STEROLS AS BIOMARKERS IN GYMNODINIUM BREVE: DISTRIBUTION IN DINOFLAGELLATES

A poorly understood feature of nostocacean growth and development is the formation of ordered macroscopic structures from microscopic cells, trichomes, and filaments. Using macro-photography, time-lapse micro-cinematography, light and electron microscopy of Nostoc species in pure culture, it has been possible to demonstrate how motility, adhesion and aggregation of photo-induced hormogonia result in macro-morphogenesis of dendroid forms. Red-light induced hormogonia from synchronized cultures aggregate rapidly on agar as tight flowing streams, in patterns responsive to the direction and quality of incident light. Unlike the even textured cell surfaces of heterocystous filaments, the cell walls of swarming hormogonia are covered with a striate mucoid layer containing pili attached to cells of adjacent hormogonia. During differentiation to an aseriate phase, cell wall fusions occur and a gelatinous matrix forms around the enlarging sub-globose cells. Liquid suspensions of hormogonia aggregate in a solid mass following the net centripetal movement of interlaced loops of curved hormogonia attached by adhesive pili. In darkness or dim white light, compressed hormogonial aggregates form erect tree-like (dendroid) macro-structures by photo-tactic reversal of streaming motility. Hormogonia within the aggregates re-organize into streams that push upward into the light, forming structured, positively phototropic protuberances, several millimeters in length. Under weak illumination, the structures become branched with crowns of waving hormogonia. The dendroid morphology is stabilized by deposit of gelatinous material derived from successive cycles of cell-filament development, liberation of heterocysts and formation of dormant cells and trichomes.     

Hormogonia in two nostocalean cyanophytes (cyanobacteria) from the genera Hapalosiphon and Fischerella

The formation of hormogonia in the nostocalean cyanophytes/cyanobacteria Hapalosiphon fontinalis (C. Agardh) Bornet and Fischerella sp. was studied in natural populations collected from the Klin peatbog, northern Slovakia. Hormogonia were produced terminally in lateral branches of filaments (both species), or also directly on the main branches (Fischerella sp.). In contrast to vegetative filaments, hormogonia were not ramified, lacked heterocytes, were embedded in mucilaginous envelopes, were able to move, and their cells contained aerotopes. They were released by gliding through an opening in the sheath at the end of lateral branches of filaments. Released hormogonia of H. fontinalis were solitary or agglomerated into common fascicles morphologically resembling planktic colonies of Aphanizomenon flos-aquae (L.) Ralfs ex Bornet et Flahault or Dolichospermum affine (Lemmermann) Wacklin, Hoffmann et Komárek (syn. Anabaena affinis Lemmermann). Occasionally, lateral or sessile Nostochopsis-like heterocytes and apical spherical monocytes were formed on the main filaments. Hormogonia of Fischerella sp. were formed not only in apical part of lateral trichomes, but also directly on the main trichomes. Their cells were markedly larger than the vegetative cells and possessed well-developed aerotopes. Released hormogonia remained solitary, and were not agglomerated into fascicles. Apical hormogonia were released by gliding through an opening in the sheath at the end of lateral branches of filaments, and basal hormogonia were released by breaking off the main axis. In contrast to filaments of H. fontinalis which were very common and represented the dominant species of the cyanophyte communities in the locality, filaments of Fischerella sp. were observed only in one sample and for a limited period. This is the first record of a representative of the genus Fischerella in Slovakia.     

Branching and intercellular communication in the Section V cyanobacterium Mastigocladus laminosus,a complex multicellular prokaryote

The filamentous Section V cyanobacterium Mastigocladus laminosus is one of the most morphologically complex prokaryotes. It exhibits cellular division in multiple planes, resulting in the formation of true branches, and cell differentiation into heterocysts, hormogonia and necridia. Here, we investigate branch formation and intercellular communication in M. laminosus. Monitoring of membrane rearrangement suggests that branch formation results from a randomized direction of cell growth. Transmission electron microscopy reveals cell junction structures likely to be involved in intercellular communication. We identify a sepJ gene, coding for a potential key protein in intercellular communication, and show that SepJ is localized at the septa. To directly investigate intercellular communication, we loaded the fluorescent tracer 5‐carboxyfluorescein diacetate into the cytoplasm, and quantified its intercellular exchange by fluorescence recovery after photobleaching. Results demonstrate connectivity of the main trichome and branches, enabling molecular exchange throughout the filament network. Necridia formation inhibits further molecular exchange, determining the fate of a branch likely to become a hormogonium. Cells in young, narrow trichomes and hormogonia exhibited faster exchange rates than cells in older, wider trichomes. Signal transduction to co‐ordinate movement of hormogonia might be accelerated by reducing cell volume.     

Role of streams in myxobacteria aggregate formation

Cell contact, movement and directionality are important factors in biological development (morphogenesis), and myxobacteria are a model system for studying cell-cell interaction and cell organization preceding differentiation. When starved, thousands of myxobacteria cells align, stream and form aggregates which later develop into round, non-motile spores. Canonically, cell aggregation has been attributed to attractive chemotaxis, a long range interaction, but there is growing evidence that myxobacteria organization depends on contact-mediated cell-cell communication. We present a discrete stochastic model based on contact-mediated signaling that suggests an explanation for the initialization of early aggregates, aggregation dynamics and final aggregate distribution. Our model qualitatively reproduces the unique structures of myxobacteria aggregates and detailed stages which occur during myxobacteria aggregation: first, aggregates initialize in random positions and cells join aggregates by random walk; second, cells redistribute by moving within transient streams connecting aggregates. Streams play a critical role in final aggregate size distribution by redistributing cells among fewer, larger aggregates. The mechanism by which streams redistribute cells depends on aggregate sizes and is enhanced by noise. Our model predicts that with increased internal noise, more streams would form and streams would last longer. Simulation results suggest a series of new experiments.     

发状念珠藻藻殖段的分化及其光合特性的研究

Hormogonia of Nostoc flagelliforme is one of the developmental stages in the life cycle of cyanobacterium. High yields of pure hormogonia were obtained by weak light (the filaments were covered by sterilized sand for blocking light), red light, white light plus DCMU (3, 4-dichlorophenyl-1, 1-dimethylurea) in the culture. These pure fractions of hormogonia allowed the study of physiological measurements in comparison to vegetative filaments. The photosynthesis in the hormogonia and the vegetative filaments was characterized by fluorescence emission spectra at 77 K, absorption spectrum and oxygen evolution. Absorption spectrum of the hormogoia and vegetative filaments did not reveal difference. The data indicated the similarity of pigment contents between hormogonia and vegetative filaments. Some differences were observed in oxygen evolution of vegetative filaments and hormogonia in the temperature range of 15 ℃ to 45 ℃ and light intensity around 110 μmol·m-2·s-1 to 1200 μmol·m-2·s-1. The fluorescence emission spectra showed that energy distribution between the two photosystems in mature colonies was more balance than in hormogonia. The absorption of light energy in phycobilisomes and the transfer to the two photosystems in the hormogonia were more effective than in the mature colonies. It may be concluded that the formation of hormogonia affected on the structure and function of phytosynthesis.     

Trichome micromorphology of the Chinese-Himalayan genus <Emphasis Type="Italic">Colquhounia</Emphasis> (Lamiaceae), with emphasis on taxonomic implications

Trichome micromorphology of leaves and young stems of nine taxa (including four varieties) of Colquhounia were examined using light and scanning microscopy. Two basic types of trichomes were recognized: eglandular and glandular. Eglandular trichomes are subdivided into simple and branched trichomes. Based on the number of cells and trichome configuration, simple eglandular trichomes are further divided into four forms: unicellular, two-celled, three-celled and more than three-celled trichomes. Based on branching configuration, the branched eglandular trichomes can be separated into three forms: biramous, stellate and dendroid. Glandular trichomes can be divided into two subtypes: capitate and peltate glandular trichomes. Results from this study of morphological diversity of trichomes within Colquhounia lend insight into infrageneric classification and species relationships. Based on the presence of branched trichomes in C. elegans, this species should be transferred from Colquhounia sect. Simplicipili to sect. Colquhounia. We provide a taxonomic key to species of Chinese Colquhounia based on trichome morphology and other important morphological traits.     

Motility in cyanobacteria: polysaccharide tracks and Type IV pilus motors

Motility in cyanobacteria is useful for purposes that range from seeking out favourable light environments to establishing symbioses with plants and fungi. No known cyanobacterium is equipped with flagella, but a diverse range of species is able to ‘glide’ or ‘twitch’ across surfaces. Cyanobacteria with this capacity range from unicellular species to complex filamentous forms, including species such as Nostoc punctiforme, which can generate specialised motile filaments called hormogonia. Recent work on the model unicellular cyanobacterium Synechocystis sp. PCC 6803 has shown that its means of propulsion has much in common with the twitching motility of heterotrophs such as Pseudomonas and Myxococcus. Movement depends on Type IV pili, which are extended, adhere to the substrate and then retract to pull the cell across the surface. Previous work on filamentous cyanobacteria suggested a very different mechanism, with movement powered by the directional extrusion of polysaccharide from pores close to the cell junctions. Now a new report by Khayatan and colleagues in this issue of Molecular Microbiology suggests that the motility of Nostoc hormogonia has much more in common with Synechocystis than was previously thought. In both cases, polysaccharide secretion is important for preparing the surface, but the directional motive force comes from Type IV pili.     

Electron Transport Regulates Cellular Differentiation in the Filamentous Cyanobacterium Calothrix

Differentiation of the filamentous cyanobacteria Calothrix sp strains PCC 7601 and PCC 7504 is regulated by light spectral quality. Vegetative filaments differentiate motile, gas-vacuolated hormogonia after transfer to fresh medium and incubation under red light. Hormogonia are transient and give rise to vegetative filaments, or to heterocystous filaments if fixed nitrogen is lacking. If incubated under green light after transfer to fresh medium, vegetative filaments do not differentiate hormogonia but may produce heterocysts directly, even in the presence of combined nitrogen. We used inhibitors of thylakoid electron transport (3-[3,4-dichlorophenyl]-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) to show that the opposing effects of red and green light on cell differentiation arise through differential excitations of photosystems I and II. Red light excitation of photosystem I oxidizes the plastoquinone pool, stimulating differentiation of hormogonia and inhibiting heterocyst differentiation. Conversely, net reduction of plastoquinone by green light excitation of photosystem II inhibits differentiation of hormogonia and stimulates heterocyst differentiation. This photoperception mechanism is distinct from the light regulation of complementary chromatic adaptation of phycobilisome constituents. Although complementary chromatic adaptation operates independently of the photocontrol of cellular differentiation, these two regulatory processes are linked, because the general expression of phycobiliprotein genes is transiently repressed during hormogonium differentiation. In addition, absorbance by phycobilisomes largely determines the light wavelengths that excite photosystem II, and thus the wavelengths that can imbalance electron transport.     

丝状体蓝藻藻殖段的分化及其调节机制

本文介绍了丝状体蓝藻(亦称蓝细菌) 的藻殖段的分化及其调节机制。藻殖段与正常藻丝体的区别在于细胞形状、细胞内存有气囊和可移动的短而直的藻丝链等。本文对许多环境因子包括光和营养因素等促进或抑制藻殖段的分化进行了讨论;还介绍了念珠藻(Nostoc) ,单歧藻(Tolypothrix) 和眉藻(Calothrix)所具有复杂的细胞发育过程,即具气囊又可移动的藻殖段分化,异形胞分化以及营养细胞的补偿性色适应。这三种细胞类型的适应形成取决于两种不同的光受体系统。藻殖段和异形胞两者的分化可能取决于光合电子传递链;而营养细胞的补偿性色适应则受光敏色素的调节。此外,谷酰胺合成酶合成和活性调节的PII蛋白,在协同藻殖段分化、异形胞分化及营养细胞的补偿色适应中起重要作用。由于蓝藻藻殖段分化及其调节机制是一个新的研究领域,关于它的知识尚不完整,亟待人们加强研究。     

丝状体蓝藻藻殖段的分化及其调节机制

本文介绍了丝状体蓝藻（亦称蓝细菌）的藻殖段的分化及其调节机制。藻殖段与正常藻丝体的区别在于细胞开状、细胞内存有气囊和可移动的短而真的藻丝链等。本文对许多环境因子包括光和营养因素等促进或抑制藻殖段的分化进行一讨论;还介绍了含球藻（Ｎｏｓｔｏｃ）,单歧藻（Ｔｏｌｙｐｏｔｈｒｉｘ）和眉藻（Ｃａｌｏｔｈｒｉｘ）所具有复杂的细胞发育过程,即具气囊又可移动的藻殖段分化,异形胞分化以及营养细胞的被偿性色适应。这     

Sacrificial cell death and trichome breakage in an oscillatoriacean blue-green alga: the role of murein

Summary Trichomes of Microcoleus vaginatus, a motile blue-green alga of the family Oscillatoriaceae, were studied by light and electron microscopy in an effort to determine the sites of trichome breakage during production of hormogonia.According to the evidence presented herein, transcellular breakage of trichomes is the only mechanism of hormogonium production in M. vaginatus. Tearing of the murein sacculus appears to be necessary and sufficient for transcellular breakage to ensue. As Fuhs and earlier investigators have correctly claimed, this process always involves the death of the cell whose wall is torn.When trichomes of M. vaginatus break across cells to produce hormogonia, the murein sacculus usually tears along a circumferential set of junctional pores. This particular mechanism of trichome breakage is not universal among members of the family Oscillatoriaceae.This report is based on a thesis submitted in partial fulfillment of the requirements for a Ph. D. degree in Biology at Harvard University.     

Short-Range Guiding Can Result in the Formation of Circular Aggregates in Myxobacteria Populations

Myxobacteria are social bacteria that upon starvation form multicellular fruiting bodies whose shape in different species can range from simple mounds to elaborate tree-like structures. The formation of fruiting bodies is a result of collective cell movement on a solid surface. In the course of development, groups of flexible rod-shaped cells form streams and move in circular or spiral patterns to form aggregation centers that can become sites of fruiting body formation. The mechanisms of such cell movement patterns are not well understood. It has been suggested that myxobacterial development depends on short-range contact-mediated interactions between individual cells, i.e. cell aggregation does not require long-range signaling in the population. In this study, by means of a computational mass-spring model, we investigate what types of short-range interactions between cells can result in the formation of streams and circular aggregates during myxobacterial development. We consider short-range head-to-tail guiding between individual cells, whereby movement direction of the head of one cell is affected by the nearby presence of the tail of another cell. We demonstrate that stable streams and circular aggregates can arise only when the trailing cell, in addition to being steered by the tail of the leading cell, is able to speed up to catch up with it. It is suggested that necessary head-to-tail interactions between cells can arise from physical adhesion, response to a diffusible substance or slime extruded by cells, or pulling by motility engine pili. Finally, we consider a case of long-range guiding between cells and show that circular aggregates are able to form without cells increasing speed. These findings present a possibility to discriminate between short-range and long-range guiding mechanisms in myxobacteria by experimentally measuring distribution of cell speeds in circular aggregates.     

Development of motility in cultures of the cyanobacterium Mastigocladus laminosus

Abstract The time course for the development of motility in cultures of the cyanobacterium Mastigocladus laminosus was established quantitatively using a slicer tool as described here. The slicer tool produces samples of trichomes from centrifuged pellets that, under identical conditions, shed comparable numbers of hormogonia. The number of hormogonia formed in liquid culture rises steeply between 24 and 31 h of incubation, returning to essentially zero in the next 24 h. The initial lag may be devoted to the cell divisions needed to form the cells of the hormogonium. The drop in motility could be due to one or more heat-stable substance(s) accumulated in the medium, since used media inhibited motility and the effect resisted autoclaving. The fact that the inoculum needed to be ground in order for motility to occur suggests that the structure of the clump inhibits the shedding of hormogonia. Some ecological implications are proposed, assuming that the clump structure interferes with light and mass transfer.     

Helical disorder and the filament structure of F-actin are elucidated by the angle-layered aggregate

Angle-layered aggregates of F-actin are net-like structures induced by Mg2+ concentrations below that used to form paracrystals. These aggregates incorporate the angular disorder of subunits, which has been described elsewhere for isolated actin filaments. Because this disorder is incorporated into the aggregates in solution at the time they are formed, the possibility of negative stain preparation being responsible for the disorder is excluded. The simple two-layered geometry of the angle-layered aggregate provides information about the shape of the component actin filaments free from the superposition of large numbers of layers. A model for the filament shape, derived from single filaments and confirmed by the angle-layered aggregate, is different from those that have previously emerged from paracrystal studies. An understanding of the interfilament bond in both the angle-layered aggregate and the paracrystal allows one to reconcile these different models. We have found a bipolar bonding rule, with staggered crossover points in the angle-layered aggregate, which we suggest is also responsible for Mg2+ paracrystals. This bonding rule can explain the apparent alignment of crossover points in adjacent filaments in paracrystals as a consequence of the superposition of staggered filaments.     

On‐demand killing of adherent cells on photo‐acid‐generating culture substrates

As a powerful tool of cell screening and cell purification, we developed a novel method to kill adherent cells as cultured on a substrate by micro‐projection of incoherent visible light. To kill the cells by the mild light irradiated by electrically controllable micro‐projection systems currently available, we introduced the assist of the photo‐responsive culture substrates functionalized with a photo‐acid‐generating polymer. In clear contrast to the existing laser‐based methods requiring point scanning, areal micro‐prjection of blue light with the wavelength 436 nm killed many CHO‐K1 cells at a time in the irradiated area on the substrate. The effect of the photo‐generated acid was so confined that selective killing of targeted cells was achieved without critical damage to the neighboring cells. Further, we demonstrated the photo‐selective killing of the adherent cells after preliminarily patterning through the photo‐induced removal of cell adhesion‐inhibiting polymer. Biotechnol. Bioeng. 2013; 110: 348–352. © 2012 Wiley Periodicals, Inc.     

Botulinum hemagglutinin‐mediated in situ break‐up of human induced pluripotent stem cell aggregates for high‐density suspension culture

Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high‐density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break‐up using botulinum hemagglutinin (HA), which specifically bound with E‐cadherin and disrupted cell–cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E‐cadherin‐mediated cell–cell connections to facilitate the break‐up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 10⁶ cells ml^?1 was obtained by aggregate break‐up into small ones, which was three times higher than that with the conventional culture without aggregate break‐up. Therefore, the temporary activity of HA for disrupting E‐cadherin‐mediated cell–cell connection was key to establishing a simple in situ method for hiPSC aggregate break‐up in bioreactors, leading to high cell density in suspension culture.     

Multiple three‐dimensional mammalian cell aggregates formed away from solid substrata in ultrasound standing waves

Single and multiple three‐dimensional cell aggregates of human red blood cells (RBCs) and HepG2 cells were formed rapidly in low mega‐Hertz ultrasound standing wave fields of different geometries. A single discoid aggregate was formed in a half‐wavelength pathlength resonator at a cell concentration sufficient to produce a 3D structure. Multiple cell aggregates were formed on the axis of a cylindrical resonator with a plane transducer (discoid aggregates); in a resonator with a tubular transducer and in the cross‐fields of plane and tubular transducers and two plane orthogonal transducers (all cylindrical aggregates). Mechanically strong RBC aggregates were obtained by crosslinking with wheat germ agglutinin (WGA, a lectin). Scanning electron microscopy showed aggregate surface porous structures when RBCs were mixed with WGA before sonication and tighter packing when ultrasonically preformed aggregates were subsequently exposed to a flow containing WGA. HepG2 cell aggregates showed strong accumulation of F‐actin at sites of cell–cell contact consistent with increased mechanical stability. The aggregates had a porous surface, and yet confocal microscopy revealed a tight packing of cells in the aggregate's inner core. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Trichome forms in Ardisia (Myrsinaceae) in relation to the bacterial leaf nodule symbiosis

Symbiotic leaf-nodule bacteria in nodulated members of Rubiaceae live in mucilage secreted by colleters located on stipules within buds. These differ from colleters on most nodule-free species. This study was undertaken to examine buds of Ardisia and the related monotypic Amblyanthus of Myrsinaceae to see if nodulated species had secretory structures dissimilar from those of nodule-free species. Buds removed from herbarium specimens (61 species) and live plants (3 species) were paraffin-sectioned. Diverse trichome forms occur, including dimorphism between adaxial and abaxial trichomes in some species. Species within each subgenus were arranged according to trichome form: peltate scale, irregularly capitate, capitate, sessile capitate, bicellular capitate, and uniseriate. Only seven Ardisia species (all in subgenus Crispardisia, widely assumed to have bacteria in marginal leaf nodules of all 30 species) have short-lived trichomes bearing one or more elongate, swollen, distal cells that appear to be secretory cells. These trichomes are analogous to the dendroid or brushlike colleters of nodulated Rubiaceae. Druses occur in most subgenera and this appears to be by far the most predominant crystal type m Ardisia.     

Swapping genes to survive - a new role for archaeal type IV pili

Type IV pili are filamentous structures that are found on the surface of many bacterial and archaeal cells, they are involved in cell motility and surface adhesion. In the crenarchaeon Sulfolobus solfataricus, type IV pili formation is strongly induced by UV irradiation and leads to cellular aggregation. The study by Ajon et al. (2011) published in this issue of Molecular Microbiology shows that UV-induced cellular aggregation greatly stimulates the exchange of chromosomal markers among irradiated cells, and that this strategy helps with cell survival. Sulfolobus knockout strains that are incapable of forming pili proved to be deficient in aggregation, and also showed decreased cellular survival after UV irradiation. The UV-induced pili of three different Sulfolobus species had distinct morphologies, and correspondingly these three species were able to aggregate only with their own kind. This work has defined a new role for type IV pili in both the transfer of genes within species and the recovery from UV-induced DNA damage.     

A puddle: an ombrophilic cyano-bacterial community

Sequential stages of formation of an ombrophilic cyano-bacterial community on clay were determined in a laboratory model of a puddle community. In a suspension of washed clay obtained from loamy soil, with montmorillonite as the predominant phase, a bacterial neustonic film is initially formed; it acts as a support for cyanobacterial hormogonia. At the next stage, the upper layer of precipitated clay (about 1 mm) is reinforced by a cyano-bacterial structure of Phormidium sp. trichomes and develops a tissue-like structure. The hormogonia and sheathless cyanobacteria remain free from mineral particles. Subsequently, gas formation results in a separation of a dense cyano-bacterial film from the underlying loose suspension and in formation of gas swellings. The mineral component of the film is differentiated: mineral particles of quartz and feldspar grains are attached to Phormidium sp. trichomes, which act as a factor of mineral selection.