Effect of divalent cations on flagellar scales in the green flagellateTetraselmis cordiformis

Summary The structure and topography of flagellar scales (underlayer scales, rodshaped scales, hair-scales) in the green flagellateTetraselmis cordiformis has been studied in detail and the effect of divalent cations and fixation conditions on scale structure and topography was followed quantitatively. Hair-scales occur in two rows on opposite sides of a flagellum and are linked to the flagellar membrane and to two axonemal doublets by B-tubule-flagellar membrane connectives. Underlayer scales form about 24 longitudinal rows along the flagellum and occur in two distinctive shapes (pentagonal and square). The square shaped underlayer scales are related in position to the attachment sites of the hair-scales. Rod-shaped scales occur in about 20 longitudinal rows along the flagellum and are characteristically positioned as double scales. Calcium in the culture medium is necessary to retain rod-shaped scales on the flagellum, absence of calcium or chelation with EGTA or pyrophosphate leads to disappearance of rod-shaped scales from the flagellum. Other divalent cations can only partially substitute for calcium. It is suggested that calcium provides the linkage between underlayer scales and rod-shaped scales inTetraselmis. Flagellar scales inTetraselmis apparently fall into two categories: a) hair-scales (not affected by fixation or absence of divalent cations, firmly bound to axonemal microtubules via the flagellar membrane), b) underlayer scales and rod-shaped scales (affected by fixation and absence of divalent cations, kept on the flagellum mainly by electrostatic forces). The function of flagellar scales inTetraselmis is discussed.     

Ultrastructure of the flagellar apparatus of the green algaTetraselmis subcordiformis

Summary The ultrastructure of the flagellar apparatus of the marine quadriflagellate green algaTetraselmis subcordiformis is described in detail. Special consideration is given to the functional significance of the contractile rhizoplast and also to a complex structure which anchors the flagellar apparatus to the cell membrane and theca. The flagellar apparatus lies at the base of a deep apical depression. Four basal bodies lie in a zigzag row with their long axes nearly parallel. Outer adjacent pairs of basal bodies are structurally linked by a Z-shaped, ribbon-like structure. A striated fiber (transfiber) connects each outer basal body with the inner basal body of the opposite, mirror image pair. A complex system of four laminated oval discs (rhizanchora), microtubule rootlets and fibrous material anchor the flagellar apparatus and rhizoplasts to the plasma membrane and theca. A 4-2-4-2 arrangement of microtubule rootlets is present. Rhizoplasts, which are contractile organelles, branch into five distinct arms and associate with the near outer basal body and each of the four rhizanchora. Rhizoplast contraction is thought to be linked to flagellar activity and may act to alter the direction of motion of the cell.     

Fine structure of the zoospore ofUlothrix belkae with emphasis on the flagellar apparatus

Summary The anterior end of the zoospore ofUlothrix belkae has been examined in detail and is compared toStigeoclonium and other filamentous green algae. The nature of the symmetry of green algal motile cells is discussed and the term, 180° rotational symmetry, is proposed to describe the type of arrangement of anterior end components seen inU. belkae, including the four basal bodies, rootlets and striated fibers. The four microtubular rootlets are cruciately arranged. A striated microtubule-associated component (SMAC) has a periodicity of 6.4 nm and extends with each 2-membered rootlet posteriorly into the cell. One 5-membered rootlet passes very near the eyespot. Phylogeny in green algal motile cells is discussed.     

The flagellar apparatus of the scaly green flagellatePyramimonas obovata: Absolute configuration

Summary The flagellar apparatus of the quadriflagellate scaly green algaPyramimonas obovata has been studied in detail and the absolute configuration of the flagellar apparatus has been determined. The flagellar root system is cruciate (4-2-4-2-system). 18 major basal body associated fibrous structures connect the four basal bodies with each other. Each basal body is linked to an adjacent basal body by a unique set of connecting fibres, i.e., the flagellar apparatus does not exhibit 180° rotational symmetry. The flagellar apparatus ofPyramimonas obovata is compared with that of quadriflagellate motile cells of theChlorophyceae sensu Stewart andMattox and the phylogenetic relationships are discussed.     

Golgi apparatus activity and membrane flow during scale biogenesis in the green flagellateScherffelia dubia (Prasinophyceae). II: Cell wall secretion and assembly

Summary Secretion of the cell wall (theca) in the scaly green flagellateScherffelia dubia (Prasinophyceae) has been examined by electron microscopy during cytokinesis. The bi-laminate wall forms by the extracellular amalgamation of two layers of scales produced in the Golgi apparatus (GA). Each mature GA cisterna contains ca. 12,000 scales of two distinct varieties arranged in two layers on the cisternal membrane. GA cisternae undergo turnover and one scale containing cisterna matures from thetransface of each dictyosome every 3–4 minutes. Cisternae then fuse with the plasma membrane at the anterior end of the cell releasing the scales onto the cell surface. The two layers of wall scales integrate on the cell surface in a time-dependent self-assembly process. The first scales deposited commence assembly at the cell posterior and the wall develops anteriorly by edge growth. The daughter cell wall is composed of ca. 1.2 million scales deposited in about 3 hours. Calculations of net membrane flow strongly indicate extensive endocytosis during wall deposition.     

The Golgi apparatus of the scaly green flagellate Scherffelia dubia: uncoupling of glycoprotein and polysaccharide synthesis during flagellar regeneration

The flagella of the green alga Scherffelia dubia are covered by scales which consist of acidic polysaccharides and glycoproteins. Experimental deflagellation results in the regeneration of flagella complete with scales. During flagellar regeneration, scales are newly synthesized in the Golgi apparatus, exocytosed and deposited on the growing flagella. Flagellar regeneration is dependent upon protein synthesis and N-glycosylation, as it is blocked by cycloheximide and partially inhibited by tunicamycin. Metabolic labeling with [³⁵S]methionine/cysteine demonstrated that scale-associated proteins were not newly synthesized during flagellar regeneration, suggesting that the proteins deposited on regenerating flagella were drawn from a pool. Quantitative immunoelectron microscopy using a monospecific antibody directed against a scale-associated protein of 126 kDa (SAP126) revealed that the pool of SAP126 was primarily located at the plasma membrane, with minor labeling of the scale reticulum and trans-Golgi cisternae, both before deflagellation and during flagellar regeneration. Since SAP126 was sequestered during flagellar regeneration into secretory vesicles together with newly synthesized scales, it is concluded that the persistent presence of SAP126 in the trans-Golgi cisternae during scale biogenesis requires retrograde transport of the protein from the plasma membrane to the Golgi apparatus. Received: 3 July 1999 / Accepted: 21 August 1999     

Ultrastructure of the motile cells of the prostrate filamentous green algaeProtoderma sarcinoidea andChamaetrichon capsulatum

The biflagellate zoospores ofProtoderma sarcinoidea and the quadriflagellate zoospores ofChamaetrichon capsulatum are each covered by an amorphous, mucous material and a single layer of square scales, and the pyrenoid matrix is traversed by one or more thylakoid membranes. In the flagellar apparatus the basal bodies ofP. sarcinoidea and the upper basal bodies ofC. capsulatum are displaced in the counterclockwise absolute orientation, while the lower basal bodies ofC. capsulatum are directly opposed. Other components of the flagellar apparatus observed in each alga include: cruciately arranged d and s rootlets, each associated with an electron-dense component; simple terminal caps comprised of large and small subunits; a terminal electron-dense mass located near the proximal end of each basal body inP. sarcinoidea and near the upper basal bodies inC. capsulatum; and two rhizoplasts. Components specific to one or the other species include a single accessory basal body inP. sarcinoidea and a fibrous, electron-opaque band that links the upper and the lower basal bodies inC. capsulatum. The flagellar apparatus architecture ofP. sarcinoidea resemblesGayralia oxysperma, while that ofC. capsulatum is similar toTrichosarcina polymorphum andUlothrix species, all of which are included in theUlothrix-group,Ulotrichales, Ulvophyceae.     

The architecture of the flagellar apparatus ofPrymnesium patellifera (Prymnesiophyta)

The flagellar apparatus of the small prymnesiophytePrymnesium patellifera has been analysed and a reconstruction is presented. Externally, the cell carries two sub-equal flagella and a short non-coiling haptonema. Within the cell, there are four microtubular roots and a number of fibrous bands, the latter interconnecting the two basal bodies and the haptonema base. One of the roots (r1) consists of a sheet of up to 25 microtubules originating close to the proximal extremity of the haptonema base, but the other three roots are composed of between 1 and 4 microtubules only. Distally, a large striated fibrous auxiliary connecting root extends across the anterior part of the cell linking root r1 and a mitochondrial profile on the opposite side of the cell. The arrangement of the components of the flagellar apparatus ofP. patellifera is commensurate with the general pattern found in many prymnesiophytes other than members of the Pavlovales, but there are a number of differences in detail from the other species described hitherto.     

广东四片藻鞭毛器的亚微结构和囊壳的形成

本文详细研究了1种海生4条鞭毛的单胞绿藻——广东四片藻鞭毛器的亚微结构和囊壳的形成。4条鞭毛着生于细胞前端凹陷的基部,鞭毛表面覆盖2层鳞片;基体呈纵向平行的“Z”字形排列;具纹纤维连接内外径向排列的两个基体;4个片层状的卵形盘(或称半桥粒(halfdesmosome))微管和纤维物质构成的复合体将鞭毛器和根丝体固着在质膜和囊壳上。根丝体通过两束交叉的微管带与两个邻近的外侧基体相连接。这种连接方式与其它已研究过的四片藻是不同的。囊壳的形成开始于内膜系统,特别是高尔基体。纤维丛和电子密集颗粒在其中合成、修饰,同时由高尔基体衍生的小泡转移到原生质体表面特定的区域,然后经若干步骤接合成完整的囊壳。这个区域与蛋白核的位置相关,表明聚合星状颗粒酶是在蛋白核位点制造或释放的,同时分泌到细胞外。囊壳沿边生长组装与细胞质发育产生特征性的前端鞭毛凹陷同时发生。     

A cryptic cytostome is present inEuglena

Summary A short cylindrical pocket arises as an infolding from the ventral surface of the reservoir near the canal in several species ofEuglena (E. mutabilis, E. gracilis strain T,E. spec.). The structure is linked to a band of microtubules which is shown to be identical to the ventral flagellar root of the euglenoid flagellar root system. An absolute configuration analysis of the flagellar root system inE. mutabilis and a comparison with the flagellar apparatus of colourlessEuglenophyceae and the bodonids (Kinetoplastida) reveals structural and positional homology between the reservoir pocket ofEuglena and the cytostome of these organisms and strongly supports the phylogenetic derivation of theEuglenophyceae from theKinetoplastida and the evolution of greenEuglenophyceae from phagotrophic colourless taxa. The functional significance of the cryptic cytostome ofEuglena is discussed in relation to the occurrence of intracellular endosymbiotic bacteria.     

Flagellar transformation in the heterokontEpipyxis pulchra (Chrysophyceae): Direct observations using image enhanced light microscopy

Summary Cells ofEpipyxis pulchra possess two heteromorphic flagella that differ markedly in function, particularly during motility and prey capture. Flagellar heterogeneity is achieved during the course of at least three cell cycles. Prior to cell division, cells produce two new long, hairy flagella while the parental long flagellum is transformed into a new short, smooth flagellum. The parental short flagellum remains a short flagellum for this and subsequent cell division cycles. Although flagellar transformation requires only two cell cycles, developmental differences exist between daughter cells and the maturation of a flagellum/basal body requires at least three cycles.     

Transformation and development of the flagellar apparatus ofCryptomonas ovata (Cryptophyceae) during cell division

Summary InCryptomonas ovata, long, dorsal flagella are produced which transform during the following cell division into short, ventral flagella. At division there is a reorientation in cell polarity, and the parental basal apparatus, which comprises the basal bodies and associated roots, is distributed to the daughter cells via a complex sequence of events. Flagellar apparatus development includes the transformation of a four-stranded microtubular root into a mature root of different structure and function. Each newly formed basal body nucleates new microtubular roots, but receives a striated fibrous root from a parental basal body. The striated roots are originally produced on the transforming basal body and are transferred to the new basal bodies at each successive division. The development of the asymmetric flagellar apparatus throughout the cell cycle is described.     

ATPase activity of the polar organelle demonstrated by cytochemical reaction in whole unstained cells of Rhodopseudomonas palustris

The polar organelle, a structure associated with the flaggelar apparatus of bacteria, has been demonstrated in whole unstained cells of the photosynthetic bacterium Rhodopseudomonas palustris. It is subpolarly located close to the surface of the bacterial cell and has a round or ellipsoidal shap. It shows a strong ATPase activity which enables its cytochemical electron microscopical visualization.     

Fractionation of suspension cultures of wild carrot and kinetics of membrane labeling

Summary Wild carrot (Daucus carota L.) cells, grown in suspension culture, were labeled with radioactive precursors and fractionated into constituent membranes to be analyzed for specific radioactivity. Results show rapid incorporation of [³H] leucine into endoplasmic reticulum (ER)-, Golgi apparatus-, and plasma membrane/tonoplast-enriched fractions. The time lag between incorporation into ER and its appearance in Golgi apparatus or plasma membrane/tonoplast were less than 5 minutes. With an average time of 3–4 minutes for cisternal formation estimated from studies with monensin, and an average of 5 cisternae per dictyosome (total transit time of 15–20 minutes), it was not possible to account for early incorporation of radioactivity into plasma membranes by passage of proteins from ER to plasma membrane via the Golgi apparatus. To account for the findings, it would appear that at least some proteins were delivered to the plasma membrane via the first membranes that exited (i.e., mature face vesicles) from the Golgi apparatus post-pulse and that some of these proteins had been translated and inserted into membranes at or near the mature face of the Golgi apparatus.     

Membrane flow in plants: Fractionation of growing pollen tubes of tobacco by preparative free-flow electrophoresis and kinetics of labeling of endoplasmic reticulum and golgi apparatus with [3H]leucine

Summary Tobacco (Nicotiana tabacum L.) pollen, germinated 4 hours in suspension culture, was labeled with radioactive leucine and fractionated into constituent membranes by the technique of preparative free-flow electrophoresis. Tubes were ruptured by sonication directly into the electrophoresis buffer. Unfortunately, the Golgi apparatus of the rapidly elongating pollen tubes did not survive the sonication step. However, it was possible to obtain useful fractions of endoplasmic reticulum and mitochondria. To obtain Golgi apparatus, glutaraldehyde was added to the homogenization buffer during sonication. Plasma membrane, which accounted for only about 3% of the total membrane of the homogenates as determined by staining with phosphotungstate at low pH, was obtained in insufficient quantity and fraction purity to permit analysis. Results show rapid incorporation of [³H]leucine into endoplasmic reticulum followed by rapid chase out. The half-time for loss of radioactivity from the pollen tube endoplasmic reticulum was about 10 minutes. Concomitant with the loss of radioactivity from endoplasmic reticulum, the Golgi apparatus fraction was labeled reaching a maximum 20 minutes post chase. The findings suggest flow of membranes from endoplasmic reticulum to the Golgi apparatus during pollen tube growth.     

Selective real-time herbicide monitoring by an array chip biosensor employing diverse microalgae

A multiple-strain algal biosensor was constructed for the detection of herbicides inhibiting photosynthesis. Nine different microalgal strains were immobilised on an array biochip using permeable membranes. The biosensor allowed on-line measurements of aqueous solutions passing through a flow cell using chlorophyll fluorescence as the biosensor response signal. The herbicides atrazine, simazine, diuron, isoproturon and paraquat were detectable within minutes at minimal LOEC (Lowest Observed Effect Concentration) ranging from 0.5 to 100μgL⁻¹, depending on the herbicide and algal strain. The most sensitive strains in terms of EC₅₀ values were Tetraselmis cordiformis and Scherffelia dubia. Less sensitive species were Chlorella vulgaris, Chlamydomonas sp. and Pseudokirchneriella subcapitata, but for most of the strains no general sensitivity or resistance was found. The different responses of algal strains to the five herbicides constituted a complex response pattern (RP), which was analysed for herbicide specificity within the linear dose-response relationship. Comparisons of herbicide-specific RP to reference RPs of the five herbicides always showed the lowest deviation of the herbicide-specific RP tested with the reference RP of the same herbicide for the triazine and phenylurea herbicides. We therefore conclude that, in principle, identification of a specific herbicide is possible employing the algal sensor chip.     

Flagellar regeneration and associated scale deposition inPyramimonas gelidicola (Prasinophyceae,Chlorophyta)

Summary During regeneration of mechanically amputated flagella, flagellar scales and the subtending membrane accumulate in a villiform scale reservoir in which the scales interact to form patterns on the villi reminiscent of the arrangement they later assume on the flagellum. The reservoir membrane is continuous with the plasmalemma, and the scales, attached directly and indirectly to the membrane, leave the reservoir and migrate toward the developing flagella where they assemble into highly ordered layers. It is proposed that scale-scale interactions induce a process of auto-assembly initiating the complex arrangement of scale tiers on the flagellum and cell body.     

Membrane flow in plants: preparation and kinetics of labelling of plasma membranes from growing pollen tubes of tobacco

Summary Growing pollen tubes of tobacco germinated in suspension culture, were labelled with [³H]leucine and after varying times of chase with unlabelled leucine at 23, 16, or 4°C, were separated into plasma membrane-enriched and plasma membrane-depleted fractions by aqueous two-phase partition. At 23°C, the specific radioactivity of the plasma membrane increased with time to a maximum at 60 min. At 16°C and 4°C, labelling of the plasma membrane was respectively 40% and 10% that at 23°C. However, if labelling was at 23°C and subsequent transfer was at 4°C, plasma membrane labelling was much less affected and labelling of the plasma membrane was 60% that at 23°C. Additionally, quantitation of various morphological parameters revealed no accumulations of 50–70 nm transition vesicles in the space between endoplasmic reticulum and cis Golgi apparatus that might suggest formation of a low temperature compartment similar to those described for mammalian cells and tissues. Similarly, growth of pollen tubes was reduced but not blocked even at temperatures of 12°C. The results suggest that tube elongation is accompanied by a steady state flow of membranes to the cell surface that is relatively insensitive to interruption by low temperatures. Whereas leucine incorporation is reduced by low temperature even at 16°C, the flow pathway to the cell surface, including the endoplasmic reticulum to Golgi apparatus transfer step, as well as elongation growth does not exhibit a pronounced low temperature block in this tip growing system.     

Golgi apparatus buds — vesicles or coated ends of tubules?

Summary Based on cell-free processing whereby membrane glycoproteins from one cell type were processed by enzymes located in Golgi apparatus from another cell type, J. Rothman and colleagues postulated that vesicles budding from one Golgi apparatus stack migrated to and fused with cisternal membranes of other Golgi apparatus stacks in the cell-free milieu. An extension of this hypothesis was that these same or similar vesicles were involved in the trafficking of membrane material from one cisterna to the next even in the same Golgi apparatus stack [W. G. Dunphy, J. E. Rothman: Compartmental organization of the Golgi stack. Cell 42: 13–21 (1985)]. A coated bud revealed by tannic acid-containing fixatives was the morphological entity associated with this intercompartment Golgi apparatus transfer. This report summarizes information from the author's laboratories that suggests that perhaps the majority of these coated buds, while associated with the Golgi apparatus, are not vesicles per se but rather coated ends of tubules. Golgi apparatus tubules have been postulated to permit interconnections among adjacent Golgi apparatus stacks but not to function in transport between contiguous cisternae of the same Golgi apparatus stack.In the interest of scientific discourse, reasoned and constructive replies to views expressed under New Ideas in Cell Biology will be considered for publication. In this case, the responsible editor, to be contacted by respondents, is E. Schnepf.