Genome sequencing and de novo assembly of the giant unicellular alga Acetabularia acetabulum using droplet MDA.

The macroscopic single-celled green alga Acetabularia acetabulum has been a model system in cell biology for more than a century. However, no genomic information is available from this species. Since the alga has a long life cycle, is difficult to grow in dense cultures, and has an estimated diploid genome size of almost 2 Gb, obtaining sufficient genomic material for genome sequencing is challenging. Here, we have attempted to overcome these challenges by amplifying genomic DNA using multiple displacement amplification (MDA) combined with microfluidics technology to distribute the amplification reactions across thousands of microscopic droplets. By amplifying and sequencing DNA from five single cells we were able to recover an estimated ~ 7-11% of the total genome, providing the first draft of the A. acetabulum genome. We highlight challenges associated with genome recovery and assembly of MDA data due to biases arising during genome amplification, and hope that our study can serve as a reference for future attempts on sequencing the genome from non-model eukaryotes.

Spectroscopic and biochemical analysis of regions of the cell wall of the unicellular 'mannan weed', Acetabularia acetabulum.

Although the Dasycladalean alga Acetabularia acetabulum has long been known to contain mannan-rich walls, it is not known to what extent wall composition varies as a function of the elaborate cellular differentiation of this cell, nor has it been determined what other polysaccharides accompany the mannans. Cell walls were prepared from rhizoids, stalks, hairs, hair scars, apical septa, gametophores and gametangia, subjected to nuclear magnetic resonance and Fourier transform infrared spectroscopy, and analyzed for monosaccharide composition and linkage, although material limitations prevented some cell regions from being analyzed by some of the methods. In diplophase, walls contain a para-crystalline mannan, with other polysaccharides accounting for 10-20% of the wall mass; in haplophase, gametangia have a cellulosic wall, with mannans and other polymers representing about a quarter of the mass. In the walls of the diplophase, the mannan appears less crystalline than typical of cellulose. The walls of both diploid and haploid phases contain little if any xyloglucan or pectic polysaccharides, but appear to contain small amounts of a homorhamnan, galactomannans and glucogalactomannans, and branched xylans. These ancillary polysaccharides are approximately as abundant in the cellulose-rich gametangia as in the mannan-rich diplophase. In the diplophase, different regions of the cell differ modestly but reproducibly in the composition of the cell wall. These results suggest unique cell wall architecture for the mannan-rich cell walls of the Dasycladales.

Development and organization of the central vacuole of Acetabularia acetabulum.

* Here we analyzed the shape of the central vacuole of Acetabularia acetabulum by visualizing its development during diplophase (from juvenility through reproduction) and haplophase (from meiosis through mating). * Light microscopy and whole-organism applications of a pH-sensitive dye, neutral red, were used to visualize the anatomy of the central vacuole. We studied connectivity within the thallus by locally applying dye to morphologically distinct regions (rhizoid, stalk, apex, hairs) and observing dye movements. * In vegetative thalli most of the rhizoid, stalk and young hairs stained with dye. In reproductive structures (caps, gametangia) dye also stained the majority of the interiors. When applied to small areas, dye moved at different rates through each region of the thallus (e.g. within the stalk). Dye moved from younger hairs, but not from older hairs, into the stalk. Errors in incorporation of central vacuole into gametangia occurred at <10(-5). * These data indicate that the central vacuole of A. acetabulum is a ramified polar organelle with, potentially, a gel-like sap that actively remodels its morphology during development.

Growth inhibition and changes in morphology and actin distribution in Acetabularia acetabulum by phalloidin and phalloidin derivatives.

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.

Poly(A)+ RNA during vegetative development of Acetabularia peniculus.

In the juvenile stage, the diploid giant-celled green algae Acetabularia spp. are differentiated into an upright stalk and an irregularly branched rhizoid. Early amputation and grafting experiments as well as biochemical and molecular analyses have shown that mRNA (as poly(A)+ RNA) is continuously supplied from the primary nucleus in the rhizoid and accumulates in the stalk apex. In the present study, localization of poly(A)+ RNA in the juvenile stage of the Acetabularia peniculus was investigated by fluorescent in situ hybridization using oligo(dT) as a probe. The signal was localized in the apical cytoplasm and, in addition, multiple longitudinal striations throughout the stalk and rhizoid cytoplasm. A large portion of the poly(A)+ RNA striations exhibited structural polarity, broadened at one end and gradually thinned toward the other end. Some of the striations in the rhizoid cytoplasm were continuous with a zone of signal in the area of the perinuclear rim. The poly(A)+ RNA striations were associated with thick bands of longitudinal actin bundles which run through the entire length of the stalk. Cytochalasin D caused fragmentation of the actin bundles and irregular distribution of the fluorescent signal. We suggest that the poly(A)+ RNA striations constitute a hitherto unknown form of packaged mRNA that is transported over large distances along the actin cytoskeleton to be stored and expressed in the growing apex.

Whorl morphogenesis in the dasycladalean algae: the pattern formation viewpoint.

The dasycladalean algae produce diverse whorled structures, among which the best known are the vegetative and reproductive whorls of Acetabularia acetabulum. In this paper, we review the literature pertaining to the origin of these structures. The question is addressed in terms of the necessary pattern-forming events and the possible mechanisms involved, an outlook we call the pattern formation viewpoint. The pattern-forming events involved in the morphogenesis of the vegetative and reproductive whorls of Acetabularia have been used to define five and six morphogenetic stages, respectively. We discuss three published mechanisms which account, at least in part, for the pattern-forming events. The mechanisms are mechanical buckling of the cell wall, reaction-diffusion of morphogen molecules along the cell membrane, and mechanochemical interactions between Ca2+ ions and the cytoskeleton in the cytosol. The numerous differences between these mechanisms provide experimental grounds to test their validity. To date, the results of these experiments point towards reaction diffusion as the most likely patterning mechanism. Finally, we consider the evolutionary origin of the vegetative and reproductive whorls and provide mechanistic explanations for some of the major evolutionary advances.

Coordination of cellular events that precede reproductive onset in Acetabularia acetabulum: evidence for a 'loop' in development.

Amputated apices from vegetative wildtype cells of the uninucleate green alga Acetabularia acetabulum can differentiate a reproductive structure of 'cap' in the absence of the nucleus (Hämmerling, J. (1932) Biologisches Zentralblatt 52, 42-61). To define the limits of the ability of wildtype cells to control reproductive differentiation, we determined when during development apices from wildtype cells first acquired the ability to make a cap in the absence of the nucleus and, conversely, when cells with a nucleus lost the ability to recover from the loss of their apices. To see when the apex acquired the ability to make a cap without the nucleus, we removed apices from cells varying either the developmental age of the cells or the cellular volume left with the apex. Cells must have attained the adult phase of development before the enucleate apex could survive amputation and make a cap. Apices removed from cells early in adult growth required more cell volume to make a cap without the nucleus than did apices removed from cells late in adult growth. To define the limits of the cell to recapitulate development when reproduction falters, we analyzed development in cells whose caps either had been amputated or had spontaneously aborted. After loss of the first cap, cells repeated part of vegetative growth and then made a second cap. The ability to make a second cap after amputation of the first one was lost 15-20 days after cap initiation. Our data suggest that internal cues, cell age and size, are used to regulate reproductive onset in Acetabularia acetabulum and add to our understanding of how reproduction is coordinated in this giant cell.

Is morphogenesis an intrinsically robust process?

Because morphogenesis is dynamically complex, involving many processes operating over different spatial and temporal scales, it is generally assumed that the stability and repeatability of development depends primarily upon accurate control over parameter values i.e. a precise genetic program. However, it is possible that the dynamic coupling between the different mechanisms involved in development reduces the choices available to the system because of bias in successive symmetry-breaking events. Evidence for such stable cascades in morphogenetic processes is described, and a general argument is presented for morphogenesis as an intrinsically robust process. The evolutionary implications of the hypothesis are considerable.

Regulation of a ribonucleoside reductase during the early generative phase in Acetabularia.

The activity of a ribonucleoside reductase was estimated during the life cycle of Acetabularia. During the early generative phase the enzyme activity was dramatically increased. Regulation of the ribonucleoside reductase was observed even in the absence of the nucleus. The increase in activity was inhibited by chloramphenicol but not by cycloheximide. These results indicate that the enzyme is translated on 70 S ribosomes.

[Mechanical and chemical patterns in the completion of isolated cell division]. / Mekhanicheskie i khimicheskie zakonomernosti zaversheniia deleniia izolirovannoi kletki.

Bicellular complex of a divided oocyte of the mollusc Lymnea stagnalis is chosen as a "testing machine" for studying mechanical and biochemical properties of the membranes of interblastomere wall. The membrane material is described by a standard rheological body. Rheological and kinetic problems are considered. Their solution allows an experimental solution of elastic, viscose and relaxation constants and chemical-mechanical parameter of the balance of the membrane synthesis and its deformations. A relationship (of the logistic type) between the growth of the wall area and time is found.

Cable properties and compartmentation in Acetabularia.

The electrical cable properties of three different compartmentation types of Acetabularia cells have been investigated. These three types were: normal cells, 'stumps' (filled with cytoplasm, no central vacuole) and 'tubes' (cytoplasm depleted vacuoles). The latter two types have been obtained by centrifugation of normal cells. Qualitatively, the characteristic biphasic voltage response upon rectangular current pulses is the same in these three types. Quantitatively, however, the two conductances which can be obtained from the biphasic voltage response as well as the apparent capacity of several F . m-2 which derives from the large time constant of the second phase, are drastically increased in stumps and decreased in tubes compared to normal cells. The resting potential is a few mV more negative in stumps, and more positive in tubes, than in normal cells. Based on the existence of the high resting potential and the apparent large capacity in the non-vacuolated stumps, it is concluded that the electrogenic Cl- pump of Acetabularia is located in the plasmalemma membrane and that the apparent large capacity is not a result of the complicated membraneous organisation of the vacuolar system. Several possibilities are discussed, in relation to the quantitative correlation between intracellular compartmentation and electrical membrane parameters.

Feulgen positive nucleoli in Epon semithin sections: fact or artifact?

Epon semithin sections from glutaraldehyde fixed tissues show stained nucleoli after treatment with the Schiff's reagent. The possibility that aldehyde groups already present could account for this positive staining reaction is briefly discussed.

[ On the absence of high-molecular polyphosphates in chloroplasts of Acetabularia mediterranea]. / Ob otsutstivii vysokomolekuliarnykh polifosfatov v khloroplastakh Acetabularia mediterranea.

High-molecular polyphosphates have been identified in a crude fraction of chloroplasts of Acetabularia mediterranea. However, after a short-term treatment of the fraction with a hypotonic salt solution and centrifugation in a sucrose density gradient it was found possible to completely separate high-molecular polyphosphates from intact chloroplasts. Consequently the chloroplasts themselves contain no high-molecular polyphosphates. It is assumed that the high-molecular polysphates found in a crude fraction of chloroplasts are constitutents of the "metachromatic" granules which can be revealed in the A. mediterranea cytoplasm by cytochemical methods.

[Deformation and morphogenesis of Acetabularia cell membrane. I. Relation of deformation to genetic and geometric factors]. / Deformatsii i morfogenez kletochnoi obolochki atsetabuliarii. I. Sviaz' deformatsii s geneticheskimi i geometricheskimi faktorami

Relationships between the changes of geometric shape, volume and deformations of the apical growth part of acetabularia cell are studied. Jung module E approximately 25 kg/cm2, viscosity coefficient of the apex cell theta 4 - 10(4) kg-sec/cm2 and intracellular pressure P approximately 1 kg/cm2 are measured. Cell shape formation according to Waddington is concerned with simple physical factors which are under the genetic control. It is shown that uneven deformations with the loss of shape stability bring about periodic cell complications.

[Deformation and morphogenesis of Acetabularia cell membrane. II. Local mechanisms of regulating viscoelastic deformations]. / Deformatsii i morfogenez kletochnoi obolochki atsetabuliarii. II. Lokal'nye mekhanizmy upravleniia viazkouprugimi deformatsiiami

Relationships between growth velocities of some parts of Acetabularia cell, ratio between the thickness of the cell membrane and apex diameter (its constructive rigidity), intracellular pressure and viscose-elastic deformations are used for physical substantiation of Waddington's concepts concerned with the existence and important search of local morphogenetic mechanisms. Their realization in a strongly deformated regions of Acetabularia cell membrane is shown. The relationship between viscosity, synthesis of the material and dimensions of region and intracellular pressure is found.

[Species specificity of morphogenetic factors of Acetabularia, localized in the cytoplasmic zone adjacent to the cell membrane]. / Vidospetsifichnost' morfogeneticheskikh faktorov Acetabularia, lokalizovannykh v prilegaiushchei k kletochnoi membrane zone tsitoplazmy

The species specificity of the factors controlling the cap development was established in the experiments with the transplantation of both the intact and centrifuged in the basal direction apical regions of Acetabularia meditteranea on nuclear basal regions of A. crenulata. These factors are found at the stage of 72 hrs of regeneration primarily in the cytoplasmic zone adjacent to the cell membrane which is not displaced during centrifugation. Using direct measurements and radiochemical method, we have shown that the accumulation of proteins proceeded in this zone due, mainly, to their transition from the cytoplasmic zone displaced during centrifugation.

The August Krogh Principle: "For many problems there is an animal on which it can be most conveniently studied".

The idea stated in the title, first clearly expressed by August Krogh, is illustrated by examples from animal biochemistry, physiology, general cell biology, experimental medicine, ethology and botany. General aspects of the concept are discussed.