Calcium-induced changes in the location and conformation of troponin in skeletal muscle thin filaments.
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Troponin is the regulatory protein of striated muscle. Without Ca2+, the contraction of striated muscle is inhibited. Binding of Ca2+ to troponin activates contraction. The location of troponin on the thin filaments and its relation to the regulatory mechanism has been unknown, though the Ca2+-induced dislocation of tropomyosin has been studied. By binding troponin(C+I) to actin in an almost stoichiometric ratio and reconstituting actin-tropomyosin-troponin(C+I) filaments, we reconstructed the three-dimensional structure of actin-tropomyosin-troponin(C+I) with or without Ca2+ from electron cryomicrographs to about 2.5 or 3 nm resolution, respectively. Without Ca2+, the three-dimensional map reveals the extra-density region due to troponin(C+I), which extends perpendicularly to the helix axis and covers the N-terminal and C-terminal regions of actin. In the presence of Ca2+, the C-terminal region of actin became more exposed, and troponin(C+I) became V-shaped with one arm extending towards the pointed end of the actin filament. This structure can be considered to show the location of troponin(C+I) in at least one of the states of skeletal muscle thin filaments. These Ca2+-induced changes of troponin(C+I) provide a clue to the regulatory mechanism of contraction. (+info)
Assembly of a novel cartilage matrix protein filamentous network: molecular basis of differential requirement of von Willebrand factor A domains.
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Cartilage matrix protein (CMP) is the prototype of the newly discovered matrilin family, all of which contain von Willebrand factor A domains. Although the function of matrilins remain unclear, we have shown that, in primary chondrocyte cultures, CMP (matrilin-1) forms a filamentous network, which is made up of two types of filaments, a collagen-dependent one and a collagen-independent one. In this study, we demonstrate that the collagen-independent CMP filaments are enriched in pericellular compartments, extending directly from chondrocyte membranes. Their morphology can be distinguished from that of collagen filaments by immunogold electron microscopy, and mimicked by that of self-assembled purified CMP. The assembly of CMP filaments can occur from transfection of a wild-type CMP transgene alone in skin fibroblasts, which do not produce endogenous CMP. Conversely, assembly of endogenous CMP filaments by chondrocytes can be inhibited specifically by dominant negative CMP transgenes. The two A domains within CMP serve essential but different functions during network formation. Deletion of the A2 domain converts the trimeric CMP into a mixture of monomers, dimers, and trimers, whereas deletion of the A1 domain does not affect the trimeric configuration. This suggests that the A2 domain modulates multimerization of CMP. Absence of either A domain from CMP abolishes its ability to form collagen-independent filaments. In particular, Asp22 in A1 and Asp255 in A2 are essential; double point mutation of these residues disrupts CMP network formation. These residues are part of the metal ion-dependent adhesion sites, thus a metal ion-dependent adhesion site-mediated adhesion mechanism may be applicable to matrilin assembly. Taken together, our data suggest that CMP is a bridging molecule that connects matrix components in cartilage to form an integrated matrix network. (+info)
We identify an actin-based protrusive structure in growth cones termed "intrapodium." Unlike filopodia, intrapodia are initiated exclusively within lamellipodia and elongate in a continuous (nonsaltatory) manner parallel to the plane of the dorsal plasma membrane causing a ridge-like protrusion. Intrapodia resemble the actin-rich structures induced by intracellular pathogens (e.g., Listeria) or by extracellular beads. Cytochalasin B inhibits intrapodial elongation and removal of cytochalasin B produced a burst of intrapodial activity. Electron microscopic studies revealed that lamellipodial intrapodia contain both short and long actin filaments oriented with their barbed ends toward the membrane surface or advancing end. Our data suggest an interaction between microtubule endings and intrapodia formation. Disruption of microtubules by acute nocodazole treatment decreased intrapodia frequency, and washout of nocodazole or addition of the microtubule-stabilizing drug Taxol caused a burst of intrapodia formation. Furthermore, individual microtubule ends were found near intrapodia initiation sites. Thus, microtubule ends or associated structures may regulate these actin-dependent structures. We propose that intrapodia are the consequence of an early step in a cascade of events that leads to the development of F-actin-associated plasma membrane specializations. (+info)
Role of actin stress fibres in the development of the intervertebral disc: cytoskeletal control of extracellular matrix assembly.
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Orientation of collagen fibrils is a key event in the development of many tissues. In the intervertebral disc, the outer annulus fibrosus comprises lamellae of parallel collagen fibres, the direction of orientation of the long axis of which alternates in angle between lamellae. In development, this organisation is preceded by the formation of sheets of oriented fibroblasts, which then deposit the oriented lamellae. Here, using fluorescent labelling, confocal and electron microscopic techniques on developmental series, we show that the orientation of cells in lamellae is associated with the formation of adherens junctions intercellularly, involving cadherins and vinculin, and longitudinal stress fibres (label for filamentous actin and tropomyosin) intracellularly. The stress fibres direct the initial elongation of cells and control the deposition of oriented extracellular matrix via junctional complexes with the matrix involving vinculin and alpha 5 beta 1 integrins, which in turn promote the formation of oriented fibronectin at the cell surface; oriented collagen is deposited between cells at the same stages. Shortly after birth, the stress fibres disappear, probably because cells now gain orientational cues from the matrix, and are undergoing differentiation-related changes to form fibrocartilage cells. Dev Dyn 1999;215:179-189. (+info)
Bending of the neural plate during mouse spinal neurulation is independent of actin microfilaments.
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To examine the role of actin microfilaments in mouse spinal neurulation, we stained cryosections of E8.5-10.5 CBA/Ca embryos with FITC-phalloidin. Microfilaments are present in the apical region of all cells throughout the neuroepithelium, irrespective of whether they are involved in bending of the neural plate. Disruption of the microfilaments with cytochalasin D inhibited closure of the cranial neural folds in cultured embryos, even at the lowest concentrations tested, and prevented the initiation of spinal neurulation (Closure 1) at higher concentrations. In contrast, closure of the posterior neuropore was resistant to cytochalasin D at the highest concentrations tested. Phalloidin staining and transmission electron microscopy confirmed that cytochalasin D is effective in disassembling microfilaments in spinal neuroepithelial cells. We conclude that spinal neural tube closure does not require microfilament function, in contrast to cranial neurulation which is strongly microfilament-dependent. Histological examination of cytochalasin D-treated embryos revealed that bending at hinge points, both in the midline (MHP) and dorsolaterally (DLHPs), continues in the absence of microfilaments, whereas the rigidity of non-bending regions of the neural plate is lost. This suggests that spinal neurulation can continue in the presence of cytochalasin D largely as a result of intrinsic bending of the neural plate at hinge points. Cytochalasin D treatment is a useful tool for revealing the localisation of hinge points in the neural plate. Analysis of treated embryos demonstrates a transition, along the spinal axis, from closure solely involving midline bending, at high levels of the spinal axis, to closure solely involving dorsolateral bending, low in the spinal region. These findings support the idea of mechanistic heterogeneity in mouse neurulation, along the body axis, and demonstrate that contraction of actin microfilaments is not obligatory for epithelial bending during embryonic morphogenesis. Dev Dyn 1999;215:273-283. (+info)
Augmented acetylcholine-induced, Rho-mediated Ca2+ sensitization of bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats.
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Treatment with acetylcholine (ACh) of a beta-escin-permeabilized intrapulmonary bronchial smooth muscle of the rat induced force when the Ca2+ concentration was clamped at 1 microM. The ACh-induced Ca2+ sensitization of myofilaments was significantly greater in antigen-induced airway hyperresponsive rats than in control rats. The ACh-induced Ca2+ sensitization was completely blocked by treatment with Clostridium botulinum C3 exoenzyme, an inactivator of Rho family of proteins. Moreover, the protein level of RhoA in the intrapulmonary bronchi was significantly increased in the airway hyperresponsive rats. Thus, increased airway smooth muscle contractility observed in asthmatics may be related to augmented agonist-induced, Rho-mediated Ca2+ sensitization of myofilaments. (+info)
Campylobacter jejuni 81-176 associates with microtubules and dynein during invasion of human intestinal cells.
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Campylobacter jejuni uptake into cultured INT407 cells was analyzed kinetically over a wide range of starting multiplicities of infection (MOI; from 0.02 to 20,000 bacteria/epithelial cell). The efficiency of internalization was the highest at MOI of 0.02 and decreased steadily at higher MOIs, presumably due to reported C. jejuni autoagglutination at higher densities. Total internalized Campylobacter CFU increased gradually from an MOI of 0.02 to a peak at an MOI of 200 (reaching an average of two bacteria internalized per epithelial cell) and decreased at higher MOIs. The invasion process was apparently saturated within 2 h at an MOI of 200, indicating stringent host cell limitations on this entry process. Furthermore, whereas control Salmonella typhi invaded all monolayer cells within 1 h, only two-thirds of monolayer cells were infected after 2 h with C. jejuni at MOIs of 200 to 2,000. The percentage of Campylobacter-infected host cells gradually increased to 85% after 7 h of infection, suggesting that C. jejuni entry may be host cell cycle dependent. Direct evidence of the involvement of microtubules in C. jejuni internalization, suggested previously by biochemical inhibitor studies, was obtained by time course immunofluorescence microscopic analyses. Bacteria initially bound to the tips of host cell membrane extensions containing microtubules, then aligned in parallel with microtubules during entry, colocalized specifically with microtubules and dynein but not with microfilaments, and moved over 4 h, presumably via microtubules to the perinuclear region of host cells. Orthovanadate, which inhibits dynein activity, specifically reduced C. jejuni 81-176 entry, suggesting that this molecular motor is involved in entry and endosome trafficking during this novel bacterial internalization process. Collectively, these data suggest that C. jejuni enters host cells in a targeted and tightly controlled process leading to uptake into an endosomal vacuole which apparently moves intracellularly along microtubules via the molecular motor, dynein, to the perinuclear region. (+info)
Citrobacter freundii invades and replicates in human brain microvascular endothelial cells.
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Neonatal bacterial meningitis remains a disease with unacceptable rates of morbidity and mortality despite the availability of effective antimicrobial therapy. Citrobacter spp. cause neonatal meningitis but are unique in their frequent association with brain abscess formation. The pathogenesis of Citrobacter spp. causing meningitis and brain abscess is not well characterized; however, as with other meningitis-causing bacteria (e.g., Escherichia coli K1 and group B streptococci), penetration of the blood-brain barrier must occur. In an effort to understand the pathogenesis of Citrobacter spp. causing meningitis, we have used the in vitro blood-brain barrier model of human brain microvascular endothelial cells (HBMEC) to study the interaction between C. freundii and HBMEC. In this study, we show that C. freundii is capable of invading and trancytosing HBMEC in vitro. Invasion of HBMEC by C. freundii was determined to be dependent on microfilaments, microtubules, endosome acidification, and de novo protein synthesis. Immunofluorescence microscopy studies revealed that microtubules aggregated after HBMEC came in contact with C. freundii; furthermore, the microtubule aggregation was time dependent and seen with C. freundii but not with noninvasive E. coli HB101 and meningitic E. coli K1. Also in contrast to other meningitis-causing bacteria, C. freundii is able to replicate within HBMEC. This is the first demonstration of a meningitis-causing bacterium capable of intracellular replication within BMEC. The important determinants of the pathogenesis of C. freundii causing meningitis and brain abscess may relate to invasion of and intracellular replication in HBMEC. (+info)