Deborah A. Barton

Potential role of tubulin acetylation and microtubule-based protein trafficking in familial dysautonomia.

Familial dysautonomia (FD), a disease of the autonomic and sensory nervous systems, involves mutations in the protein IκB kinase complex‐associated protein, which is a component of the human Elongator acetylase complex. We suggest a hypothesis in which defects in tubulin acetylation and impairment of microtubule‐based protein trafficking may be an underlying cause of FD. In addition, an Arabidopsis homolog of the Elongator subunit ELP3 has been found to bind to the αβ‐tubulin heterodimer, suggesting that α‐tubulin may be a cytoplasmic target of Elongator acetylase activity. Studies of synergistic double mutants in yeast indicate a novel role for Elongator in cytoskeletal dynamics, although this is probably because of an effect on actin rather than microtubules. Finally, we suggest that tubulin deacetylase inhibitors may prove useful in the treatment of FD.

The cytoskeleton in plasmodesmata: a role in intercellular transport?

Actin and myosin are components of the plant cell cytoskeleton that extend from cell to cell through plasmodesmata (PD), but it is unclear how they are organized within the cytoplasmic sleeve or how they might behave as regulatory elements. Early work used antibodies to locate actin and myosin to PD, at the electron microscope level, or to pitfields (aggregations of PD in the cell wall), using immunofluorescence techniques. More recently, a green fluorescent protein (GFP)-tagged plant myosin VIII was located specifically at PD-rich pitfields in cell walls. Application of actin or myosin disrupters may modify the conformation of PD and alter rates of cell-cell transport, providing evidence for a role in regulating PD permeability. Intriguingly, there is now evidence of differentiation between types of PD, some of which open in response to both actin and myosin disrupters, and others which are unaffected by actin disrupters or which close in response to myosin inhibitors. Viruses also interact with elements of the cytoskeleton for both intracellular and intercellular transport. The precise function of the cytoskeleton in PD may change during cell development, and may not be identical in all tissue types, or even in all PD within a single cell. Nevertheless, it is likely that actin- and myosin-associated proteins play a key role in regulating cell-cell transport, by interacting with cargo and loading it into PD, and may underlie the capacity for one-way transport across particular cell and tissue boundaries.

Neurotrophic Support and Oxidative Stress: Converging Effects in the Normal and Diseased Nervous System

Oxidative stress and loss of neurotrophic support play major roles in the development of various diseases of the central and peripheral nervous systems. In disorders of the central nervous system such as Alzheimers, Parkinsons, and Huntingtons diseases, oxidative stress appears inextricably linked to the loss of neurotrophic support. A similar situation is seen in the peripheral nervous system in diseases of olfaction, hearing, and vision. Neurotrophic factors act to up-regulate antioxidant enzymes and promote the expression of antioxidant proteins. On the other hand, oxidative stress can cause down-regulation of neurotrophic factors. We propose that normal functioning of the nervous systems involves a positive feedback loop between antioxidant processes and neurotrophic support. Breakdown of this feedback loop in disease states leads to increased oxidative stress and reduced neurotrophic support.

Analysis of Cortical Arrays from Tradescantia virginiana at High Resolution Reveals Discrete Microtubule Subpopulations and Demonstrates That Confocal Images of Arrays Can Be Misleading

Cortical microtubule arrays are highly organized networks involved in directing cellulose microfibril deposition within the cell wall. Their organization results from complex interactions between individual microtubules and microtubule-associated proteins. The precise details of these interactions are often not evident using optical microscopy. Using high-resolution scanning electron microscopy, we analyzed extensive regions of cortical arrays and identified two spatially discrete microtubule subpopulations that exhibited different stabilities. Microtubules that lay adjacent to the plasma membrane were often bundled and more stable than the randomly aligned, discordant microtubules that lay deeper in the cytoplasm. Immunolabeling revealed katanin at microtubule ends, on curves, or at sites along microtubules in line with neighboring microtubule ends. End binding 1 protein also localized along microtubules, at microtubule ends or junctions between microtubules, and on the plasma membrane in direct line with microtubule ends. We show fine bands in vivo that traverse and may encircle microtubules. Comparing confocal and electron microscope images of fluorescently tagged arrays, we demonstrate that optical images are misleading, highlighting the fundamental importance of studying cortical microtubule arrays at high resolution.

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.

Membrane–wall attachments in plasmolysed plant cells

Summary.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.

Differential Growth in Periclinal and Anticlinal Walls during Lobe Formation in Arabidopsis Cotyledon Pavement Cells

During epidermal cell development in Arabidopsis cotyledons, lobes form by uneven expansion of the outer periclinal wall and microtubules, not actin filaments, persist at lobes during this time. Lobe development in the epidermal pavement cells of Arabidopsis thaliana cotyledons and leaves is thought to take place via tip-like growth on the concave side of lobes driven by localized concentrations of actin filaments and associated proteins, with a predicted role for cortical microtubules in establishing the direction of restricted growth at the convex side. We used homologous landmarks fixed to the outer walls of pavement cells and thin-plate spline analysis to demonstrate that lobes form by differential growth of both the anticlinal and periclinal walls. Most lobes formed within the first 24 h of the cotyledons unfurling, during the period of rapid cell expansion. Cortical microtubules adjacent to the periclinal wall were persistently enriched at the convex side of lobes during development where growth was anisotropic and were less concentrated or absent at the concave side where growth was promoted. Alternating microtubule-enriched and microtubule-free zones at the periclinal wall in neighboring cells predicted sites of new lobes. There was no particular arrangement of cortical actin filaments that could predict where lobes would form. However, drug studies demonstrate that both filamentous actin and microtubules are required for lobe formation.

Cell-to-cell transport via the lumen of the endoplasmic reticulum

Plasmodesmata are plasma membrane-lined channels through which cytoplasmic molecules move from cell-to-cell in plants. Most plasmodesmata contain a desmotubule, a central tube of endoplasmic reticulum (ER), that connects the ER of adjacent cells. Here we demonstrate that molecules of up to 10.4 kDa in size can move between the ER lumen of neighbouring leaf trichome or epidermal cells via the desmotubule lumen. Fluorescent molecules of up to 10 kDa, microinjected into the ER of Nicotiana trichome cells, consistently moved into the ER and nuclei of neighbouring trichome cells. This movement occurred more rapidly than movement via the cytoplasmic pathway. A fluorescent 3-kDa dextran microinjected into the ER of a basal trichome cell moved into the ER and nuclei of epidermal cells across a barrier to cytoplasmic movement. We constructed a 10.4-kDa recombinant ER-lumenal reporter protein (LRP) from a fragment of the endogenous ER-lumenal binding protein AtBIP1. Following transient expression of the LRP in the ER of Tradescantia leaf epidermal cells, it often moved into the nuclear envelopes of neighbouring cells. However, green fluorescent protein targeted to the ER lumen (ER-GFP) did not move from cell to cell. We propose that the ER lumen of plant cells is continuous with that of their neighbours, and allows movement of small ER-lumenal molecules between cells.

High resolution scanning electron microscopy of plasmodesmata.

Symplastic transport occurs between neighbouring plant cells through functionally and structurally dynamic channels called plasmodesmata (PD). Relatively little is known about the composition of PD or the mechanisms that facilitate molecular transport into neighbouring cells. While transmission electron microscopy (TEM) provides 2-dimensional information about the structural components of PD, 3-dimensional information is difficult to extract from ultrathin sections. This study has exploited high-resolution scanning electron microscopy (HRSEM) to reveal the 3-dimensional morphology of PD in the cell walls of algae, ferns and higher plants. Varied patterns of PD were observed in the walls, ranging from uniformly distributed individual PD to discrete clusters. Occasionally the thick walls of the giant alga Chara were fractured, revealing the surface morphology of PD within. External structures such as spokes, spirals and mesh were observed surrounding the PD. Enzymatic digestions of cell wall components indicate that cellulose or pectin either compose or stabilise the extracellular spokes. Occasionally, the PD were fractured open and desmotubule-like structures and other particles were observed in their central regions. Our observations add weight to the argument that Chara PD contain desmotubules and are morphologically similar to higher plant PD.

Chilling to zero degrees disrupts pollen formation but not meiotic microtubule arrays in Triticum aestivum L.

Throughout the wheat-growing regions of Australia, chilling temperatures below 2 °C occur periodically on consecutive nights during the period of floral development in spring wheat (Triticum aestivum L.). In this study, wheat plants showed significant reductions in fertility when exposed to prolonged chilling temperatures in controlled environment experiments. Among the cultivars tested, the Australian cultivars Kite and Hartog had among the lowest levels of seed set due to chilling and their responses were investigated further. The developmental stage at exposure, the chilling temperature and length of exposure all influenced the level of sterility. The early period of booting, and specifically the +4 cm auricle distance class, was the most sensitive and corresponded to meiosis within the anthers. The response of microtubules to chilling during meiosis in Hartog was monitored, but there was little difference between chilled and control plants. Other abnormalities, such as plasmolysis and cytomixis increased in frequency, were associated with death of developing pollen cells, and could contribute to loss of fertility. The potential for an above-zero chilling sensitivity in Australian spring wheat varieties could have implications for exploring the tolerance of wheat flower development to chilling and freezing conditions in the field.