structural investigation of amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations, which are different from those of in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo α-synuclein filaments extracted from multiple system atrophy patients adopt distinct molecular structures from those of in vitro α-synuclein filaments, suggesting the presence of co-factors for α-synuclein aggregation in vivo. Here, we report structural characterizations of α-synuclein filaments formed in the presence of a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted α-synuclein filaments reveals some similarities to one of the previously reported polymorphs of in vitro α-synuclein filaments in the core region, while illustrating distinct conformations in the N- and C-terminal regions. The structural study highlights the conformational plasticity of α-synuclein filaments and the importance of the co-factors, requiring additional structural investigation of not only more ex vivo α-synuclein filaments, but also in vitro α-synuclein filaments formed in the presence of diverse co-factors. The comparative structural analyses will help better understand molecular basis of diverse structures of α-synuclein filaments and possible relevance of each structure to the disease phenotype.

The myosin II coiled-coil domain atomic structure in its native environment.

The atomic structure of the complete myosin tail within thick filaments isolated from Lethocerus indicus flight muscle is described and compared to crystal structures of recombinant, human cardiac myosin tail segments. Overall, the agreement is good with three exceptions: the proximal S2, in which the filament has heads attached but the crystal structure doesn't, and skip regions 2 and 4. At the head-tail junction, the tail α-helices are asymmetrically structured encompassing well-defined unfolding of 12 residues for one myosin tail, ∼4 residues of the other, and different degrees of α-helix unwinding for both tail α-helices, thereby providing an atomic resolution description of coiled-coil "uncoiling" at the head-tail junction. Asymmetry is observed in the nonhelical C termini; one C-terminal segment is intercalated between ribbons of myosin tails, the other apparently terminating at Skip 4 of another myosin tail. Between skip residues, crystal and filament structures agree well. Skips 1 and 3 also agree well and show the expected α-helix unwinding and coiled-coil untwisting in response to skip residue insertion. Skips 2 and 4 are different. Skip 2 is accommodated in an unusual manner through an increase in α-helix radius and corresponding reduction in rise/residue. Skip 4 remains helical in one chain, with the other chain unfolded, apparently influenced by the acidic myosin C terminus. The atomic model may shed some light on thick filament mechanosensing and is a step in understanding the complex roles that thick filaments of all species undergo during muscle contraction.

CryoEM structure of Drosophila flight muscle thick filaments at 7 Å resolution.

Striated muscle thick filaments are composed of myosin II and several non-myosin proteins. Myosin II's long α-helical coiled-coil tail forms the dense protein backbone of filaments, whereas its N-terminal globular head containing the catalytic and actin-binding activities extends outward from the backbone. Here, we report the structure of thick filaments of the flight muscle of the fruit fly Drosophila melanogaster at 7 Å resolution. Its myosin tails are arranged in curved molecular crystalline layers identical to flight muscles of the giant water bug Lethocerus indicus Four non-myosin densities are observed, three of which correspond to ones found in Lethocerus; one new density, possibly stretchin-mlck, is found on the backbone outer surface. Surprisingly, the myosin heads are disordered rather than ordered along the filament backbone. Our results show striking myosin tail similarity within flight muscle filaments of two insect orders separated by several hundred million years of evolution.

Identification of interfaces involved in weak interactions with application to F-actin-aldolase rafts.

Macromolecular interactions occur with widely varying affinities. Strong interactions form well defined interfaces but weak interactions are more dynamic and variable. Weak interactions can collectively lead to large structures such as microvilli via cooperativity and are often the precursors of much stronger interactions, e.g. the initial actin-myosin interaction during muscle contraction. Electron tomography combined with subvolume alignment and classification is an ideal method for the study of weak interactions because a 3-D image is obtained for the individual interactions, which subsequently are characterized collectively. Here we describe a method to characterize heterogeneous F-actin-aldolase interactions in 2-D rafts using electron tomography. By forming separate averages of the two constituents and fitting an atomic structure to each average, together with the alignment information which relates the raw motif to the average, an atomic model of each crosslink is determined and a frequency map of contact residues is computed. The approach should be applicable to any large structure composed of constituents that interact weakly and heterogeneously.

Coupling between myosin head conformation and the thick filament backbone structure.

The recent high-resolution structure of the thick filament from Lethocerus asynchronous flight muscle shows aspects of thick filament structure never before revealed that may shed some light on how striated muscles function. The phenomenon of stretch activation underlies the function of asynchronous flight muscle. It is most highly developed in flight muscle, but is also observed in other striated muscles such as cardiac muscle. Although stretch activation is likely to be complex, involving more than a single structural aspect of striated muscle, the thick filament itself, would be a prime site for regulatory function because it must bear all of the tension produced by both its associated myosin motors and any externally applied force. Here we show the first structural evidence that the arrangement of myosin heads within the interacting heads motif is coupled to the structure of the thick filament backbone. We find that a change in helical angle of 0.16° disorders the blocked head preferentially within the Lethocerus interacting heads motif. This observation suggests a mechanism for how tension affects the dynamics of the myosin heads leading to a detailed hypothesis for stretch activation and shortening deactivation, in which the blocked head preferentially binds the thin filament followed by the free head when force production occurs.

The structure of the actin-smooth muscle myosin motor domain complex in the rigor state.

Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography postulated that higher actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM structures of F-actin bound myosin-I, -II and -V have provided details of this model. The smooth muscle myosin II interaction with F-actin may differ from those for striated and non-muscle myosin II due in part to different lengths of important surface loops. Here we report a ∼6 Šresolution reconstruction of F-actin decorated with the nucleotide-free recombinant smooth muscle myosin-II motor domain (MD) from images recorded using a direct electron detector. Resolution is highest for F-actin and the actin-myosin interface (3.5-4 Å) and lowest (∼6-7 Å) for those parts of the MD at the highest radius. Atomic models built into the F-actin density are quite comparable to those previously reported for rabbit muscle actin and show density from the bound ADP. The atomic model of the MD, is quite similar to a recently published structure of vertebrate non-muscle myosin II bound to F-actin and a crystal structure of nucleotide free myosin-V. Larger differences are observed when compared to the cryoEM structure of F-actin decorated with rabbit skeletal muscle myosin subfragment 1. The differences suggest less closure of the 50 kDa domain in the actin bound skeletal muscle myosin structure.

Structure of myosin filaments from relaxed Lethocerus flight muscle by cryo-EM at 6 Å resolution.

We describe a cryo-electron microscopy three-dimensional image reconstruction of relaxed myosin II-containing thick filaments from the flight muscle of the giant water bug Lethocerus indicus. The relaxed thick filament structure is a key element of muscle physiology because it facilitates the reextension process following contraction. Conversely, the myosin heads must disrupt their relaxed arrangement to drive contraction. Previous models predicted that Lethocerus myosin was unique in having an intermolecular head-head interaction, as opposed to the intramolecular head-head interaction observed in all other species. In contrast to the predicted model, we find an intramolecular head-head interaction, which is similar to that of other thick filaments but oriented in a distinctly different way. The arrangement of myosin's long α-helical coiled-coil rod domain has been hypothesized as either curved layers or helical subfilaments. Our reconstruction is the first report having sufficient resolution to track the rod α helices in their native environment at resolutions ~5.5 Å, and it shows that the layer arrangement is correct for Lethocerus. Threading separate paths through the forest of myosin coiled coils are four nonmyosin peptides. We suggest that the unusual position of the heads and the rod arrangement separated by nonmyosin peptides are adaptations for mechanical signal transduction whereby applied tension disrupts the myosin heads as a component of stretch activation.

The Structure of a Full-length Membrane-embedded Integrin Bound to a Physiological Ligand.

Increased ligand binding to integrin ("activation") underpins many biological processes, such as leukocyte trafficking, cell migration, host-pathogen interaction, and hemostasis. Integrins exist in several conformations, ranging from compact and bent to extended and open. However, the exact conformation of membrane-embedded, full-length integrin bound to its physiological macromolecular ligand is still unclear. Integrin αIIbß3, the most abundant integrin in platelets, has been a prototype for integrin activation studies. Using negative stain electron microscopy and nanodisc-embedding to provide a membrane-like environment, we visualized the conformation of full-length αIIbß3 in both a Mn(2+)-activated, ligand-free state and a Mn(2+)-activated, fibrin-bound state. Activated but ligand-free integrins exist mainly in the compact conformation, whereas fibrin-bound αIIbß3 predominantly exists in a fully extended, headpiece open conformation. Our results show that membrane-embedded, full-length integrin adopts an extended and open conformation when bound to its physiological macromolecular ligand.

Psychometric assessment of the Correa-Barrick Postpartum Depression Scale.

INTRODUCTION: Empirical evidence has suggested an association between mood and color sensitivity. The purpose of this study is to report on the psychometric properties of a new postpartum depression rating scale while at the same time showing a correlation between depression severity and impaired color sensitivity. METHODS: Using a survey design of a sample of 17 postpartum patients, color sensitivity was assessed by the patient's response to a self-report depression scale item, "I notice that everything seems gray/cloudy/drab/lacking color". RESULTS: There was a statistically significant finding between color sensitivity and depression, r=.50, P<.05. Based on classical test theory, findings also supported the validity and reliability of the new scale. LIMITATIONS: The limitation of the study was the small sample size, but it also suggests that the findings seem particularly robust in view of this. CONCLUSIONS: This scale could be used in clinical practice to detect early signs of depression for prompt intervention. Referral should be made to mental health providers for follow-up to insure that the mother is not psychotic or so depressed that she cannot care for the baby. Color sensitivity impairment and depression severity has been replicated several times with different samples. It is worthwhile pursuing the pathophysiological basis for this.

Phosphorylated smooth muscle heavy meromyosin shows an open conformation linked to activation.

Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions between motor domains. The "free head" can bind to actin, but the actin binding interface of the "blocked head" is involved in interactions with the free head. We report here a three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional arrays. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the "blocked" head of dephosphorylated smHMM. We hypothesize that regulatory light chain phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.

Recreation of the terminal events in physiological integrin activation.

Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.

The 3D structure of villin as an unusual F-Actin crosslinker.

Villin is an F-actin nucleating, crosslinking, severing, and capping protein within the gelsolin superfamily. We have used electron tomography of 2D arrays of villin-crosslinked F-actin to generate 3D images revealing villin's crosslinking structure. In these polar arrays, neighboring filaments are spaced 125.9 +/- 7.1 A apart, offset axially by 17 A, with one villin crosslink per actin crossover. More than 6500 subvolumes containing a single villin crosslink and the neighboring actin filaments were aligned and classified to produce 3D subvolume averages. Placement of a complete villin homology model into the average density reveals that full-length villin binds to different sites on F-actin from those used by other actin-binding proteins and villin's close homolog gelsolin.

On the freezing and identification of lipid monolayer 2-D arrays for cryoelectron microscopy.

Lipid monolayers provide a convenient vehicle for the crystallization of biological macromolecules for 3-D electron microscopy. Although numerous examples of 3-D images from 2-D protein arrays have been described from negatively stained specimens, only six structures have been done from frozen-hydrated specimens. We describe here a method that makes high quality frozen-hydrated specimens of lipid monolayer arrays for cryoelectron microscopy. The method uses holey carbon films with patterned holes for monolayer recovery, blotting and plunge freezing to produce thin aqueous films which cover >90% of the available grid area. With this method, even specimens with relatively infrequent crystals can be screened using automated data collection techniques. Though developed for microscopic examination of 2-D arrays, the method may have wider application to the preparation of single particle specimens for 3-D image reconstruction.