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At least 361 records · Page 20Linked to original sources
PubMed2001

Myosin II-dependent cylindrical protrusions induced by quinine in Dictyostelium: antagonizing effects of actin polymerization at the leading edge.

We found that amoeboid cells of Dictyostelium are induced by a millimolar concentration of quinine to form a rapidly elongating, cylindrical protrusion, which often led to sustained locomotion of the cells. Formation of the protrusion was initiated by fusion of a contractile vacuole with the cell membrane. During protrusion extension, a patch of the contractile vacuole membrane stayed undiffused on the leading edge of the protrusion for over 30 seconds. Protrusion formation was not inhibited by high osmolarity of the external medium (at least up to 400 mosM). By contrast, mutant cells lacking myosin II (mhc(-) cells) failed to extend protrusions upon exposure to quinine. When GFP-myosin-expressing cells were exposed to quinine, GFP-myosin was accumulated in the cell periphery forming a layer under the cell membrane, but a newly formed protrusion was initially devoid of a GFP-myosin layer, which gradually formed and extended from the base of the protrusion. F-actin was absent in the leading front of the protrusion during the period of its rapid elongation, and the formation of a layer of F-actin in the front was closely correlated with its slowing-down or retraction. Periodical or continuous detachment of the F-actin layer from the apical membrane of the protrusion, accompanied by a transient increase in the elongation speed at the site of detachment, was observed in some of the protrusions. The detached F-actin layers, which formed a spiral layer of F-actin in the case of continuous detachment, moved in the opposite direction of protrusion elongation. In the presence of both cytochalasin A and quinine, the protrusions formed were not cylindrical but spherical, which swallowed up the entire cellular contents. The estimated bulk flux into the expanding spherical protrusions of such cells was four-times higher than the flux into the elongating cylindrical protrusions of the cells treated with quinine alone. These results indicate that the force responsible for the quinine-induced protrusion is mainly due to contraction of the cell body, which requires normal myosin II functions, while actin polymerization is important in restricting the direction of its expansion. We will discuss the possible significance of tail contraction in cell movement in the multicellular phase of Dictyostelium development, where cell locomotion similar to that induced by quinine is often observed without quinine treatment, and in protrusion elongation in general. Movies available on-line

Actins↗
PubMed2004

Osteocyte dendrogenesis in static and dynamic bone formation: an ultrastructural study.

The present ultrastructural investigation into osteocyte dendrogenesis represents a continuation of a previous study (Ferretti et al., Anat. Embryol., 2002; 206:21-29), in which we pointed out that, during intramembranous ossification, the well-known dynamic bone formation (DBF), performed by migrating osteoblast laminae, is preceded by static bone formation (SBF), in which cords of stationary osteoblasts transform into osteocytes in the same site where they differentiated. The research was carried out on the perichondral center of ossification surrounding the mid shaft level of various long bones of chick embryos and newborn rabbits. Transmission electron microscope observations showed that the formation of osteocyte dendrites is quite different in the two types of osteogenesis, mainly depending on whether or not osteoblast movement occurs. In DBF, osteoblasts transform into small ovoidal/ellipsoidal osteocytes and their dendrites form in an asynchronous and asymmetrical manner in concomitance with, and depending on, the advancing mineralizing surface and the receding osteogenic laminae. In SBF, stationary osteoblasts give rise to big globous osteocytes, located inside confluent lacunae, with short and symmetrical dendrites that can radiate simultaneously all around their cell body because they are completely surrounded by unmineralized matrix. Contacts and gap junctions were observed between all osteocytes (both SBF- and DBF-derived) and between osteocytes and osteoblasts. Finally, a continuous osteocyte network extends throughout the bone, regardless of its static or dynamic origin. This network has the characteristic of a functional syncytium, potentially capable of modulating, by wiring transmission, the cells of the osteogenic lineage covering the bone surfaces.

Animals↗
PubMed2002

Localised depletion of polymerised actin at the front of Walker carcinosarcoma cells increases the speed of locomotion.

Spontaneously migrating Walker carcinosarcoma cells usually form lamellipodia at the front. Combined treatment with 10(-5)M colchicine and 10(-7)M latrunculin A produces large defects in the cortical F-actin layer at the leading front and suppresses lamellipodia. However, the cortical actin layer at the rear is intact and shows myosin IIA accumulation. These cells, showing no or little detectable cortical F-actin at the front and no morphologically recognisable protrusions, migrate faster than control cells with lamellipodia and an intact cortical actin layer. This documents that the cortical actin layer or actin-powered force generation at the front is redundant for locomotion. Colchicine and latrunculin A have synergistic effects in compromising the cortical layer at the front and in increasing the speed of locomotion, but antagonistic effects on the relative amount of F-actin per cell. Colchicine but not latrunculin A, can increase the proportion of polarised and locomoting cells under appropriate conditions. Locomotion and polarity of cells treated with latrunculin A and colchicine is inhibited at latrunculin A concentrations >10(-7)M, by the myosin inhibitor BDM or the ROCK inhibitor Y-27632. Colchicine and Y-27632 have antagonistic effects on polarity and the speed of locomoting cells. The data show that locomotion of metazoan cells, which normally form lamellipodia, can be driven by actomyosin contraction behind the front (cell body, uropod). They are best compatible with a cortical contraction/frontal expansion model, but they are not compatible with models implying that actin polymerisation or actomyosin contraction at the front drive locomotion of the cells studied.

Actinin↗
PubMed2001

Hair-forming activity of human lymphocyte specific protein 1 requires cooperation between its caldesmon-like domains and the villin headpiece-like domains.

LSP1 is an F-actin binding with multiple F-actin binding domains. Overexpression of LSP1 in NAD 47/89 patient's neutrophils created hair-like projections on the patient's neutrophil cell surfaces and inhibited neutrophil cell motility and transfection of LSP1 in serial cell lines recreate the NAD 47/89 phenotype and produce branching hair-like surface projections. Although LSP1 contains hair-forming ability and LSP1 F-actin binding domains have been defined, the LSP1 domains responsible for its hair-forming activity, the relationship to the F-actin binding domains, and the required domain interactions, if any, for hair formation are not well understood. To define the hair-forming domains of LSP1, the relationship to the known F-actin binding domains, and binding domain interactions, LSP1 truncates, which include or exclude the different F-actin binding domains, were created by PCR. LSP1 mutants were created by site-directed mutagenesis to define the amino acids important for hair formation. Sf9 cells were infected with recombinant baculovirus expressing the cDNA of LSP1 truncates and mutants, and the morphology of infected Sf9 cells was documented by DIC optics. Results show that (1) the hair-forming activity of LSP1 is localized to the basic C-terminal half of the molecule, which contains all of the F-actin binding domains; (2) both the caldesmon-like domains and the villin headpiece-like domains are required for the hair-forming activity of LSP1; (3) basic amino acids in the villin headpiece regions are crucial for the hair-forming activity of LSP1 molecule. The results suggest cooperation between the caldesmon-like domains and the villin headpiece-like domains are required for the hair-forming activity of human LSP1 in cells.

Amino Acid Motifs↗
PubMed2006

Effects of chemoattractant pteridines upon speed of D. discoideum vegetative amoebae.

Movements of D. discoideum vegetative amoebae responding to pteridine chemoattractants, folate acid and pterin, were recorded. A vector analysis of these images was performed to partition the speed and orientation components of these motility patterns. This study demonstrates that in addition to orientation (chemotaxis), stimulated speed (chemokinesis) is an important component of the directed migration of these amoebae. Furthermore, the primary difference in their response to folate versus pterin is in speed rather than orientation. The data support a model of directed migration of these cells in which there are (1) separate signal translation pathways consequent from folate versus pterin reception and (2) specific pathways leading to increase in orientation versus speed.

Amoeba↗
PubMed2006

Static magnetic fields affect cell size, shape, orientation, and membrane surface of human glioblastoma cells, as demonstrated by electron, optic, and atomic force microscopy.

BACKGROUND: It is common knowledge that static magnetic fields (SMF) do not interact with living cells; thus, fewer studies of SMF compared with variable magnetic fields are carried out. However, evidence demonstrated that SMF affect cellular structures. To investigate the effect of exposure to increasing doses of SMF on cell morphology, human glioblastoma cells were exposed to SMF ranging between 80 and 3,000 G (8 and 300 mT). METHODS: Cell morphology of human glioblastoma cells, derived from a primary culture, was studied by electron and optic microscopy. FITC-phalloidin staining of actin filaments was also investigated. Finally, cell surface structure changes were detected by atomic force microscopy. RESULTS: Scanning electron microscopy demonstrated a dose-dependent cell shape modification, progressive cell detachment, loss of the long villi, and appearance of membrane roughness and blebs. FITC-phalloidin staining confirmed the villi retention and cell dimension decrease. At 3,000 G, the appearance of apoptotic morphology was also observed by transmission electron microscopy. Cell exposed to SMF showed different orientation and alignment when compared with nonexposed cells. The atomic force microscopy of the exposed cells' membrane surfaces demonstrated the disappearance of the ordered surface ripples and furrows typical of the unexposed cells, and the occurrence of surface membrane corrugation at increasing dose exposure CONCLUSIONS: Our experimental procedures demonstrated that exposure to SMF affects not only cell size, shape, and orientation but also human glioblastoma cells' membrane surfaces.

Apoptosis↗
PubMed2004

Evolutionary transformation from muscular to hydraulic movements in spider (Arachnida, Araneae) genitalia: a study based on histological serial sections.

The male genitalia of 107 spider species representing 73 families were serially sectioned and studied with an emphasis on muscles moving the genital bulb. As a rule, most non-Entelegynae have two bulbal muscles, most Entelegyne have none, but many exceptions occur. Variation also occurs with regard to origin and attachment of bulbal muscles. There appears to be a trend towards a shift of the origin from proximal (Liphistius, Atypus) to more distal palpal segments (Haplogynae). In most Entelegynae the muscular movement is replaced by hydraulic movement caused by expanding membranes (hematodochae). Hematodochae probably permit increased bulbal rotation and movements of higher complexity. New evidence is presented arguing against Palpimanidae being representatives of Entelegynae. Bulbal glands other than those discharging into the sperm duct (previously known in Amaurobiidae and Dictynidae only) are described in several entelegyne families.

Animals↗
PubMed2003

Protection of jejunal crypts by RH-3 (a preparation of Hippophae rhamnoides) against lethal whole body gamma irradiation.

RH-3, an alcoholic extract of whole berries of Hippopheae rhamnoides, has been demonstrated to provide radioprotective activity in terms of survival of mice against whole body lethal irradiation (10 Gy). It was, therefore, investigated for its mode of action by monitoring crypt survival, cellularity of crypts and villi and the magnitude of apoptosis in the GI tract. Administration of RH-3 before irradiation (-30 min) increased the number of surviving crypts in the jejunum by a factor of 2.02 (p < 0.05) and villi cellularity by 2.5 fold (p < 0.05) in comparison to the irradiated control. RH-3 administration before irradiation also reduced the incidence of apoptotic bodies in the crypts (p < 0.05) in a time dependent manner and increased cellularity in the crypts and villi (84 h post irradiation) as compared to control. Caspase-3 activity was also significantly lower in the mice administered RH-3 before irradiation as compared to irradiated control. This study indicates that reduction in the radiation induced loss of cellularity of crypts and villi and also decrease in frequency of apoptosis could have contributed towards protection of mice treated with RH-3 before irradiation. The cellular and molecular mechanisms of radioprotection by Rh-3 need to be investigated further in detail.

Animals↗
PubMed2002

Osteoclasts from mice deficient in tartrate-resistant acid phosphatase have altered ruffled borders and disturbed intracellular vesicular transport.

Tartrate-resistant acid phosphatase (TRAP) is an enzyme highly expressed in osteoclasts (OC) and chondroclasts. As an approach to pinpoint the function of TRAP in bone-resorbing osteoclasts, the morphological phenotypic alterations of bone and osteoclasts in mice with targeted disruption of the TRAP gene were assessed by quantitative histomorphometry and immunocytochemistry at the light microscopic and ultrastructural levels. TRAP-deficient mice display alterations in the epiphyseal growth plates as evidenced by increased height with disorganized columns of chondrocytes, in particular affecting the zone of hypertrophic chondrocytes, consistent with a disturbance of chondrocyte maturation and chondroclastic resorption at the epiphyseal/metaphyseal junction. TRAP -/- mice express an early onset osteopetrotic bone phenotype, apparent already at 4 weeks of age. The differentiation of OCs was apparently normal; however, the osteoclasts in TRAP-deficient mice were less active in terms of degradation or release of the resorption marker C-terminal type I collagen cross-linked peptide, indicative of an intrinsic defect. Ultrastructural morphometry disclosed that OCs from TRAP-deficient young mice exhibited an increased relative area of ruffled borders. Moreover, mutant OC accumulated cytoplasmic vesicles 200-500 nm in size in both ruffled border and basolateral parts of the cytoplasm, reflecting disturbed intracellular transport. The accumulated vesicles were not likely derived from the secretory pathway, since cathepsin K was detected at normal levels in the ruffled border area and matrix in TRAP -/- mice. In summary, the resorptive defect in TRAP-deficient OCs is reflected by a disturbance at the level of ruffled borders and intracellular transport vesicles. Consequently, accumulation of vesicles in the cytoplasm of mutant OCs indicates a novel function for TRAP in modulating intracellular vesicular transport in osteoclasts.

Acid Phosphatase↗
PubMed2001

Calsyntenin-1, a proteolytically processed postsynaptic membrane protein with a cytoplasmic calcium-binding domain.

In a screen for proteins released from synapse-forming spinal cord neurons, we found the proteolytically cleaved N-terminal fragment of a transmembrane protein localized in the postsynaptic membrane of both excitatory and inhibitory synapses. We termed this protein calsyntenin-1, because it binds synaptic Ca2+ with its cytoplasmic domain. By binding Ca2+, calsyntenin-1 may modulate Ca2+-mediated postsynaptic signals. Proteolytic cleavage of calsyntenin-1 in its extracellular moiety generates a transmembrane stump that is internalized and accumulated in the spine apparatus of spine synapses. Therefore, the synaptic Ca2+ modulation by calsyntenin-1 may be subject to regulation by extracellular proteolysis in the synaptic cleft. Thus, calsyntenin-1 may link extracellular proteolysis in the synaptic cleft and postsynaptic Ca2+ signaling.

Animals↗
PubMed2003

Application of proteomics to Pseudomonas aeruginosa.

The recent completion of the Pseudomonas Genome Project, in conjunction with the Pseudomonas Community Annotation Project (PseudoCAP) has fast-tracked our ability to apply the tools encompassed under the term 'proteomics' to this pathogen. Such global approaches will allow the research community to answer long-standing questions regarding the ability of Pseudomonas aeruginosa to survive diverse habitats, its high intrinsic resistance to antibiotics and its pathogenic nature towards humans. Proteomics provides an array of tools capable of confirming the expression of Open Reading Frames (ORF), the relative levels of their expression, the environmental conditions required for this expression and the sub-cellular location of the encoded gene-products. Since proteins are important cellular effectors, the biological questions we pose can be defined in terms of changes in protein expression detectable by separation to purity using two-dimensional gel electrophoresis (2-DGE) and relation to gene sequences via mass spectrometry. As such, we can compare strains with well-characterized phenotypic differences, growth under a variety of stresses, protein interactions and complexes and aid in defining proteins of unknown function. While the complete genome has only recently been finished, a number of studies have already utilized this information and examined various protein gene-products using proteomics. This review summarizes the application of proteomics to P. aeruginosa and highlights potential areas of future research, including overcoming the traditional technical limitations associated with 2-DGE. More focused approaches that target sub-cellular fractions ('sub-proteomes') prior to 2-DGE can provide further functional information. A review of current and previous proteomic projects on P. aeruginosa is presented, as well as theoretical considerations of the importance of sub-proteomic approaches to enhance these investigations.

Amino Acid Sequence↗
PubMed2001

The vacuole as central element of the lytic system and sink for lipid droplets in maturing appressoria of Magnaporthe grisea.

Histochemical and ultrastructural studies were carried out on a wild-type strain (Guy11) and a melanin-deficient mutant (buf1) of the rice-blast pathogen, Magnaporthe grisea (= Pyricularia oryzae), in order to investigate the destination of lipid storage reserves during appressorium development. Lipid droplets were abundant in conidia and were mobilised upon germination, accumulating in the appressorial hook which developed at the tip of each germ tube. Following the formation of a septum at the base of the nascent appressorium, one or a few closely appressed central vacuoles became established and were observed to enlarge in the course of appressorium maturation. On unyielding artificial surfaces such as glass or plastic, appressoria matured to completion within 36-48 h, by which time the enlarged vacuole filled most of the inside volume of the appressorium. Light and transmission electron microscopical observations revealed that the lipid droplets entered the vacuole by autophagocytosis and were degraded therein. Histochemical approaches confirmed the vacuole as the key lytic element in maturing appressoria. Endocytosis of a vital dye, Neutral Red, progressed via endosomes which migrated into the vacuole and lysed there, releasing their dye content into the vacuolar lumen. Furthermore, activity of the lysosomal marker enzyme, acid phosphomonoesterase, was strongly localised in the vacuole at all stages of appressorium maturation. It is therefore envisaged that vacuoles are involved in the degradation of lipid storage reserves which may act as sources of energy and/or osmotically active metabolites such as glycerol, which generate the very high turgor pressure known to be crucial for penetration of hard surfaces. On softer surfaces such as onion epidermis, appressoria of M. grisea were able to penetrate before degradation of lipid droplets had been completed.

Acid Phosphatase↗
PubMed2005

Surface-enhanced Raman scattering detection of lysophosphatidic acid.

Surface-enhanced Raman scattering using silver nanoparticles was applied to detect various forms of lysophosphatidic acid (LPA) to examine its potential application as an alternative to current detection methods of LPA as biomarkers of ovarian cancer. Enhancement of the Raman modes of the molecule, especially those related to the acyl chain within the 800-1300 cm(-1) region, was observed. In particular, the C-C vibration mode of the gauche-bonded chain around 1100 cm(-1) was enhanced to allow the discrimination of two similar LPA molecules. Given the molecular selectivity of this technique, the detection of LPA using SERS may eliminate the need for partial purification of samples prior to analysis in cancer screening.

Biomarkers, Tumor↗
PubMed2002

The integrin alpha(v)beta(3) and CD44 regulate the actions of osteopontin on osteoclast motility.

In the studies reported here we demonstrate that osteopontin is secreted from the basolateral surfaces of osteoclasts where it binds to the avb3-integrin, suggesting that it may be an autocrine factor. Osteopontin stimulation of osteoclasts produced changes in cell shape by causing disruption of peripheral podosome structures and formation of actin filaments at the leading edge of the migrating osteoclasts. The latter was part of the assumption of a motile phenotype prior to cells reforming peripheral ring type podosome containing clear zones. It is well established in our laboratory as well as in others that osteopontin stimulated osteoclast motility and bone resorption. The effect of osteopontin was mimicked by RGD containing peptides and blocked by a avb3 antibody, demonstrating that signals generated by integrin ligation contributed to the actions of osteopontin. In addition, the migratory effects of osteopontin on osteoclasts were also mediated through CD44 receptors since blocking antibodies to CD44 blocked stimulation of motility. Our data strongly suggest that osteopontin is an osteoclast autocrine motility factor binding to avb3 and CD44 during stimulation of osteoclast migration.

Actins↗
PubMed2005

Resource level affects relative performance of the two motility systems of Myxococcus xanthus.

The adventurous (A) and social (S) motility systems of the microbial predator Myxococcus xanthus show differential swarming performance on distinct surface types. Under standard laboratory conditions, A-motility performs well on hard agar but poorly on soft agar, whereas the inverse pattern is shown by S-motility. These properties may allow M. xanthus to swarm effectively across a greater diversity of natural surfaces than would be possible with one motility system alone. Nonetheless, the range of ecological conditions under which dual motility enhances effective swarming across distinct surfaces and how ecological parameters affect the complementarity of A-motility and S-motility remain unclear. Here we have examined the role of nutrient concentration in determining swarming patterns driven by dual motility on distinct agar surfaces, as well as the relative contributions of A-motility and S-motility to these patterns. Swarm expansion rates of dually motile (A+S+), solely A-motile (A+S-), and solely S-motile (A-S+) strains were compared on hard and soft agar across a wide range of casitone concentrations. At low casitone concentrations (0-0.1%), swarming on soft agar driven by S-motility is very poor, and is significantly slower than swarming on hard agar driven by A-motility. This reverses at high casitone concentration (1-3.2%) such that swarming on soft agar is much faster than swarming on hard agar. This pattern greatly constrained the ability of M. xanthus to encounter patches of prey bacteria on a soft agar surface when nutrient levels between the patches were low. The swarming patterns of a strain that is unable to produce extracellular fibrils indicate that these appendages are responsible for the elevated swarming of S-motility at high resource levels. Together, these data suggest that large contributions by S-motility to predatory swarming in natural soils may be limited to soft, wet, high-nutrient conditions that may be uncommon. Several likely benefits of S-motility to the M. xanthus life cycle are discussed, including synergistic interactions with A-motility across a wide variety of conditions.

Agar↗
PubMed2005

Role of lipid protrusions in the function of interfacial enzymes.

Secretory phospholipase A(2) (sPLA(2)) is a class of interfacially active enzymes that selectively hydrolyze lipid molecules organized at interfaces like membranes. We present a simple theoretical model that relates the sPLA(2) action to the protrusions of the lipid molecules. The model explains (1) the observed enhancement of enzymatic activity by lipids with flexible, neutral, water-soluble polymers linked to their head groups and (2) the lag-burst kinetics of sPLA(2). It yields qualitative predictions of the effect of the initial composition of the membrane, the molecular weight of the polymer, and the composition of the hydrolysis products.

Cell Surface Extensions↗
PubMed2004

Localization of selenium-binding protein at the tips of rapidly extending protrusions.

Cell protrusive motility underlies cell fundamental biological processes such as cell growth, locomotion, and migration. Here I showed that selenium-binding protein (SBP) was exclusively located at the leading edges of rapidly growing protrusions in newly plated T98G glioma cells, and at the growing tips of the neurites in SH-SY5Y neuroblastoma cells. Double staining by anti-SBP antibody and deoxyribonuclease (DNase I) that labels monomeric G-actin or phalloidin that labels filamentous F-actin showed that the SBP-positive area was overstained by DNase I but, surprisingly, was not stained by phalloidin. When the cells were incubated with chemicals which block actin polymerization or activity of phosphatidylinositol 3-kinase, recruitment of SBP and G-actin at the cell margin was still observed, showing that their recruitment precedes actin polymerization. Taken together, I suggest that SBP may be involved in the initial sequential events in rapid cell outgrowth, such as determining direction of cell outgrowth and recruitment of actin monomer.

Actins↗