Lipids trigger changes in the elasticity of the cytoskeleton in plant cells: a cell optical displacement assay for live cell measurements.

An assay has been developed to quantitatively measure the tension and elasticity of the cytoskeleton in living plant cells. The cell optical displacement assay (CODA) uses a focused laser beam to optically trap and displace transvacuolar and cortical strands through a defined distance within the cell. Results from these experiments provide evidence for the classification of at least two rheologically distinct cytoskeletal assemblies, cortical and transvacuolar, that differ in their tension and response to both signaling molecules and reagents that perturb the cytoskeleton. It is further demonstrated that the tension of the transvacuolar strands can be significantly decreased by the addition of either linoleic acid, 1,2 dioctanoyl-sn-glycerol, or 1,3 dioctanoylglycerol. These decreases in tension could also be induced by lowering the cytoplasmic pH. In contrast, addition of Ca2+, Mg2+, or the ionophore A23187 to the cells caused a considerable increase in the tension of the transvacuolar strands. The data provides evidence that: (a) linoleic acid may be a signaling molecule in plant cells; (b) diacylglycerol functions as a signaling molecule through a protein kinase C-independent pathway mediated by PLA2; and (c) Ca2+ and pH have regulatory roles for controlling cytoskeleton tension and organization.

Re-establishment of the interphase microtubule array in vacuolated plant cells, studied by confocal microscopy and 3-D imaging

There are two conflicting ideas about the site of reassembly of cortical microtubules following cytokinesis. Some observations indicate that microtubules (MTs) radiate from the surface of the postcytokinetic nuclear envelope, before becoming organized at the cortex. On the other hand, results of regrowth experiments, following MT depolymerization by drugs, suggest that the array may assemble directly upon the cortex. In this study, we have taken advantage of the significant separation between nucleus and cortex, in large, vacuolated epidermal cells, to determine which of these two potential sites supports the earliest stages of regrowth in the undrugged, native state. MTs in stem epidermis of Datura stramonium L. were stained by indirect immunofluorescence. This was performed on hand-cut sections of tissue in which the cells were not separated by enzymes or distorted by air- drying. Epidermal cells with these sheets were optically sectioned by confocal laser scanning microscopy and three-dimensional images reconstructed, rotated and viewed stereoscopically using computer methods. During metaphase, no MTs can be detected at the cortex but MTs begin to re-colonize the cell surface during early cytokinesis. Thick cables of MTs radiate from the nucleus parallel to the cell plate as well as in other directions, along transvacuolar strands, out to the cortex. Microtubules grow out over the cortex where the thick bundles make contact, as well as from the edges of the fully developed phragmoplast. These early cortical MTs do not form regular transverse arrays: they either appear to be random or to grow in branching V- shaped patterns. The cortical array is therefore not organized immediately but at a later stage. It is concluded that MT bundles, radiating from the nucleus, are involved in the earliest stages of cortical array formation.

An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus.

We have studied the F-actin network in cycling suspension culture cells of carrot (Daucus carota L.) using rhodaminyl lysine phallotoxin (RLP). In addition to conventional fixation with formaldehyde, we have used two different nonfixation methods before adding RLP: extracting cells in a stabilizing buffer; inducing transient pores in the plasma membrane with pulses of direct current (electroporation). These alternative methods for introducing RLP revealed additional features of the actin network not seen in aldehyde-fixed cells. The three-dimensional organization of this network in nonflattened cells was demonstrated by projecting stereopairs derived from through-focal series of computer-enhanced images. F-actin is present in interphase cells in four interconnected configurations: a meshwork surrounding the nucleus; thick cables in transvacuolar strands and deep in the cytoplasm; a finer network of bundles within the cortical cytoplasm; even finer filaments that run in ordered transverse array around the cell periphery. The actin network is organized differently during division but it does not disappear as do the cortical microtubules. RLP stains a central filamentous cortical band as the chromatin begins to condense (preprophase); it stains the mitotic spindle (as recently shown by Seagull et al. [Seagull, R. W., M. Falconer, and C. A. Weerdenburg, 1987, J. Cell Biol., 104:995-1004] for aldehyde fixed suspension cells) and the cytokinetic apparatus (as shown by Clayton, L., and C. W. Lloyd, 1985, Exp. Cell Res., 156:231-238). However, it is now shown that an additional network of F-actin persists in the cytoplasm throughout division associating in turn with the preprophase band, the mitotic spindle, and the cytokinetic phragmoplast.

Microfilaments: dynamic arrays in higher plant cells

By using fluorescently labeled phalloidin we have examined, at the light microscope level, the three-dimensional distribution and reorganization of actin-like microfilaments (mfs) during plant cell cycle and differentiation. At interphase, mfs are organized into three distinct yet interconnected arrays: fine peripheral networks close to the plasma membrane; large axially oriented cables in the subcortical region; a nuclear "basket" of mfs extending into the transvacuolar strands. All these arrays, beginning with the peripheral network, disappear at the onset of mitosis and reappear, beginning with the nuclear basket, after cytokinesis. During mitotic and cytokinetic events, mfs are associated with the spindle and phragmoplast. Actin staining in the spindle is localized between the chromosomes and the spindle poles and changes in a functionally specific manner. The nuclear region appears to be the center for mf organization and/or initiation. During differentiation from rapid cell division to cell elongation, mf arrays switch from an axial to a transverse orientation, thus paralleling the microtubules. This change in orientation reflects a shift in the direction of cytoplasmic streaming. These observations show for the first time that actin-like mfs form intricate and dynamic arrays in plant cells which may be involved in many as yet undescribed cell functions.

Osmotically induced volume changes in protoplasts isolated from Cucurbita ficifolia

Free membrane-bound protoplasts are formed when turgid stem segments are cross-sectioned into 2-mm-thick slices and placed in a nonplasmolysing solution of electrolyte or mannitol. The protoplasts range in diameter from less than 5 μm to a few more than 80 μm. The larger protoplasts contain a fuller complement of organelles than the small ones, and some large protoplasts form transvacuolar strands and restart cyclosis after 2 to 3 days. The protoplasts change diameter when they are exposed to fusicoccin or 2,4-dinitrophenol; from this we conclude that some of the solute transporters in the bounding membranes are still functioning. The pattern of swelling and shrinking of the protoplasts in response to the change in concentration and composition of the bathing medium leads us to believe that the membranes are very leaky to solutes causing a large coupling between solute and water flow. In addition high capacity net ion transport occurs at rates of up to 2 μosmol∙s−1∙m−2 causing shrinkage of the protoplasts in hypotonic KCl solution even though osmotic considerations force the prediction that the protoplasts will swell. Ion transporters working at the net rate of 0.5 μosmol∙s−1∙m−2 also appear to be responsible for swelling of protoplasts that previously shrank in hypertonic KCl or NaCl.

Growth and metabolism of cells and tissue of jack pine (Pinus banksiana). 7. Observations on cytoplasmic streaming and effects of L-glutamine and its analogues on subcellular activities

Cells from jack pine (Pinus banksiana Lamb.) callus and suspension cultures were observed by cinemicrography to evaluate cytoplasmic streaming, motion of organelles, and nuclear behaviour under glutamine stress. Streaming rates were greatest in the largest cells. Rates were reduced in smaller cells, where transvacuolar strands were not as prominent and displacements of organelles were not strongly affected by cyclosis. During increased rates of cytoplasmic streaming, chloroplasts tended to associate with each other and mitochondria often changed direction and shape.The addition of 400 ppm L-glutamine to a defined basal medium increased the linear velocity of organelles from 2-7 microns (μ)/s to 7–12 μ/s, whereas D-glutamine eventually stopped cytoplasmic streaming, with a concomitant increase in nucleolar size. While additions of low levels of α-N-acetyl-L-glutamine and isoglutamine stimulated streaming, this occurred usually without other significant morphological effects. Cytoplasmic movements that were induced by exposure of cells to L-glutamine were intimately involved with the fine strands and with nuclear and nucleolar behavior. Movements ceased when strands were disrupted under nutritional stress and by inhibitory responses to D-glutamine.