Isolation of a cell-fusion hormone from Griffithsia pacifica kylin,a red alga

Filaments of Griffithsia pacifica replace dead cells by the process of cell repair. When an intercalary cell is killed, but its cell wall remains intact holding the two halves of the plant together, the cell above it produces a repair rhizoid cell; the cell below it produces a specialized, rhizoid-like repair shoot cell. The repair rhizoid and shoot grow towards each other, meet, and fuse to form a single shoot cell. Evidence from observations of cell repair in vivo has indicated that the repair rhizoid produces a hormone or hormones which induce the production of the repair shoot, maintain the rhizoid-like morphology and growth of the repair shoot, and attract it to the repair rhizoid for fusion. This hormone has been named rhodomorphin. Using an artificial cell-fusion system we show that repair rhizoids and normal rhizoids, but no shoot cell, can induce decapitated filaments to form repair shoot cells. Decapitated filaments form repair shoot cells only when they are exposed to the hormone within 4–6 h after decapitation; after this time they lose their sensitivity to the hormone. A method has been developed for isolating, and assaying for, the cell-fusion hormone. Rhodomorphin retains its activity for several days at room temperature and for at least two years at-16° C.     

MORPHOLOGICAL REVERSION OF SPIRULINA (ARTHROSPIRA) PLATENSIS (CYANOPHYTA): FROM LINEAR TO HELICAL1

The cyanobacterium Spirulina Turpin is characterized by its regularly coiled trichomes. Under some conditions, its helical filaments can convert to abnormal morphologies, such as irregularly curved and even linear shapes, that had been considered as a permanent degeneration that could not be reversed. However, here we found that the linear filaments of Spirulina platensis Geitler could spontaneously revert to the helical form with the same morphology as the original filaments. Further studies showed that the ultrastructural, physiological, and biochemical characteristics of linear filaments were different from those of the original filaments, whereas they were the same for the reverted and the original filaments. The SDS‐PAGE analysis revealed at least four proteins or subunits related to Spirulina morphogenesis: The 21.9‐kDa and the 20.3‐kDa proteins were highly expressed in the helical filaments, whereas the 52.0‐kDa and the 31.8‐kDa proteins were highly expressed in the linear filaments. The random amplified polymorphic DNA analysis with 96 random primers showed that the genetic background of the reverted filaments was the same as that of the original filaments but distinct from that of the linear filaments. The results indicated that linear filaments of Spirulina could revert to the original morphology under certain conditions, and their other distinctive traits were regained.     

SYSTEMATIC REVISION OF THE GENERA LIAGORA AND IZZIELLA (LIAGORACEAE,RHODOPHYTA) FROM TAIWAN BASED ON MOLECULAR ANALYSES AND CARPOSPOROPHYTE DEVELOPMENT,WITH THE DESCRIPTION OF TWO NEW SPECIES1

Some Liagora and Izziella distributed in Taiwan display a wide range of morphological variation and can be difficult to distinguish. To clarify species concepts, we applied DNA sequence analyses and examined carposporophyte development in detail. These studies revealed two new species, which are described herein as Izziella hommersandii sp. nov. and Izziella kuroshioensis sp. nov. I. kuroshioensis superficially resembles Izziella formosana and Izziella orientalis in that its involucral filaments subtend rather than surround the lower portion of the gonimoblast mass (= Izziella type) and a fusion cell is formed from cells of the carpogonial branch, but it can be separated by differences in the cell numbers and branching pattern of the involucral filaments, as well as thallus morphology. In contrast to other species that also bear short lateral branchlets, I. hommersandii is unique in possessing a mixture of short and long involucral filaments, a phenomenon not reported before. The length of the involucral filaments is species specific among species of Izziella and contrasts to the behavior of the involucral filaments after fertilization in species such as “Liagora”setchellii [= Titanophycus setchellii comb. nov.], in which the filaments completely envelop the gonimoblast. In addition, the cells of the carpogonial branch in Titanophycus do not fuse after fertilization to form a fusion cell. Thus, a combination of characters with respect to the behavior of the carpogonial branch and the involucral filaments after fertilization is very useful for delineating species boundaries in Izziella and for separating Titanophycus from Izziella and Liagora.     

Immunocytochemical electron microscopic study and Western blot analysis of paramyosin in different invertebrate muscle cell types of the fruit flyDrosophila melanogaster, the earthwormEisenia foetida, and the snailHelix aspersa

Summary The presence and distribution pattern of paramyosin have been examined in different invertebrate muscle cell types by means of Western blot analysis and electron microscopy immunogold labelling. the muscles studied were: transversely striated muscle with continuous Z lines (flight muscle fromDrosophila melanogaster), transversely striated muscle with discontinuous Z lines (heart muscle from the snailHelix aspersa), obliquely striated body wall muscle from the earthwormEisenia foetida, and smooth muscles (retractor muscle from the snail and pseudoheart outer muscular layer from the earthworm). Paramyosin-like immunoreactivity was localized in thick filaments of all muscles studied. Immunogold particle density was similar along the whole thick filament length in insect flight muscle but it predominated in filament tips of fusiform thick filaments in both snail heart and earthworm body wall musculature when these filaments were observed in longitudinal sections. In obliquely sectioned thick filaments, immunolabelling was more abundant at the sites where filaments disappeared from the section. These results agree with the notion that paramyosin extended along the whole filament length, but that it can only be immunolabelled when it is not covered by myosin. In all muscles examined, immunolabelling density was lower in cross-sectioned myofilaments than in longitudinally sectioned myofilaments. This suggests that paramyosin does not form a continuous filament. The results of a semiquantitative analysis of paramyosin-like immunoreactivity indicated that it was more abundant in striated than in smooth muscles, and that, within striated muscles, transversely striated muscles contain more paramyosin than obliquely striated muscles.     

The flagellar protein of Clostridium tetani.

Clostridium tetani ATCC 19406 was investigated with regard to the flagellar filaments produced by this anaerobic species. Flagellar filaments were removed from the cell bodies by hydrodynamic shear forces and purified by differential centrifugation. Exposure to acid was shown to result in disaggregation of the flagellar filaments into a preparation of flagellar protein containing 3.5% carbohydrate. The protein was judged homogeneous after examination by acrylamide gel electrophoresis in the presence of 4 M urea at several pH levels, and was shown to have a molecular weight of 35,000 daltons. Amino acid analyses indicated the absence of cysteine and tryptophan and a preponderance of acidic residues, epsilon-N-methyllysine was shown to be absent and the N-terminal amino acid was identified as alanine. Analysis of the C-terminal region indicated the sequence -Leu-Leu-Arg. These findings indicated that the obligate anaerobe C. tetani produced flagella filaments similar to previously studied filaments of aerobic and facultatively anaerobic bacteria.     

DEVELOPMENT OF THE CARPOSPOROPHYTE OF KALLYMENIA RENIFORMIS (TURNER) J. AGARDH1

The development of the carposporophyte in Kallymenia reniformis involves an elaborate series of interactions between reproductive and vegetative tissues. Following fertilization, the inner cells of the carpogonial branches form processes that unite with the supporting cell and with each other, giving rise to a large fusion cell. A number of medullary filaments are enveloped and incorporated within the developing fusion cell. Secondary filaments may be produced from medullary cells outside the fusion cell after connecting filaments have been initiated. Connecting filaments are nonseptale and wind their way through the medulla. The presence of a connecting filament in the vicinity of an auxiliary cell appears to initiate a complex sequence of responses. Vegetative filaments are produced in the medulla and inner cortex that grow centripetally toward the auxiliary cell and fuse with one another, forming a network of secondary tissue. One of the vegetative cells may penetrate the auxiliary cell, while others intrude into subsidiary cells connecting them with the network. An ostiolate pericarp is initiated in the cortex above the auxiliary cell. The connecting filament fuses with the auxiliary cell and also with some of the surrounding vegetative cells, forming an irregular lobed structure. Gonimoblast is initiated in scattered pockets from vegetative cells in the vicinity of the auxiliary cell apparatus.     

Lithophyllum cuneatum sp. nov. (Corallinaceae, Rhodophyta), a new species of non-geniculate coralline alga semi-endophytic in Hydrolithon onkodes and Neogoniolithon sp. from Fiji, South Pacific

A new species of semi-endophytic coralline alga, Lithophyllum cuneatum (Corallinaceae: Lithophylloideae), is described from Fiji. The species is characterized by a wedge-like thallus that is partially buried in the thallus of the host coralline, Hydrolithon onkodes (Heydrich) Penrose et Woelkerling or occasionally Neogoniolithon sp., and that appears at the surface of the host as a small pustule that is usually paler in color than the host. The thallus consists of erect filaments that are derived from a single cell. The basal cell, when visible, is non-palisade, and areas of bistratose margin are absent. Cells of contiguous erect filaments are joined by secondary pit connections. Epithallial cells are present in 2–3 layers, and individual trichocytes are common. Gametangial plants are dioecious. Male conceptacles have simple spermatangial systems that are confined to the floors of their elliptical chambers. Carposporangial conceptacles contain 5–8 celled gonimoblast filaments that are borne at the margin of a more-or-less discoid fusion cell, and so occupy the periphery of the elliptical conceptacle chambers. Tetrasporangial conceptacles are uniporate, with roofs formed from peripheral filaments, and chambers lack a central columella of sterile filaments. Despite its semi-endophytic nature, haustorial cells are absent, and plastids and pigmentation are present.     

Defective cell division in thermosensitive mutants of Salmonella typhimurium

Summary Two temperature-sensitive mutants ofSalmonella typhimurium defective in cell division (divA anddivC) have been isolated. Cell division in these mutants is arrested at elevated temperature while DNA and protein synthesis are unaffected. This results in formation of long filaments. Filaments returned to permissive temperature divide after a short lag. Inhibition of DNA synthesis by nalidixic acid does not block these divisions. This suggests that the thermosensitive step is required late in the division cycle. Chloramphenicol prevents the division of filaments shifted back to permissive temperature in one of these mutants (divA) and allows limited division to take place in the other mutant (divC). The arrest of cell division at elevated temperature may be phenotypically cured by high osmolarity of the medium. The mutationdivA has been mapped betweenrha andmetB and the mutationdivC betweenleu andaziA.If the filaments ofdivA are starved for thymine and then returned to permissive temperature with the simultaneous restoration of thymine the start of their division is delayed in comparison with the division of the control (unstarved) filaments. The argument is raised that a proper ratio of terminated chromosomes to cell mass must be attained to allow division.     

Studies on the growth and development of protoplasts of the moss,Physcomitrella patens,and its control by light

Protoplasts prepared from protonemal cultures of the moss Physcomitrella patens begin to regenerate a new cell wall within 1 h of removal from cellulase. The wall is seen as a gradually thickening mat of fibres when examined by scanning electron microscopy. Development of filaments from protoplasts takes place in the majority of cases only after one or more cell divisions have occurred. The direction of emergence of filaments is random in uniform light, but strongly negatively phototropic in bright unidirectional horizotal light. Filament growth is also strongly negatively phototropic. The influence of unidirectional light can be destroyed by incubating protoplasts in the presence of colchicine. Filaments growing in unidirectional light have cytoplasmic microtubules running along their long axes and in close association with large organelles. These results are discussed in terms of the potential for this system for the study of polarity in plants.     

Bumble bee-initiated vibration release mechanism of rhododendron pollen

Although Rhododendron spp. anthers have apical pores and should be expected to be buzz pollinated, bees do not normally sonicate them to release pollen. Stamens were examined to determine their pollen release mechanism; the filaments were shown to have natural vibration modes that cause pollen to be forcibly ejected by vibration, providing the pollen is mature. The first natural vibration mode of the stamens was found to generate centrifugal force sufficient to throw the pollen toward the apical pore and out of the anther, so that it adheres to the anther tip and hangs in the bee's path to the nectaries. These vibrations may be triggered by bees brushing past the long stamens when foraging for nectar, so that the flowers retain their pollen until insect vectors begin foraging on them. The low frequency vibration modes of the viscin threads, which hold the pollen together, may contribute towards the pollen ejection by low frequency stamen vibration. Vibration transmission of dehiscent anthers containing pollen was good from 50 Hz to 850 Hz, suggesting that sonication should be an efficient method of pollen collection, although this rarely occurs. Vibratory ejection occurs at a constant energy level, rather than the constant force levels found in previous research on Actinidia.     

Thigmonasticity of thistle staminal filaments

Summary Touching stimulates thistle (Cirsium horridulum Michx.) staminal filaments to rapidly shorten to approximately 70% of their original length. The filaments subsequently re-elongate and regain contractibility within 10min. This thigmonastic movement can be repeated at least 10 times in detached flowers. Filament length is reduced by bathing in 1 M sorbitol, indicating that length change depends on variation in turgor pressure, as is also indicated by plasmolysis observed in contracted filaments. Contraction also depends upon elastic properties of the cuticle. These properties of the cuticle are resistant to boiling in water, chloroform or acid, and treatment with proteases, protein denaturants, sulfatases, and many cell wall degrading enzymes, but are sensitive to cutinase, -glucuronidase, and boiling in 1 M NaOH. Analysis of carbohydrates from flowers boiled in 1 N NaOH showed that most galacturonic acid residues were extracted from filaments but not from petals (which are inelastic). The filament galacturonic acid residues may form a part of the cuticular contractile mechanism, and/or they may facilitate the bending of the vascular and cortical cells that occurs during contraction. Our results indicate that the elasticity of the stretched cuticle provides the force for the contraction of the filaments and the build-up of turgor causes re-elongation.     

Immunocytochemical demonstration of a new vimentin-associated protein in 3T3 fibroblasts

Summary Using a xanthophore cytoskeletal preparation as immunogen, we have produced a monoclonal antibody, A2, which recognized a 160 kDa protein in 3T3 fibroblasts. This protein makes up a cytoplasmic filamentous system, which colocalizes with vimentin filaments. When microtubules and actin filaments are dissolved by high salt extraction, staining with antibody A2 is unaffected. Immunoblot analysis confirms that the 160 kDa protein is co-isolated with vimentin duringin vivo high salt extraction. Following vinblastine treatment, both the 160 kDa protein and vimentin become localized to perinuclear caps, as do other intermediate filaments and their associated proteins; after vinblastine removal, the immunostaining produced by A2 becomes filamentous. Immunoelectron microscopy demonstrates that antibody A2 stains a filament system with a diameter of about 10 nm. Our observations suggest that the 160 kDa protein may be a new vimentin-associated protein which differs from the intermediate filament-associated proteins previously reported, and is widely distributed in several cell types.     

Fertilization in Torenia fournieri: Actin organization and nuclear behavior in the central cell and primary endosperm

Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct actin network. At this stage the secondary nucleus, which is located in the central region of the central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin filaments become fragmented in the micropylar end and the secondary nucleus is located next to the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that actin is involved in the migration of the nucleus in the central cell. Our data also suggest that the dynamics of actin cytoskeleton may be responsible for the reorganization of the central cell and primary endosperm cytoplasm during fertilization.     

Phytochrome-controlled phototropism of protonemata of the moss Ceratodon purpureus: physiology of the wild type and class 2 ptr–mutants

Phototropism and polarotropism in protonemata of the moss Ceratodon purpureus are controlled by the photoreceptor phytochrome. One class of phototropism mutants is characterised by growing randomly when kept for a prolonged time (5 d or longer) in unilateral red light. It was found that a subclass of these mutants grows faster than the wild type, the rate of cell division and the length of the cells being increased. This difference is found for light-grown and dark-grown filaments. It is therefore suggested that the mutant phenotype neither results from a defect in phytochrome photoconversion nor from a defect in phytochrome-gradient formation. Instead, it is possible that a factor which is involved in both signal transduction of phototropism and regulation of cell size and cell division is deregulated. If dark-grown mutant filaments are phototropically stimulated for 24 h, they show a weak phototropic response. Phototropism and polarotropism fluence-rate effect curves for mutants were flattened and shifted to higher fluence rates compared with those for the wild type. With wild-type filaments, a previously unreported response was observed. At a low fluence rate, half of the filaments grew positively phototropically, while the other half grew negatively phototropically. It seems that under these conditions, a phytochrome gradient with two maxima for the far-red-absorbing form of phytochrome (Pfr) within the cross-section of the cell is displayed by the response of the filaments. At higher fluence rates, all filaments of the wild type grew towards the light. These data and results from microbeam irradiation experiments and from phototropism studies with filaments growing within agar, indicate that light refraction plays an important role in the formation of the Pfr gradient in phototropism of Ceratodon. Received: 10 September 1998 / Accepted: 30 December 1998     

PLICATE STAMINAL FILAMENTS IN TILLANDSIA SUBGENUS ANOPLOPHYTUM (BROMELIACEAE)

Plication of staminal filaments is an important diagnostic character for Tillandsia subgenus Anoplophytum (ca. 45 species). The monophyletic integrity of subgenus Anoplophytum has recently been questioned, and we conducted an anatomical investigation of plicate staminal filaments to better characterize this putative synapomorphy. Developmental studies show that the filament plications, or folds, become visible during or soon after anthesis. Serial sections of preplication filaments and filaments in sequential stages of plication were prepared and observed with light microscopy. A uniform sequence of parenchyma cell collapse begins three to four cell layers out from the vascular bundle and proceeds centrifugally to the epidermis. Eventually the epidermal cells collapse, leaving only the vascular bundle and a few surrounding parenchyma cells intact. Above and below the zone of plication, all parenchyma and epidermal cells in the filament remain intact. Species traditionally placed in subgenera Tillandsia and Allardtia have been found with plicate staminal filaments that are anatomically and develop-mentally identical to those studied from subgenus Anoplophytum. Alone, staminal filament plication does not appear to be a good diagnostic character for subgenus Anoplophytum, and doubts concerning the monophylesis of this subgenus are reinforced. The functional significance of stamen filament plication remains unknown.     

Vimentin Filaments Follow the Preexisting Cytokeratin Network during Epithelial–Mesenchymal Transition of Cultured Neonatal Rat Hepatocytes

Changes in cell cytoskeleton are known to play an important role in differentiation and embryogenesis and also in carcinogenesis. Previous studies indicated that neonatal hepatocytes undergo an epithelial–mesenchymal transition when cultured in a serum-free medium for several days. Here we show by Western blotting of neonatal rat liver cells cultured for 3 days that vimentin and cytokeratin were expressed by these cells. Epidermal growth factor treatment induced high coexpression of vimentin and cytokeratin filaments in hepatocytes from neonatal livers, as detected by double immunofluorescence microscopy. Confocal scanning laser microscopy was used to determine the spatial and cell distribution of cytokeratin and vimentin intermediate filament networks. Vimentin-expressing hepatocytes were mainly located on the periphery of epithelial clusters and presented a migratory morphology, suggesting that vimentin expression was related to the loss of cell–cell contact. Short vimentin filaments were mainly located at the cytoplasmic sites behind the extending lamella. Horizontal and vertical dual imaging of double immunofluorescence with anti-vimentin and anti-cytokeratin antibodies indicated that both filaments colocalize strongly. Three-dimensional reconstruction of serial optical sections revealed that newly synthesized vimentin distributed following the preexisting cytokeratin network and, when present, both filament scaffolds codistributed inside cultured hepatocytes. Immunoelectron microscopy performed in whole-mount-extracted cultured cells revealed that both filaments are closely interrelated but independent. However, a high degree of immunogold colocalization was found in the knots of the filament network. Further experiments with colce- mide and cytochalasin treatment indicated that vimentin filament distribution, but not cytokeratin, was dependent on an intact microtubule network. These results are consistent with a mechanism of vimentin assembly, whereby growth of vimentin intermediate filaments is dependent on microtubules in topographically restricted cytoplasmic sites, in close relation to the cytokeratin cytoskeleton and to changes in cell–cell contact and cell shape.     

New Insights in the Cryptonemiales–Rhodymeniales Complex and Resurrection of the Allied Red Algal Order Nemastomatales Kylin 1925 as Inferred From rbcL Sequence Analysis and Comparative Reproductive Morphology

In a global molecular phylogeny of florideophycean red algae inferred from chloroplast‐encoded rbcL sequence analysis, a major monophyletic assemblage comprises the Cryptonemiales (=Halymeniales), the Rhodymeniales, the Schizymeniaceae (Schizymenia, Titanophora, Platoma) and the Nemastomataceae (Nemastoma, Predaea). The phylogenetic significance of the auxiliary cell and its interaction with the fertilized egg cell in this assemblage is discussed in relation to established and newly proposed classification schemes. The order Nemastomatales Kylin 1925 is reinstated and emended to contain the nonprocarpic Schizymeniaceae and Nemastomataceae. Unifying characters of the Nemastomatales include fertilized carpogonia that may establish fusions with carpogonial nutritive cells prior to the formation of septate connecting filaments, and simple gonimoblasts developing outwardly from auxiliary cells or from connecting filaments in their vicinity. The auxiliary cell is a transformed vegetative intercalary cell (Sebdeniaceae), that becomes surrounded by either clusters of nutritive cells (Nemastomataceae), involucral filaments (Schizymeniaceae) or by three‐dimensional ampullary filaments (Halymeniaceae including the Corynomorphaceae), or is part of a procarp (Rhodymeniales). The homology of outward gonimoblast initiation and maturation into a simple ball of carposporangia in the Cryptonemiales, Rhodymeniales and Nemastomatales will be illustrated.