Ultrastructural studies of microtubules and microtubule organizing centers of the vertebrate olfactory neuron.

The olfactory neuron is specialized along its length into highly determined morphological regions. These regions include the dendritic cilia, dendritic vesicle, dendritic shaft proper, perikaryon, axon, zone of transition where the axon widens as it approaches its termination, and the axon terminal. Except for the zone of transition and the terminal, characteristic populations of microtubules occur in these compartments. In the olfactory vesicle, three discrete microtubule organizing centers (MTOCs) nucleate microtubules: the basal body, the lateral foot associated with the body, and dense masses of nearby material. Little is known about MTOCs elsewhere in the neuron, although the polarity of the axonal microtubules indicate that they originate at or near the perikaryon. An attempt is made to summarize what is known of the origin, structure, distribution, and function of microtubules in vertebrate olfactory neurons, which are useful model systems in which to study microtubules. Information about olfactory neuron microtubules may be applicable to neurons in general (e.g., the discovery that axons contain microtubules of uniform polarity was first made in the olfactory neuron) or to microtubules in other eukaryotic cells.     

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.     

Higher plant cells: gamma-tubulin and microtubule nucleation in the absence of centrosomes

The assembly of the higher plant cytoskeleton poses several fundamental questions. Since different microtubule arrays are successively assembled during the cell cycle in the absence of centrosomes, we can ask how these arrays are assembled and spatially organized. Two hypotheses are under debate. Either multiple nucleation sites are responsible for the assembly and organization of microtubule arrays or microtubule nucleation takes place at one site, the nuclear surface. In the latter case, microtubule nucleation and organization would be two distinct but coregulated processes. During recent years, novel approaches have provided entirely new insights to understand the assembly and dynamics of the plant cytoskeleton. In the present review, we summarize advances made in microscopy and in molecular biology which lead to novel hypotheses and open up new fields of investigation. From the results obtained, it is clear that the higher plant cell is a powerful model system to investigate cytoskeletal organization in acentrosomal eukaryotic cells.     

Microtubule and cellulose microfibril orientation during plant cell and organ growth

In this review, I ask the question of what is the relationship between growth and the orientations of microtubules and cellulose microfibrils in plant cells. This should be a relatively simple question to answer considering that text books commonly describe microtubules and cellulose microfibrils as hoops that drive expansion perpendicular to their orientation. However, recent live imaging techniques, which allow microtubules and cellulose synthase dynamics to be imaged simultaneously with cell elongation, show that cells can elongate with nonperpendicular microtubule arrays. In this review, I look at the significance of these different microtubule arrangements for growth and cell wall architecture and how these resultant walls differ from those derived from perpendicular arrays. I also discuss how these divergent arrays in stems may be important for coordinating growth between the different cell layers. This role reveals some general features of microtubule alignment that can be used to predict the growth status of organs. In conclusion, nonperpendicular arrays demonstrate alternative ways of cell elongation that do not require hooped arrays of microtubules and cellulose microfibrils. Such nonperpendicular arrays may be required for optimal growth and strengthening of tissues.     

Microtubule organizing centers and the origin of centrioles during spermatogenesis in the pteridophyte Phylloglossum

Spermatogenesis in the lycophyte Phylloglossum is characterized by profound ultrastructural changes that involve complex microtubule arrays and discrete microtubule organizing centers (MTOCs). The first visible MTOC is an electron-opaque acentriolar centrosome that organizes the mitotic spindles in late spermatogeneous cells. In the spermatid mother cell, centrioles arise de novo within the pericentriolar matrix of the MTOC. Approximately 20 centrioles, which ultimately function as basal bodies, originate in each of two branched "blepharoplasts." Constituent centrioles of each organelle radiate from a central region where they are interconnected by cartwheel cylinders, each with nine-fold symmetry. The development and structure of this novel centriolar-generating organelle suggests a direct evolutionary link with the bicentriole of other lycophytes, and are consistent with the concept that multiflagellated spermatozoids in Phylloglossum evolved independently of those in other pteridophytes. During spermiogenesis, two additional structurally defined MTOCs organize the ton and locomotory apparatus, which comprises 20 staggered flagella over a multilayered structure. An MTOC that overlies the multilayered structure and consists of a cloud of electron-opaque material is involved in repositioning basal bodies and generating flagella. The spline, a band of up to 200 microtubules, provides the architectural framework for development and maintenance of cell shape and is organized by the lamellar strip, a highly structured MTOC. Microtubule arrays during spermatogenesis in Phylloglossum are diverse and include mitotic, cytokinetic, cytoskeletal, and locomotory assemblages. MTOCs responsible for the nucleation and organization of these arrays are among the most elaborate and morphologically distinct of any described in land plants.     

Morphological studies on the mechanisms of pigmentary organelle transport in fish xanthophores and melanophores

Pigmentary organelle translocations within fish chromatophores undergo physiological color changes when exposed to external signals. Chromatophores can be isolated in high yields, and their pigmentary organelles can be tracked readily by microscopy. The combined efforts of morphology and biomolecular chemistry have led to the identification of and determination of the interrelationships between cytoskeletal elements and accessory proteins, motor molecules, cytomatrix, and pigmentary organelles of various sizes. Fish chromatophores have been classified as fast, intermediate, and slow translocators, based on the relative numbers of microtubules. Studies on cultured goldfish (Carassius auratus L.) xanthophores for over 20 years have demonstrated that in this slow translocator, tubulovesicular structures of the smooth endoplasmic reticular (SER) cisternae are involved in the disperson and aggregation of associated carotenoid droplets (CD) with some involvement of cytoskeletal elements. Killifish (Fundulus heteroclitus L.) melanophore, a fast translocator, was also examined. Recent work demonstrates a bright fluorescent "starburst"-like spot that we call an actin filament-organizing center (AFOC) with radiating microfilaments, akin to the microtubule-organizing center (MTOC) with radiating microtubules. Melanosomes translocate single-file on microtubules and are not associated with SER cisternae. Slower CD dispersion or aggregation in goldfish xanthophores seems to be predominantly microfilament-based transport, or microfilament- and microtubule-based transport, respectively. Faster melanosome translocations in killifish melanophores are based on microtubules, with our evidence indicating microfilament involvement. Neural crest-derived chromatophores are models for vesicular transport in axons, and immunocytochemical and imaging technologies may help to elucidate the cellular transport mechanisms.     

Carbon-dioxide sensing structures in terrestrial arthropods

Sensory structures that detect atmospheric carbon dioxide have been identified and described to the subcellular level in adults of Lepidoptera, Diptera, Hymenoptera, Isoptera, Chilopoda, and Ixodidae, as well as in lepidopteran larvae. The structures are usually composed of clusters of wall-pore type sensilla that may form distinct sensory organs, often recessed in pits or capsules. In insects, they are located on either the palps or the antennae, in chilopods on the head capsule, and in ixodids on the forelegs. In the two cases where the central projections have been examined (Lepidoptera and mosquitoes), the clustering is preserved to the level of second order neurons, which are located in the deutocerebrum. Individual sensilla usually contain a single receptor neuron that is sensitive to CO(2); it may be accompanied by other neurons that respond to other olfactory qualities. The distal dendritic processes of CO(2)-sensitive neurons invariably show an increased surface area, dividing into many cylindrical branches or into lamellar structures. Lamellar membranes are often closely linked to arrays of microtubules. Fine pore canal tubules are usually associated with the cuticular pores.     

MAP1B expression and microtubule stability in growing and regenerating axons

MAP1B is a microtubule-associated phosphoprotein that is particularly highly expressed in developing neurons. There is experimental evidence that it plays an important role in neuronal differentiation, especially the extension of axons and dendrites, but exactly what role is unclear. Recent experiments have shed light on the gene structure of MAP1B and identified some of the kinases that phosphorylate the protein. Implicit in these findings is the idea that MAP1B regulates the organisation of microtubules in neurites and is itself regulated in a complex way and at a number of levels.     

Mechanism of the process formation; podocytes vs. neurons

In this review article we discuss the common mechanism for cellular process formation. Besides the podocyte, the mechanism of process formation, including cytoskeletal organization and signal transduction, etc., has been studied using neurons and glias as model systems. There has been an accumulation of data showing common cell biological features of the podocyte and the neuron: 1) Both cells possess long and short cell processes equipped with highly organized cytoskeletal systems; 2) Both show cytoskeletal segregation; microtubules (MTs) and intermediate filaments (IFs) in podocyte primary processes and in neurites, while actin filaments (AFs) are abundant in podocyte foot processes in neuronal synaptic regions; 3) In both cells, process formation is mechanically dependent on MTs, whose assembly is regulated by various microtubule- associated proteins (MAPs); 4) In both cells, process formation is positively regulated by PP2A, a Ser/Thr protein phosphatase; 5) In both cells, process formation is accelerated by laminin, an extracellular matrix protein. In addition, recent data from our and other laboratories have shown that podocyte processes share many features with neuronal dendrites: 1) Podocyte processes and neuronal dendrites possess MTs with mixed polarity, namely, plus-end-distal and minus-end-distal MTs coexist in these processes; 2) To establish the mixed polarity of MTs, both express CHO1/MKLP1, a kinesin-related motor protein, and when its expression is inhibited formation of both podocyte processes and neuronal dendrites is abolished; 3) The elongation of both podocyte processes and neuronal dendrites is supported by rab8-regulated basolateral-type membrane transport; 4) Both podocyte processes and neuronal dendrites express synaptopodin, an actin-associated protein, in a development-dependent manner; interestingly, in both cells, synaptopodin is localized not in the main shaft of processes but in thin short projections from the main shaft. We propose that the podocyte process and the neuronal dendrite share many features, while the neuronal axon should be thought of as an exceptionally differentiated cellular process.     

The role of microtubules in cellular organization and endocytosis in the plant pathogen Ustilago maydis

Microtubules are an important part of the eukaryotic cytoskeleton, which participates in numerous essential cellular processes. In fungi interphase microtubules mediate cell polarity and participate in polar growth. However, our understanding of their detailed role in fungal growth is just at the beginning. In growing cells of the plant pathogenic fungus Ustilago maydis microtubules are organized by polar microtubule organizing centres that focus the microtubule minus ends at the small bud. Two opposing motor complexes utilize this microtubule polarity. Cytoplasmic dynein and a kinesin of the Unc104/Kif1A family of kinesins mediate rapid bi‐directional transport of early endosomes. A balance of their activity is required for cell cycle‐dependent accumulation of early endosomes at the growth site, the rear cell pole and the region of cell cleavage. Mutant phenotypes suggest that these endosomes participate in polar growth, bud site selection and cell separation. Therefore, our data suggest that endocytotic membrane recycling participates in local exocytosis, and that the microtubule cytoskeleton has a crucial role in this process.     

Regulatory cell interactions between retinal ganglion cells and radial glia during axonal and dendritic outgrowth

Neuronal differentiation and the formation of cell polarity are crucial events during the development of the nervous system. Cell polarity is a prerequisite for directed information flux within neuronal networks. In this article, we focus on neuro-glial cell interactions that influence the establishment of neural cell polarity and the directed outgrowth of axons versus dendrites. The cellular model discussed in detail is the retinal ganglion cell (RGC) of the chick retina, which is investigated by a comprehensive set of in vitro assays. The experiments demonstrate that retinal microenvironment determines axon vs. dendrite formation of RGCs. The instructive differences in different retinal microenvironments are substantially influenced by radial glia. Different glial domains support or inhibit axon vs. dendrite outgrowth. The data support the notion that neuro-glial interactions are crucial for directed neurite outgrowth.     

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.     

Microtubule dynamics in Xenopus egg extracts

The organization and function of microtubules change dramatically during the cell cycle. At the onset of mitosis, a radial array of microtubules is broken down and reorganized into a bipolar spindle. This event requires changes in the dynamic behavior of individual microtubules. Through the use of Xenopus laevis egg extracts, a number of proteins affecting microtubule behavior have been identified. Recently, progress has also been made towards understanding how the activities of such microtubule-affecting proteins are regulated in a cell cycle-dependent manner. It is hoped that understanding how microtubule behavior is controlled during the cell cycle in vitro may illuminate the role of microtubule dynamics in various cellular processes.     

Important factors determining the nanoscale tracking precision of dynamic microtubule ends

Tracking dynamic microtubule ends in fluorescence microscopy movies provides insight into the statistical properties of microtubule dynamics and is vital for further analysis that requires knowledge of the trajectories of the microtubule ends. Here we analyse the performance of a previously developed automated microtubule end tracking routine; this has been optimized for comparatively low signal‐to‐noise image sequences that are characteristic of microscopy movies of dynamic microtubules growing in vitro. Sequences of simulated microtubule images were generated assuming a variety of different experimental conditions. The simulated movies were then tracked and the tracking errors were characterized. We found that the growth characteristics of the microtubules within realistic ranges had a negligible effect on the tracking precision. The fluorophore labelling density, the pixel size of the images, and the exposure times were found to be important parameters limiting the tracking precision which could be explained using concepts of single molecule localization microscopy. The signal‐to‐noise ratio was found to be a good single predictor of the tracking precision: typical experimental signal‐to‐noise ratios lead to tracking precisions in the range of tens of nanometres, making the tracking program described here a useful tool for dynamic microtubule end tracking with close to molecular precision.     

Herpes simplex virus-mediated expression of the axonal protein tau in human model neurons (NT2-N cells)

The establishment of axonal-somatodendritic polarity is an important event during neuronal development. The analysis of the underlying molecular events requires experimental models that display characteristic steps in the development of polarity and that are accessible for experimental manipulations. Here we show that human model neurons (NT2-N cells) can be efficiently infected with an amplicon-based herpes simplex virus (HSV) system that expresses the axonal microtubule-associated protein tau. We demonstrate that the neurons express a high level of exogenous tau, which persists for several days, thus allowing us to analyze the morphological effects of the expressed protein. The intracellular interactions of tau and the effects on the microtubule structure of infected neurons, which were processed for immunocytochemistry, were determined using laser scanning microscopy (LSM). Exogenous tau expression does not result in an increased axon growth of the neurons but promotes neuronal microtubule assembly as indicated by an increased amount of total microtubule polymer as well as a labile, detyrosinated microtubule subpopulation. In contrast, tau expression does not induce a significant microtubule stabilization as judged from the quantitation of acetylated microtubule staining 24 hours after infection. The data demonstrate that HSV-mediated expression of proteins in human model neurons provides a useful system for analysis of the effect of neuronal proteins on the morphology and cytoskeletal organization of terminally differentiated polar neurons. In addition, it suggests a role for tau as a factor which locally promotes tubulin polymerization while the dynamics of axonal microtubules are preserved.     

Cortical microtubule reorientation in higher plants: dynamics and regulation

This paper aims to review aspects of cortical microtubule reorientation in higher plant cells. First, we look at the divergent environmental and developmental signals that can elicit the realignment of microtubules in interphase cells. Second, the regulatory factors that might orchestrate microtubule reorientation are examined. In particular, we address the questions of how these extracellular signals are perceived, by what mechanisms this information might be transmitted to the cortical microtubules, and what molecular factors regulate the process of realignment. We put forward an hypothesis of how electric fields reorientate microtubules in plant cells, focusing on the role of transmembrane proteins which might link cortical microtubules in the cytoplasm to the extracellular matrix. Finally, the need to examine microtubule reorientation in live cells is discussed, and we describe the novel visualization of microtubules in live cells of an intact plant. We conclude with our perspective of the future path of research which will be necessary to broaden our understanding of how microtubules undergo rapid reorientation in plant cells.     

Relationships between the central spindle and the contractile ring during cytokinesis in animal cells

During late anaphase and telophase, animal cells develop a bundle of antiparallel, interdigitating microtubules between the two daughter nuclei. Recent data indicate that this structure, called the central spindle, plays an essential role during cytokinesis. Studies in Drosophila and on vertebrate cells strongly suggest that the molecular signals for cytokinesis specifically emanate from the central spindle midzone. Moreover, the analysis of Drosophila mutants defective in cytokinesis has revealed a cooperative interaction between the central spindle microtubules and the contractile ring: when either of these structures is perturbed, the proper assembly of the other is disrupted. Based on these results we propose a model for the role of the central spindle during cytokinesis. We suggest that the interaction between central spindle microtubules and cortical actin filaments leads to two early events crucial for cytokinesis: the positioning of the contractile ring, and the stabilization of the plus ends of the interdigitating microtubules that comprise the central spindle. The latter event would provide the cell with a specialized microtubule scaffold that could mediate the translocation of plus-end-directed molecular motors to the cell's equator. Among the cargoes transported by these motors could be proteins involved in the regulation and execution of cytokinesis.     

Adrenergic regulation of bone metabolism: possible involvement of sympathetic innervation of osteoblastic and osteoclastic cells

It has been demonstrated that human osteoblastic as well as osteoclastic cells are equipped with adrenergic receptors and neuropeptide receptors and that they constitutively express diffusible axon guidance molecules that are known to function as a chemoattractant and/or chemorepellent for growing nerve fibers. These findings suggest that the extension of axons of sympathetic and peripheral sensory neurons to osteoblastic and osteoclastic cells is required for the dynamic neural regulation of local bone metabolism. Recently, bone resorption modulated by sympathetic stimulation was demonstrated to be associated with ODF (osteoclast differentiation factor) and OCIF (osteoclastogenesis inhibitory factor) produced by osteoblasts/stromal cells. This review summarizes the evidence implicating sympathetic neuron action in bone metabolism. The possible function of osteoclastogenesis, which could result in the initiation of sympathomimetic bone resorption, is also discussed.     

Analysis of efficiency of two-photon versus single-photon absorption for fluorescence generation in biological objects

By microinjecting rhodamine-conjugated porcine tubulin into pea epidermis we recently showed how cortical microtubules reorientate from transverse to longitudinal in living cells (Yuan et al ., 1994, Proc. Nat. Acad. Sci, USA 91, 6050–6053). In the present paper we compare this reorientation with the contrary longitudinal to transverse realignment induced by adding gibberellic acid to pre-injected cells on the microscope slide. Both kinds of reorientation are initiated by the appearance of 'discordant' microtubules which do not share the existing alignment but anticipate the new direction. These increase in number as the existing microtubules depolymerize, one alignment apparently replacing the other in a continuous process.
By rotating stacks of confocal sections by computer methods we have previously shown that microtubules at the outer tangential cell wall do not necessarily have the same orientation as microtubules at the adjoining anticlinal walls of the same cell (Yuan et al ., 1995, Plant J. 7, 17–23). This suggests that microtubule reorientations in these epidermal cells occur mainly (or, at least, first) at the outer wall, indicating that the array may not reorientate as a whole. Collectively, these data emphasize the discontinuous nature of the realignment process, the importance of new microtubule polymerization, and the special property of the outer epidermal surface as a sensitive domain.     

New polarized light microscope with precision universal compensator

A new type of polarized light microscope (‘new pol-scope’) for fast and orientation-independent measurement of birefringent fine structure has been developed. The design of the new pol-scope incorporates a precision universal compensator made from two liquid crystal variable retarders. A video camera and digital image processing system provide fast measurements of specimen anisotropy (retardance magnitude and azimuth) at all points of the image forming the field of view. The images document fine structural and molecular organization within a thin optical section of the specimen. The sensitivity of the current instrument is 0·1 nm of specimen retardance, measured with data gathered in 0·43 s at all 640 × 480 image points. Examples of birefringence measurements in biological (microtubule arrays) and industrial (magneto-optical disc substrate) specimens are presented.