The three-dimensional morphology of aggregates of native cotton cellulose microfibrils

The three-dimensional morphology of native bacterial cellulose is confirmed by scanning electron microscopy. In addition, it is shown by scanning electron microscopy, and transmission electron microscopy with positive staining by phosphotungstic acid ions that aggregates of microfibrils of native cotton cellulose have a similar structure. The results are consistent with previous reports on microfibrils of algal cellulose. These observations exclude a simple spinneret process as a mechanism of formation of the microfibrils of these sources of cellulose.     

Regeneration of the cell wall of protoplasts of the fission yeastSchizosaccharomyces versatilis in liquid media and their reversion to cells

Regeneration of the cell wall and reversion of protoplasts with a completely regenerated cell wall to cells were studied by light and electron microscopy in protoplasts of the fission yeastsSchizosaccharomyces versatilis. On their surface the protoplasts regenerated a complete new wall even m liquid media The wall regeneration began with the formation of a thin irregular net of flat bundles of long microfibrils and the net was gradually filled with aggregates of short straight microfibrils and small piles of amorphous material. Osmotically resistant organisms with regenerated walls were detected after a 4–6 h cultivation Depending on the nutrient medium used 10–80 % of protoplasts with the regenerated wall were obtained that reverted subsequently to cells. The high percentage of the wall regeneration and reversion to cells was reached by combining cultivation in a poor medium with that in a rich medium Reversion to cells could only occur after the protoplasts had regenerated rigid cell walls These walled protoplasts underwent septation, and, by polar growth, produced cylindrical cells, further dividing by fission.     

Observations on the time course of wall development at the surface of isolated protoplasts

The formation of cell wall fibres at the surface of isolated leaf protoplasts has been studied by scanning electron microscopy. Fibres are not formed on incubated protoplasts until a lag period has elapsed. This period is about 8 h for leaf protoplasts of Nicotiana tabacum and about 45 h for leaf protoplasts of Antirrhinum majus. In the case of Antirrhinum protoplasts the length of the lag period is dependent on the concentration of osmoticum present during the incubation period. If regenerating protoplasts are briefly treated with dilute cellulase, the newly formed wall is completely digested. Such protoplasts are capable of producing new fibres at the surface within minutes of their return to a nutrient medium. These results are discussed in terms of the likely source of the lag period and its significance in wall regeneration studies.Abbreviations MS culture medium used at full strength - 0.1 MS culture medium used at one tenth full strength     

The role of microtubules and cell-wall deposition in elongation of regenerating protoplasts of Mougeotia

Protoplasts of the filamentous green alga Mougeotia sp. are spherical when isolated and revert to their normal cylindrical cell shape during regeneration of a cell wall. Sections of protoplasts show that cortical microtubules are present at all times but examination of osmotically ruptured protoplasts by negative staining shows that the microtubules are initially free and become progressively cross-bridged to the plasma membrane during the first 3 h of protoplast culture. Cell-wall microfibrils areoobserved within 60 min when protoplasts are returned to growth medium; deposition of microfibrils that is predominantly transverse to the future axis of elongation is detectable after about 6 h of culture. When regenerating protoplasts are treated with either colchicine or isopropyl-N-phenyl carbamate, drugs which interfere with microtubule polymerization, they remain spherical and develop cell walls in which the microfibrils are randomly oriented.     

Xylan deposition on secondary wall of Fagus crenata fiber

Summary. Delignified and/or xylanase-treated secondary walls of Fagus crenata fibers were examined by field emission scanning electron microscopy. Microfibrils with a smooth surface were visible in the innermost surface of the differentiating fiber secondary wall. There was no ultrastructural difference between control and delignified sections, indicating that lignin deposition had not started in the innermost surface of the cell wall. There was no ultrastructural difference between control and xylanase-treated sections. Microfibrils on the outer part of the differentiating secondary wall surface had globular substances in delignified sections. These globular substances disappeared following xylanase treatment, indicating that these globules are xylan. The globular substances were not visible near the inner part of the differentiating secondary wall but gradually increased toward the outer part of the secondary wall, indicating that xylan penetrated into the cell wall and continuously accumulated on the microfibrils. Mature-fiber secondary walls were also examined by field emission scanning electron microscopy. Microfibrils were not apparent in the secondary wall in control specimens. Microfibrils with many globular substances were observed in the delignified specimens. Following xylanase treatment, the microfibrils had a smooth surface without any globules, indicating that the globular substance is xylan. These results suggest that cellulose microfibrils synthesized on the plasma membrane are released into the innermost surface of the secondary wall and coated with a thin layer of xylan. Successive deposition of xylan onto the cell wall increases the microfibril diameter. The large amounts of xylan that accumulated on microfibrils appear globular but are covered with lignin after they are deposited. Received February 20, 2001/Accepted September 1, 2001     

Papulacandin B: Inhibitor of biogenesis of (1→3)-β-d-glucan fibrillar component of the cell wall ofSaccharomyces cerevisiae protoplasts

The effect of papulacandin B on regenerating protoplasts ofSaccharomyces cerevisiae was studied by light and electron microscopy. In liquid media it inhibited the biogenesis of (1→3)-β-d-glucan fibrillar nets; as a result, the protoplasts did not grow polarly but only spherically. The effect was reversible. Instead of the nets the inhibited protoplasts synthesized only individual microfibrils soluble in hydroxide; these were not joined in the nets and were partially masked by amorphous material. The microfibrils disintegrated after lysis and did not maintain the shape of protoplasts. Protoplasts inhibited in solid media grew spherically up to 25 μm but they did not divide or revert or revert, in spite of forming cell walls. These walls were amorphous and fragile and they disintegrated during preparation. Papulacandin B did not decrease the viability of protoplasts and did not interfere with their growth, biogenesis of alkali-soluble glucan microfibrils or amorphous wall matrix. It inhibited specifically the synthesis of alkali-insoluble branched (1→3)-β-d-glucan, a necessary building unit required for the formation of the fibrillar component of the cell wall responsible for the cell wall shape, its rigidity and tensile strength.     

Patch clamping corn protoplasts

Summary Cell wall regeneration around protoplasts from Black Mexican Sweet corn suspension cells has been observed using scanning electron microscopy. A coherent array of cellulose microfibrils can be seen around protoplasts two hours after they have been isolated. This array does not form in the presence of 15 mg/l Congo Red. The frequency and electrical resistance of seals made between patch clamp pipettes and the plasmalemma around corn protoplasts is not significantly affected by the presence or absence of these fibrils (p₀=0.75); it remains relatively low. Some single channel records from BMS corn protoplasts are shown.     

Effekte der Phospholipase D auf den Elektronentransport und die Lipidzusammensetzung isolierter Spinatchloroplasten

Summary The process of cell wall regeneration around two species of higher plant protoplasts has been studied using reflection scanning electron microscopy. The first stage in the process is the formation of short fibres from randomly spaced centres. With protoplasts of tobacco leaf (Nicotiana tabacum L., cv White Burley) these fibres then elongate and interlace apparently at random to give rise to a matted continuous layer of wall. Protoplasts of a suspension culture of grapevine cells (Vitis vinifera L. cv Müller Thurgau) produce short fibres but these fail to elongate. Budding is observed during wall regeneration around vine protoplasts. The results are discussed in terms of the mechanical properties of the wall and its relationship to changes in plasmalemma morphology which are observed during wall formation.Abbreviation SEM scanning electron microscopy     

High-resolution microscopy of cell wall formation in regenerating Mougeotia (Chlorophyceae) protoplasts

Field emission scanning electron microscopy (FESEM) preparation techniques have been successfully adapted for visualization of the internal and external ultrastructure of Mougeotia filaments and protoplasts. FESEM of the innermost layer of cell wall in Mougeotia filaments revealed that microfibrils are deposited parallel to each other in an interconnected mesh and are oriented perpendicular to the direction of elongation. For the first time, the surface of protoplasts at different stages of regeneration has been observed using FESEM. Nascent microfibril deposition occurs between 1 and 2 h after isolation and arrangement of these microfibrils is random for at least 8 h. Observation of the inner surface of the plasma membrane in burst protoplasts showed that microtubules are not strongly attached for at least 3 h after protoplast isolation.     

Fine mapping of tropoelastin-derived components in the aorta of developing chick embryo

Summary Affinity-purified antitropoelastin antibodies have been used to localize tropoelastin-derived components in aortas from chick embryos of different age by immunoelectron microscopy. Staining in the matrix is first noted at day 3 associated with irregular bundles of filaments resembling microfibrils, in the absence of amorphous elastin deposits. Amorphous material, which rapidly accumulates at later stages, is heavily labelled, while surrounding microfibrils are only poorly labelled. By contrast, a more intense staining of microfibrils persists in regions in which amorphous material is not morphologically evident. These observations indicate that the initial accumulation of elastin requires microfibrils, while the two components are not in close association in the subsequent growth of the amorphous core of the fibre. Intracellular staining is evident in the secretory apparatus of the cell and in peripheral large vesicles. Differentiated cells also show regions of close contact with elastic fibres in which immunological staining for elastin is very close to the cell membrane.     

3,4-Dehydroproline inhibits cell wall assembly and cell division in tobacco protoplasts.

We investigated the function of cell wall hydroxyproline-rich glycoproteins by observing the effects of a selective inhibitor of prolyl hydroxylase, 3,4-dehydro-L-proline (Dhp), on wall regeneration by Nicotiana tabacum mesophyll cell protoplasts. Protoplasts treated with micromolar concentrations of Dhp do not develop osmotic stability and do not initiate mitosis. The architecture of regenerated cell walls was examined using deep-etch, freeze-fracture electron microscopy of rapidly frozen tobacco cells. Untreated protoplasts assemble a dense fibrillar cell wall consisting of laterally associating subelementary fibrils. In contrast, treatment of protoplasts with Dhp alters the structure of the regenerated wall fibrils in several ways: first, the microfibrils are coated with globular knobs; second, some larger fiber bundles have an open ribbon-like appearance; and third, the smallest subelementary fibrils were not visible. Tobacco cells develop an abnormal morphology as a consequence of this abnormal cell wall structure. Thus, inhibition of prolyl hydroxylase results in the regeneration of a cell wall with abnormal structural and functional properties. These data provide experimental evidence that hydroxyproline-rich glycoproteins are important for the structural integrity of primary cell walls and for the correct assembly of other wall polymers, and that wall structure is an important regulator of cell division and cell morphology.     

Taxol maintains organized microtubule patterns in protoplasts which lead to the resynthesis of organized cell wall microfibrils

Summary We have investigated the effects of microtubule stabilizing conditions upon microtubule patterns in protoplasts and developed a new method for producing protoplasts which have non-random cortical microtubule arrays. Segments of elongating pea epicotyl tissue were treated with the microtubule stabilizing drug taxol for 1 h before enzymatic digestion of the cell walls in the presence of the drug. Anti-tubulin immunofluorescence showed that 40 M taxol preserved regions of ordered microtubules. The microtubules in these regions were arranged in parallel arrays, although the arrays did not always show the transverse orientation seen in the intact tissue. Protoplasts prepared without taxol had microtubules which were random in distribution. Addition of taxol to protoplasts with random microtubule arrangements did not result in organized microtubule arrays. Taxol-treated protoplasts were used to determine whether or not organized microtubule arrays would affect the organization of cell wall microfibrils as new walls were regenerated. We found that protoplasts from taxol-treated tissue which were allowed to regenerate cell walls produced organized arrays of microfibrils whose patterns matched those of the underlying microtubules. Protoplasts from untreated tissue synthesized microfibrils which were disordered. The synthesis of organized microfibrils by protoplasts with ordered microtubules arrays shows that microtubule arrangements in protoplasts influence the arrangement of newly synthesized microfibrils.Abbreviations DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - IgG immunoglobulin G - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - PBS phosphate buffered saline     

Changes in the Arrangement of Microtubules and Microfibrils in Differentiating Conifer Tracheids During the Expansion of Cells

The arrangements of microtubules and the cellulose microfibrilsof radial walls in tracheids of Abies sachalinensis Mastersduring the expansion of cells were examined by immunofluorescenceand field-emission scanning electron microscopy. The radialdiameter of tracheids increased to three to four times thatof cambial initial cells. Microfibrils on the innermost surfaceof primary walls of conifer tracheids at early stages were notwell ordered and most of the microfibrils were oriented longitudinally.As each cell expanded, microfibrils in the process of depositionwere still not well ordered but their orientation changed fromlongitudinal to transverse. When cell expansion ceased, microfibrilswere well ordered and oriented transversely. Cortical microtubulesshowed a change in orientation similar to that of the microfibrils.These results indicate that the orientation of cortical microtubulesis correlated with that of microfibrils as they are being laiddown and with cell morphogenesis in conifer tracheids.Copyright1995, 1999 Academic Press Microfibril, microtubule, tracheid, cell expansion, Abies sachalinensis Masters, field-emission scanning electron microscopy, immunofluorescence microscopy     

Plasmolysis of Pteridium protoplasts: A study using light and scanning-electron microscopy

A study was undertaken using gametophytes of the fern Pteridium aquilinum to examine the effects of plasmolysis on the topography of protoplasts. Methods are described whereby the surfaces of non-isolated protoplasts can be observed in the plasmolysed condition using scanning electron microscopy. Plasmolysed gametophytes were also examined in the light microscope using differential interference contrast and ultra-violet fluorescence microscopy after staining with fluorescein diacetate. With scanning electron microscopy, plasmolysed protoplast surfaces appeared smooth with no evidence of wrinkling or infolding of excess membrane. The formation of irregular-shaped protoplasts, protoplasmic threads, subprotoplasts, and protoplasmic networks covering internal wall surfaces all provided evidence for strong wall adhesion of the protoplasm. The availability of membrane for uptake into folds or vesicles is therefore thought to be minimal. Transmission electron microscopy showed some protoplasmic threads to be plasmodesmata, the remainder being cell-wall contact points. Remnants of these threads were occasionally observed on isolated protoplasts in both the light and electron microscopes.     

STRUCTURAL BIOLOGY OF THE FIBRES OF THE COLLAGENOUS AND ELASTIC SYSTEMS

The different types of fibres of the collagenous and elastic systems can be demonstrated specifically in tissue sections by comparing the typical ultrastructural picture of each of the fibre types with studies using selective staining techniques for light microscopy. A practicalmodus operandi, which includes the recommended staining procedures and interpretation of the results, is presented. Micrographs and tables are provided to summarize the differential procedures. Reticulin fibres display a distinct argyrophilia when studied by means of silver impregnation techniques, and show up as a thin meshwork of weakly birefringent, greenish fibres when examined with the aid of the Picrosirius-polarization method. In addition, electron-microscopic studies showed that reticulin fibres are composed of a small number of thin collagen fibrils, contrasting with the very many thicker fibrils that could be localized ultrastructurally to the sites where non-argyrophilic, coarse collagen fibres had been characterized by the histochemical methods used. The three different fibre types of the elastic system belong to a continuous series: oxytalan—elaunin—elastic (all of the fibre types comprising collections of microfibrils with, in the given sequence, increasing amounts of elastin). The three distinct types of elastic system fibres have different staining characteristics and ultrastructural patterns. Ultrastructurally, a characteristic elastic fibre consists of two morphologically different components: a centrally located solid cylinder of amorphous and homogeneous elastin surrounded by tubular microfibrils. An oxytalan fibre is composed of a bundle of microfibrils, identical to the elastic fibre microfibrils, without amorphous material. In elaunin fibres, dispersed amorphous material (elastin) is intermingled among the microfibrils.     

Two separate zones of helicoidally orientated microfibrils are present in the walls ofNitella internodes during growth

Summary The dynamic changes in microfibril architecture in the internode cell walls of the giant unicellular algaNitella translucens were studied during cell expansion. Thin section electron microscopy in conjunction with mild matrix polysaccharide extraction techniques revealed three distinct architectural zones in the walls of fully grown cells. These zones were related to distinct phases of growth by monitoring changes in cell wall architecture of internodes during active cell expansion. The initial microfibril deposition before the onset of active cell growth is helicoidal. A helicoid is a structurally complex but ordered arrangement of microfibrils that has been detected increasingly often in higher plant cell walls. During active cell elongation microfibrils are deposited transversely to the direction of cell elongation as shown in earlier studies by birefringence measurements in the polarizing microscope. The gradual decline in cell elongation corresponds with a final helicoidal deposition which continues after cell expansion ceases entirely.The continual presence of the initial helicoidal zone in the outer wall region during the whole growth process suggests that these microfibrils do not experience strain reorientation and are continually reorganized, or maintained, in a well ordered helicoidal arrangement.     

Wall architecture in the cellulose-deficientrsw1 mutant ofArabidopsis thaliana: Microfibrils but not microtubules lose their transverse alignment before microfibrils become unrecognizable in the mitotic and elongation zones of roots

Summary Thersw1 mutant ofArabidopsis thaliana has a single amino acid substitution in a putative glycosyl transferase that causes a temperature-dependent reduction in cellulose production. We used recently described methods to examine root growth by surface marker particles, cell wall structure by field emission scanning electron microscopy and microtubule alignment by immunofluorescence after the mutant is transferred to its restrictive temperature. We find that raising the temperature quickly accelerates root elongation in both wild type and mutant, presumably as a result of general metabolic stimulation, but that in the mutant, the rate declines within 7–8 h and elongation almost ceases after 24 h. Radial swelling begins at about 6 h in the mutant and root diameter continues to increase until about 24 h. The normal transverse alignment of microfibrils is severely impaired in the mutant after 8 h, and chemical inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile causes a similar loss of orientation. After 24 h, microfibrils are not clearly visible in the walls of cells that would have been in the mitotic and early-elongation zone of wild-type roots. Changes in older cells are less marked; loss of transverse microfibril orientation occurs without disruption to the transverse orientation of cortical microtubules. The wild type shows none of the changes except for acceleration of elongation, which in its case is sustained. We conclude that microfibril alignment requires the normal functioning of RSW1 and that, in view of the effects of dichlorobenzonitrile, there may be a more general linkage between the rate of cellulose production and its proper alignment.Abbreviations DCB 2,6-dichlorobenzonitrile - REGR relative elemental growth rate Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Structure of Acetobacter cellulose composites in the hydrated state

The structure of composites produced by the bacterium Acetobacter xylinus have been studied in their natural, hydrated, state. Small-angle X-ray diffraction and environmental scanning electron microscopy has shown that the ribbons have a width of 500 A and contain smaller semi-crystalline cellulose microfibrils with an essentially rectangular cross-section of approximately 10 x 160 A(2). Incubation of Acetobacter in xyloglucan or pectin results in no changes in the size of either the microfibrils or the ribbons. Changes in the cellulose crystals are seen upon dehydration of the material, resulting in either a reduction in crystal size or an increase in crystal disorder.     

Distribution and organization of the elastic system fibres in healthy human gingiva

Summary The ultrastructural distribution and organization of the elastic system fibres, i.e. oxytalan, elaunin and elastic fibres, were studied by transmission electron microscopy and by an immunohistochemical method for the detection of elastin in healthy human gingiva. The morphological distribution of these fibres was characterized by the presence of oxytalan, elaunin and elastic fibres, respectively, in the upper, medium, and deep layers of gingival connective tissue. Anti-elastin antibody reacted with microfibrils and amorphous material of the elastic system fibres throughout the gingival connective tissue. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin at their surface.     

Regulation of cell division in pea root tips after wounding: A possible role for ethylene

Summary Calcofluor White ST is a fluorescent brightener that has previously been shown to alter cellulose ribbon assembly in the bacteriumAcetobacter xylinum. In this report, we demonstrate that Calcofluor also disrupts cell wall assembly in the eukaryotic algaOocystis apiculata. When observed with polarization microscopy, walls altered by Calcofluor show reduced birefringence relative to controls. Electron microscopy has shown that these altered walls contain regions which consist primarily of amorphous material and which generally lack organized microfibrils. We propose that wall alteration occurs because Calcofluor binds with the glucan chains polymerized by the cellulose synthesizing enzymes as they are produced. As a consequence, the glucan chains are prevented from co-crystallizing to form microfibrils. Synthesis of normal walls resumes when Calcofluor is removed, which is consistent with our proposal that Calcofluor acts by direct physical interaction with newly synthesized wall components.Several types of fluorescent patterns at the cell wall/plasmalemma interface have also been observed following Calcofluor treatment. Fluorescent spots, striations; helical bands, and lens-shaped thickenings have been documented. Each of these patterns may be the result of the interaction of Calcofluor with cellulose at different spatial or temporal levels or from varying concentrations of the brightener itself. Helical bands and lens-shaped thickenings also have been examined with the electron microscope. Like other regions of wall alteration, they are found to contain primarily amorphous material. Finally, we note that cells with severely disrupted walls are unable to complete their normal life cycle.