The functional significance of the ultrastructure of the vestibular end organs
Functional significance of ultrastructure of sensory hair cells in vestibular end organs
LABYRINTH↗
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Proliferation, differentiation and apoptosis in connexin43-null osteoblasts
Osteoblasts are highly coupled by gap junctions formed primarily by connexin43 (Cx43). We have shown that interference with Cx43 expression or function disrupts transcriptional regulation of osteoblast genes, and that deletion of Cx43 in the mouse causes skeletal malformations, delayed mineralization, and osteoblast dysfunction. Here, we studied the mechanisms by which genetic deficiency of Cx43 alters osteoblast development. While cell proliferation rates were similar in osteoblastic cells derived from calvaria of Cx43-null and wild type mice, camptothecin-induced apoptosis was 3-fold higher in mutant compared to wild type osteoblasts. When grown in mineralizing medium, Cx43-null cells were able to produce mineralized matrix but it took one week longer to reach the same mineralization levels as in normal cells. Likewise, expression of alkaline phosphatase activity per cell--a marker of osteoblast differentiation--was maximal only 2 weeks later in Cx43-null relative to wild-type cells. These observations suggest that Cx43 is important for a normal and timely development of the osteoblastic phenotype. Delayed differentiation and increase programmed cell death may explain the skeletal phenotype of Cx43-null mice.
NASA Discipline Cell Biology↗
Structural analysis of photosystem I polypeptides using chemical crosslinking
Thylakoid membranes, obtained from leaves of 14 d soybean (Glycine max L. cv. Williams) plants, were treated with the chemical crosslinkers glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to investigate the structural organization of photosystem I. Polypeptides were resolved using lithium dodecyl sulfate polyacrylamide gel electrophoresis, and were identified by western blot analysis using a library of polyclonal antibodies specific for photosystem I subunits. An electrophoretic examination of crosslinked thylakoids revealed numerous crosslinked products, using either glutaraldehyde or EDC. However, only a few of these could be identified by western blot analysis using subunit-specific polyclonal antibodies. Several glutaraldehyde dependent crosslinked species were identified. A single band was identified minimally composed of PsaC and PsaD, documenting the close interaction between these two subunits. The most interesting aspect of these studies was a crosslinked species composed of the PsaB subunit observed following EDC treatment of thylakoids. This is either an internally crosslinked species, which will provide structural information concerning the topology of the complex PsaB protein, a linkage with a polypeptide for which we do not yet have an immunological probe, or a masking of epitopes by the EDC linkage at critical locations in the peptide which is linked to PsaB.
NASA Discipline Number 93-10↗
Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds
Culture of seeded osteoblastic cells in three-dimensional osteoconductive scaffolds in vitro is a promising approach to produce an osteoinductive material for repair of bone defects. However, culture of cells in scaffolds sufficiently large to bridge critical-sized defects is a challenge for tissue engineers. Diffusion may not be sufficient to supply nutrients into large scaffolds and consequently cells may grow preferentially at the periphery under static culture conditions. Three alternative culturing schemes that convect media were considered: a spinner flask, a rotary vessel, and a perfusion flow system. Poly(DL-lactic-co-glycolic acid) (PLGA) foam discs (12.7 mm diameter, 6.0 mm thick, 78.8% porous) were seeded with osteoblastic marrow stromal cells and cultured in the presence of dexamethasone and L-ascorbic acid for 7 and 14 days. Cell numbers per foam were found to be similar with all culturing schemes indicating that cell growth could not be enhanced by convection, but histological analysis indicated that the rotary vessel and flow system produced a more uniform distribution of cells throughout the foams. Alkaline phosphatase (ALP) activity per cell was higher with culture in the flow system and spinner flask after 7 days, while no differences in osteocalcin (OC) activity per cell were observed among culturing methods after 14 days in culture. Based on the higher ALP activity and better cell uniformity throughout the cultured foams, the flow system appears to be the superior culturing method, although equally important is the fact that in none of the tests did any of the alternative culturing techniques underperform the static controls. Thus, this study demonstrates that culturing techniques that utilize fluid flow, and in particular the flow perfusion system, improve the properties of the seeded cells over those maintained in static culture.
NASA Discipline Cell Biology↗
Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure
We report here that living cells and nuclei are hard-wired such that a mechanical tug on cell surface receptors can immediately change the organization of molecular assemblies in the cytoplasm and nucleus. When integrins were pulled by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoskeleton and nucleus. Actin microfilaments mediated force transfer to the nucleus at low strain; however, tearing of the actin gel resulted with greater distortion. In contrast, intermediate filaments effectively mediated force transfer to the nucleus under both conditions. These filament systems also acted as molecular guy wires to mechanically stiffen the nucleus and anchor it in place, whereas microtubules acted to hold open the intermediate filament lattice and to stabilize the nucleus against lateral compression. Molecular connections between integrins, cytoskeletal filaments, and nuclear scaffolds may therefore provide a discrete path for mechanical signal transfer through cells as well as a mechanism for producing integrated changes in cell and nuclear structure in response to changes in extracellular matrix adhesivity or mechanics.
NASA Discipline Cell Biology↗
Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix
This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In contrast, cell spreading could be completely inhibited by combining suboptimal doses of cytochalasin D and nocodazole, suggesting that intact microtubules can stabilize cell form when the microfilament lattice is partially compromised. The physiological relevance of the cytoskeleton and cell shape in hepatocyte physiology was highlighted by the finding that a short exposure (6 hour) of cells to nocodazole resulted in production of smaller cells 42 hours later that exhibited enhanced production of a liver-specific product (albumin). These data demonstrate that spreading and flattening of the entire cell body is not driven directly by net polymerization of either microfilaments or microtubules.(ABSTRACT TRUNCATED AT 400 WORDS).
NASA Discipline Cell Biology↗
Three distinct cell phenotypes of induced-TNF cytotoxicity and their relationship to apoptosis
We have identified three distinct cell phenotypes with respect to the conditions under which cells became susceptible to TNF-mediated lysis. These conditions include: 1) treatment with the protein synthesis inhibitor, cycloheximide; 2) contact with activated macrophages, and 3) infection with vaccinia virus. Whereas vaccinia virus-infected 3T3 cells became sensitive to soluble TNF, F5b cells required contact with activated macrophages. We showed that the "macrophage-resistant" F5m cells did not become sensitive to TNF or to killing by activated macrophages after infection with vaccinia virus. Therefore, vaccinia infection does not sensitize all cells to TNF. We also determined the pathways of lysis for cells after sensitization. Whereas 3T3, LM929, and F5b cells were killed by the process of necrosis, F5m cells lysis was characterized by the release of low mol wt DNA fragments (apoptosis).
Non-NASA Center↗
The role of microtubules in contractile ring function
During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.
NASA Discipline Cell Biology↗
Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells
A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.
Non-NASA Center↗
Cell prestress. II. Contribution of microtubules
The tensegrity model hypothesizes that cytoskeleton-based microtubules (MTs) carry compression as they balance a portion of cell contractile stress. To test this hypothesis, we used traction force microscopy to measure traction at the interface of adhering human airway smooth muscle cells and a flexible polyacrylamide gel substrate. The prediction is that if MTs balance a portion of contractile stress, then, upon their disruption, the portion of stress balanced by MTs would shift to the substrate, thereby causing an increase in traction. Measurements were done first in maximally activated cells (10 microM histamine) and then again after MTs had been disrupted (1 microM colchicine). We found that after disruption of MTs, traction increased on average by approximately 13%. Because in activated cells colchicine induced neither an increase in intracellular Ca(2+) nor an increase in myosin light chain phosphorylation as shown previously, we concluded that the observed increase in traction was a result of load shift from MTs to the substrate. In addition, energy stored in the flexible substrate was calculated as work done by traction on the deformation of the substrate. This result was then utilized in an energetic analysis. We assumed that cytoskeleton-based MTs are slender elastic rods supported laterally by intermediate filaments and that MTs buckle as the cell contracts. Using the post-buckling equilibrium theory of Euler struts, we found that energy stored during buckling of MTs was quantitatively consistent with the measured increase in substrate energy after disruption of MTs. This is further evidence supporting the idea that MTs are intracellular compression-bearing elements.
Non-NASA Center↗
Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target
Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 microm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.
NASA Program Fundamental Space Biology↗
Suppressed PHA activation of T lymphocytes in simulated microgravity is restored by direct activation of protein kinase C
Utilizing clinostatic rotating wall vessel (RWV) bioreactors that simulate aspects of microgravity, we found phytohemagglutinin (PHA) responsiveness to be almost completely diminished. Activation marker expression was significantly reduced in RWV cultures. Furthermore, cytokine secretion profiles suggested that monocytes are not as adversely affected by simulated microgravity as T cells. Reduced cell-cell and cell-substratum interactions may play a role in the loss of PHA responsiveness because placing peripheral blood mononuclear cells (PBMC) within small collagen beads did partially restore PHA responsiveness. However, activation of purified T cells with cross-linked CD2/CD28 and CD3/CD28 antibody pairs was completely suppressed in the RWV, suggesting a defect in signal transduction. Activation of purified T cells with PMA and ionomycin was unaffected by RWV culture. Furthermore, sub-mitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture. Thus our data indicate that during polyclonal activation the signaling pathways upstream of PKC activation are sensitive to simulated microgravity.
NASA Discipline Cell Biology↗
Van slyke's buffer values for cell secretions
Van Slykes buffer values for Chlorella cell secretions into surrounding medium
BUFFER↗
Anatomical aspects on functional organization of the vestibular nuclei
Vestibular nuclear complex of cat, afferent and efferent fiber connections and synaptic arrangements, and dissimilarity in functioning of cell groups and parts of nuclei
SYNAPSE↗
Studies of liver and gastrointestinal tract irradiated with protons Progress report
Proton irradiation effects on cells of gastrointestinal tract and liver
LIVER↗
The NASA Specialized Center of Research and Training (NSCORT) in Gravitational Biology
The Life Sciences Division of NASA has initiated a NASA Specialized Centers of Research and Training (NSCORT) program. Three Centers were designated in late 1990, as the culmination of an in-depth peer review analysis of proposals from universities across the nation and around the world. Kansas State University was selected as the NSCORT in Gravitational Biology. This Center is headquartered in the KSU Division of Biology and has a research, training, and outreach function that focuses on cellular and developmental biology.
NASA Discipline Cell Biology↗
Studies on the effects of microgravity on the ultrastructure and functions of cultured mammalian cells (L-6)
The human body consists of 10(exp 13) cells. Understanding the mechanisms by which the cells sense and respond to microgravity is very important as the basis for space biology. The cells were originally isolated aseptically from mammalian bodies and cultured in vitro. A set of cell culture vessels was developed to be applied to three kinds of space flight experiments. Experiment 1 is to practice the cell culture technique in a space laboratory and obtain favorable growth of the cells. Aseptic handling in tryspin treatment and medium renewal will be tested. The cells, following space flight, will be returned to the ground and cultured continuously to investigate the effects of space flight on the cellular characteristics. Experiment 2 is to examine the cytoskeletal structure of the cells under microgravity conditions. The cytoskeletal structure plays essential roles in the morphological construction, movements, axonal transport, and differentiation of the cells. The cells fixed during space flight will be returned and the cytoskeleton and ultrastructure observed using electron microscopy and fluorescence microscopy. Experiment 3 is to study the cellular productivity of valuable substances. The waste medium harvested during space flight are returned and quantitated for the cellular products. The effects of microgravity on mammalian cells will be clarified from the various aspects.
Sato, Atsushige↗
Furrow microtubules and localized exocytosis in cleaving Xenopus laevis embryos
In dividing Xenopus eggs, furrowing is accompanied by expansion of a new domain of plasma membrane in the cleavage plane. The source of the new membrane is known to include a store of oogenetically produced exocytotic vesicles, but the site where their exocytosis occurs has not been described. Previous work revealed a V-shaped array of microtubule bundles at the base of advancing furrows. Cold shock or exposure to nocodazole halted expansion of the new membrane domain, which suggests that these microtubules are involved in the localized exocytosis. In the present report, scanning electron microscopy revealed collections of pits or craters, up to approximately 1.5 micro m in diameter. These pits are evidently fusion pores at sites of recent exocytosis, clustered in the immediate vicinity of the deepening furrow base and therefore near the furrow microtubules. Confocal microscopy near the furrow base of live embryos labeled with the membrane dye FM1-43 captured time-lapse sequences of individual exocytotic events in which irregular patches of approximately 20 micro m(2) of unlabeled membrane abruptly displaced pre-existing FM1-43-labeled surface. In some cases, stable fusion pores, approximately 2 micro m in diameter, were seen at the surface for up to several minutes before suddenly delivering patches of unlabeled membrane. To test whether the presence of furrow microtubule bundles near the surface plays a role in directing or concentrating this localized exocytosis, membrane expansion was examined in embryos exposed to D(2)O to induce formation of microtubule monasters randomly under the surface. D(2)O treatment resulted in a rapid, uniform expansion of the egg surface via random, ectopic exocytosis of vesicles. This D(2)O-induced membrane expansion was completely blocked with nocodazole, indicating that the ectopic exocytosis was microtubule-dependent. Results indicate that exocytotic vesicles are present throughout the egg subcortex, and that the presence of microtubules near the surface is sufficient to mobilize them for exocytosis at the end of the cell cycle.
Non-NASA Center↗