Polyglucosan body density in the aged mouse hippocampus is controlled by a novel modifier locus on chromosome 1.

Aging can be associated with the accumulation of hypobranched glycogen molecules (polyglucosan bodies, PGBs), particularly in astrocytes of the hippocampus. While PGBs have a detrimental effect on cognition in diseases such as adult polyglucosan body disease and Lafora disease, the underlying mechanism and clinical relevance of age-related PGB accumulation remains unknown. Here, we have investigated the genetic basis and functional impact of age-related PGB accumulation in 32 fully sequenced BXD-type strains of mice which exhibit a 400-fold variation in PGB burden in 16-18 month old females. We mapped a major locus controlling PGB density in the hippocampus to chromosome 1 at 72-75 Mb (linkage of 4.9 -logP), which we defined as the Pgb1 locus. To identify potentially causal gene variants within Pgb1, we generated extensive hippocampal transcriptome datasets and identified two strong candidate genes for which mRNA correlates with PGB density-Smarcal1 and Usp37. In addition, both Smarcal1 and Usp37 contain non-synonymous allele variations likely to impact protein function. A phenome-wide association analysis highlighted a trans-regulatory effect of the Pgb1 locus on expression of Hp1bp3, a gene known to play a role in age-related changes in learning and memory. To investigate the potential impact of PGBs on cognition, we performed conditioned fear memory testing on strains displaying varying degrees of PGB burden, and a phenome-wide association scan of ~12,000 traits. Importantly, we did not find any evidence suggesting a negative impact of PGB burden on cognitive capacity. Taken together, we have identified a major modifier locus controlling PGB burden in the hippocampus and shed light on the genetic architecture and clinical relevance of this strikingly heterogeneous hippocampal phenotype.

Correlative microscopy: providing new understanding in the biomedical and plant sciences.

Correlative microscopy is the application of two or more distinct microscopy techniques to the same region of a sample, generating complementary morphological, structural and chemical information that exceeds what is possible with any single technique. As a variety of complementary microscopy approaches rather than a specific type of instrument, correlative microscopy has blossomed in recent years as researchers have recognised that it is particularly suited to address the intricate questions of the modern biological sciences. Specialised technical developments in sample preparation, imaging methods, visualisation and data analysis have also accelerated the uptake of correlative approaches. In light of these advances, this critical review takes the reader on a journey through recent developments in, and applications of, correlative microscopy, examining its impact in biomedical research and in the field of plant science. This twin emphasis gives a unique perspective into use of correlative microscopy in fields that often advance independently, and highlights the lessons that can be learned from both fields for the future of this important area of research.

Cryofixation rapidly preserves cytoskeletal arrays of leaf epidermal cells revealing microtubule co-alignments between neighbouring cells and adjacent actin and microtubule bundles in the cortex.

Accurate preservation of microtubule and actin microfilament arrays is crucial for investigating their roles in plant cell development. Aldehyde fixatives such as paraformaldehyde or glutaraldehyde preserve cortical microtubule arrays but, unless actin microfilaments are stabilized with drugs such as m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), ethylene glycol bis[sulfosuccinimidylsuccinate] (sulfo-EGS) or phalloidin, their arrays are often poorly preserved. Cryofixation, used primarily for electron microscopy, preserves actin microfilaments well but is used rarely to fix plant cells for optical microscopy. We developed a novel whole-mount cryofixation method to preserve microtubule and microfilament arrays within Tradescantia virginiana leaf epidermal cells for investigation using confocal microscopy. Cortical microtubule arrays were often oriented in different directions on the internal and external faces of the epidermal cells. A number of arrays were aligned in several directions, parallel to microtubules of neighbouring cells. Actin microfilaments were particularly well preserved possibly due to the speed with which they were immobilized. No transverse cortical microfilament arrays were observed. On occasion, we observed co-aligned microfilament and microtubule bundles lying adjacent to the plasma membrane and positioned side by side suggesting a potential direct interaction between the cytoskeletal filaments at these locations. Cryofixation is therefore a valuable tool to investigate the interactions between cytoskeletal arrays in plant cells using confocal microscopy.

Towards correlative imaging of plant cortical microtubule arrays: combining ultrastructure with real-time microtubule dynamics.

There are a variety of microscope technologies available to image plant cortical microtubule arrays. These can be applied specifically to investigate direct questions relating to array function, ultrastructure or dynamics. Immunocytochemistry combined with confocal laser scanning microscopy provides low resolution "snapshots" of cortical microtubule arrays at the time of fixation whereas live cell imaging of fluorescent fusion proteins highlights the dynamic characteristics of the arrays. High-resolution scanning electron microscopy provides surface detail about the individual microtubules that form cortical microtubule arrays and can also resolve cellulose microfibrils that form the innermost layer of the cell wall. Transmission electron microscopy of the arrays in cross section can be used to examine links between microtubules and the plasma membrane and, combined with electron tomography, has the potential to provide a complete picture of how individual microtubules are spatially organized within the cortical cytoplasm. Combining these high-resolution imaging techniques with the expression of fluorescent cytoskeletal fusion proteins in live cells using correlative microscopy procedures will usher in an radical change in our understanding of the molecular dynamics that underpin the organization and function of the cytoskeleton.

The phospholipase A inhibitor, aristolochic acid, disrupts cortical microtubule arrays and root growth in Arabidopsis.

The role of phospholipase A(2) in Arabidopsis root growth and microtubule organisation was investigated using a specific inhibitor, aristolochic acid. At 0.5-1.5 microm concentrations, this inhibitor reduced root elongation and caused radial swelling of the root tip. The normally transverse cortical microtubules in root tip cells became progressively more disorganised with increasing concentrations of the inhibitor. Microtubule disorganisation also occurred in leaf epidermal cells of Allium porrum. We propose that phospholipase A(2) is involved in microtubule organisation and anisotropic growth in a manner similar to that reported previously for phospholipase D, thus broadening the significance of phospholipid signalling in microtubule organisation in plants.

Reflection across plant cell boundaries in confocal laser scanning microscopy.

The fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFP-tagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finite-difference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal localization due to the effects of the cell wall on the reflection and scattering of light.

Are histones, tubulin, and actin derived from a common ancestral protein?

Histones and the cytoskeletal components tubulin and actin all act as thermal ratchets, using the energy present in Brownian motion to do work. All three also bind to nucleotides. Here we suggest that histones, tubulin, and actin derive from a common ancestral protein. There is some sequence similarity between histone 2A and the bacterial tubulin homologue FtsZ. Histones and actin also share some sequence similarity in the nucleotides and at phosphate-binding sites. Thus, actin and tubulin may also be related, although this is not obvious from sequence analysis. Indeed, actin and tubulin are closely functionally related and cooperate in many cellular processes. Interestingly, recent advances in nanotechnology suggest that thermal ratchets may be able to impart lifelike properties; thus, the evolution of the ancestral histone, tubulin, and actin thermal ratchet may have been crucial in the development of complexity in living organisms.

Effects of chilling on male gametophyte development in rice.

Chilling during male gametophyte development in rice inhibits development of microspores, causing male sterility. Changes in cellular ultrastructure that have been exposed to mild chilling include microspores with poor pollen wall formation, abnormal vacuolation and hypertrophy of the tapetum and unusual starch accumulation in the plastids of the endothecium in post-meiotic anthers. Anthers observed during tetrad release also have callose (1,3-beta-glucan) wall abnormalities as shown by immunocytochemical labelling. Expression of rice anther specific monosaccharide transporter (OsMST8) is greatly affected by chilling treatment. Perturbed carbohydrate metabolism, which is particularly triggered by repressed genes OsINV4 and OsMST8 during chilling, causes unusual starch storage in the endothecium and this also contributes to other symptoms such as vacuolation and poor microspore wall formation. Premature callose breakdown apparently restricts the basic framework of the future pollen wall. Vacuolation and hypertrophy are also symptoms of osmotic imbalance triggered by the reabsorption of callose breakdown products due to absence of OsMST8 activity.

Membrane-wall attachments in plasmolysed plant cells.

Field emission scanning electron microscopy of plasmolysed Tradescantia virginiana leaf epidermal cells gave novel insights into the three-dimensional architecture of Hechtian strands, Hechtian reticulum, and the inner surface of the cell wall without the need for extraction. At high magnification, we observed fibres that pin the plasma membrane to the cell wall after plasmolysis. Treatment with cellulase caused these connecting fibres to be lost and the pinned out plasma membrane of the Hechtian reticulum to disintegrate into vesicles with diameters of 100-250 nm. This suggests that the fibres may be cellulose. After 4 h of plasmolysis, a fibrous meshwork that labelled with anti-callose antibodies was observed within the space between the plasmolysed protoplast and the cell wall by field emission scanning electron microscopy. Interestingly, macerase-pectinase treatment resulted in the loss of this meshwork, suggesting that it was stabilised by pectins. We suggest that cellulose microfibrils extending from strands of the Hechtian reticulum and entwining into the cell wall matrix act as anchors for the plasma membrane as it moves away from the wall during plasmolysis.

Cytoplasmic acidification with butyric acid does not alter the ionic conductivity of plasmodesmata.

The effect of lowering cytoplasmic pH on the ionic conductivity of higher-plant plasmodesmata was investigated with corn (Zea mays L. cv. Black Mexican Sweet) suspension culture cells. Exposure to butyric acid decreased the cytoplasmic pH by 0.8 units. Intercellular communication was monitored by electrophysiological techniques that allowed the measurement of membrane resistances of sister cells and the electrical resistance of the plasmodesmata connecting them. The decrease in cytoplasmic pH did not affect the resistance of plasmodesmata, despite the fact that the butyric acid treatment more than doubled the concentration of cytoplasmic calcium. This is discussed in light of previous findings that increases in cytoplasmic calcium increase the electrical resistance of plasmodesmata.

Dystrophin in adult zebrafish muscle.

Mutations in the human dystrophin gene are implicated in the fatal muscle wasting disease Duchenne Muscular Dystrophy (DMD). This gene expresses a sarcolemmal-associated protein that is evolutionarily conserved, underpinning its important role in the architecture of muscle. In terms of DMD modelling, the mouse has served as a suitable vertebrate species but the pathophysiology of the disease in the mouse does not entirely mimic human DMD. We have examined the zebrafish in order to expand the repertoire of vertebrate species for muscle disease modelling, and to dissect further the functional interactions of dystrophin. We report here the identification of an apparent zebrafish orthologue of the human dystrophin gene that expresses a 400-kDa protein that is localised to the muscle membrane surface. These data suggest that the zebrafish may prove to be a beneficial vertebrate model to examine the role and functional interactions of dystrophin in disease and development.

Changes in symplastic permeability during adventitious shoot regeneration in tobacco thin cell layers.

Thin cell layer (TCL) explants of tobacco (Nicotiana tabacum L.) were cultured in either a regeneration medium that resulted in formation of adventitious vegetative shoots or a non-regeneration (control) medium that maintained the TCLs but did not promote shoot formation. Microinjections were conducted on epidermal cells at 1- or 2-day intervals during the culture period (14 days) and also on meristematic regions as they appeared in regenerating TCLs. A fluorescein isothiocyanate-labelled peptide (F(Glu)3 MW 799) was used to assess the permeability of the symplast during adventitious shoot regeneration. A period of increased symplastic movement of F(Glu)3 was detected during day 2 of culture and was significantly greater in regenerating TCLs than in non-regenerating TCLs. This corresponded to the period of the first cell divisions and represents the re-initiation of a meristematic type of symplastic linkage between epidermal cells. A smaller increase in cell-to-cell movement within non-regenerating TCLs indicated a possible stress response as a factor in these changes. Movement of F(Glu)3 throughout the epidermal symplast of regenerating TCLs returned to pre-culture levels by the time of shoot primordia formation. F(Glu)3 movement was further down-regulated in non-regenerating TCLs, with a high degree of cell isolation observed. Within newly formed shoots, symplastic movement of F(Glu)3 cycled between high and low levels.

Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata.

The concentration of cytoplasmic free calcium ([Ca(2+)](cyt)) required to close higher plant plasmodesmata was investigated using corn (Zea mays L. cv. Black Mexican Sweet) suspension-culture cells. Physiological elevations of [Ca(2+)](cyt) were applied by cold treatment, and ion injection was also used to increase [Ca(2+)](cyt), by diffusion (for small increases) or by iontophoresis (for larger increases). The impact of such treatments on [Ca(2+)](cyt) was measured by ratiometric ion imaging. Intercellular communication during treatments was monitored using our recently developed electrophysiological technique that allows the electrical resistance of plasmodesmata and the plasma membranes of a sister-cell pair to be measured. A 4-fold increase in the calculated resistance of single plasmodesmata was observed in response to cold treatment that caused a 2-fold increase in average [Ca(2+)](cyt) (from 107 to 210 nM). In response to iontophoresis of Ca(2+), plasmodesmata were observed to go from "open" (low resistance) to "shut" (high resistance) and then back "open" within 10 s. Our results thus indicate that higher plant plasmodesmata respond quickly to physiological changes in [Ca(2+)](cyt).

Localization of a centrin-like protein to higher plant plasmodesmata.

Antibodies against centrin, the ubiquitous calcium-binding contractile protein, recognized a 17 kDa protein in extracts of onion root tips and cauliflower florets. Using immunofluorescence microscopy, anti-centrin antibodies were localized to the developing cell plate of onion and cauliflower root tip cells. In cauliflower florets, these antibodies localized to the walls in a punctate manner, consistent with the distribution of plasmodesmata as shown by colocalization with callose. Anti-centrin antibodies were localized to plasmodesmata of onion root tips and cauliflower florets with immunogold electron microscopy. Furthermore, this label was concentrated around the necks of plasmodesmata. In contrast, an antibody against calmodulin, which is a closely related calcium-binding protein, did not label plasmodesmata. We propose that centrin is a component of calcium-sensitive contractile nanofilaments in the neck region of plasmodesmata and facilitates the calcium-induced regulation of intercellular transport.

Combined immunofluorescence and field emission scanning electron microscope study of plasma membrane-associated organelles in highly vacuolated suspensor cells of white spruce somatic embryos.

Highly vacuolated suspensor cells of spruce somatic embryos were examined by immunofluorescence light microscopy using butyl-methyl-methacrylate (BMM) and polyethylene glycol (PEG) embedded sections, transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM). The use of PEG embedded embryos provided a rapid method for light microscope detection of antigens before committing to FESEM analysis. BMM embedded specimens provided well preserved suspensor cells for immunofluorescence. FESEM permitted high resolution observation of large areas of the inner surface of the plasma membrane and associated cell organelles. Suspensor cells contained mostly transversely oriented cortical microtubules linked to the plasma membrane and adjacent microtubules by cross- bridges. Light and electron microscopy revealed numerous clathrin coated structures on the plasma membrane. These included flat patches of clathrin, coated pits and coated vesicles. Many coated vesicles were associated with microtubules. Both tubular and lamellar endoplasmic reticulum were observed on the plasma membrane by FESEM.

Cell-to-cell communication via plant endomembranes.

Cell-to-cell communication was investigated in epidermal cells cut from stem internodal tissue of Nicotiana tabacum and Torenia fournieri. Fluorescently labelled peptides and dextrans were microinjected using iontophoresis into the cytoplasm andcortical endomembrane network of these cells. The microinjected endomembrane network was similar in location and structure to the endoplasmic reticulum (ER) as revealed by staining with 3, 3'-dihexyloxacarbocyanine iodide (DiOC(6)). No cell-to-cell movement of dextrans was observed following cytoplasmic injections but injection of dextrans into the endomembrane network resulted in rapid diffusion of the probes to neighbouring cells. It is proposed that the ER acts as a pathway for intercellular communication via the desmotubule through plasmodesmata.

A simple and rapid technique for the immunofluorescence confocal microscopy of intact Arabidopsis root tips.

Visualisation of immunofluorescence labelling of Arabidopsis roots has previously been limited to single cell layers. A simple, rapid method has been devised in which the whole root can be processed to allow antibody penetration into several cell layers. When optically sectioned using confocal microscopy, cellular arrangements of microtubules, callose, calmodulin and a phosphoprotein epitope have been visualised using this technique. As the root is not physically sectioned, information regarding the three-dimensional position of individual cells in relation to each other and the tissue as a whole is retained. Using this technique, we have assessed the effect of brefeldin A on the frequency of mitotic arrays in root tip cortical and epidermal cells, and found that the occurrence of phragmoplasts increases significantly with brefeldin A treatment. This study demonstrates the possible future use of the whole root technique to assess rapidly the developmental, mutational and inhibitor-induced changes in the organisation of cellular components in Arabidopsis.

Ionic Current Changes Associated with the Gravity-Induced Bending Response in Roots of Zea mays L.

A vibrating probe was used to measure the changes in ionic currents around gravistimulated roots of Zea mays L. in an effort to determine whether these currents are involved in stimulus transduction from the root cap to the elongation zone. We did not observe a migration of the previously reported auxin-insensitive current efflux associated with gravity sensing (T. Björkman, A.C. Leopold [1987] Plant Physiol 84:841-846) back from the root cap. Instead, beginning 10 to 15 min after gravistimulation, an asymmetry in current developed simultaneously along the root around the meristem and apical regions of the elongation zone. This asymmetry comprised a proton efflux from the upper surface, which was superimposed on the symmetrical pattern around the vertical root. The gravity-induced proton efflux was inhibited by the application of the auxin transport inhibitor, 2,3,5-triiodobenzoic acid, whereas the calcium channel blocker, lanthanum, had little effect. Because the onset of the gravity-induced current asymmetry coincided both spatially and temporally with the onset of the differential growth response, we suggest that this current efflux may result from auxin-requiring acid-growth phenomena in the upper root tissue. The implications of this simultaneous onset of both proton efflux and elongation for theories about gravity stimulus transduction are discussed.

Ionic currents traversing cell clusters from carrot suspension cultures reveal perpetuation of morphogenetic potential as distinct from induction of embryogenesis.

Patterns of ionic currents accompanying shape and surface changes during growth of cell clusters from carrot suspension cultures were examined using a vibrating probe. Electrical polarity was established in clusters undergoing apparently disorganized proliferation in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D). This electrical polarity is similar to that found in organized somatic embryos which form upon removal of 2,4-D, and implies that the proliferating clusters are actually suppressed embryos. This implication is further supported by observations using scanning electron microscopy. We conclude that the potential to undergo embryogenesis is present even before the 2,4-D is removed, but cannot be realized in the presence of the auxin.