The challenge of the post-genomic era is functional genomics, i.e. understanding how the genome is expressed to produce myriad cell phenotypes. A phenotype is the result of selective expression of the genome in response to the microenvironment. to use genomic information to understand the biology of complex organisms, the biological responses and signaling pathways in cells need to be studied in context, i.e. within the proper tissue structure. Nonetheless, most current biology is conducted using cells cultured in monolayers on traditional tissue culture plastic. These non-physiological models impede the ability to predict in vivo responses from model systems. The same cells cultured in 2-dimensions (i.e. monolayers) vs. 3-dimensions (e.g. multicellular tumor spheroids) differ in their responses to external stimuli such as ionizing radiation, viral infection, cytotoxic drugs, and chemotherapeutic agents. Our laboratory has led the way in promoting and developing 3-dimensional cell culture models that more accurately reflect in vivo biology, beginning with the establishment 15 years ago of physiologically functional reconstituted mammary acini in culture.

Quantitation of spatial and temporal concurrent behavior of multiple markers in these 3-dimensional cell cultures is hampered by the currently routine mode of sequential image acquisition, measurement and analysis of specific targets. This precludes the detailed analysis of multi-dimensional, time sequence responses and fails to relate features in novel and meaningful ways that will further our understanding of basic biology. Thus new methodology was needed for high-throughput, dynamic evaluations of large numbers of live multicellular specimens. Rather than using confocal microscopy methods, which interfere with live cell systems due to photo-damage, optical sectioning of the 3-dimensional structures is achieved with structured light illumination using the Wilson grating in an implementation described by Lanni.

Post-Golgi sorting of different classes of newly synthesized proteins and lipids is central to the generation and maintenance of cellular polarity. to directly visualize the dynamics and location of apical/basolateral sorting and trafficking we used fast time-lapse multicolor video microscopy in living cells. Specifically, green fluorescent protein color variants (cyan, CFP; yellow, YFP) of apical cargo (GPI-anchored) and basolateral cargo (vesicular stomatitis virus glycoprotein, VSVG) were generated; see FIG 1. Fast dual color fluorescence video microscopy allowed visualization with high temporal and spatial resolution. Our studies revealed that apical and basolateral cargo progressively segregated into large domains in Golgi/TGN structures, excluded resident proteins, and exited in separate transport containers. These carries remained distinct and did not merge with endocytic structures en route to the plasma membrane. Interestingly, our data suggest that the primary sorting occurs by lateral segregation in the Golgi, prior to budding (FIG 2). Further characterization of morphological differences of apical versus basolateral transport carriers was achieved using a specialized microscopy technique called total internal reflection (TIR) microscopy. with this approach only the bottom of the cell (<100 nm) was illuminated by an exponentially decaying evanescent “wave” of light. A series of images, taken at ∼1 second intervals, shows a bright “flash” of fluorescence when the vesicle fuse with the plasma membrane and the fluorophore diffuses into the plasma membrane (FIG 3).

We present a new method of fluorescence imaging, which yields an unprecedented nm scale vertical resolution. The method uses the unique spectral signature of the fluorescent emission intensity above a reflecting surface to determine vertical position unambiguously. Emission from several heights could be resolved by deconvoluting the spectrum, so that three dimensional imaging is possible. The related technique of standing wave microscopy uses the spatial intensity variation for increased resolution. Applications of this technique include intracellular imaging and screening for specific bacteria, virus or proteins, where discrimination between raised fluorescently labeled specifically bound markers from non-specific binding is crucial.

The emission spectrum of fluorescent markers is modified in the presence of a reflecting surface. Within the wavelength range of the fluorescent emission, the selfinterference of the emitted photons from the direct and reflected path results in enhanced or suppressed emission (constructive or destructive interference) depending on the height and wavelength.

Microscopy is an essential tool for developing strategies to use genetic therapy in the treatment of diseases. Negative staining (fig. 1) and cryo-electron microscopy are critical in understanding the structure of native and hybrid vectors to be used as transfer agents. Enzyme cytochemistry is widely used for the detection of markers that aid in determining the rate of gene transfer to cells and tissues. Immunocytochemistry is necessary to identify new gene products. Light, confocal and electron microscopy are used to evaluate pathogenesis, inflammation and cellular changes. For much of the past decade, we have been working to identify the specific gene and cell structure (chloride channel) that is compromised in Cystic Fibrosis patients. Subsequently, appropriate vectors had to be developed for treatment. A significant amount of time and effort has been expended to identify effective viral and other vectors to be used in the transfer of the normal gene construct.

In order to facilitate experimentation, we have developed a cell culture system utilizing normal and cystic fibrosis airway epithelia. These cultures are maintained at an air interface and the cells differentiate into the same types found in vivo.

The formation of memory and the process of learning are believed to be regulated, at least in part, by the expression, distribution, and redistribution of glutamate receptors. The expression of these receptors at synaptic sites has a major impact on the strength of synaptic connections, and the AMPA receptor subunit GluR1 appears to play a key role within this system. Increasing evidence suggests that previously silent synapses become activated through rapid AMPA receptor insertion upon appropriate stimulation, and thus the trafficking of this receptor subunit from cellular stores to the synapse is of prime interest.

We are using a transgenic mouse expressing a GFP-tagged form of GluR1 (GluR1-GFP) in order to study the dynamic changes in GluR1 expression and distribution occurring during brain development and following induction of long-term potentiation (LTP). The fusion protein is transcribed under the control of the CaMKIIα promoter, which restricts the localization to forebrain neurons and is coupled to a tetrepressible system, thus allowing for the control of transcription with doxycycline. The anatomical distribution of GluR1-GFP on the light level is consistent with that of wild-type GluR1 (Fig. 1).

We are combining molecular biology with quantitative 3D image analysis on the light and electron microscopic level. The highly fluorescent polar tracer Alexa Fluor 568 (Molecular Probes Inc.) is being used to fill GluR1-GFP expressing pyramidal neurons in tissue slices fixed with aldehydes

Cooling significantly affects cellular ultrastructure [1], as well as structural and functional state of histohematic barrier [2]. The aim of the given work was to study ultrastructural mechanisms of the permeability of brain cortex vascular endothelium under the effect of rhythmic hypothermia. Experiments were conducted on 180-220 g white males rats. Cooling of caudal part of animals was conducted in an automatic regimen by an ice water with 0.1 Hz frequency during 40 and 80 min. Experimental animals were narcotized by intraperitoneal injection of thiopenthal sodium and sodium hydroxybutyrate mixture in respect of 30mg/kg, 100 mg/kg of mass, correspondingly. Material from parietal lobe of brain cortex for electron-microscopic investigations was taken immediately after the effect finishing. Rectal temperature, which made 36-35C, was measured during the experiment. For electron microscopic investigations the samples were fixed by immerging into 4% glutar aldehyde on a phosphate buffer and were additionally fixed by 1% osmium fixative on a phosphate buffer. After dehydration in ethanol, samples were placed into the mixture of epoxy resins Epon-Araldit (Fluka, Sweden). Colloid carbon was used as a tracer for the estimation of permeability of brain capillary endothelium. with this aim 3-5 ml of 10% colloid carbon solution, prepared on 0,85% NaCl solution, were injected into a tail vein of the animals immediately after their decapitation. Preliminary estimation of the material quality was done on semi-fine tissue sections after the staining by toluidine blue.

The long bones of vertebrates, such as the tibiae and femurs of humans, extend in length by means of genotypic and phenotypic changes orchestrated by the chondrocytes comprising growth plate cartilage. Among the constituents synthesized by these cartilage cells, osteopontin (OPN), a phosphorylated glycoprotein, is thought to play a critical role in events leading to normal growth plate function and ultimate mineralization of the deeper zones of this cartilage region. The precise role of OPN, however, is uncertain with regard to mineralization, and present evidence supports the alternative possibilities that the protein may be either facilitative or inhibitory to mineral deposition. in order to investigate OPN function in a model growth plate, cartilage from normal 1-11 day old postnatal mice was examined by the novel techique of laser capture microdissection (LCM) followed by RT-PCR to obtain a measure of OPN gene expression by chondrocytes of known age and specific location in the plate. LCM permits identification of individual or clusters of cells within a tissue section and subsequent unique isolation (“capture”) of such cells for a variety of molecular analyses.

In this study, mouse tibiae were dissected, placed in RNAlater (Ambion, Austin, TX) to preserve message, and stored at −20°C. Sections (5 μm thick) of fresh frozen developing epiphyseal growth plates were obtained in a cryostat maintained at −20°C, fixed briefly in 70% ethanol, and stained with eosin. Sections were examined in a Pixcell LCM system (Arcturus Engineering, Mountain View, CA) where chondrocytes were attached to the surface of polymer film substrates and lifted free of sections. in separate experiments, ∼200-1200 cells were captured and analyzed. Substrates were transferred to Eppendorf tubes containing RNA extraction buffer. RNA was extracted from cells by microisolation (Stratagene, La Jolla, CA), DNAse-treated, reverse-transcribed, and then subjected to PCR (40 cycles) with AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA). Ethidium bromide agarose gels revealed OPN mRNA from groups of chondrocytes isolated from whole cartilage and from resting, proliferating, and hypertrophic growth plate zones from the mouse tibiae. Brain cells captured by LCM from the same mouse sections served as positive controls and reactions containing no reverse transcriptase were negative controls. 18S rRNA was used as a marker for semiquantitation and standardization of expressed message from captured cells.

For industrial-scale culture of anchorage-dependent mammalian cell, such as vero cell, growth on the microcarriers offers certain advantages. As previous research reported, growth process can be illustrated by a growth curve - cell density versus time, which shows the change of cell density through whole growth process including: first adhesion stage; spreading and division stage; and last confluent stage. The growth curves are controlled by the culture condition (culture medium, temperature, pH value, inoculation concentration, etc.) and space condition (kind of microcarrier, microcarrier size, and surface property, etc.). Therefore, the cell growth curve is not only the important basis for studying growth kinetics, but also for designing biological reactors.

Traditionally, total cell concentration for the microcarrier cultures were determined using the crystal violet method, and the cell nuclei were counted with a hemacytometer. in this method, the cells attached on each microcarrier were trypsinized and dyed by crystal violet dye, so that the number of nuclei counted was statistically representative. in this work, the cells and microcarriers were examined by using an environmental SEM - KYKY1500. As a result, the moist cells and microcarriers in SEM images show their natural characteristics, because this environmental SEM permits biological sample to be observed directly in an environmental specimen chamber without normal preparation for biological sample. As for the normal preparation, it needs a long duration, e.g. 2 days normally, to do fixing, desiccating and gold-coating. in contrast, the operation for observation of the biological samples in this SEM is much simpler. in view of this, a statistical counting method based on the SEM images for determining cell concentration during microcarrier culture was developed.

In the embryologic development of the nephron, the glomerular tuft (renal corpuscle) and the excretory tubules develop from two distinct embryologic origins or anlagen. Nephrotomes constitute the earliest formation of the excretory tubules and appear first as segmentally arranged cell clusters, which grow and become canalized, forming nephric tubules. Formation of the glomerulus begins when the dorsal aorta gives rise to small branches, consisting of tufts of capillaries. Bowman’s capsule forms as a blind terminal expansion at the proximal end of the nephron. in the final stage of development, Bowman’s capsule becomes deeply indented by the glomerular tuft. Which leads to a differentiation of it into 2 layers: 1) an inner, visceral layer and 2) an outer, or external, parietal layer. The two layers are separated by a crescent shaped space, known as Bowman’s or the urinary space.

The inner, visceral layer encloses, and, comes into close apposition with the glomerular capillaries. At the vascular pole of the glomerulus, the visceral layer is reflected off these vessels and becomes continuous with the external, or parietal layer of Bowman’s capsule, which is lined by a very flat polygonal epithelium. Traditionally, it has been depicted that there is a direct opening of the urinary space into the tubule and that the flat cells of the parietal layer of Bowman’s capsule become continuous with the cuboidal epithelium in the neck of the proximal convoluted tubule.

This investigation shows that the junction of the renal corpuscle and the proximal convoluted tubule is a more complicated one. Scanning electron microscopic studies of microdissected nephrons enabled the visualization of the glomerulus, with the two vessels at the vascular pole and the proximal convoluted tubule at the urinary pole. An unexpected finding, was the presence of a gourd-like out-pouching at the urinary pole. (Fig. 1, “A”) Higher magnification showed a saucer shaped depression (50 μm × 25 μm) on its free border. (Fig. 2). This was a consistent finding in the material that was studied.

The Chlamydomonas zygospore is formed after gametic cell fusion by a progression of ultrastructural and physiological changes, including the synthesis of a protective and elaborately sculptured secondary wall (Figure 1). Assembly of the secondary wall occurs within the confines of a transient primary zygote wall, synthesized immediately after gamete fusion, and assembled beneath the residual gamete walls. Upon completion of secondary wall assembly, the primary zygote wall is shed from the maturing zygospore (Figure 2). This primary zygote wall, composed primarily of a β-1,3 glucan (callose), may be important in the regulation of secondary zygospore wall formation.

A temperature-sensitive mutant strain of C. monoica, zym30, has been isolated that shows marked abnormalities in both primary and secondary wall ultrastructure. The degree of wall formation in the mutant strain is inversely proportional to temperature. At 25° C most zygotes fail to synthesize either the primary or the secondary wall. At 15° C both primary and secondary wall assembly occurs to some extent and as many as 50% of the zygotes develop a “pseudo-wildtype” phenotype (Figure 3). Although release of the primary wall rarely occurs in the zym30 strain (even at permissive temperatures), those walls that are released appear thinner than the walls shed from wildtype zygospores, as viewed by phase contrast microscopy. Fluorescence images incorporating aniline blue, a fluorochrome specific for β-1,3 glucans, show decreased fluorescence suggesting reduced callose synthesis (Figure 4).

Several permanent, ultrastructurally distinct layers comprise the secondary wall, the assembly of which may be controlled, in part, by the primary wall. One or more of these secondary wall layers contain the highly durable, autofluorescent biopolymer, sporopollenin. Autofluorescence of zym30 zygospores produced at the permissive temperature is enhanced relative to wildtype zygospores. This correlates with ultrastructural changes in the primary wall and overproduction and/or misassembly of the surface secondary wall layer (Figure 5). Work is in progress to determine whether the environmental resistance, and/or viability of the zym30 “pseudowildtype” zygospores are reduced relative to wildtype zygospores.

The concept of prion (infectious protein) originated in studies of transmissible spongioform encephalopathies (TSE) in mammals, but more recently, two nonchromosomal genes of yeast- [PSI] and [URE3] - were identified as prions. While the agents of TSEs kill infected cells and [URE3] or [PSI+] only slow growth at most, these infections are believed to have similar mechanisms, i.e. self-propagating amyloids. TSEs are often associated with amyloid deposition in infected tissues, and both Sup35p and Ure2p have been shown to form amyloid in vitro.

[URE3] is a prion of the Ure2 protein, that normally regulates nitrogen catabolism. Its ‘prion’ domain (residues 1-65) is necessary and sufficient for propagation of the prion, whereas the C-terminal portion (residues 81-354) is sufficient to carry out the nitrogen regulation function. The prion domain peptide spontaneously forms amyloid filaments in vitro. Full-length native Ure2p is a stable soluble dimer, but forms co-filaments when the prion domain peptide is added. This in vitro amyloid formation is highly specific and self-propagating, thus providing a possible explanation for the [URE3] prion. We have sought to clarify this hypothesis by examining the state of Ure2p in [URE3] cells by thin-section electron microscopy.

Yeast cells with the [URE3] prion and control [ure-o] cells were thin-sectioned after fixation and embedding in an epoxy resin. We found distinctive filamentous aggregates in the cytoplasm of [URE3] cells of a strain that overexpresses Ure2p (Fig. 1). These aggregates were seen in some cell profiles represented in 50-70 nm sections, and were never seen in control sections of [ure-o] cells. in cell sections showing these structures, there was typically one such aggregate, which could be quite large - up to several μm across - and approximately globular in outline. They contain irregularly associated filaments about 25 nm in diameter.

Shells of agglutinated foraminiferan protists are composed of mineral grains bound by secreted adhesives. As such, they are useful models for examining the evolution of “primitive” exoskeletons. Previous studies revealed the ultrastructure of shells in the giant Antarctic foraminiferan Astrammina rara and demonstrated that shucked specimens would reconstruct shells using glass beads. Here we further investigate shell architecture in this model species.

For micromechanical testing, an intact A. rara shell was placed between a fixed plate and a facing plate in series with a calibrated load cell. Displacement was effected by a high-precision drive, and 2-3 loading cycles were used to determine shell material properties. to assay tensile properties of the adhesive matrix, a network of pseudopodia and extracellular matrix fibers (i.e., the shell adhesive component) was obtained by incubating shucked cell bodies on 200-mesh gold grids. Pseudopodia were subsequently removed by detergent washes. Fibers in the resultant isolated matrix were severed with a Nd: YAG laser using an inverted DIC light microscope equipped with a 60× objective lens. Preliminary loading experiments using glass needles showed that Sepharose 2B beads were suitable strain gauges to assess compression within reconstructed shells (Fig. 1). in this assay, shucked cell bodies were incubated with a mixture of glass and Sepharose beads, and the reconstructed shells were examined by SEM.

Repeated loading and unloading demonstrated the elastic behavior of intact shells (Fig. 2). Adhesive matrix fibers snapped towards their attachment sites within 2 sec after cutting with a laser (Fig. 3), demonstrating that they are deployed under tension. SEM images of shells reconstructed with Sepharose show compressed particle profiles (Fig. 4).

In this study a combination of scanning (SEM) and transmission electron microscopy (TEM) was used to examine leaf cells of the common weed Indian strawberry (Duchesnea indica) infected by the biotrophic rust fungus Frommeëla mexicana var. indicae. For SEM, free-hand sections of infected leaves cut with a razor blade were placed in a 5% (wt/vol) aqueous solutions of Ariel (Proctor and Gamble, Cincinnati, OH), a washing powder containing a bacterial protease. This treatment removed exposed host cell cytoplasm so as to reveal details of fungal haustoria and host cell wall responses to infection (Figs. 1, 2). Samples then were processed for study according to procedures described previously. For TEM, samples were prepared for study using high pressure freezing followed by freeze substitution. This fixation procedure has been used with excellent results to study hostpathogen interactions in rust-infected plants.

Frommeëla mexicana var. indicae produced specialized hyphal branches known as haustoria that extended through the walls of infected cells. Each haustorium possessed a long, slender neck region (Fig. 3) and an expanded body (Figs. 1, 4) that was separated from the host cell cytoplasm by the invaginated host cell plasma membrane. At maturity the neck region was surrounded by a extensive labyrinth cell wall in-growth or elaboration (Figs. 1-3) that appeared to be identical to wall elaborations that characterize plant transfer cells. Such cells are thought to be involved in intensive solute transfer over short distances. While transfer cells have been reported within cells of the haustoria of various plant parasitic higher plants and in plant giant cells induced by certain plant parasitic nematodes, this appears to be the first report of their formation in response to infection by a plant pathogenic fungus. Not only did these elaborations form in the immediate vicinity of haustoria, they also arose apart from haustoria in infected cells. However, such elaborations were not found in cells of uninfected leaves.

Type 1 pili are outer membrane organelles formed by 80% of all E. coli strains. They are up to 2 μm long, rod-like appendages covering the bacterial body like spines on a hedgehog (Fig. 1). Type 1 pili expressed by uropathogenic E. coli strains mediate adhesion to specific receptors of the bladder epithelium. They allow colonization of the cell surface as a prelude to infection. Type 1 pili are composed of a rigid rod and a fibrillar tip (Fig. 2a). The minor subunit FimH mediates adhesion and is located at the fibrillar tip as demonstrated by immunolabeling (Fig. 2b). The pilus rod is built from a structural protein, the major subunit FimA. Brinton analyzed parameters describing the helix of the type 1 pilus rod based on paracrystalline arrays. Accordingly, the data predicted one helical repeat to be composed of 25 subunits in 8 turns with a helical pitch of either 2.32 or 2.4 nm.

Here, the 3-D helical structure of the pilus rod has been determined from isolated negatively stained type 1 pili imaged in the annular dark-field mode by scanning transmission electron microscopy (STEM) (Fig. 3a). The power spectra of unbent rods (Fig. 3b) exhibit the strongest layerline at a spacing of (2.42 ± 0.04 nm)−1 representing the pitch of the putative one-start genetic helix (Fig. 3c). The axial distance to its nearest layerline corresponds to the helical repeat (Fig. 3c). Averaging by a linear superposition, as employed for structural analysis of F18 fimbriae, another type of adhering E. coli appendages, confirmed the helical repeat to be 19.31 ± 0.34 nm. Mass-per-length (MPL) determination of unstained type 1 pili by STEM revealed one helical repeat to be composed of 26 ±1 15.8 kDA FimA molecules. All possible helical selection rules were tested by D(Z,k) filtering. Thereby, any data inconsistent with the employed helical selection rule are excluded (Fig. 3d). Hence, the helical selection rule yielding the most power recovery represents the closest selection rule describing the helix. We found l = 8n + 21m to be the optimum helical selection rule. Fig. 3e represents the filtered image. Accordingly, one helical repeat is made of 8 turns containing 27 FimA monomers occurring with an axial rise of 0.72 ± 0.01 nm. D(Z,k) filtering with the helical selection rule assumed by Brinton resulted in significant loss of structure. Hence, we conclude to have refined the previous indexation. The resulting 3-D structure of the type 1 pilus unveils a 6.8 nm wide right handed helical tube harboring an axial cavity (Fig. 4). The tube wall is formed by a coiled fiber comparable to the metal thread of a spring.

Napsin is an aspartic proteinase which has two essential asparagine residues in the active peptide (Tatnell et al., 1997). Napsin may participate in the processing of pulmonary surfactant protein B (Weaver et al., 1992; Chuman et al., 1999). in addition, changes in napsin expression have been linked to primary adenocarcinoma as well as kidney dysfunction (Hirano et al., 2000; Schauer-Vukasinovic et al., 2000). Localization studies by light microscope immunocytochemistry and in situ hybridization detected napsin in the lungs as well as in the kidney (Tatnell et al., 1997; Chuman et al, 1999; Schauer- Vukasinovic et al., 1999). However, these studies did not resolve the ultrastructure localization of napsin. in this study, EM cryoimmunogold labeling was used to characterize the subcellular distribution of napsin in lung and kidney cells.

A truncated murine napsin propeptide construct (1212 b.p.) without the signal peptide sequence (47 b.p.) was cloned into pProEX™HTb plasmid vector for the generation of recombinant protein. Polyclonal antibodies were generated against purified recombinant protein. The specificity of polyclonal antibodies was determined by immunoprecipitation and Western blotting using isolated mouse alveolar type 2 epithelial cells and kidney homogenates. The napsin antibodies detected a single protein with Mr= 43 kD.

Localization of napsin by EM cryoimmunogold labeling was performed using protein A gold probes as previously described (Na et al, 2000). in the lungs, napsin was localized in both alveolar type 2 epithelial cells and in alveolar macrophages. in alveolar type 2 epithelial cells, napsin was detected in multivesicular bodies and in lamellar bodies; internalized BSA gold colocalized with napsin in the lysosomes of alveolar macrophages (Figure 1A and 1B), consistent with the observation that napsin is active at low pH (Schauer-Vukasinovic et al., 1999). Although Schauer-Vukasinovic et al. (1999) reported that napsin was expressed by some bronchiolar cells, we did not observe any significant napsin labeling in Clara cells or in ciliated epithelial cells.

Zanichellia palustris is a submerged halophyte which occurs in waters of low salinity (5%o to 10%o). It is not capable of surviving at high salinity. Reasons for its inability to tolerate hypersaline conditions is important to our understanding of its biology. in the present study, leaf ultrastructure of plants growing at normal salinity was compared with plants growing at high salinity so as to assess the effects of hypersalinity. Attention was focused on ultrstructural changes occurring in leaf blade epidermal cells.

Plants were grown in seawater at two salinities : 10%o (control) and 20%o (high salinity). Pieces of mature leaf blades from both treatments were harvested and prepared for Transmission Electron Microscopy (TEM) following standard procedures. in addition, the overall distribution and concentration of chlorine (Cl) in the leaves was ascertained since it is the most abundant anion in seawater and is important in considerations of salt tolerance in submerged halophytes. Cl was determined by means of a nuclear microprobe. Pieces of sectioned leaves were rapidly frozen, freeze dried and carbon coated. Elemental distribution maps of Cl were obtained using the true elemental imaging system (Dynamic Analysis). Two complementary techniques, Proton Induced X-Ray Emission (PLXE) and Proton Back Scattering (BS) were performed simultaneously using the GeoPIXE suite of programmes. Sample thickness and composition of major elements were obtained from the BS spectra.

The ultrastructural morphology of leaf blade epidermal cells, which possess transfer cell characteristics differed at each salinity. While the cytoplasm was well preserved in epidermal cells of plants at the control salinity : 10%o (Fig 1), considerable cytoplasmic damage occurred in epidermal cells of plants at high salinity, in the form of breakdown of membranes such as the plasmamembrane and tonoplast, and senescence of various organelles including chloroplasts and mitochondria (Fig. 2).

Growth is “anisotropic” when growth rates in different directions are not equal. Anisotropic elongation is controlled by cortical microtubules and cellulose microfibrils of the cell wall. Distorted anisotropic growth results when there are aberrations in either the cellulose network or microtubule cytoskeleton. in this ultrastructural study, the roots of wild type (control) and mutants of Arabidopsis thaliana (L.) Heynh, ecotype Columbia, were compared to determine the role of microtubule organisation, cellulose synthesis and cytokinesis on root expansion.

Three mutations, obtained by treating seeds of A. thaliana with ethane-methylsulfonate and backcrossing once to wild type after four selfed generations, were isolated and designated as rsw (radially swollen). These phenotypes are all temperature sensitive, growing and appearing as wild type at 19°C but expressing the mutant phenotype at or above 30°C. Segments of intact root tips from the three mutants and wild type were prepared and examined for TEM with a Jeol 1200EX. The overall appearance of rsw seedlings at 19°C is the same as wild type. At 30°C these phenotypes lead to extensive swelling of the root apex. Despite considerable alteration in the morphology of the root apex, the appearance of the root cap and root hairs seems essentially normal. When mutant roots expressing these phenotypes at 30°C are returned to 19°C, a wild type appearance of the root apex and elongation gradually returns.

The primary root of the wild type has a remarkably uniform cellular organisation with regularly arranged dense cytoplasmic cells (Fig. 1). At 19°C, rsw4, rsw6 and rsw7 were indistinguishable from wild type; however, at 30°C, the well-defined anatomy was distorted in all tissues by additional cell walls in unusual positions (Fig. 2).