Mary C. Reedy

Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells

Enterotoxigenic Escherichia coli (ETEC) is a prevalent cause of travelers diarrhea and infant mortality in third‐world countries. Heat‐labile enterotoxin (LT) is secreted from ETEC via vesicles composed of outer membrane and periplasm. We investigated the role of ETEC vesicles in pathogenesis by analyzing vesicle association and entry into eukaryotic cells. Fluorescently labeled vesicles from LT‐producing and LT‐nonproducing strains were compared in their ability to bind adrenal and intestinal epithelial cells. ETEC‐derived vesicles, but not control nonpathogen‐derived vesicles, associated with cells in a time‐, temperature‐, and receptor‐dependent manner. Vesicles were visualized on the cell surface at 4°C and detected intracellularly at 37°C. ETEC vesicle endocytosis depended on cholesterol‐rich lipid rafts. Entering vesicles partially colocalized with caveolin, and the internalized vesicles accumulated in a nonacidified compartment. We conclude that ETEC vesicles serve as specifically targeted transport vehicles that mediate entry of active enterotoxin and other bacterial envelope components into host cells. These data demonstrate a role in virulence for ETEC vesicles.

Drosophila as a model for the identification of genes causing adult human heart disease.

Drosophila melanogaster genetics provides the advantage of molecularly defined P-element insertions and deletions that span the entire genome. Although Drosophila has been extensively used as a model system to study heart development, it has not been used to dissect the genetics of adult human heart disease because of an inability to phenotype the adult fly heart in vivo. Here we report the development of a strategy to measure cardiac function in awake adult Drosophila that opens the field of Drosophila genetics to the study of human dilated cardiomyopathies. Through the application of optical coherence tomography, we accurately distinguish between normal and abnormal cardiac function based on measurements of internal cardiac chamber dimensions in vivo. Normal Drosophila have a fractional shortening of 87 ± 4%, whereas cardiomyopathic flies that contain a mutation in troponin I or tropomyosin show severe impairment of systolic function. To determine whether the fly can be used as a model system to recapitulate human dilated cardiomyopathy, we generated transgenic Drosophila with inducible cardiac expression of a mutant of human δ-sarcoglycan (δsgS151A), which has previously been associated with familial dilated cardiomyopathy. Compared to transgenic flies overexpressing wild-type δsg, or the standard laboratory strain w1118, Drosophila expressing δsgS151A developed marked impairment of systolic function and significantly enlarged cardiac chambers. These data illustrate the utility of Drosophila as a model system to study dilated cardiomyopathy and the applicability of the vast genetic resources available in Drosophila to systematically study the genetic mechanisms responsible for human cardiac disease.

An Escherichia coli Mutant Defective in Lipid Export

Escherichia coli phospholipids and lipopolysaccharide, made on the inner surface of the inner membrane, are rapidly transported to the outer membrane by mechanisms that are not well characterized. We now report a temperature-sensitive mutant (WD2) with an A270T substitution in a trans-membrane region of the ABC transporter MsbA. As shown by32Pi and 14C-acetate labeling, export of all major lipids to the outer membrane is inhibited by ∼90% in WD2 after 30 min at 44 °C. Transport of newly synthesized proteins is not impaired. Electron microscopy shows reduplicated inner membranes in WD2 at 44 °C, consistent with a key role for MsbA in lipid trafficking.

Tomographic 3D Reconstruction of Quick-Frozen, Ca2+-Activated Contracting Insect Flight Muscle

Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active cross-bridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (approximately 30% of all heads) depart markedly in axial and azimuthal angles from Rayments rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from approximately 105 degrees to approximately 70 degrees. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an approximately 35 degrees axial angle, giving an approximately 13 nm interaction distance and an approximately 4-6 nm working stroke.

Molecular Modeling of Averaged Rigor Crossbridges from Tomograms of Insect Flight Muscle

Electron tomography, correspondence analysis, molecular model building, and real-space refinement provide detailed 3-D structures for in situ myosin crossbridges in the nucleotide-free state (rigor), thought to represent the end of the power stroke. Unaveraged tomograms from a 25-nm longitudinal section of insect flight muscle preserved native structural variation. Recurring crossbridge motifs that repeat every 38.7 nm along the actin filament were extracted from the tomogram and classified by correspondence analysis into 25 class averages, which improved the signal to noise ratio. Models based on the atomic structures of actin and of myosin subfragment 1 were rebuilt to fit 11 class averages. A real-space refinement procedure was applied to quantitatively fit the reconstructions and to minimize steric clashes between domains introduced during the fitting. These combined procedures show that no single myosin head structure can fit all the in situ crossbridges. The validity of the approach is supported by agreement of these atomic models with fluorescent probe data from vertebrate muscle as well as with data from regulatory light chain crosslinking between heads of smooth muscle heavy meromyosin when bound to actin.

The Yb protein defines a novel organelle and regulates male germline stem cell self-renewal in Drosophila melanogaster.

Yb regulates the proliferation of both germline and somatic stem cells in the Drosophila melanogaster ovary by activating piwi and hh expression in niche cells. In this study, we show that Yb protein is localized as discrete cytoplasmic spots exclusively in the somatic cells of the ovary and testis. These spots, which are different from all known cytoplasmic structures in D. melanogaster, are evenly electron-dense spheres 1.5 µm in diameter (herein termed the Yb body). The Yb body is frequently associated with mitochondria and a less electron-dense sphere of similar size that appears to be RNA rich. There are one to two Yb bodies/cell, often located close to germline cells. The N-terminal region of Yb is required for hh expression in niche cells, whereas the C-terminal region is required for localization to Yb bodies. The entire Yb protein is necessary for piwi expression in niche cells. A double mutant of Yb and a novel locus show male germline loss, revealing a function for Yb in male germline stem cell maintenance.

Morphology and ultrastructure of differentiating three-dimensional mammalian skeletal muscle in a collagen gel.

Because previous studies of three‐dimensional skeletal muscle cultures have shown limited differentiation, the goal of this study was to establish conditions that would produce mature sarcomeres in a mammalian‐derived skeletal muscle construct. We evaluated the differentiation of bioartificial muscles generated from C2C12 myoblasts in a collagen gel cultured under steady, passive tension for up to 36 days. Staining for alpha‐actinin, myosin, and F‐actin indicated the presence of striated fibers as early as 6 days post‐differentiation. Electron microscopy at 16 days post‐differentiation revealed multinucleated myotubes with ordered, striated myofibers. At 33 days, the cultures contained collagen fibers and showed localization of paxillin at the fiber termini, suggesting that myotendinous junctions were forming. The present study demonstrates mature muscle synthesis in a three‐dimensional system using a pure mammalian myoblast cell line. Our results suggest that this culture model can be used to evaluate the effects of various mechanical and biochemical cues on muscle development under normal and pathological conditions. Muscle Nerve, 2007

A mighty small heart: the cardiac proteome of adult Drosophila melanogaster.

Drosophila melanogaster is emerging as a powerful model system for the study of cardiac disease. Establishing peptide and protein maps of the Drosophila heart is central to implementation of protein network studies that will allow us to assess the hallmarks of Drosophila heart pathogenesis and gauge the degree of conservation with human disease mechanisms on a systems level. Using a gel-LC-MS/MS approach, we identified 1228 protein clusters from 145 dissected adult fly hearts. Contractile, cytostructural and mitochondrial proteins were most abundant consistent with electron micrographs of the Drosophila cardiac tube. Functional/Ontological enrichment analysis further showed that proteins involved in glycolysis, Ca2+-binding, redox, and G-protein signaling, among other processes, are also over-represented. Comparison with a mouse heart proteome revealed conservation at the level of molecular function, biological processes and cellular components. The subsisting peptidome encompassed 5169 distinct heart-associated peptides, of which 1293 (25%) had not been identified in a recent Drosophila peptide compendium. PeptideClassifier analysis was further used to map peptides to specific gene-models. 1872 peptides provide valuable information about protein isoform groups whereas a further 3112 uniquely identify specific protein isoforms and may be used as a heart-associated peptide resource for quantitative proteomic approaches based on multiple-reaction monitoring. In summary, identification of excitation-contraction protein landmarks, orthologues of proteins associated with cardiovascular defects, and conservation of protein ontologies, provides testimony to the heart-like character of the Drosophila cardiac tube and to the utility of proteomics as a complement to the power of genetics in this growing model of human heart disease.

X-Ray Diffraction Indicates That Active Cross-Bridges Bind to Actin Target Zones in Insect Flight Muscle

We report the first time-resolved study of the two-dimensional x-ray diffraction pattern during active contraction in insect flight muscle (IFM). Activation of demembranated Lethocerus IFM was triggered by 1.5-2.5% step stretches (risetime 10 ms; held for 1.5 s) giving delayed active tension that peaked at 100-200 ms. Bundles of 8-12 fibers were stretch-activated on SRS synchrotron x-ray beamline 16.1, and time-resolved changes in diffraction were monitored with a SRS 2-D multiwire detector. As active tension rose, the 14.5- and 7.2-nm meridionals fell, the first row line dropped at the 38.7 nm layer line while gaining a new peak at 19.3 nm, and three outer peaks on the 38.7-nm layer line rose. The first row line changes suggest restricted binding of active myosin heads to the helically preferred region in each actin target zone, where, in rigor, two-headed lead bridges bind, midway between troponin bulges that repeat every 38.7 nm. Halving this troponin repeat by binding of single active heads explains the intensity rise at 19.3 nm being coupled to a loss at 38.7 nm. The meridional changes signal movement of at least 30% of all myosin heads away from their axially ordered positions on the myosin helix. The 38.7- and 19.3-nm layer line changes signal stereoselective attachment of 7-23% of the myosin heads to the actin helix, although with too little ordering at 6-nm resolution to affect the 5.9-nm actin layer line. We conclude that stretch-activated tension of IFM is produced by cross-bridges that bind to rigors lead-bridge target zones, comprising < or = 1/3 of the 75-80% that attach in rigor.

Co-ordinated electron microscopy and X-ray studies of glycerinated insect flight muscle. II. Electron microscopy and image reconstruction of muscle fibres fixed in rigor, in ATP and in AMPPNP.

SummaryThis paper presents electron microscopy, supported by optical diffraction and filtering of images from 100 nm and 25 nm sections, to complement the companion report of X-ray diffraction monitoring (immediately preceding this article) performed on the same insect flight muscle specimens during fixation, dehydration and embedding. GlycerinatedLethocerus fibre bundles initially fixed in rigor, in ATP relaxing buffer, or in 1mM AMPPNP at 2° C, gave thin-section images from each state whose optical transforms match the distinctive X-ray diffraction patterns from the embedded samples. For rigor and relaxed states, this extends and confirms a long-known correlation between X-ray patterns and EM image regularities. For the AMPPNP state, such correlation is here fully developed for the first time, and involves a new and distinctive EM image pattern of the crossbridge array, clearly different from a previously reported structure in AMPPNP-treated muscles that appears identical to fixed relaxed muscle. We found this latter artifact of ‘AMPPNP-relaxed structure’ in many fibres from our best AMPPNP specimen, but could identify other fibres which retained the distinctive AMPPNP structure, known to be dominant in this specimen from the X-ray pattern. The true AMPPNP structure shows features of both the ATP-relaxed and rigor crossbridge patterns, not as separate patches, but hybridized uniformly along each filament and throughout each affected sarcomere and fibre. It presents a 14.5 nm repeat of striping and lateral projections along thick filaments, together with variously angled crossbridge attachments to actin that form a 38.7 nm repeat of diffuse chevrons or deltoids replacing the more clearly delinated rigor double chevrons. The associated optical transform has the typical AMPPNP features, that is, it has in common with rigor a strong 19.3 nm layer line and strong second to fourth row line sampling on the 38.7 nm layer line, it has in common with relaxed patterns a strong 14.5 nm meridional and layer line, but it uniquely shows no intensity at the first row line on the 38.7 nm layer line (the 10.3 X-ray reflection), where rigor and relaxed transforms always show high intensity. The processing artifacts which intensify the 10.3 reflection, and produce the weak 19.3 nm layer line (a gain of intensity for ATP but a loss for the AMPPNP state), throughout ATP specimens and in those analogue-treated fibres showing AMPPNP-relaxed structure, might indicate trapping and accumulation of minority populations within the native equilibrium distribution of crossbridge conformations in each nucleotide state.