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The present work is focused on the influence of the high-gradient-magnetic field (HGMF) on spatial distribution of ion fluxes along the roots (a), cytoplasmic streaming (b), and the processes of plant cell growth connected with intracellular mass and charge transfer (c).
When one pseudopod of an amoeba is sucked into a capillary connected to a partial vacuum and subjected to a pressure reduction of 30 to 35 centimeters of water, extension of other pseudopods, exposed to atmospheric pressure, is not prevented. This result is interpreted to mean that cytoplasmic streaming cannot be the result of a positive pressure gradient generated along the length of the stream, for if it were, streaming would have reversed its direction under the applied pressure gradient of opposite sign and supposedly greater magnitude.
Programmed cell death (PCD) functions in the developmental remodeling of leaf shape in higher plants, a process analogous to digit formation in the vertebrate limb. In this study, we provide a cytological characterization of the time course of events as PCD remodels young expanding leaves of the lace plant. Tonoplast rupture is the first PCD event in this system, indicated by alterations in cytoplasmic streaming, loss of anthocyanin color, and ultrastructural appearance. Nuclei become terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive soon afterward but do not become morphologically altered until late stages of PCD. Genomic DNA is fragmented, but not into internucleosomal units. Other cytoplasmic changes, such as shrinkage and degradation of organelles, occur later. This form of PCD resembles tracheary element differentiation in cytological execution but requires unique developmental regulation so that discrete panels of tissue located equidistantly between veins undergo PCD while surrounding cells do not.
Cellular and intracellular motile events in plants are susceptible to SH reagents such as N-ethylmaleimide (NEM). It has long been believed that the target of the reagent is myosin. We compared the effect of NEM on the motile and ATPase activities of skeletal muscle myosin with that on plant myosin using characean algal myosin. It was found that the motile activity of myosin prepared from NEM-treated C. corallina decreased to a level accountable for the decrease in the velocity of cytoplasmic streaming but it was also found that Chara myosin was far less susceptible to NEM than skeletal muscle myosin.
Solutions containing heavy meromyosin, actin, native tropomyosin, and Mg-ATP exhibited streaming in horizontally placed glass microcapillaries. Up-hill streaming could also be observed when the capillaries were at an inclined position; this served for the clear distinction between active and passive streaming provided surface tension effects were eliminated. The presence of native tropomyosin and actin-activation of the ATPase activity of HMM were essential for the reconstitution of active streaming. The significance of the results for cytoplasmic streaming and muscle contraction is discussed.
Motile extracts from D, discoideum and A. proteus have been characterized in order to compare the structural dynamics and chemical regulation of movement in 2 different types of amoeboid cells. The structural dynamics of both extracts involve the formation of a nonmotile cytoskeleton followed by the contraction of actin and myosin to generate both direct contractile force and cytoplasmic streaming. The contractions are regulated by calcium ions and a threshold of ca. 1.0 X 10(-6) M calcium induces a transformation of actin to the free F-actin state which is capable of interacting with myosin. Furthermore, 3 low molecular weight proteins are concentrated along with actin and myosin during contraction and might play a regulatory role in movement. Several common characteristics of amoeba cytoplasm have been exhibited by these two types of amoeboid cells. The major contractile and "associated" proteins are similar, actin and associated proteins are structurally dynamic, and movement is regulated by calcium. The different modes of movement observed in different types of amoeboid cells could result from the site, rate, and extent of actin transformation followed in some regions by contractions.
The ubiquitin/proteasome pathway represents one of the most important proteolytic systems in eukaryotes and has been proposed as being involved in pollen tube growth, but the mechanism of this involvement is still unclear. Here, we report that proteasome inhibitors MG132 and epoxomicin significantly prevented Picea wilsonii pollen tube development and markedly altered tube morphology in a dose- and time-dependent manner, while hardly similar effects were detected when cysteine-protease inhibitor E-64 was used. Fluorogenic kinetic assays using fluorogenic substrate sLLVY-AMC confirmed MG132-induced inhibition of proteasome activity. The inhibitor-induced accumulation of ubiquitinated proteins (UbPs) was also observed using immunoblotting. Transmission electron microscopy revealed that MG132 induces endoplasmic reticulum (ER)-derived cytoplasmic vacuolization. Immunogold-labeling analysis demonstrated a significant accumulation of UbPs in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescence labeling with fluorescein isothiocyanate-phalloidin and beta-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules and consequently affects cytoplasmic streaming in pollen tubes. However, tip-focused Ca2+ gradient, albeit reduced, seemingly persists after MG132 treatment. Finally, fluorescence labeling with antipectin antibodies and calcofluor indicated that MG132 treatment induces a sharp decline in pectins and cellulose. This result was confirmed by Fourier transform infrared analysis, thus demonstrating for the first time the inhibitor-induced weakening of tube walls. Taken together, these findings suggest that MG132 treatment promotes the accumulation of UbPs in pollen tubes, which induces ER-derived cytoplasmic vacuolization and depolymerization of cytoskeleton and consequently strongly affects the deposition of cell wall components, providing a mechanistic framework for the functions of proteasome in the tip growth of pollen tubes.
Through the injection of f-aequorin (a calcium-specific luminescent reporter), and the use of an imaging photon detector, transient localized elevations of free cytosolic calcium in the forming blastodisc (BD) and animal hemisphere cortex were visualized that correlated with ooplasmic segregation. The introduction of an appropriate concentration of the weak (KD = 1.5 micromol/L) calcium buffer 5,5'-dibromo-BAPTA results in the dissipation of these calcium domains, and inhibits cytoplasmic streaming and the subsequent formation of a BD at the animal pole. These inhibitory actions are dependent on the final cytosolic concentration of buffer within the egg: > or = 1.3 mmol/L blocks ooplasmic streaming; < 1.3 mmol/L eggs segregate normally. Injection of 5,5'-dimethyl-BAPTA (KD = 0.15 micromol/L) to a final concentration of 1.5 mmol/L as a control has no effect on ooplasmic streaming. These results suggest that localized domains of elevated free cytosolic calcium are essential for ooplasmic segregation in zebrafish. Furthermore, a hypothetical model is presented linking these calcium transients to the contraction of a cortically located actin microfilament network as a possible mechanism providing the driving force for segregation.
Mycelial fungi play a central role in element cycling in nature by degrading dead organic material such as wood. Fungal colonization of a substrate starts with the invasion of exploring hyphae. These hyphae secrete enzymes that convert the organic material into small molecules that can be taken up by the fungus to serve as nutrients. Using green fluorescent protein (GFP) as a reporter, we show for the first time that exploring hyphae of Aspergillus niger differentiate with respect to enzyme secretion; some strongly express the glucoamylase gene glaA, while others hardly express it at all. When a cytoplasmic GFP was used, 27% of the exploring hyphae of a 5-day-old colony belonged to the low expressing hyphae. By fusing GFP to glucoamylase and by introducing an ER retention signal, this number increased to 50%. This difference is due to cytoplasmic streaming of the reporter in the former case, as was shown by using a photo-activatable GFP. Our findings indicate that a fungal mycelium is highly differentiated, especially when taking into account that hyphae in the exploration zone were exposed to the same nutritional conditions.
Myosin from the slime mold Physarum polycephalum contains three sizes of polypeptides: a heavy chain and two light chains, LC-1 and LC-2. Using a simple qualitative test for calcium binding by comparing electrophoretic migration of the polypeptides in sodium dodecyl sulfate (SDS) acrylamide gels in the presence and absence of calcium, we have found that Physarum myosin light chain LC-2 migrates with an apparent molecular weight of 16,900 daltons in the presence of the metal ion chelator ethylene glycol bis (B-aminoethyl ether) N,N'-tetraacetic acid (EGTA). However, if calcium chloride is added to the sample prior to electrophoresis, the apparent molecular weight decreases to 16,100. Lanthanide and cadmium ions, but not magnesium, can substitute for calcium. Because the ionic radii of Ca2+, La3+, and Cd2+ are almost identical, we conclude that Physarum myosin LC-2 possesses a very size-specific binding site for calcium. Physarum myosin LC-1 and the heavy chain give no evidence for binding calcium by this test. Since cytoplasmic streaming in the plasmodium of Physarum requires calcium, our evidence indicates that the calcium-binding property of Physarum myosin LC-2 may be important in regulating the production of force by actomyosin in the ectoplasm. Unexpectedly, the myosin light chain in Physarum capable of binding calcium, LC-2, is the essential light chain, while LC-1 is a member of the regulatory class of myosin light chains [V. T. Nachmias, personal communication]. Until now, essential myosin light chains have not been shown to have high affinity divalent cation binding sites. This means a new version of the myosin-based model for actomyosin regulation by calcium may be required to explain cytoplasmic movement in Physarum, and perhaps in other motile systems involving cytoplasmic myosins as well.
Evidence for actin-dependent organelle movement was first obtained from studies of cytoplasmic streaming in plants. These studies, together with cell-free organelle motility studies and biophysical analyses of muscle myosin, support a model whereby organelle-associated motor molecules utilize the energy of adenosine triphosphate binding and hydrolysis to drive movement along F-actin tracks. Recent studies indicate that this mechanism for organelle movement may be responsible for organelle and vesicle movement during secretion, endocytosis and mitochondrial inheritance in a variety of eukaryotes.
Using a newly developed vibrating electrode, we have explored the electric fields around lily pollen germinating in vitro. From these field measurements, we infer that each weeted pollen drives a steady current of a few hundred picoamperes through itself. Considered as a flow of positive ions, this current enters an ungerminated grain's prospective growth site and leaves it opposite end. After a grain germinates and forms a tube, this current enters most of the growing tube and leaves the whole grain. The current densities over both of these extended surface regions are relatively uniform, and the boundary zone, near the tube's base, is relatively narrow. This current continues as long as the tube grows, and even continues when elongation, as well as cytoplasmic streaming, are blocked by 1 mug/ml of cytochalasin B. After a otherwise indistinguishable minority of tubes have grown to lengths of a millimeter or more, their current comes to include an endless train of discrete and characteristic current pulses as well as a steady component. These pulses are about 30s long, never overlap, recur every 60-100s, and seem to enter a region more restricted to be growing tip than the steady current's sink. In most ways, the current through growing lily pollen resembles that known to flow through focoid eggs.
Data and theories about the identity of the mass that acts in gravitropic sensing are reviewed. Gravity sensing may have evolved several times in plants and algae in processes such as gravitropism of organs and tip-growing cells, gravimorphism, gravitaxis, and the regulation of cytoplasmic streaming in internodal cells of Chara. In the latter and in gravitaxis, the mass of the entire cell may function in sensing. But gravitropic sensing appears to rely upon the mass of amyloplasts that sediment since (i) the location of cells with sedimentation is highly regulated, (ii) such cells contain other morphological specializations favoring sedimentation, (iii) sedimentation always correlates with gravitropic competence in wild-type plants, (iv) magnetophoretic movement of rootcap amyloplasts mimics gravitropism, and (v) starchless and intermediate starch mutants show reduced gravitropic sensitivity. The simplest interpretation of these data is that gravitropic sensing is plastid-based.
At first sight the alpha-helix appears as a rigid scaffold braced by hydrogen bonds nearly parallel to the helix axis. Looked at more closely it turned out to be highly dynamic and able to transform chemical into mechanical energy. The hydrogen bonds are fairly weak and compliant bonds. Their length, usually between 0.267 and 0.291 nm (mean value, 0.28 nm), depends on the interaction of the side chains. The most important strong interaction is the electrostatic repelling force between equally charged side chains (Glu-, Asp-, Lys+, Arg+), well known by experiments with polyamino acids. In proteins with different amino acids, repelling forces between charged side chains work in the axial direction and stretch the hydrogen bonds. Extreme shortening of the hydrogen bonds occurs when ions, e.g., Ca2+, H+, or PO3-, are added and discharge side chains. This means a cooperative pitch decrease of the alpha-helix (pitch range between 0.52 and more than 0.55 nm; mean value, 0.54 nm). This pitch change is absolutely connected by steric reasons with torque generation and torsional rotations, as demonstrated by molecular and tubular alpha-helix models. Thus, charged alpha-helices are molecular motors propelled by the electrostatic energy of added ions. The motor effect is most striking with highly charged alpha-helical coiled coils, e.g., tropomyosin, myosin, and alpha-actinin that can rotate actin filaments by winding and unwinding. For example, the shortening of muscle depends on the sliding (drilling) motion of the Ca2+-activated helical actin filaments into the cross-bridges of the A-band. Here, models are presented for the in vitro sliding of actin filaments and for cytoplasmic streaming by winding and unwinding of myosin chains, and for membrane proteins that contain nonhelical domains between membrane-penetrating alpha-helices. They may transport molecules by the described torsional rotations if they perform supercoiling. Winding and supercoiling can lead to displacement of bound ions and to a feed-back-regulated oscillation between two different coiling stages E1 and E2 that explain "eversion". The models need the torque for 1-2 rotations. They explain active and passive transports, the driving-effects of ion gradients, ATP hydrolysis by unwinding, ATP synthesis by winding up of the supercoils, etc.
In mesophyll cells of Vallisneria gigantea Graebner, an aquatic angiosperm, the association of the plasma membrane with the cell wall at the end wall has been reported to be indispensable for the mechanism that maintains the stationary organization of the bundles of microfilaments (MFs) [Masuda et al. (1991) Protoplasma 162: 151]. To identify putative extracellular components that might play a crucial role in this mechanism, we examined the effects of two exogenously applied synthetic hexapeptides, GRGDSP and ARYDEI, which include an RGD and an RYD motif, respectively. The RGD motif is known as a recognition site in molecules required for adhesion to the substratum at sites of focal contacts. Within 24 h, both peptides (at concentrations of 1-15 mM) induced extremely abnormal patterns of cytoplasmic streaming, as well as the striking disruption of the arrangement of bundles of MFs. GRGESP and ARYEEI peptides, used as controls, had no detectable effects. Immunofluorescence microscopy revealed that polyclonal antibodies against the ARYDEI peptide bound to the cell walls of mesophyll cells while a preimmune serum did not. Western blotting analysis demonstrated that the antibodies recognized polypeptides of 54 kDa and 27 kDa in an extract of total proteins from the leaves of Vallisneria. The results suggest that some extracellular proteins(s), with a conserved RGD or RYD motif in its amino acid sequence, might be involved in the maintenance of the stationary organization of the bundles of MFs.
Reticulomyxa is a large multinucleated freshwater protozoan that provides a new model system in which to study intracellular transport and cytoskeletal dynamics. Within the cell body and reticulopodial network, rapid, visually striking saltatory organelle motility as well as bulk cytoplasmic streaming can be readily observed. In addition, the cytoskeletal elements within these strands undergo dynamic splaying and fusing rearrangements, which can be visualized by video-enhanced light microscopy. A reactivatable lysed cell model has been developed that appears to preserve, and therefore permits examination of, these three forms of motility in a more controlled environment. Individual organelle movements are microtubule-based and have similarities to, but also differences from, the recently described kinesin-based transport. This lysed cell model can be further manipulated to provide native, ordered, completely exposed networks of either microtubules or microfilaments, or a combination of both, and thus may serve as a versatile motility assay system in which to examine the movement of exogenously added isolated organelles or latex beads.
Subcellular localization of gene products and cell migration are both critical for pattern formation during development. The bullwinkle gene is required in Drosophila for disparate aspects of these processes. In females mutant at the bullwinkle locus, the follicle cells that synthesize the dorsal eggshell filaments do not migrate properly, creating short, broad structures. Mosaic analyses demonstrate that wild-type BULLWINKLE function is required in the germ line for these migrations. Since the mRNA for gurken, the putative ligand that signals dorsal follicle cell fate, is correctly localized in bullwinkle mutants, we conclude that our bullwinkle alleles do not affect the dorsoventral polarity of the oocyte and thus must be affecting the follicle cell migrations in some other way. In addition, the embryos that develop from bullwinkle mothers are bicaudal. A KINESIN:beta-GALACTOSIDASE fusion protein is correctly localized to the posterior pole of bullwinkle oocytes during stage 9. Thus, the microtubule structure of the oocyte and general transport along it do not appear to be disrupted prior to cytoplasmic streaming. Unlike other bicaudal mutants, oskar mRNA is localized correctly to the posterior pole of the oocyte at stage 10. By early embryogenesis, however, some oskar mRNA is mislocalized to the anterior pole. Consistent with the mislocalization of oskar mRNA, a fraction of the VASA protein and nanos mRNA are also mislocalized to the anterior pole of bullwinkle embryos. Mislocalization of nanos mRNA to the anterior is dependent on functional VASA protein. Although the mirror-image segmentation defects appear to result from the action of the posterior group genes, germ cells are not formed at the anterior pole. The bicaudal phenotype is also germ-line dependent for bullwinkle. We suspect that BULLWINKLE interacts with the cytoskeleton and extracellular matrix and is necessary for gene product localization and cell migration during oogenesis after stage 10a.
We have investigated the role of the actin cytoskeleton during mid-oogenesis and have found that disrupting the actin cytoskeleton with cytochalasin D induces microtubule bundling and microtubule-based cytoplasmic streaming within the oocyte, similar to that which occurs prematurely in cappuccino and spire mutant oocytes. After examining a number of mutants that affect the actin cytoskeleton, we have found that chickadee, which encodes the actin-binding protein, profilin, shares this phenotype. In addition to the microtubule misregulation, mutants in chickadee resemble cappuccino in that they fail to localize STAUFEN and oskar mRNA to the posterior pole of the developing oocyte. Also, a strong allele of cappuccino has multinucleate nurse cells, similar to those previously described in chickadee. In an independent line of experiments, we have identified profilin as a CAPPUCCINO interactor in a two-hybrid screen for proteins that bind to CAPPUCCINO. This, together with the similarity of mutant phenotypes, suggests that profilin and CAPPUCCINO may interact during development.