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PubMed1983

Cell surface heparan sulfate mediates some adhesive responses to glycosaminoglycan-binding matrices, including fibronectin.

Proteins with affinities for specific glycosaminoglycans (GAC's) were used as probes for testing the potential of cell surface GAG's to mediate cell adhesive responses to extracellular matrices (ECM). Plasma fibronectin (FN) and proteins that bind hyaluronate (cartilage proteo-glycan core and link proteins) or heparan sulfate (platelet factor 4 [PF4]) were adsorbed to inert substrata to evaluate attachment and spreading of several 3T3 cell lines. Cells failed to attach to hyaluronate-binding substrata. The rates of attachment on PF4 were identical to those on FN; however, PF4 stimulated formation of broad convex lamellae but not tapered cell processes fibers during the spreading response. PF4-mediated responses were blocked by treating the PF4-adsorbed substratum with heparin (but not chondroitin sulfate), or alternatively the cells with Flavobacter heparinum heparinase (but not chondroitinase ABC). Heparinase treatment did not inhibit cell attachment to FN but did inhibit spreading. Cells spread on PF4 or FN contained similar Ca2+-independent cell-substratum adhesions, as revealed by EGTA-mediated retraction of their substratum-bound processes. Microtubular networks reorganized in cells on PF4 but failed to extend into the broadly spread lamellae, where fine microfilament bundles had developed. Stress fibers, common on FN, failed to develop on PF4. These experiments indicate that (a) heparan sulfate proteoglycans are critical mediators of cell adhesion and heparan sulfate-dependent adhesion via PF4 is comparable in some, but not all, ways to FN-mediated adhesion, (b) the uncharacterized and heparan sulfate-independent "cell surface" receptor for FN permits some but not all aspects of adhesion, and (c) physiologically compatible and complete adhesion of fibroblasts requires binding of extracellular matrix FN to both the unidentified "cell surface" receptor and heparan sulfate proteoglycans.

Animals↗
PubMed1968

Analysis of glycerol-3H transport in the frog oocyte by extractive and radioautographic techniques.

The efflux of glycerol-(3)H from mature R. pipiens oocytes was studied by extractive analysis and by quantitative radioautography using techniques suitable for diffusible solutes. Extractive analysis was used to determine the total cellular concentration of tracer, and radioautography, regional intracellular concentrations, at equilibrium and as a function of efflux time, t(E). The efflux was resolvable into four kinetic fractions: cytoplasmic fast and slow fractions, and nuclear fast and slow fractions. The fast fractions represent freely diffusible glycerol in the two compartments; the solvent space accessible to glycerol is unity in the nucleus (germinal vesicle), but only 0.73 in the cytoplasm. The efflux of both fast fractions from the cell is determined by the permeability of the cortical membrane, with neither the nuclear membrane nor diffusion in the cytoplasm detectably slowing the flux. The permeability at 13.6 degrees C is 2.2 x 10(-5) cm/sec. The slow fractions leave the cell at about one-tenth the rate of the fast; the interpretation is that these fractions represent glycerol bound to impermeant cellular constituents. The size of these constituents is below the radioautographic resolution; in the cytoplasm, they appear not to be the yolk platelets. The extent of binding is about fourfold greater, per milliliter of compartment water, in the cytoplasm than in the germinal vesicle.

Animals↗
PubMed1999

Microtubule-based endoplasmic reticulum motility in Xenopus laevis: activation of membrane-associated kinesin during development.

The endoplasmic reticulum (ER) in animal cells uses microtubule motor proteins to adopt and maintain its extended, reticular organization. Although the orientation of microtubules in many somatic cell types predicts that the ER should move toward microtubule plus ends, motor-dependent ER motility reconstituted in extracts of Xenopus laevis eggs is exclusively a minus end-directed, cytoplasmic dynein-driven process. We have used Xenopus egg, embryo, and somatic Xenopus tissue culture cell (XTC) extracts to study ER motility during embryonic development in Xenopus by video-enhanced differential interference contrast microscopy. Our results demonstrate that cytoplasmic dynein is the sole motor for microtubule-based ER motility throughout the early stages of development (up to at least the fifth embryonic interphase). When egg-derived ER membranes were incubated in somatic XTC cytosol, however, ER tubules moved in both directions along microtubules. Data from directionality assays suggest that plus end-directed ER tubule extensions contribute approximately 19% of the total microtubule-based ER motility under these conditions. In XTC extracts, the rate of ER tubule extensions toward microtubule plus ends is lower ( approximately 0.4 microm/s) than minus end-directed motility ( approximately 1.3 microm/s), and plus end-directed motility is eliminated by a function-blocking anti-conventional kinesin heavy chain antibody (SUK4). In addition, we provide evidence that the initiation of plus end-directed ER motility in somatic cytosol is likely to occur via activation of membrane-associated kinesin.

Animals↗
PubMed1998

The myosin I SH3 domain and TEDS rule phosphorylation site are required for in vivo function.

The class I myosins play important roles in controlling many different types of actin-based cell movements. Dictyostelium cells either lacking or overexpressing amoeboid myosin Is have significant defects in cortical activities such as pseudopod extension, cell migration, and macropinocytosis. The existence of Dictyostelium null mutants with strong phenotypic defects permits complementation analysis as a means of exploring important functional features of the myosin I heavy chain. Mutant Dictyostelium cells lacking two myosin Is exhibit profound defects in growth, endocytosis, and rearrangement of F-actin. Expression of the full-length myoB heavy chain in these cells fully rescues the double mutant defects. However, mutant forms of the myoB heavy chain in which a serine at the consensus phosphorylation site has been altered to an alanine or in which the C-terminal SH3 domain has been removed fail to complement the null phenotype. The wild-type and mutant forms of the myoB heavy chain appeared to be properly localized when they were expressed in the myosin I null mutants. These results suggest that the amoeboid myosin I consensus phosphorylation site and SH3 domains do not play a role in the localization of myosin I, but are absolutely required for in vivo function.

Actins↗
PubMed2001

Dynamics of intracellular granules with CD63-GFP in rat basophilic leukemia cells.

CD63 is located on the basophilic granule membranes in resting basophils, mast cells, and platelets, and is also located on the plasma membranes of the cells. We constructed a CD63-GFP (green fluorescent protein) plasmid and introduced it into rat basophilic leukemia (RBL-2H3) cells to observe the movements of CD63 on degranulation. The movements of CD63-GFP were studied in living RBL cells by confocal laser scanning microscopy (CLSM). CD63-GFP, in which GFP was conjugated to the C-terminus of CD63, was located on both the granule membranes and the plasma membranes of RBL cells. The diameter of the fluorescent granules in the cytoplasm varied from 0.5 to 1.5 microm. Before antigen stimulation most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation the plasma membranes ruffled violently and the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. Analysis of the movement of each granule provided a new insight into the elementary process of degranulation. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.1+/-0.02 microm/s. This shows that the granules are able to reach the plasma membranes in 2 or 3 min if the diameter of the cells is 20 microm.

Animals↗
PubMed2000

Blue light-induced chloroplast relocation in Arabidopsis thaliana as analyzed by microbeam irradiation.

Chloroplast relocation in mesophyll cells of Arabidopsis thaliana was observed microscopically and analyzed by microbeam irradiation. Chloroplasts located along the anticlinal walls in dark-adapted cells. When part of a cell was irradiated with a microbeam of high fluence rate blue light (B) simultaneously with background red light (R) on the whole cell, the chloroplasts moved towards the B-irradiated area, but did not enter the beam. The background R illumination activated cytoplasmic motility as well as chloroplast movement. Without R illumination, there was little chloroplast relocation. In light-adapted cells in which the chloroplasts were spread over the cell surface perpendicular to the incident light, R-illumination had the same effect. Under background R, the chloroplasts moved out of the area irradiated with a B microbeam of 8 or 30 W m(-2) (avoidance response), but chloroplasts outside the beam moved towards the area irradiated with the B microbeam (accumulation response). These results suggest that the signals for accumulation and avoidance responses were generated in a single cell by high fluence rate B. cry1cry2, npq1 and nph1 mutants showed B-induced chloroplast relocation. Both the accumulation and avoidance responses were observed in all the mutants, although in the nph1 mutant, the sensitivity of accumulation movement was slightly lower than that of the wild type. We discuss the possible photoreceptor for B-induced chloroplast relocation.

Adaptation, Physiological↗
PubMed2002

Temporal relationship between cytosolic free Ca(2+) and membrane potential during hypotonic turgor regulation in a brackish water Charophyte Lamprothamnium succinctum.

Internodal cells of a brackish water charophyte, Lamprothamnium succinctum, regulate turgor pressure in response to changes in external osmotic pressure by modifying vacuolar concentrations of KCl. An increase in cytosolic concentration of free Ca(2+) ([Ca(2+)](c)) is necessary for the progress of turgor regulation induced by hypotonic treatment. Initial changes in membrane potential and [Ca(2+)](c) upon hypotonic treatment were measured to examine the temporal relationship between the two parameters. Fura-dextran (potassium salt, M(r) 10,000, anionic) that had been injected into the cytosol was used to measure [Ca(2+)](c). Membrane potential and membrane conductance under a current-clamp condition were also measured. Decrease in external osmotic pressure by 0.16 Osm induced a simultaneous increase in [Ca(2+)](c) with both depolarization of the membrane and increase in the membrane conductance. Decrease in external osmotic pressure by 0.05 Osm induced a simultaneous increase in [Ca(2+)](c) with membrane depolarization but the increase in membrane conductance started later than the other two processes. There was a close temporal relationship between the increase in [Ca(2+)](c) and membrane depolarization on the initial response of turgor regulation induced by hypotonic treatment.

Biological Transport↗
PubMed2004

Obtaining multiple separate food sources: behavioural intelligence in the Physarum plasmodium.

To evaluate performance in a complex survival task, we studied the morphology of the Physarum plasmodium transportation network when presented with multiple separate food sources. The plasmodium comprises a network of tubular elements through which chemical nutrient, intracellular signals and the viscous body are transported and circulated. When three separate food sources were presented, located at the vertices of a triangle, the tubular network connected them via a short pathway, which was often analogous to the mathematically shortest route known as Steiner's minimum tree (SMT). The other common network shape had high fault tolerance against accidental disconnection of the tubes and was known as cycle (CYC). Pattern selection appeared to be a bistable system involving SMT and CYC. When more than three food sources were presented, the network pattern tended to be a patchwork of SMT and CYC. We therefore concluded that the plasmodium tube network is a well designed and intelligent system.

Animals↗
PubMed2003

Regulation of actin-dependent cytoplasmic motility by type II phytochrome occurs within seconds in Vallisneria gigantea epidermal cells.

The effects of light on actin-dependent cytoplasmic motility in epidermal cells of green leaves of the aquatic angiosperm Vallisneria gigantea were investigated quantitatively using a custom-made dynamic image analyzer. Cytoplasmic motility was measured by monitoring changes in the brightness of individual pixels on digitized images taken sequentially under infrared light. Acceleration and deceleration of cytoplasmic motility were regulated photoreversibly by type II phytochrome(s). This phytochrome-dependent induction of cytoplasmic motility did not occur uniformly in cytoplasm but took place as scattered patches in which no particular organelles, including nucleus, existed. The induction became detectable at 2.5 s after the start of irradiation with pulsed red light. In cells exposed to microbeam irradiation, cytoplasmic motility was induced only in sites in the cytoplasm that were irradiated directly, whereas nonirradiated neighboring areas were unaffected. The effect was short-lived, disappearing within a few minutes, and no signal was transmitted from an irradiated cell to its neighbors. Anti-phytochrome antibody-responsive protein(s) was detectable in the leaf extract by immunoblot and zinc blot analyses and in cryosections of the epidermis by immunocytochemistry. Although the phytochrome-dependent cytoplasmic motility was blocked by exogenously applied latrunculin B or cytochalasins, treatment of the dark-adapted cells with Ca(2+)-chelating reagents induced the cytoplasmic motility. We have proposed a model for the phytochrome regulation of cytoplasmic motility as one of the earliest responses to a light stimulus.

Actin Cytoskeleton↗
PubMed1989

Estimation of the energy of cytoplasmic movements by magnetometry: effects of temperature and intracellular concentration of ATP.

Ferrimagnetic particles (gamma-Fe2O3) were spontaneously phagocytized by pulmonary macrophages obtained from the lungs of hamsters. By the magnetometric method proposed earlier, changes in the energy of the movements of the intracellular structures and also that of the apparent viscosity of the cytoplasm were estimated. The changes were created by lowering the incubation temperature or by decreasing the intracellular concentration of ATP. While the low temperature decreased the energy and increased the apparent viscosity, the low concentration of ATP decreased the energy but had little effects on the apparent viscosity. Estimation of the these parameters was based on the model previously proposed.

Adenosine Triphosphate↗
PubMed1975

The effects of hypobaric atmospheres on living cells in layers of thin media and implications for electron microscopy.

At present, the only possibility for improved resolution of intracellular movement appears to be electron microscopy using a hydration chamber (EMC) at reduced gas pressures with thinned medium around the cells. The environmental constraints of examining cells under such conditions were examined in the absence of ionizing radiation by using an EMC-analogue fitted to a light microscope. White blood cells and baby hamster kidney cells were examined. A 50% survival pressure value, for hypobaric atmospheres, was determined for these cells. Morphological changes of the hypobarically-exposed cells are described.

Air Pressure↗
PubMed2006

Ca2+ transient induced by extracellular changes in osmotic pressure in Arabidopsis leaves: differential involvement of cell wall-plasma membrane adhesion.

We investigated the mechanism underlying the perception of extracellular changes in osmotic pressure in Vallisneria gigantea Graebner and transgenic Arabidopsis thaliana (L.) Heynh. expressing cytoplasmic aequorin. Hypertonic and hypotonic treatments of A. thaliana leaves each rapidly induced a Ca2+ transient. Both responses were essentially dependent on the presence of extracellular Ca2+ and were sensitive to Gd3+ a potential blocker of stretch-activated Ca2+ channels. Immediately after plasmolysis caused by hypertonic treatment and subsequent deplasmolysis caused by hypotonic treatment, the cells did not respond to a second hypertonic treatment and exhibited an impaired adhesion of the plasma membrane (PM) to the cell wall (CW). Recovery of the responsiveness required about 6 h. By contrast, no refractory phenomenon was observed in response to hypotonic treatment. Pretreatment with cellulase completely inhibited the Ca2+ transient induced by hypertonic treatment, but it did not affect the response to hypotonic treatment. V. gigantea mesophyll cells pretreated with cellulase exhibited an impaired adhesion of the PM to the CW. The leaf cells of multicellular plants can respond to both hypertonic and hypotonic treatments through the stretch-activated Ca2+ channels, whereas cellulase-sensitive adhesion of the PM to the CW is involved only in the response to hypertonic treatment.

Actin Cytoskeleton↗