Endoplasmic reticulum targeted GFP reveals ER organization in tobacco NT-1 cells during cell division.

The endoplasmic reticulum (ER) of plant cells undergoes a drastic reorganization during cell division. In tobacco NT-1 cells that stably express a GFP construct targeted to the ER, we have mapped the reorganization of ER that occurs during mitosis and cytokinesis with confocal laser scanning microscopy. During division, the ER and nuclear envelope do not vesiculate. Instead, tubules of ER accumulate around the chromosomes after the nuclear envelope breaks down, with these tubules aligning parallel to the microtubules of the mitotic spindle. In cytokinesis, the phragmoplast is particularly rich in ER, and the transnuclear channels and invaginations present in many interphase cells appear to develop from ER tubules trapped in the developing phragmoplast. Drug studies, using oryzalin and latrunculin to disrupt the microtubules and actin microfilaments, respectively, demonstrate that during division, the arrangement of ER is controlled by microtubules and not by actin, which is the reverse of the situation in interphase cells.

Investigation of factors affecting the adsorption of functional molecules onto gel silicas. 1. Flow microcalorimetry and infrared spectroscopy.

Flow microcalorimetry and infrared spectroscopy were used to study the surface structure and adsorptive properties of a series of calcined and uncalcined porous silicas. The adsorbates DL-menthol, (R)-(+)-limonene, (+/-)-citronellal and carvone were selected for their functionality, that included carbonyl, vinylic and hydroxyl groups. The amounts of probe retained by the silicas together with the energy exchange involved in the adsorption/desorption process were determined by flow microcalorimetry. The functional groups involved in these interactions were studied by means of infrared spectroscopy. It was observed that the strongest interactions with the silica surface took place through hydrogen bonding onto the surface silanol. The most retentive probes were found to be those with a carbonyl group in their structure. Adsorption onto calcined silicas was found to be less energetic than onto the equivalent calcined samples. The adsorption densities were compared with theoretical predictions based on molecular models. In all cases apart from citronellal monolayer coverage were not observed due to steric effects.

Cytoplasmic pH dynamics in maize pulvinal cells induced by gravity vector changes.

In maize (Zea mays) and other grasses, changes in orientation of stems are perceived by pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. The amyloplast-containing bundle sheath cells are the sites of gravity perception, although the initial steps of gravity perception and transmission remain unclear. In columella cells of Arabidopsis roots, we previously found that cytoplasmic pH (pH(c)) is a mediator in early gravitropic signaling (A.C. Scott, N.S. Allen [1999] Plant Physiol 121: 1291-1298). The question arises whether pH(c) has a more general role in signaling gravity vector changes. Using confocal ratiometric imaging and the fluorescent pH indicator carboxy seminaphtorhodafluor acetoxymethyl ester acetate, we measured pH(c) in the cells composing the maize pulvinus. When stem slices were gravistimulated and imaged on a horizontally mounted confocal microscope, pH(c) changes were only apparent within the bundle sheath cells, and not in the parenchyma cells. After turning, cytoplasmic acidification was observed at the sides of the cells, whereas the cytoplasm at the base of the cells where plastids slowly accumulated became more basic. These changes were most apparent in cells exhibiting net amyloplast sedimentation. Parenchyma cells and isolated bundle sheath cells did not show any gravity-induced pH(c) changes although all cell types responded to external stimuli in the predicted way: Propionic acid and auxin treatments induced acidification, whereas raising the external pH caused alkalinization. The results suggest that pH(c) has an important role in the early signaling pathways of maize stem gravitropism.

Demonstration of prominent actin filaments in the root columella.

The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

Aluminum accumulation at nuclei of cells in the root tip. Fluorescence detection using lumogallion and confocal laser scanning microscopy.

The mechanistic basis for Al toxicity effects on root growth is still a matter of speculation, but it almost certainly involves decreased cell division at the root apex. In this series of experiments, we attempt to determine whether Al enters meristematic cells and binds to nuclei when roots are exposed to a low Al(3+) activity in solution. The methodology involved the use of the Al-sensitive stain lumogallion (3-[2,4 dihydroxyphenylazo]-2-hydroxy-5-chlorobenzene sulfonic acid), the DNA stain 4',6-diamino-phenylindole, and confocal laser scanning microscopy. Soybean (Glycine max L. Merr.) cv Young (Al-sensitive) and PI 416937 (Al-tolerant) genotypes were exposed to 1.45 microM Al(3+) for periods ranging from 30 min to 72 h, and then washed with 10 mM citrate to remove apoplastic Al. Fluorescence images show that within 30 min Al entered cells of the sensitive genotype and accumulated at nuclei in the meristematic region of the root tip. Substantial Al also was present at the cell periphery. The images indicated that the Al-tolerant genotype accumulated lower amounts of Al in meristematic and differentiating cells of the root tip and their cell walls. Collectively, the results support an important role for exclusion in Al tolerance.

Plant nuclei can contain extensive grooves and invaginations.

Plant cells can exhibit highly complex nuclear organization. Through dye-labeling experiments in untransformed onion epidermal and tobacco culture cells and through the expression of green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The substantial increase in nuclear surface area resulting from these grooves and invaginations, their apparent preference for association with nucleoli, and the presence in them of actin bundles that support vesicle motility suggest that the structures might function both in mRNA export from the nucleus and in protein import from the cytoplasm to the nucleus.

Changes in cytosolic pH within Arabidopsis root columella cells play a key role in the early signaling pathway for root gravitropism.

Ratiometric wide-field fluorescence microscopy with 1',7'- bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran demonstrated that gravistimulation leads to rapid changes in cytoplasmic pH (pHc) in columella cells of Arabidopsis roots. The pHc of unstimulated columella cells in tiers 2 and 3, known sites of graviperception (E.B. Blancaflor, J.B. Fasano, S. Gilroy [1998] Plant Physiol 116: 213-222), was 7.22 +/- 0.02 pH units. Following gravistimulation, the magnitude and direction of pHc changes in these cells depended on their location in the columella. Cells in the lower side of tier 2 became more alkaline by 0.4 unit within 55 s of gravistimulation, whereas alkalinization of the cells on the upper side was slower (100 s). In contrast, all cells in tier 3 acidified by 0.4 pH unit within 480 s after gravistimulation. Disrupting these pHc changes in the columella cells using pHc modifiers at concentrations that do not affect root growth altered the gravitropic response. Acidifying agents, including bafilomycin A1, enhanced curvature, whereas alkalinizing agents disrupted gravitropic bending. These results imply that pHc changes in the gravisensing cells and the resultant pH gradients across the root cap are important at an early stage in the signal cascade leading to the gravitropic response.

Model system for plant cell biology: GFP imaging in living onion epidermal cells.

The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

Modified LDLs induce and bind to membrane ruffles on macrophages.

Macrophage foam cell formation in vitro requires uptake of modified low density lipoproteins (LDL) such as acetylated LDL (AcLDL) and moderately oxidized LDL (OxLDL), or beta-migrating very low density lipoprotein (betaVLDL), a naturally occurring lipoprotein. Incubation ofmacrophages with AcLDL and OxLDL resulted in stimulation of membrane ruffle formation, while betaVLDL primarily resulted in increased numbers of microvilli. Time-lapse Allen video enhanced contrast differential interference contrast (AVEC-DIC) light microscopy and correlative whole mount intermediate-voltage transmission electron microscopy (IVEM) was used to examine the dynamics ofAcLDL stimulated membrane ruffling and membrane ruffle ultrastructure. Stereo 3D surface replicas confirmed that AcLDL bound to these AcLDL-induced membrane ruffles. Quantification of the plasma membrane surface area after incubation with AcLDL, betaVLDL or LDL confirmed that AcLDL stimulated membrane ruffling, while betaVLDL and LDL stimulated microvilli formation. These studies suggest that modified LDLs induce circular membrane ruffles and modified LDLs bind to these ligand-induced membrane ruffles.

Growth dynamics and cytoskeleton organization during stem maturation and gravity-induced stem bending in Zea mays L.

Characterization of gravitropic bending in the maize stem pulvinus, a tissue that functions specifically in gravity responses, demonstrates that the pulvinus is an ideal system for studying gravitropism. Gravistimulation during the second of three developmental phases of the pulvinus induces a gradient of cell elongation across the non-growing cells of the pulvinus, with the most elongation occurring on the lower side. This cell elongation is spatially and temporally separated from normal internodal cell elongation. The three characterized growth phases in the pulvinus correspond closely to a specialized developmental sequence in which structural features typical of cells not fully matured are retained while cell maturation occurs in surrounding internodal and nodal tissue. For example, the lignification of supporting tissue and rearrangement of transverse microtubules to oblique that occur in the internode when cell elongation ceases are delayed for up to 10 d in the adjacent cells of the pulvinus, and only occurs as a pulvinus loses its capacity to respond to gravistimulation. Gravistimulation does not modify this developmental sequence. Neither wall lignification nor rearrangement of transverse microtubules occurs in the rapidly elongating lower side or non-responsive upper side of the pulvinus until the pulvinus loses the capacity to bend further. Gravistimulation does, however, lead to the formation of putative pit fields within the expanding cells of the pulvinus.

Electro-optical imaging of F-actin and endoplasmic reticulum in living and fixed plant cells.

Confocal and video micrographs of living and fixed alfalfa roots, onion epithelial and pear pollen cells illustrate the architecture of the cytoskeleton and endoplasmic reticulum in plant cells. Fixation of plant tissues to preserve cytoplasmic structure poses special problems. When possible, emphasis should be placed on the imaging of structures in stained living cells over time. The early events that occur when Nod factors or bacteria elicit nodule formation in alfalfa roots will illustrate several approaches to plant cell fixation, staining and imaging. The first observable events after Nod factor stimulation occur in root hairs and are changes in rates of cytoplasmic streaming, nuclear movements, and changes in the shape of the vacuole. Within ten minutes, the endoplasmic reticulum shifts position towards the tip of the root hair. For comparison, the endoplasmic reticulum localization in pollen tubes and onion epithelial cells will be illustrated. The actin cytoskeleton undergoes a series of changes over a twelve hour period. These changes in the cytoskeleton are spatially and temporally correlated with the observed growth changes of the root hairs. This dynamic change of the actin filament and endoplasmic reticulum and associated secretory vesicles in these root hairs suggests a mechanism for the observed root hair growth changes.

Beta VLDL uptake by pigeon monocyte-derived macrophages: correlation of binding dynamics with three-dimensional ultrastructure.

Endocytosis of pigeon beta migrating very-low-density lipoprotein (beta VLDL) by monocyte-derived macrophages (monocyte/macrophages), cultured from Random Bred White Carneau (RBWC) pigeons, occurs by both coated and non-coated regions of the plasma membrane (Henson et al.: Exp. Mol. Pathol. 51:243-263, 1989). Secondary to binding, the beta VLDL is translocated to lysosomes for degradation. Ultimately these events lead to foam cell formation in vitro. Utilizing video-enhanced contrast light microscopy in conjunction with whole mount intermediate-voltage transmission electron microscopy (IVEM) and high-resolution scanning EM, the dynamics of beta VLDL binding have been correlated with ultrastructure. Beta VLDL conjugated to gold colloids was visualized at the surface of living cells by using Allen video-enhanced contrast-differential interference contrast microscopy (AVEC-DIC). Subsequent to AVEC-DIC, direct observation of the identical cells by IVEM and SEM was facilitated through the use of gold finder grids, and these EM observations confirmed identification of the video-observed beta VLDL particles. Upon addition of beta VLDL, pigeon monocyte/macrophages underwent gross morphological changes. These changes were recorded by video as movements at the cytoplasmic periphery, and the movements involved extension of microvilli, expression of retraction fibers, and elaboration of membrane ruffles. When secondarily observed by stereo (3-D) IVEM and SEM, the identification of microvilli, retraction fibers, and membrane ruffles was confirmed and the lipoprotein-gold conjugates were associated with these ligand-induced membrane structures. Beta VLDL-gold conjugates were also associated with pit-like regions at the base of microvilli, while at the base of ruffles, beta VLDL-gold conjugates were located in membrane invaginations and cytoplasmic vesicles.

Macrophages form circular zones of very close apposition to IgG-coated surfaces.

When phagocytes spread on surfaces coated with ligands such as IgG, they form a tight seal with the substrate. This seal excludes soluble macromolecules in the medium from the interface between the cell and substrate. In contrast, when cells spread on control surfaces that are not coated with ligands, the underside of the cell remains freely accessible to soluble proteins (Wright and Silverstein: Nature 309:359, 1984). We employed reflection-interference microscopy (RIM) to determine where the seal forms during interaction with ligand (IgG)-coated surfaces. Human monocyte-derived macrophages (MO) were plated at 37 degrees C on dinitrophenylated (DNP)-glass coverslips (control substrate), IgM anti-DNP-DNP-coated glass (control substrate), or on IgG anti-DNP-DNP-coated glass (phagocytosis-promoting substrate). Live or fixed cells were examined by RIM. Spreading on control surfaces at 37 degrees C was complete in 25 minutes, whereas spreading on IgG-coated surfaces was maximal within 15 minutes and resulted in cell-substrate contact area 1.6 X that of control cells. Within 1 h at 37 degrees C, 90% of MO that spread on IgG-coated substrates, but not on control substrates, excluded macromolecules from their underside. A minor population of cells (19%) exhibited a uniform iron gray RIM appearance indicating an even, close approach to the substrate. These cells may represent early stages of frustrated phagocytosis. In contrast to cells on control substrates, 70% of cells on IgG-coated substrates developed continuous peripheral dark rings in RIM indicative of close association with the substrate. Essentially all cells with peripheral dark rings in RIM excluded macromolecules from their underside. Enclosed within this ring was an area of greater separation between the cell membrane and the substrate, as indicated by the lighter grey of this region in RIM and by the accessibility of substrate to anti-substrate antibody when breaks in the dark ring occur. Thus, MO can create a closed compartment between plasma membrane and substrate that excludes proteins in the surrounding medium, thereby protecting substances secreted into this space from potentially inhibitory substances in the medium.

Dyke Award. Europium-DTPA: a gadolinium analogue traceable by fluorescence microscopy.

A lanthanide series chelate, europium(Eu)-DTPA, was synthesized to serve as a histochemical analogue for the widely used MR contrast agent gadolinium(Gd)-DTPA. Eu and Gd, being neighboring elements on the periodic table, share many fundamental properties, including ionic radius, valence, and chemical reactivity. Eu-DTPA, however, possesses one important physical property not shared by Gd-DTPA: luminescence under ultraviolet light. The feasibility of detecting Eu-DTPA in animal tissues under fluorescence microscopy was systematically evaluated and documented. Distinctive orange-red luminescence of Eu-DTPA could be observed in the kidneys, livers, dura, choroid, and pituitary glands of rats after intravascular injection. No luminescence was detected in areas of brain beyond an intact blood-brain barrier. When the brain was locally injured by an experimental laceration, however, leakage of Eu-DTPA was detected. Electron probe microanalysis confirmed the parallel presence or absence of simultaneously injected Eu-DTPA and Gd-DTPA in all tissues studied. Fluorescence microscopy with Eu-DTPA has thus been validated as a method for tracing the distribution of Gd-DTPA at the microscopic level.

Video-enhanced microscopy with a computer frame memory.

Video-enhanced microscopy combined with the use of a computer frame memory extends considerably the useful range of our video enhanced contrast (AVEC) methods for polarizing, double-beam interference and differential interference contrast microscopy. Increased visual contrast is achieved by two stages of amplifications: the first optical, by using high bias retardation settings, and the second electronic. These steps are followed by a reduction of background brightness by means of a clamp voltage applied to a DC restoration circuit of the video camera. One of the limitations of the AVEC method alone is the inevitable appearance under high gain conditions of a pattern of mottle due to inaccessible dirt and defects in the lenses even of high quality. This limitation has been circumvented by storing the mottle pattern in the frame memory (frame store) and continuously subtracting it from each succeeding frame to clear the image. A major gain in image quality has resulted. In polarizing microscopy, the frame memory can be used also to subtract the image at one compensator setting from that at the equivalent setting of opposite sign, thus removing from the final image not only most of the mottle pattern but also the contrast due to the bright-field contrast. In the polarizing microscope, these manipulations of the raw video image make it possible to observe and measure the birefringence of various organelles and elements such as microtubules, intermediate filaments and bundles of as few as a half dozen actin filaments. Since scattered light is also removed from the image, features hidden from view in the unprocessed image become visible. In differential interference microscopy, the AVEC method makes visible (i.e. detectable) many linear elements and particles that are an order of magnitude smaller than the resolution limit and not visible in the optical image. Such features are inflated by diffraction, however, to Airy disk size.

Video-enhanced contrast polarization (AVEC-POL) microscopy: a new method applied to the detection of birefringence in the motile reticulopodial network of Allogromia laticollaris.

A new method is described for recording rapid processes of cell motility in polarized light. The Allen video-enhanced contrast (AVEC-POL) method of polarization microscopy achieves significant improvements in resolution, contrast, and the visibility of fine detail by a combination of novel adjustments to a standard (unrectified) polarizing microscope and video camera. Using the full working aperture of a high-power planapochromatic objective lens and compensator setting of lambda/9-lambda/4, visible images appear lacking in contrast. However, the same images viewed with an appropriate video camera equipped with an electronic offset adjustment can be made to appear with as much contrast as desired, revealing a significantly greater amount of fine detail in the image than can be seen by high extinction visual microscopy alone. At bias retardations between one-ninth and one-quarter wave, the diffraction anomaly observed near extinction disappears. Consequently, polarizing rectifiers are not required with the AVEC-POL method, and images previously requiring photographic exposures of around 20 seconds are sufficiently bright to be registered on the video monitor in 1/60 second. Using an intensity monitor, quantitative measurements of cellular birefringence can be retrieved from live or videotaped images displaying a linear relationship between contrast and phase retardation due to birefringence. The AVEC-POL method also renders accessible to polarized light analysis a number of objects that scatter or depolarize too much light to be studied by high extinction methods. The method is demonstrated on model objects and applied to the highly motile reticulopodial network of Allogromia laticollaris. Rapid motion in close association with microtubules can now be analyzed in greater detail at a significant reduction in the cost of recording.