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Band growth and localization of vesicle exocytosis in the red alga Antithamnion nipponicum (Ceramiales)
Abstract
In uniseriate ceramiacean red algae, intercalary cells grow in a longitudinal direction by the formation of new cell surface in narrow, transverse bands of the cell. This localized growth mode is known as ‘band growth’. In this study, the distribution of vesicle exocytosis in the axial cells of the red alga Antithamnion nipponicum was investigated by transmission electron microscopy in specimens prepared by freeze-substitution. Two zones of band growth, which were detected as dark, horizontal bands after fluorescent cell wall staining followed by 24-h incubation in stain-free medium, were observed in the apical and basal regions of the cell. The width of the band was much greater in the basal region than in the apical region. Comparisons between the total elongation of the cells and the total width of the apical and basal bands during a 24-h incubation indicated that all of the cell elongation could be attributed to band growth. A short incubation in stain-free medium revealed that cell elongation in the basal band occurred in a narrower zone, less than 5 µm wide. Small depressions in the plasma membrane and small vesicles located beneath the plasma membrane were observed in actively growing axial cells. The distribution of these structures was limited to the narrow zones near the bases of the cells (the basal bands) where cell elongation occurred. A significant longitudinal elongation of the axial cells was still detectable after 24-h treatment of the plants with the microtubule inhibitor griseofulvin or the actin filament inhibitor cytochalasin D, but there was scarcely any discernible localization of cell growth to the band. The inhibitory treatments also caused dispersion of the plasma membrane depressions and vesicles along the plasma membrane. These results suggest that these membranous structures are related to the vesicle exocytosis that occurs during longitudinal elongation of the axial cells of Antithamnion and that cytoskeletal elements, such as microtubules and actin filaments, control the localization of vesicle exocytosis and the mode of cell growth.
... Mitosis is arrested at metaphase because these drugs inhibit the formation of microtubules (Komaki and Schnittger 2016). In contrast, other drugs such as amiprophos methyl (APM), griseofulvin and oryzalin have been used to inhibit microtubule polymerization in some algae (e.g., McNaughton and Goff 1990;Takahashi et al. 2001;Mine et al. 2011;Sommer et al. 2015). Mitotic inhibitors are useful for examining the details of the process of mitosis and counting the number of chromosomes. ...
Background:
Some marine algae exhibit several characteristics of mitosis (e.g., the timing of mitosis such as diurnal periodicity) that are unique from those of land plants. Not only the timing but also other characteristics of mitosis, including the process itself and the number of chromosomes involved, are largely unknown in ulvophycean marine green algae. Effective mitotic inhibitors are useful for observing mitosis and identifying the number of chromosomes. However, few such inhibitors are available for ulvophycean algae. Here, we examined the timing and process of mitosis and the number of chromosomes with several mitotic inhibitors in the haploid gametophyte cells of the Ulvophyceae alga Monostroma angicava.
Results:
Mitosis did not occur during the light period but began immediately after the onset of the dark period. The typical process of mitosis was observed. The mitotic inhibitors colchicine and 8-hydroxyquinoline, which generally arrest mitosis in land plants, were ineffective in M. angicava. We found that three other mitotic inhibitors, amiprophos methyl, griseofulvin and oryzalin, are effective to arrest mitosis. With three-dimensional fluorescence microscopy, we demonstrated that there were nine chromosomes in each cell.
Conclusions:
In the gametophyte cells of M. angicava, mitosis occurs diurnally. It is triggered by the onset of the dark period. We identified the number of chromosomes as N = 9. Our study shows effective inhibitors to observe mitosis in ulvophycean algae.
... The modes of plant cell growth are classified into diffuse and localized growth, and the latter includes tip growth (Braidwood et al. 2014;Franciosini et al. 2017) and band growth (Mine et al. 2011). In tip-growing cells, new cell surfaces are produced only in the dome-like end of a cylindrical cell, for example, the pollen tube, root hair, hypha, or protonema, of a variety of organisms. ...
The cell wall of the tip‐growing cells of the giant‐cellular xanthophycean alga Vaucheria frigida is mainly composed of cellulose microfibrils (CMFs) arranged in random directions and the major matrix component into which the CMFs are embedded throughout the cell. The mechanical properties of a cell‐wall fragment isolated from the tip‐growing region, which was inflated by artificially applied pressure, were measured after enzymatic removal of the matrix component by using a protease; the results showed that the matrix component is involved in the maintenance of cell wall strength. Since glucose and uronic acid are present in the matrix component of Vaucheria cell walls, we measured the mechanical properties of the cell wall after treatment with endo‐1,3‐ß‐glucanase and observed the fine structures of its surfaces by atomic force microscopy. The major matrix component was partially removed from the cell wall by glucanase, and the enzyme treatment significantly weakened the cell wall strength without affecting the pH dependence of cell wall extensibility. The enzymatic removal of the major matrix component by using a protease released polysaccharide containing glucose and glucuronic acid. This suggests that the major matrix component of the algal cell walls contains both proteins (or polypeptides) and polysaccharides consisting of glucose and glucuronic acid as the main constituents.
... Fluorescent-tagged lectins were secreted from the tips of cells involved in repair (Kim et al., 1995). The growth bands identified by fluorescence microscopy were subsequently studied by transmission electron microscopy of fine longitudinal sections of the cell filaments (Mine et al., 2011). ...
Traditions of Eastern thought conceptualised life in a holistic sense, emphasising the processes of maintaining health and conquering sickness as manifestations of an essentially spiritual principle that was of overriding importance in the conduct of living. Western science, which drove the overriding and partial eclipse of Eastern traditions, became founded on a reductionist quest for ultimate realities which, in the modern scientific world, has embraced the notion that every living process can be successfully modelled by a digital computer system. It is argued here that the essential processes of cognition, response and decision-making inherent in living cells transcend conventional modelling, and microscopic studies of organisms like the shell-building amoebae and the rhodophyte alga Antithamnion reveal a level of cellular intelligence that is unrecognised by science and is not amenable to computer analysis.
Cellulose synthases form rosette terminal complexes in the plasma membranes of Streptophyta and various linear terminal complexes in other taxa. The sequence of a putative CESA from Griffithsia monilis (Rhodophyta, Floridiophyceae) was deduced using a cloning strategy involving degenerate primers, a cDNA library screen, and 5' and 3' rapid amplification of cDNA ends (RACE). RACE identified two alternative transcriptional starts and four alternative polyadenylation sites. The first translation start codon provided an open reading frame of 2610 bp encoding 870 amino acids and was PCR amplified without introns from genomic DNA. Southern hybridization indicated one strongly hybridizing gene with possible weakly related genes or pseudogenes. Amino acid sequence analysis identified a family 48 carbohydrate-binding module (CBM) upstream of the protein's first predicted transmembrane domain. There are broad similarities in predicted 3D structures of the family 48 modules from CESA, from several glycogen- and starch-binding enzymes, and from protein kinases, but there are substitutions at some residues thought to be involved in ligand binding. The module in G. monilis CESA will be on the cytoplasmic face of the plasma membrane so that it could potentially bind either low molecular weight ligands or starch which is cytosolic rather than inside membrane-bound plastids in red algae. Possible reasons why red algal CESAs have evolved family 48 modules perhaps as part of a system to regulate cellulose synthase activity in relation to cellular carbohydrate status are briefly discussed.
Cortical microtubules (CMT) were first observed in plant cells (Ledbetter and Porter 1963) by transmission electron microscopy. In intercalary growing cells, CMT have always been found oriented with a right angle to the elongation axis of the cell (reviewed in Cyr 1994). Moreover, if CMT are depolymerized by addition of pharmalogical agents, cells expand isotropically (colchicine: Green 1962; dinitroaniline herbicides: Upadhyaya and Noodén 1977; Baskin et al. 1994). Stabilization of CMT by taxol also leads to more isotropic growth (Baskin et al. 1994). Furthermore, growth regulators affect the orientation of the CMT (e.g., van Spronsen et al. 1995). Most researchers agree that the orientation of the CMT determines the direction of cell expansion.
Cells with polyploid nuclei are generally larger than cells of the same organism or species with nonpolyploid nuclei. However, no such change of cell size with ploidy level is observed in those red algae which alternate isomorphic haploid with diploid generations. The results of this investigation reveal the explanation. Nuclear DNA content and other parameters were measured in cells of the filamentous red alga Griffithsia pacifica. Nuclei of the diploid generation contain twice the DNA content of those of the haploid generation. However, all cells except newly formed reproductive cells are multinucleate. The nuclei are arranged in a nearly perfect hexagonal array just beneath the cell surface. When homologous cells of the two generations are compared, although the cell size is nearly identical, each nucleus of the diploid cell is surrounded by a region of cytoplasm (a "domain") nearly twice that surrounding a haploid nucleus. Cytoplasmic domains associated with a diploid nucleus contain twice the number of plastids, and consequently twice the amount of plastid DNA, than is associated with the domain of a haploid nucleus. Thus, doubling of ploidy is reflected in doubling of the size and organelle content of the domain associated with each nucleus. However, cell size does not differ between homologous cells of the two generations, because total nuclear DNA (sum of the DNA in all nuclei in a cell) per cell does not differ. This is the solution to the cytological paradox of isomorphy.
Apical cell wall fragments isolated from the giant-cellular xanthophycean alga Vaucheria terrestris sensu Götz were inflated with silicone oil by applying internal pressure ranging from 0.1 to 0.7 MPa, and the time-course of cell wall deformation was recorded and analyzed by videomicroscopy. Cell wall extensibility in the tip-growing region was estimated by the pressure required for cell wall extension, the amount of total extension until cell wall rupture and the rate of cell wall extension. Apical cell walls exhibited gradual extension, or creep, during inflation, which was eventually followed by rupture at the apical portion, whereas no appreciable extension was found in the cylindrical basal portion of the cell wall fragment. Besides the largest extension observed around the tip, substantial extension was also observed along the subapical region of the cell wall. The wall extensibility was dependent on the buffer pH used for infiltration before inflation. The optimum pH for the extension was about 8.0, but the cell wall was much less extensible after infiltration with an acidic buffer. Cell wall extensibility was dependent on the pH of the buffer used before inflation, regardless of that used in the previous infiltration. Moreover, pretreatment of the cell wall with a protease caused considerable loosening of cell walls, but affected the pH dependence of cell wall extensibility little. These results indicate that the extensibility of the cell walls in the giant tip-growing cells of the alga is distinct from that of plant cells that exhibit "acid growth" in its dependence on environmental pH and the role of cell wall proteins.
The multiaxial stress of turgor pressure was stimulated in vitro by inflating isolated Nitella cell walls with mercury. The initial in vitro extension at pH 6.5, 5 atmospheres pressure, returned the wall approximately to the in vivo stressed length, and did not induce any additional extension during a 15-minute period. Upon release of pressure, a plastic deformation was observed which did not correlate with cell growth rates until the final stages of cell maturation. Since wall plasticity does not correlate with growth rate, a metabolic factor(s) is implicated. Walls at all stages of development exhibited a primary yield stress between 0 and 2 atmospheres, while rapidly growing cells (1-3% per hour) exhibited a secondary yield stress of 4 to 5 atmospheres. The creep rate and plastic deformation of young walls were markedly enhanced by acid buffers (10 millimolar, pH </= 5.3).Nitella cells produce acid and base "bands" along their length due to localized excretion of protons and hydroxyl ions. Marking experiments showed that growth is largely restricted to the acid regions. Growth in the acid bands was inhibited by alkaline buffers, and growth in the base bands was stimulated by acidic buffers. The two zones have similar mechanical properties. When the proton-binding capacity of the wall was taken into account, the pH of the solution in contact with inner wall surface in the acid band was estimated to be about 4.3, well within the threshold of acid-enhanced creep. Since the inner 25% of the wall controls extensibility, we conclude that growth in the acid band is caused by the action of protons on the wall.
Recent observations of pollen tubes show that these tubes may grow in a pulsatory fashion (Pierson et al., 1995; Plyushch et al., 1995; Li et al., 1996; Geitmann et al., 1996a, 1996b), in which phases of fast and slow growth alternate regularly. The occurrence of pulsatory growth has been used by Geitmann and coworkers (1996b) to study factors that might control growth. Their results emphasize the role of the cell wall and secretory events in regulating pollen tube growth. Here we will briefly review recent results related to the role of exocytosis, cytoskeleton, calcium and the cell wall in pollen tube growth.
The filamentous red alga Anotrichium tenue C. Aghard (Naegeli) (formerly Griffithsia tenuis C. Aghard; Baldock, 1976, Aust. T. Bot. 24, 509-593) has large (1-2 mm long), cylindrical, multinucleate cells that exhibit a daily, cyclic redistribution of chloroplasts. Chloroplasts accumulate in the mid-region of each growing cell during the day; consequently, filaments appear banded with a light apical end-band, a dark mid-band and a light basal end-band in each growing cell. Chloroplasts disperse at night so that the bands are no longer visible and the cells appear evenly pigmented. Anotrichium tenue also has a type of cell elongation, known as bipolar band growth, in which new material is added to the microfibrillar part of the wall in bands located at the apical and basal poles of elongating cells. This site of wall growth corresponds to the position of the light-colored end-bands present during the day. Here we examine the structural relationship between the cytoplasmic bands and the wall-growth bands. Our results show that, in addition to the previously described bipolar wall bands, there is a non-microfibrillar wall band in the mid-region of the cell. This wall component apparently branches from near the top of the microfibrillar outer wall and terminates near but not at the bottom of the cell. It contains nodules of sulphated polysaccharide material secreted from a band of vesicles, which co-localize with the chloroplasts in the mid-band. The outer wall appears to enclose the entire cell. Nuclei do not redistribute with the chloroplasts or wall vesicles into the mid-band but remain evenly distributed throughout the cytoplasm. Each wall component grows by a different mechanism. We show that two types of wall growth, diffuse and the bipolar-type of tip growth, occur in the same cell and we propose that the observed segregation of the cytoplasm supports localized growth of the unique inner wall component. Additionally, we show that A. tenue is an excellent model for study of the role and mechanism of cytoplasmic compartmentalization and cell polarity during plant cell growth.
Cell elongation in the Ceramiaceae typically occurs by means of one or two bands located apically and (or) basally in each cell. In axial cells of Antithamnion defectum two bands are present; however, most cell elongation occurs as a result of new wall deposition in bands at the base of each axial cell. In cells of determinate branches, only the basal band is present. In experimental conditions in which apical cells of indeterminate branches are differentially excised, location of the primary elongation band can be reestablished in relation to remaining indeterminate axes. Thus, the primary elongation band in axial cells is always basal with respect to indeterminate apical cells. When all indeterminate apices are removed, band growth becomes highly disrupted, and diffuse, irregularly located bands are formed. These results suggest that regulation of band position and elongation is through apical control.
Summary In view of the importance of the lily pollen tube as an experimental model and the improvements in ultrastructural detail that can now be attained by the use of rapid freeze fixation and freeze substitution (RF-FS), we have reexamined the ultrastructure of these cells in material prepared by RF-FS. Several previously unreported details have been revealed: (1) the cytoplasm is organized into axial “slow” and “fast” lanes, each with a distinct structure; (2) long, straight microtubule (MT) and microfilament (MF) bundles occur in the cytoplasm of the fast lanes and are coaligned with every organelle present; (3) the cortical cytoplasm contains complexes of coaligned MTs, MFs, and endoplasmic reticulum (ER); (4) the cortical ER is arranged in a tight hexagonal pattern and individual elements are closely appressed to the plasma membrane with no space between; (5) mitochondria and ER extend into the extreme apex along the flanks of the pollen tube, and vesicles and ER are packed into an inverted cone-shaped area at the center of the apex; (6) MF bundles in the tip region are fewer, finer, and in random orientation in comparison to those of the fast lanes; (7) the generative cell (GC) cell wall complex contains patches of plasmodesmata; (8) The GC cytoplasm contains groups of spiny vesicles that are closely associated with and seem to be fusing with or pinching off from mitochondria, and (9) the vegetative nucleus (VN) contains internal MT-like structures as well as numerous cytoplasmic MTs associated with its membrane and also located between the VN and GC.
Summary In the ceramiacean red alga Antithamnion nipponicum Yamada et Inagaki, the structure of the spermatial covering and appendages was examined using confocal laser scanning microscopy, scanning and transmission electron microscopy. The liberated spermatium was subspherical, ca 4.5 µm in size with a colorless covering 2.7−3.0-µm thick. Two flexible, ribbon-like appendages arose from the periphery of the spermatial covering. The appendages averaged 80 µm in length and were 0.5−0.6 µm width in most parts. Each appendage consisted of a number of thin longitudinal fibrils. Concanavalin A conjugated with fluorescein isothiocyanate, colloidal gold or ferritin, bound specifically with the inner layer of spermatial covering and spermatial appendages. When the liberated spermatia were incubated with mature female gametophytes, the spermatial appendages entangled around the trichogyne.
Using rhodamine-phalloidin to detect F-actin/microfilaments and indirect immunofluorescence to detect tubulin/microtubules, we studied the cytoskeleton in axial cells of Ceramium strictum Harv., especially microfilaments and microtubules associated with cytoplasmic strands (trabeculae) that extend longitudinally through the central vacuole. As axial cells attained mature size, trabeculae became progressively thinner, branched, and then broke down. An extensive microfilament array was present in peripheral parts of axial cells as well as in trabeculae. Microfilament arrays were highly disrupted by cytochalasin-B; this resulted in small irregular actin structures in axial cell peripheries and occasional dense aggregations at the base of cells. No actin-fluorescence was detected in intact trabeculae after cytochalasin-B treatment. Microtubules formed a primary structural component in trabeculae, which were disrupted by griseofulvin (5 to 0.005 μM) but reformed after two days in griseofulvin-free medium.
The supramolecular organization of the plasma membrane of apical cells in shoot filaments of the marine red alga Porphyra yezoensis Ueda (conchocelis stage) was studied in replicas of rapidly frozen and fractured cells. The protoplasmic fracture (PF) face of the plasma membrane exhibited both randomly distributed single particles (with a mean diameter of 9.2 ± 0.2 nm) and distinct linear cellulose microfibril-synthesizing terminal complexes (TCs) consisting of two or three rows of linearly arranged particles (average diameter of TC particles 9.4 plusmn; 0.3 nm). The density of the single particles of the PF face of the plasma membrane was 3000 μm−2, whereas that of the exoplasmic fracture face was 325 μm−2. TCs were observed only on the PF face. The highest density of TCs was at the apex of the cell (mean density 23.0 plusmn; 7.4 TCs μm−2 within 5 μm from the tip) and decreased rapidly from the apex to the more basal regions of the cell, dropping to near zero at 20 μm. The number of particle subunits of TCs per μm2 of the plasma membrane also decreased from the tip to the basal regions following the same gradient as that of the TC density. The length of TCs increased gradually from the tip (mean length 46.0 plusmn; 1.4 nm in the area at 0–5 μm from the tip) to the cell base (mean length 60.0 plusmn; 7.0 μm in the area at 15–20 μm). In the very tip region (0–4 μm from the apex), randomly distributed TCs but no microfibril imprints were observed, while in the region 4–9 μm from the tip microfibril imprints and TCs, both randomly distributed, occurred. Many TCs involved in the synthesis of cellulose microfibrils were associated with the ends of microfibril imprints. Our results indicate that TCs are involved in the biosynthesis, assembly, and orientation of cellulose microfibrils and that the frequency and distribution of TCs reflect tip growth (polar growth) in the apical shoot cell of Porphyra yezoensis. Polar distribution of linear TCs as “cellulose synthase” complexes within the plasma membrane of a tip cell was recorded for the first time in plants.
Antimicrotubule agents, colchicine, vinblastine, and griseofulvin, induced conspicuous morphological anomalies inBryopsis plumosa. First, following cessation of protoplasmic streaming within 15 minutes, elongation stopped in a few hours. Second, innumerable protrusions or new growth points generated over the cell flank in a few days. Similar phenomena were observed in the cells which were subjected to high pressure or low temperature both of which are known to disrupt microtubule.
These phenomena were investigated with light and electron microscopy. It is suggested that inhibition of microtubule dependent protoplasmic streaming which may function as an intracellular transport system causes such morphological anomalies.
The tip-growth ofVaucheria geminata was analyzed. The elemental rate of surface expansion (RERE) at the very apex of this alga cell reaches ca. 100% min−1. The expansion is almost isotropic; i.e. the both meridional and latitudinal components of RERE are almost equal. An antimicrotubular
reagent, colchicine, caused expansion at the actively growing cell apex of this alga. This drug did not change the surface
expansion rate, but altered the polarity of cell wall expansion from isotropic to transversally anisotropic. The orientation
of cell wall microfibrils is random at the apex but axial at the basal cylindrical part of the cell. Colchicine did not change
the fluence-response relationship for the first positive phototropism.
The mechanism of cell elongation in five red algae, Griffithsia pacifica Kylin, G. tenuis C. Agardh, G. globulifera Harvey, Antithamnion kylinii Gardner, and Callithamnion sp. was studied using Calcofluor White ST as a vital, fluorescent cell-wall stain. In each alga elongation of intercalary shoot cells occurs primarily by the localized addition of new cell-wall material rather than by extension of pre-existing cell wall. Cell extension is localized in narrow bands in the lateral walls of a cell; there may be one or two bands per cell and these may be located at the top or bottom of the lateral wall. The number and location of bands of elongation are constant within a species but vary from species to species. Cell walls of elongating intercalary cells of each of these algae are essentially isotropic, indicating a net random orientation of cell-wall microfibrils.
The growth of pollen tubes is characterized by intense secretory activity in the tip region. This process of vesicle-mediated secretion and tip growth is strongly influenced by calcium gradients. The cytoskeletal apparatus is also critically involved, as it is required for the translocation of organelles along the tube (a prerequisite for tube extension) and for the transport of the generative/sperm cells. The microtubules and actin filaments probably have distinct functions that relate to different, but related, cytological events within the pollen tubes. Both systems, as well as cytoskeleton-based motor proteins, are necessary for the proper development and growth of the pollen tubes. Different approaches have allowed the roles of several cytoskeletal components to be deciphered, and it is now possible to speculate how they might interact.
The plant actin cytoskeleton is a structural cell element that connects cytoplasmic components with each other, with the cell surface and with neighbouring cells through plasmodesmata. It is the dynamic backbone of cytoplasmic strands. Along this backbone, cell components move and are targeted to specific sites in the cell. The specific targeting of cell organelles is instrumental in cell morphogenesis. Moreover, the actin cytoskeleton may connect to receptors, transduce signals and mediate cell responses. Actin monomers and filaments are bound to actin binding proteins that determine actin’s function in place and time. Since root hairs are accessible for the application of signal molecules and drugs, and can be visualized and manipulated in the living state, these tip-growing cells are ideal for the study of the functions of the actin cytoskeleton.
A low-viscosity embedding medium based on ERL-4206 is recommended for use in electron microscopy. The composition is: ERL-4206 (vinyl cyclohexene dioxide) 10 g, D.E.R. 736 (diglycidyl ether of polypropylene glycol) 6 g, NSA (nonenyl succinic anhydride) 26 g, and S-1 (dimethylaminoethanol or DMAE) 0.4 g. The medium is easily and rapidly prepared by dispensing the components, in turn by weight, into a single flask. The relatively low viscosity of the medium (60 cP) permits rapid mixing by shaking and swirling. The medium is infiltrated into specimens after the use of any one of several dehydrating fluids, such as ethanol, acetone, dioxan, hexylene glycol, isopropyl alcohol, propylene oxide, and tert.-butyl alcohol. It is compatible with each of these in all proportions. After infiltration the castings are polymerized at 70°C in 8 hours. Longer curing does not adversely affect the physical properties of the castings. Curing time can be reduced by increasing the temperature or the accelerator, S-1, or both; and the hardness of the castings is controlled by changes in the D.E.R. 736 flexibilizer. The medium has a long pot life of several days and infiltrates readily because of its low viscosity. The castings have good trimming and sectioning qualities. The embedding matrix of the sections is very resistant to oxidation by KMnO4 and Ba(MnO4)2, compared with resins containing NADIC methyl anhydride. Sections are tough under the electron beam and may be used without a supporting membrane on the grids. The background plastic in the sections shows no perceptible substructure at magnifications commonly used for biological materials. The medium has been used successfully with a wide range of specimens, including endosperms with a high lipid content, tissues with hard, lignified cell walls, and highly vacuolated parenchymatous tissues of ripe fruits.
To elucidate the detailed process of exocytosis at the highest possible accuracy, we dissociated the pancreatic acinus of the guinea pig and observed zymogen granules under a video-enhanced contrast differential interference contrast (VEC-DIC) microscope. The preparation was thin enough to resolve each zymogen granule with the best clarity. When acinar cells were stimulated with ACh (20 microM), many zymogen granules near the lumen showed an abrupt light intensity change. For a period of 10 s immediately before exocytosis, zymogen granules neither shifted their position nor altered their shape within an accuracy of 38 nm. The time required for individual granules to change the light intensity (the releasing time) ranged from 0.15 to 0.70 s. After each response, the granule maintained its altered contrast for a few seconds until it was retrieved to a planar membrane. No compound exocytosis including granule-granule fusion was observed. We concluded that the exocytosis is not directly initiated by any supramolecular change but by a purely molecular event.
Characean cells develop alternating alkaline and acid bands on their surface upon illumination. However, the mechanism of band formation is not fully understood. In the present study, we succeeded in inducing a new alkaline band at an original acid band in internodal cells of Chara corallina. Chloroplasts in an acid band were locally removed by wounding the cell in the absence of the cell turgor pressure. The chloroplast-removed area was observed as a white belt in a green cylindrical internodal cell. This internodal cell developed a new alkaline band on the surface at the chloroplast-removed area. The narrower the chloroplast-removed area, the less significant the extent of OH(-) extrusion. This is the first success in inducing a new alkaline band at a target position in Characeae.
Effects on morphology and microfilament structure caused by phalloidin, phallacidin, and some semisynthetic phalloidin derivatives were studied in vegetative cells of the green alga Acetabularia acetabulum (L.) Silva. All phalloidin derivatives (except for phalloidin itself) caused growth stop of the alga after 1 day and (except for the fluorescein-labeled phalloidin) death of the cells after 4-7 days. Hair whorl tip growth and morphology as screened by light microscopy, as well as microfilament structure in tips, suggested that growth stop is correlated with a disorganization of actin filaments similar to that recently described for jasplakinolide (H. Sawitzky, S. Liebe, J. Willingale-Theune, D. Menzel, European Journal of Cell Biology 78: 424-433, 1999). Using rabbit muscle actin as a model target protein, we found that the toxic effects in vivo did not correlate with actin affinity values, suggesting that permeation through membranes must play a role. Indeed, the most lipophilic phalloidin derivatives benzoylphalloidin and dithiolanophalloidin were the most active in causing growth stop at ca. 100 microM. In comparison to the concentration of jasplakinolide required to cause similar effects (<3 microM), the two most active phalloidin derivatives exhibited an activity ca. 30 times lower. Nonetheless, lipophilic phalloidin derivatives can be used in algae, and probably also other cells, to modulate actin dynamics in vivo. In addition, we found that the fluorescent fluorescein isothiocyanate-phalloidin is able to enter living algal cells and stains actin structures brightly. Since it does not suppress actin dynamics, we suggest fluorescein isothiocyanate-phalloidin as a tool for studying rearrangements of actin structures in live cells, e.g., by confocal laser scanning microscopy.
The giant-celled algae, which consist of cells reaching millimeters in size, some even centimeters, exhibit unique cell architecture and physiological characteristics. Their cells display a variety of morphogenetic phenomena, that is, growth, division, differentiation, and reproductive cell formation, as well as wound-healing responses. Studies using immunofluorescence microscopy and pharmacological approaches have shown that microtubules and/or actin filaments are involved in many of these events through the generation of intracellular movement of cell components or entire protoplasmic contents and the spatial control of cell activities in specific areas of the giant cells. A number of environmental factors including physical stimuli, such as light and gravity, invoke localized but also generalized cellular reactions. These have been extensively investigated to understand the regulation of morphogenesis, in particular addressing cytoskeletal and endomembrane dynamics, electrophysiological elements affecting ion fluxes, and the synthesis and mechanical properties of the cell wall. Some of the regulatory pathways involve signal transduction and hormonal control, as in other organisms. The giant unicellular green alga Acetabularia, which has proven its usefulness as an experimental model in early amputation/grafting experiments, will potentially once again serve as a useful model organism for studying the role of gene expression in orchestrating cellular morphogenesis.