The adult heart lacks reserve cardiocytes and cannot regenerate. Therefore, a large acute myocardial infarction often develops into congestive heart failure. To attempt to prevent this progression, we transplanted skeletal myoblasts into cryoinfarcted myocardium of the same rabbits (autologous transfer), monitored cardiac function in vivo for two to six weeks and examined serial sections of the hearts by light and electron microscopy. Islands of different sizes comprising elongated, striated cells that retained characteristics of both skeletal and cardiac cells were found in the cryoinfarct. In rabbits in which myoblasts were incorporated, myocardial performance was improved. The ability to regenerate functioning muscle after autologous myoblast transplantation could have a important effect on patients after acute myocardial infarction.

FtsZ assembles in vitro into protofilaments that can adopt two conformations-the straight conformation, which can assemble further into two-dimensional protofilament sheets, and the curved conformation, which forms minirings about 23 nm in diameter. Here, we describe the structure of FtsZ tubes, which are a variation of the curved conformation. In the tube the curved protofilament forms a shallow helix with a diameter of 23 nm and a pitch of 18 or 24°. We suggest that this shallow helix is the relaxed structure of the curved protofilament in solution. We provide evidence that GTP favors the straight conformation while GDP favors the curved conformation. In particular, exclusively straight protofilaments and protofilament sheets are assembled in GMPCPP, a nonhydrolyzable GTP analog, or in GTP following chelation of Mg, which blocks GTP hydrolysis. Assembly in GDP produces exclusively tubes. The transition from straight protofilaments to the curved conformation may provide a mechanism whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division.FtsZ is a major cytoskeletal protein in all bacteria and archaea, where it forms the framework for the cell-division machinery at the site of septation (3,9,17). Light microscopy shows FtsZ localized in a ring at the site of septation, apparently just under the cell membrane; the ring constricts as septation proceeds (1,14,18). The role of FtsZ as a structural protein is indicated by its abundance (15,000 molecules per average Escherichia coli cell [6,16]) and its assembly into protofilaments in vitro (11,16,20,21,27). In addition to providing the structural framework for the division apparatus, we suggest that FtsZ may also generate the force that powers constriction of the FtsZ ring. A possible mechanism for generating force is the transition of the protofilament from the straight to the curved conformation (reference 9; also see Discussion).The structure of FtsZ polymers in bacteria has never been visualized, but much has been learned from polymers assembled in vitro. These in vitro polymers show the range of structures that are possible, providing insight into potential in vivo structures. Four polymer forms assembled by E. coli FtsZ are shown in Fig. 1. Single, straight protofilaments (16, 21), sheets of straight protofilaments (11, 27), and minirings (11) have been described previously. Tubular polymers of FtsZ have also been reported (4,8,20,25), but their substructure has not been determined previously. Here, we describe the structure of FtsZ tubes and demonstrate that they are a variation of the curved protofilament conformation. We then investigate how GTP and GDP favor the straight and curved conformations, respectively. MATERIALS AND METHODSThe nonhydrolyzable GTP analog GMPCPP was generously provided by John J. Correia, University of Mississippi Medical Center, Jackson, Miss. Other reagents were purchased from Sigma (St. Louis, Mo.) or as noted below.Purification of FtsZ. Wild-type FtsZ was expressed in E...

Enterotoxigenic Escherichia coli (ETEC) is a prevalent cause of traveler's diarrhea and infant mortality in thirdworld 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 nonpathogenderived vesicles, associated with cells in a time-, temperature-, and receptor-dependent manner. Vesicles were visualized on the cell surface at 41C and detected intracellularly at 371C. 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.

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 by 32 P i and 14 C-acetate labeling, export of all major lipids to the outer membrane is inhibited by ϳ90% 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.The envelope of Gram-negative bacteria consists of an inner membrane, the peptidoglycan cell wall, and an outer membrane ( Fig. 1A) (1). The latter is an asymmetric bilayer with glycerophospholipids ( Fig. 1B) on its inner surface and lipid A (Fig. 1B), the hydrophobic anchor of lipopolysaccharide (2, 3), on the outside (1). The lipid A moiety is a hexa-acylated disaccharide of glucosamine unique to Gram-negative bacteria (4) and is a potent activator of innate immunity in animals via the receptor TLR-4 (5-7). The enzymes that make phospholipids and lipid A are well characterized in Escherichia coli (3,8,9). They are located in the cytoplasm or inner membrane and are targets for the design of novel antibacterial agents (10,11).How E. coli lipids cross the inner membrane and are transported to the outer membrane is unknown (Fig. 1A). A clue to lipopolysaccharide transport has recently emerged from studies of E. coli htrB mutants (12) and their suppression by msbA (13-15). HtrB is a lauroyl transferase that functions late in lipid A biosynthesis ( Fig. 1A) (12). Lipopolysaccharides bearing tetra-acylated lipid A species accumulate in the inner membrane of htrB mutants at 44°C, inhibiting growth (15). MsbA is an essential ABC transporter (Fig. 2), closely related to eucaryotic Mdr proteins (13-15). MsbA overexpression restores growth of htrB mutants at 44°C without restoring laurate incorporation, resulting in export of lipopolysaccharide with tetra-acylated lipid A anchors to the outer membrane (15).E. coli msbA knockouts are lethal. However, their biochemical analysis is complicated by long times (4 -8 h) needed to dilute out pre-existing MsbA supplied in trans from a temperaturesensitive plasmid (15) and by the fact that the lpxK gene, which is immediately downstream in an operon with MsbA, is also essential for cell growth (16).We now report the isolation and characterization of a novel temperature-sensitive point mutant of E. coli that harbors a single amino acid substitution in MsbA. Rapid inhibition of MsbA function in vivo following a shift of mid log phase cells from 30 to 44°C results in the immediate arrest of the export of all newly made lipopolysaccharide and phospholipids, demonstrating that MsbA is an essential component of a general lipid transport system in E. coli. EXPERIMENTAL PROCEDURESIsolation of Mutant WD2 and Growth Conditions-WD2 was isolated...

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 ␦-sarcoglycan (␦sg S151A ), which has previously been associated with familial dilated cardiomyopathy. Compared to transgenic flies overexpressing wild-type ␦sg, or the standard laboratory strain w 1118 , Drosophila expressing ␦sg S151A 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.optical coherence tomography ͉ cardiomyopathy

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 Rayment's 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.

Abstract. The functional relationship between three Dictyostelium myosin Is, myoA, myoB, and myoC, has been examined through the creation of double mutants.Two double mutants, myoA-/B-and myoB-/C-, exhibit similar conditional defects in fluid-phase pinocytosis. Double mutants grown in suspension culture are significantly impaired in their ability to take in nutrients from the medium, whereas they are almost indistinguishable from wild-type and single mutant strains when grown on a surface. The double mutants are also found to internalize gp126, a ll6-kD membrane protein, at a slower rate than either the wild-type or single mutant cells. Ultrastructural analysis reveals that both double mutants possess numerous small vesicles, in contrast to the wild-type or myosin I single mutants that exhibit several large, clear vacuoles. The alterations in fluid and membrane internalization in the suspension-grown double mutants, coupled with the altered vesicular profile, suggest that these cells may be compromised during the early stages of pinocytosis, a process that has been proposed to occur via actin-based cytoskeletal rearrangements. Scanning electron microscopy and rhodamine-phalloidin staining indicates that the myosin I double mutants appear to extend a larger number of actin-filled structures, such as filopodia and crowns, than wild-type cells. Rhodamine-phalloidin staining of the F-actin cytoskeleton of these suspension-grown cells also reveals that the double mutant cells are delayed in the rearrangement of cortical actinrich structures upon adhesion to a substrate. We propose that myoA, myoB, and myoC play roles in controlling F-actin filled membrane projections that are required for pinosome internalization in suspension.

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