Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5–2 m) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.Abbreviations BrdU 5-bromodeoxyuridine - DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether) - N,N,N,N tetraacetic acid We thank Ann Cork for technical assistance, Professor B.E.S. Gunning (Australian National University) and Drs. A.R. Hardham (A.N.U.) and R.E. Williamson (A.N.U.) for intellectual and material support, Dr D. McCurdy (A.N.U.) for the purified anti-actin antibody, and Professor B. Stone (La Trobe University, Melbourne, Australia) for generously providing the anti-callose antibody. We also thank the Electron Microscopy Unit of A.N.U. for the use of facilities. L.C.F. gratefully acknowledges financial support from the National Sciences and Engineering Research Council of Canada.     

Effects of ethylene on the orientation of microtubules and cellulose microfibrils of pea epicotyl cells with polylamellate cell walls

Summary Following a 5 hours ethylene treatment, cortical cells of Pea (Pisum sativum L. var Alaska) epicotyl third internode showed a change in the orientation of both microtubules near the plasma membrane and recently deposited cellulose microfibrils. Control cortical cells had mostly transverse microtubules. The ratio of the average frequency of transverse to longitudinal microtubules was 6.0. After 5 hours of ethylene treatment, cortical cells had mostly longitudinal microtubules, with the ratio of transverse to longitudinal microtubules equal to 0.1. Epidermal cells were more variable than cortical cells with regard to the frequency of longitudinal and transverse microtubules. Observation of cortical cell walls in conventionally stained thin sections revealed that recent deposition of microfibrils had been primarily transverse in almost all of the control cortical cells sampled. In contrast, more than half of the ethylene-treated cortical cells had recent deposition oriented primarily longitudinally. This change in microtubule and microfibril orientation may be early enough to constitute the primary effect of ethylene leading to radial cell expansion.Research supported by NSF grant PCM 78-03244, A1, 2 to PBG and by a Research Corporation grant to WRE.     

Extraction and immunochemical assays of a tubulin-like factor in cotton seedlings

Antibodies to tubulin were prepared in rabbits by immunization with reduced-carboxymethylated calf-brain tubulin. In immunodiffusion tests the antibodies showed full cross reactivity with the immunogen as well as with native calf-brain tubulin. The same antibodies showed cross reactivity with a factor in extract of cotton (Gossypium hirsutum L.) cotyledons but there was no full immunological identity between calf-brain tubulin and this factor. A solid-phase radioimmunoassay for quantitative estimation of this plant tubulin-like factor was developed. It measured the binding of antibodies to immobilized calf-native tubulin. Competition between the unknown soluble tubulin-like factor, and immobilized tubulin was assayed at serum dilution of 1:50. Extraction conditions which preserved the antigenic properties of the tubulin-like factor from cotton cotyledons were defined. The radioimmunoassay measured quantities of the tubulin-like factor in the range of 0.1–10 g-equivalents of calf-brain tubulin. Immediately after homogenization of the tissue only 25% of the total amount of tubulin-like activity was present in soluble form, while most of it remained in the insoluble fraction. Apparent maximal solubilization was achieved spontaneously 10 h after homogenization or by treatment with guanidine hydrochloride. These results indicate that in this material, tubulin is not released immediately by homogenization but remains assembled in microtubules and-or in a bound or sequestered form.Abbreviations NRS normal rabbit serum - RCM reduced-carboxymethylated     

Toward a biophysical theory of organogenesis: Birefringence observations on regenerating leaves in the succulent,Graptopetalum paraguayense E. Walther

Polarity shifts occur during organogenesis. The histological criterion for polarity is the direction of cell division. The biophysical criterion is the orientation of reinforcing cellulose microfibrils which lie normal to the organ axis and which determine the preferred growth direction. Using cell pattern to deduce cell lineage, and polarized light to study cellulose alignment, both aspects of polarity were examined in the epidermis of regenerating G. paraguayense. In this system new leaves and a stem arise from parallel cell files on a mature leaf. Large (90°) shifts in polarity occur in regions of the epidermis to give the new organs radial symmetry in the surface plane (files radiating from a pole). Study of the shifts in the epidermis showed that, during certain stages, shifts in the division direction are accompanied by shifts in the cellulose deposition direction, as expected. The new cellulose orientation is parallel to the new cross wall. During normal organ extension, however, shifts in division direction do not bring on changes in cellulose pattern. Thus the coupling between the two kinds of polarity is facultative. This variable relation is used in a biophysical model which can account for the reorganization of cell file pattern and cellulose reinforcement pattern into the radial symmetry of the new organ.     

Associations between membranes and microtubules during mitosis and cytokinesis in caulonema tip cells of the mossFunaria hygrometrica

Summary The interphase nucleus in theFunaria caulonema tip cells is associated with many non-cortical microtubules (Mts). In prophase, the cortical Mts disappear in the nuclear region; in contrast to moss leaflets, a preprophase band of Mts is not formed in the caulonema. The Mts of the early spindle are associated with the fragments of the nuclear envelope. Remnants of the nucleolus remain in the form of granular bodies till interphase. The metaphase chromosomes have distinct kinetochores; the kinetochore Mts are intermingled with non-kinetochore Mts running closely along the chromatin. Each kinetochore is associated with an ER cisterna. ER cisternae also accompany the spindle fibers in metaphase and anaphase. In telophase, Golgi vesicles accumulate in the periphery of the developing cell plate where no Mts are found. The reorientation of the cell plate into an oblique position can be inhibited by colchicine. It is concluded that the ER participates in controlling the Mt system, perhaps via calcium ions (membrane-bound calcium ions have been visualized by staining with chlorotetracycline) but that, on the other hand, the Mt system also influences the distribution of the ER. The occurrence and function of the preprophase band of Mts is discussed.     

Effects of microtubule-inhibitors on nuclear migration and rhizoid differentiation in germinating fern spores (Onoclea sensibilis)

Summary Germinating spores of the sensitive fern,Onoclea sensibilis L., undergo premitotic nuclear migration before a highly asymmetric cell division partitions each spore into a large protonemal cell and a small rhizoid initial. Nuclear movement and subsequent rhizoid formation were inhibited by the microtubule (MT) inhibitors, colchicine, isopropyl-N-3-chlorophenyl carbamate (CIPC) and griseofulvin. Colchicine prevented polar nuclear movement and cell division so that spores developed into enlarged, uninucleate single cells. CIPC and griseofulvin prevented nuclear migration, but not cell division, so that spores divided into daughter cells of approximately equal size. In colchicine-treated spores, MT were not observed at any time during germination. CIPC prevented MT formation at a time coincident with nuclear movement in the control and caused a disorientation of the spindle MT. Both colchicine and CIPC appeared to act at a time prior to the onset of normal nuclear movement. The effects of colchicine were reversible but those of CIPC were not. Cytochalasin b had no effect upon nuclear movement or rhizoid differentiation. These results suggests that MT mediate nuclear movement and that a highly asymmetric cell division is essential for rhizoid differentiation.     

Microtubules,dendritic spines and spine apparatuses

Summary Using techniques for enhanced microtubular preservation, including albumin pretreatment (Gray, 1975), occipital cortex of rats was studied electron microscopically at various ages of development. A close structural relationship was seen between microtubules, sacs of SER and the postsynaptic thickening in primordial spines and with the dense plate material of spine apparatuses. Stereoscopic preparations in addition show a more complicated substructure than previously described for the plate. Microtubules may contribute to the formation of the plate of the spine apparatus which in turn is associated with the postsynaptic thickening of the mature spine. Possible functional correlates are discussed.Dr. L.E. Westrum is an affiliate of the CDMRC at the University of Washington and a recipient of a Burroughs-Wellcome (USA) — Wellcome Trust (U.K.) Research Travel Grant. The research was also supported in part by NIH Grants NS 09678, NS 04053 (NINCDS) and DE 04942 (NIDR), DHHS     

Distribution and orientation of microtubules in milk secreting epithelial cells of rat mammary gland

Summary Quantitative ultrastructural analysis of mid-lactation rat mammary gland demonstrated that cytoplasmic microtubules were present in nearly all secretory epithelial cells examined. Most microtubules were oriented perpendicular to the apical membrane and were found in the apical and medial portions of the cell cytoplasm. There was no statistical difference between the number of microtubules associated with vesicles and the number that were not. Most vesicles which were in contact with microtubules were small (50 to 150 nm), appeared electron lucent and were located in a supra-Golgi complex position. Many of these vesicles were seen to be aligned along the axis of longitudinally sectioned microtubules oriented perpendicular to the apical plasma membrane. As measured by a colchicine binding assay, the total tubulin content of mammary tissue from mid-lactation rats was about 107 g/100 mg wet weight. Approximately 19% of the total tubulin was in polymerized form. This study provides evidence that microtubules may be involved in guiding transport of small secretory vesicles to the apical regions of cells for exocytosis.     

Microtubule-synaptic vesicle associations in cultured rat spinal cord neurons

Summary This paper describes new ultrastructural features of neural processes and of synapses in cultured CNS tissue treated with albumin before fixation using a modification of the technique recently introduced by Gray (1975). Nerve fibre bundles in explants of foetal spinal cord grown in vitro for 15–18 days were transected microsurgically. After transection the cultures were exposed to 20% albumin in distilled water and then fixed in unbuffered osmium tetroxide followed by unbuffered glutaraldehyde.In this material, but not in controls (injured but not exposed to albumin; exposed to albumin without injury) microtubules were found within many axonal varicosities, often situated close to presynaptic membrane specializations. These microtubules were closely associated with vesicles resembling synaptic vesicles, which were occasionally aligned in rows along the microtubules. Similar vesicle-microtubule associations were also found in non-terminal axons. Microtubules were also observed very close to some postsynaptic densities.The possibility that the microtubule-vesicle associations are involved in vesicle movements (along axons and/or within axon terminals) is discussed. A more direct involvement of microtubules in terminals in the mechanism of transmitter release is also considered.The author wishes to thank Dr. A.R. Lieberman for his help and advice, Mr. Derek Fraser and Mr. Peter Felton for their technical assistance, Mr. Stuart Waterman for the photographic prints, and Professor D.W. James for laboratory facilities     

New ultrastructural features on the cream hamster thyroid with special reference to the second kind of follicle

The thyroid cells of the cream hamster, characterized by abundance of microtubules and stratification of the organelles, undergo a particular evolution when the animals grow older. These changes are characterized by an increase of the number of lysosomes which in extreme cases become so prominent that they occupy the whole cytoplasm of the cell which thus loses its organelle stratification. As in other species, cream hamster thyroid contains so-called ultimobranchial follicles made up of at least six cell types: fibrillar dark and light cells, parafollicular cells, ciliated cells, vesicular cells, and cells with myelinic inclusions. The ultrastructure of these follicles in the cream hamster represents a mixture of the ultrastructural characteristics of the same follicles encountered in the rat and the mouse thyroid. Here also mixed follicles are seen. Nevertheless vesicular cells present such abundant "secretion granules" that the question arises as to whether these follicles produce a special secretion and perhaps a new hormone. Incubation of cream hamster thyroids in the prescence of vincristine induces vanishing of microtubules, formation of paracrystalline structures, and loss of stratification of the organelles. Although these last effects might be due to some specific toxic effect of the drug, it is suggested that the disappearing of the organelle stratification might result from a specific vincristine-induced disaggregation of the microtubules acting as a cytoskeleton.