Abstract. We have studied the F-actin network in cycling suspension culture cells of carrot (Daucus carom 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.

KEY w O R D s. Confocal microscopy, three-dimensional fluorescence microscopy, pointspread function, deconvolution, computer image processing. SUMMARY We have measured the point-spread function (PSF) for an MRC-500 confocal scanning laser microscope using subresolution fluorescent beads. PSFs were measured for two lenses of high numerical aperture-the Zeiss plan-neofluar 63 x water immersion and Leitz plan-apo 63 x oil immersion-at three different sizes of the confocal detector aperture. The measured PSFs are fairly symmetrical, both radially

A prominent feature of tip growth in filamentous plant cells is that the nucleus often migrates in step with the tip as it extends. We have studied this long‐recognized but unexplained relationship in root hairs of the legume Vicia hirsuta by a variety of microscopic techniques. Using rhodaminyl lysine phallotoxin, and antitubulin antibodies, root hairs are shown to contain axial bundles of F‐actin and a complex microtubular system. To the basal side of the nucleus the microtubules are cortical and net axial but in the region between nucleus and tip the arrangement is more complicated. Electron microscopic thin sections demonstrate that internal bundles of microtubles exist in addition to the plasma membrane‐associated kind. Computerized deblurring of through‐focal series of antitubulin stained hairs clarifies the three‐dimensional organization: bundles of endoplasmic microtubules progress from the nuclear region toward the apical dome where they can be seen to fountain out upon the cortex. The relationship between nucleus and tip can be uncoupled with antimicrotubule herbicides. Time lapse video microscopy shows that these agents cause the nucleus to migrate toward the base. This contrary migration can be inhibited by adding cytochalasin D, which fragments the F‐actin bundles. It is concluded that microtubules connect the nucleus to the tip but that F‐actin is involved in basipetal migration as is known to occur when symbiotic bacteria uncouple the nucleus from the tip.

Abstract. To investigate the spatial relationship between the nucleus and the cortical division site, epidermal cells were selected in which the separation between these two areas is large. Avoiding enzyme treatment and air drying, Datura stramonium cells were labeled with antitubulin antibodies and the threedimensional aspect of the cytoskeletons was reconstructed using computer-aided optical sectioning.In vacuolated cells preparing for division, the nucleus migrates into the center of the cell, suspended by transvacuolar strands. These strands are now shown to contain continuous bundles of microtubules which bridge the nucleus to the cortex. These nucleus-radiating microtubules adopt different configurations in cells of different shape. In elongated cells with more or less parallel side walls, oblique strands radiating from the nucleus to the long side walls are presumably unstable, for they are progressively realigned into a transverse disc (the phragmosome) as broad, cortical, preprophase bands (PPBs) become tighter. The phragmosome and the PPB are both known predictors of the division plane and our observations indicate that they align simultaneously in elongated epidermal cells. These observations suggest another hypothesis: that the PPB may contain microtubules polymerized from the nuclear surface. In elongated cells, the majority of the radiating microtubules, therefore, come to anchor the nucleus in the transverse plane, consistent with the observed tendency of such cells to divide perpendicular to the long axis.In nonrectangular isodiametric epidermal cells, which approximate regular hexagons in section, the radial microtubular strands emanating from the nucleus tend to remain associated with the middle of each subtending cell wall. The strands are not reorganized into a single dominant transverse bar, but remain as a starlike array until mitosis. PPBs in these cells are not as tight; they may only be a sparse accumulation of microtubules, even forming along nondiametrical radii. This arrangement is consistent with the irregular division patterns observed in epidermal mosaics of isodiametric D. stramonium cells.The various conformations of the radial strands can be modeled by springs held in two-dimensional hexagonal frames, and by soap bubbles in three-dimensional hexagonal frames, suggesting that the division plane may, by analogy, be selected by minimal path criteria. Such behavior offers a cytoplasmic explanation of long-standing empirically derived "rules" which state that the new cell wall tends to meet the maternal wall at right angles. The radial premitotic strands and their analogues avoid taking the longer path to the vertex of an angle where a cross wall is already present between neighboring cells. The resultant tendency to form three-rayed vertices rather than intercrossing septa helps explain the hexagonal packing of plant cells.

No abstract