VCRPs,small cysteine-rich proteins,might be involved in extracellular signaling in the green alga Volvox

The sex-inducer of the spherical green alga Volvox carteri is one of the most potent biological effector molecules known: it is released into the medium by sexual males and triggers the switch to the sexual cleavage program in the reproductive cells of vegetatively grown males and females even at concentrations as low as 10^-16 M. In an adult Volvox alga, all cells are embedded in an extensive extracellular matrix (ECM), which constitutes >99% of the volume of the spheroid. There exist no cytoplasmic connections between the cells in an adult alga, so any signal transduction between different cells or from the organism''s environment to a reproductive cell must involve the ECM. Recently, a small cysteine-rich extracellular protein, VCRP, was identified in Volvox and shown to be quickly synthesized by somatic cells in response to the sex-inducer. Due to its characteristics, VCRP was speculated to be an extracellular second messenger from somatic cells to reproductive cells. Here a related protein, VCRP2, is presented, exhibiting a 56% amino acid sequence identity with VCRP. Two possible scenarios for signal transduction from the sex-inducer to the reproductive cell are discussed.Key words: cell wall, extracellular matrix, extracellular second messenger, green algae, sex-inducer, sex inducing pheromone, sexual development, stress response, Volvocaceae, wounding     

The Protoplasmic Connection in Volvox

SYNOPSIS. Electron-microscopic observations were performed on 2 species of Volvox , one similar to V. globator , the other to V. aureus. The former has distinct protoplasmic connections in the adult coenobium and specific structures, named "medial bodies," in the connections just at the intersection with the middle lamella. The medial body is disk shaped, about 800 mμ in diameter, and is composed of 3 parts, 2 dense outer layers and an intermediate less dense zone. In the latter species, the connection and medial body were not seen. On the other hand, it was commonly seen in both of them that in younger, dividing gonidia neighboring protoplasts were connected with each other by protoplasmic bridges. The bridges are undoubtedly formed due to incomplete cell separation in the division of a gonidium. The structural difference in the adult coen***bium between the 2 species emerges just after inversion of the coenobium. In the globator type the medial body appears just after inversion, and the connection remains unruptured all thru life. In the aureus type, it seems that the connections are withdrawn or degenerate immediately after inversion. It is discussed whether protoplasmic continuity is really maintained by the connection or not in the freeswimming coenobium of Volvox.     

Polyadenylated RNA of Volvox: isolation and partial characterization

Polyadenylated RNA from Volvox carteri has been isolated and partially characterized. Electrophoretic profiles of total cellular poly(A)-associated RNA of Volvox spheroids indicate a hetero-disperse distribution of size classes with the range extending from an apparent sedimentation value of approximately 10S to greater than 38S. The radioactive labelling kinetics of this material are typical for rapidly-turning-over RNA. The profiles of poly(A) RNA from different cell types show marked differences in average migration rate. Terminally-differentiated somatic cells contain a greater proportion of material of higher molecular weight than either gonidia (germ cells) or cleaving embryos. The poly(A) segments associated with cellular RNA, obtained by selective RNase digestion are heterogeneous in size as determined by gel electrophoresis with the largest tracts estimated to be 75-80 nucleotides long. Gonidia and embryos display the greatest degree of size heterogeneity, while somatic cells show predominantly the largest classes of poly(A) tract. It is apparent that gross changes in poly(A) RNA metabolism accompany development and cellular differentiation in Volvox.     

Ultrastructure of Volvox carteri

Summary Somatic cells of mature asexual colonies of Volvox carteri do not possess a true cell wall, but are otherwise similar in ultrastructure to Chlamydomonas. Somatic cells are embedded in multilayered fibrillar material of the colonial matrix. The reproductive cells (gonidia) of Volvox carteri lie internal to the somatic cell layer of the colony matrix in an apparently structureless portion of the colony matrix. Mature gonidia are large vacuolate cells with a central nucleus and parietal chloroplasts and mitochondria. They are non-flagellated at maturity, but each contains a pair of kinetosomes.     

The DNA of Volvox carteri: a biophysical and biosynthetic characterization.

Various biophysical and biosynthetic characteristics of deoxyribonucleic acid (DNA) from Volvox carteri are examined. The DNA from three strains (HK-10,NB-7 and KA-1) is compared, and all strains are shown to contain at least two distinct DNA species which band at densities of 1.714-1.715 and 1.704-1.705 g/cm3 in neutral CsCl and correspond to nuclear and "cytoplasmic" DNA, respectively. Base compositions calculated from these densities, 55-56% G+C for nuclear DNA, and 45-46% G+C for cytoplasmic DNA, are in close agreement with % G+C values estimated from thermal denaturation data. DNA from strain KA-1 has a third component with a buoyant density of 1.693 g/cm3. DNA synthesis is analysed using radioactively labelled heterogeneously grown strains of Volvox carteri and profiles obtained following preparative CsCl density gradient centrifugation are presented. In addition, dissimilarities in patterns of DNA synthesis at various periods in the asexual life cycle are reported for synchronous cultures of strain HK-10. These differences in temporal patterns of DNA synthesis clearly indicate that while nuclear DNA is make to some degree throughout the life cycle, cytoplasmic DNA synthesis appears to occur only at discrete intervals.     

Flagellar Activity of the Colony Members of Volvox aureus Ehrbg. during Light Stimulation*

SYNOPSIS. The flagellar behavior of the colonial Volvox aureus Ehrbg. was examined by placing 1.01 μ polystyrene particles in suspension with Volvox, and recording particle movement photomicrographically. When directional light stimulation was given, flagellar activity ceased in the anterior cells of the stimulated side. Such responses create unequal driving forces on the 2 sides of the colony, so that the colony turns toward the stimulated side. Dose response studies indicated a photoresponse gradient from front to rear in the colony, anterior cells being most responsive. The mechanism of gradient formation has yet to be determined.     

Morphogenesis in Volvox: analysis of critical variables.

Inversion, the process by which Volvox embryos turn inside out, was analyzed by a combination of geometrical and experimental techniques. It was shown that simple geometric figures are adequate to represent cell shapes during inversion and that cell volumes remain constant as cell shapes change and the embryo inverts. The first stage of inversion, phialopore opening, results from the release of compressive forces as the embryo withdraws from its surrounding vesicle during a two-stage contraction of each cell around its radial axis. Premature phialopore opening occurs when withdrawal of the embryo from the vesicle is elicited artificially by exposure to either calcium ionophore or hypertonic solutions. The major event of inversion, generation of negative curvature, requires both microtubule-driven elongation of cells (to produce a classical "flask" shape) and cytochalasin-sensitive active migration of cytoplasmic bridges to the outermost ends of flask cells. Colchicine, cyclic GMP and isobutyl methyl xanthine (individually) block both normal elongation and bridge migration; cytochalasin D blocks bridge migration selectively. Flask cell formation and bridge migration are adequate to account for the negative curvature observed. An asymmetric bending of flask cell stalks along the ring of maximum curvature accounts for the fact that the embryo is not constricted in a "purse-string" fashion as negative curvature is generated. Inversion of the posterior hemisphere involves an elastic snap-through resulting from a combination of compressive stresses generated by inversion of the anterior hemisphere and the circumferential restraint imposed by cells at the equator. We conclude that the observed changes in cell shape and the migration of cytoplasmic bridges are the result of an ordered process of membrane-cytoskeletal interactions, and both necessary and sufficient to account for the morphogenetic process of inversion in Volvox.     

Genetic control of somatic cell differentiation in Volvox analysis of somatic regenerator mutants

The somatic regenerator (reg) mutants of Volvox carteri affect the ability of the normally terminally differentiated somatic cells to establish and/or maintain the differentiated state. Thirty-nine reg mutants of four phenotypic classes have been mapped to two, unlinked genes, regA and regB. Mutants at the regA locus have one of three phenotypes: All somatic cells regenerate new spheroids, somatic cells in the spheroid posterior region regenerate while those in the anterior region differentiate as somatic cells, or regenerating and nonregenerating cells are randomly intermixed. The regB mutant has a random intermixture of regenerating and nonregenerating cells. Somatic cells regenerate new Volvox spheroids in two ways; the cells lose their characteristic shape, become immotile, enlarge and undergo cleavage similar to that of normal reproductive cells or undergo cell division without prior enlargement or loss of cell shape. Temperature shift experiments on a cold-sensitive reg mutant suggest that the gene product acts after the somatic cell initials are formed at the end of cleavage.     

Do Protoplasmic Connections Function in the Phototactic Coordination of the Volvox Colony During Light Stimulation?*

SYNOPSIS. The flagellar behavior of the colonial flagellates Volvox carteri Stein and Volvox perglobator Powers was examined by placing 1.01 μm polystyrene particles in solution with swimming colonies, and photographing these particle movements. When directional light stimulation was administered to individual colonies, a cessation of flagellar activity occurred in the anterior cells of the stimulated side in both species. Since Volvox perglobator possesses prominent intercellular connections and Volvox carteri does not, the results of these experiments suggest that the connections linking colony members in some species do not function in the coordination of flagellar activity associated with light orientation behavior.     

Photosynthetic characteristics of a multicellular green alga Volvox carteri in response to external CO2 levels possibly regulated by CCM1/CIA5 ortholog

When CO(2) supply is limited, aquatic photosynthetic organisms induce a CO(2)-concentrating mechanism (CCM) and acclimate to the CO(2)-limiting environment. Although the CCM is well studied in unicellular green algae such as Chlamydomonas reinhardtii, physiological aspects of the CCM and its associated genes in multicellular algae are poorly understood. In this study, by measuring photosynthetic affinity for CO(2), we present physiological data in support of a CCM in a multicellular green alga, Volvox carteri. The low-CO(2)-grown Volvox cells showed much higher affinity for inorganic carbon compared with high-CO(2)-grown cells. Addition of ethoxyzolamide, a membrane-permeable carbonic anhydrase inhibitor, to the culture remarkably reduced the photosynthetic affinity of low-CO(2) grown Volvox cells, indicating that an intracellular carbonic anhydrase contributed to the Volvox CCM. We also isolated a gene encoding a protein orthologous to CCM1/CIA5, a master regulator of the CCM in Chlamydomonas, from Volvox carteri. Volvox CCM1 encoded a protein with 701 amino acid residues showing 51.1% sequence identity with Chlamydomonas CCM1. Comparison of Volvox and Chlamydomonas CCM1 revealed a highly conserved N-terminal region containing zinc-binding amino acid residues, putative nuclear localization and export signals, and a C-terminal region containing a putative LXXLL protein-protein interaction motif. Based on these results, we discuss the physiological and genetic aspects of the CCM in Chlamydomonas and Volvox.     

Genetic engineering of the multicellular green alga Volvox: a modified and multiplied bacterial antibiotic resistance gene as a dominant selectable marker

The green alga Volvox represents the simplest multicellular organism: Volvax is composed of only two cell types, somatic and reproductive. Volvox, therefore, is an attractive model system for studying various aspects of multicellularity. With the biolistic nuclear transformation of Volvox carteri, the powerful molecular genetic manipulation of this organism has been established, but applications have been restricted to an auxotrophic mutant serving as the DNA recipient. Therefore, a dominant selectable marker working in all strains and mutants of this organism is required. Among several gene constructs tested, the most advantageous results were obtained with a chimeric gene composed of the coding sequence of the bacterial ble gene, conferring resistance to the antibiotic zeocin, modified with insertions of two endogenous introns from the Volvox arylsulfatase gene and fused to 5' and 3' untranslated regions from the Volvox beta 2-tubulin gene. In the most suitable plasmid used, the gene dosage was increased 16-fold by a technique that allows exponential multiplication of a DNA fragment. Co-transformation of this plasmid and a non-selectable plasmid allowed the identification of zeocin resistant transformants with nuclear integration of both selectable and non-selectable plasmids. Stable expression of the ble gene and of genes from several non-selectable plasmids is demonstrated. The modified ble gene provides the first dominant marker for transformation of both wild-type and mutant strains of Volvox.     

Cell shape changes and the mechanism of inversion in Volvox

Inversion is a dominant aspect of morphogenesis in Volvox. In this process, the hollow, spheroidal Volvox embryo turns inside-out through a small opening called the phialopore to bring flagella from its inner to its outer surface. Analyses of intact, sectioned, and fragmented embryos by light, scanning electron, and transmission electron microscopy, suggest that shape changes preprogrammed into the cells cause inversion. First, cells throughout the embryo change from pear to spindle shape, which causes the embryo to contract and the phialopore to open. Then cells adjacent to the phialopore become flask-shaped, with long, thin stalks at their outer ends. Simultaneously, the cytoplasmic bridges joining all adjacent cells migrate from the midpoint of the cells to the stalk tips. Together, these changes cause the lips of cells at the phialopore margin to curl outward. Now cells progressively more distal to the phialopore become flask-shaped while the more proximal cells become columnar, causing the lips to curl progressively further over the surface of the embryo until the latter has turned completely inside-out. Fine structural analysis reveals a peripheral cytoskeleton of microtubules that is apparently involved in cellular elongation. Cell clusters isolated before inversion undergo a similar program of shape changes; this suggests that the changes in cellular shape are the cause rather than an effect of the inversion process.     

Spontaneous frequency of a developmental mutant in Volvox

Two types of mutants, those resistant to the base analog 5-bromo-2′-deoxyuridine (BrdU) and somatic regenerator (SR) mutants, have been analyzed in Volvox carteri. In somatic regenerator mutants, the somatic cells which are normally terminally differentiated dedifferentiate and regenerate gonidia [Sessoms, A., and Huskey, R. J. (1973). Proc. Nat. Acad. Sci. USA70, 1335–1338; Starr, R. C. (1970). Develop. Biol. Suppl.4, 59–100]. The SR phenotype allows recovery of SR mutations arising in somatic cells, since such somatic cells would regenerate gonidia and give rise to mutant clones. Mutants of any phenotype other than SR can only be recovered if the mutation first appears in a gonidium. Since the somatic cells are 100-fold more numerous than reproductive cells (gonidia), we have determined the spontaneous frequency of both somatic regenerator mutants and mutations to BrdU resistance in order to determine if the SR mutation exerts its effect in the gonidium or in the somatic cell. The two frequencies were found to be nearly identical, suggesting that the SR mutation must first appear in a gonidium in order to be expressed.     

The problem of germ layers in sponges (Porifera) and some issues concerning early metazoan evolution

Nowadays the formation of germ layers (endoderm and mesoderm) is associated with gastrulation. The question of whether the cell movements during early embryonic development in sponges (Porifera) are gastrulation as in eumetazoans remains in dispute. Recent data on the histological organization, digestion and embryonic morphogenesis in sponges are analyzed here in an attempt to answer this question. Unique features of these basal Metazoa are the lack of intestinal epithelium, digestive parenchyma or any cell population specialized in digestion. Food particles are captured by cells of almost all types. These data show that sponges have no embryonic layers such as ectoderm or endoderm, characteristic to eumetazoans, and, consequently, no gastrulation. We make an assumption that the formation of germ layers cannot be considered as a recapitulation of events that took place in the common ancestor of Porifera and Eumetazoa. The unity of Metazoa is expressed not in the presence of gastrulation processes per se, but in the universal nature of cell movement mechanisms ensuring various types of morphogenesis, including those underlying gastrulation. It is concluded that metazoan mechanisms of morphogenetic movements must have emerged in the course of evolution prior to the separation of the germ layers like endoderm and ectoderm.     

Synthesis and possible role of proteoglycans during Volvox development

During the embryonic development of Volvox the cells synthesize proteoglycans of relatively small molecular size up to 14 h after inversion. In the following stages there is a gradual transition towards larger forms, due to an increase in sulfated sugar moieties. The presence of sulfate in the medium was obligatory for this transition. None of these molecules could be dissociated in 4.0 M GuHCl, neither could they be digested by glycosidases. Of all proteases examined subtilisin, a bacterial protease, digested the proteoglycans. The possible role of proteoglycans during Volvox morphogenesis has been examined. Somatic proteoglycans caused spheroids to start their swimming earlier, although they inhibited their expansion. Spheroids cultured in sulfate-free medium start their swimming 24 h later than controls, but the addition of somatic proteoglycans to the sulfate-free medium could induce motility almost as well as in the normal forms. Embryonic weight proteoglycans caused aggregation of the spheroids, an effect that also appeared in the sulfate-free medium.     

The relationship between cell size and cell fate in Volvox carteri

In Volvox carteri development, visibly asymmetric cleavage divisions set apart large embryonic cells that will become asexual reproductive cells (gonidia) from smaller cells that will produce terminally differentiated somatic cells. Three mechanisms have been proposed to explain how asymmetric division leads to cell specification in Volvox: (a) by a direct effect of cell size (or a property derived from it) on cell specification, (b) by segregation of a cytoplasmic factor resembling germ plasm into large cells, and (c) by a combined effect of differences in cytoplasmic quality and cytoplasmic quantity. In this study a variety of V. carteri embryos with genetically and experimentally altered patterns of development were examined in an attempt to distinguish among these hypotheses. No evidence was found for regionally specialized cytoplasm that is essential for gonidial specification. In all cases studied, cells with a diameter > approximately 8 microns at the end of cleavage--no matter where or how these cells had been produced in the embryo--developed as gonidia. Instructive observations in this regard were obtained by three different experimental interventions. (a) When heat shock was used to interrupt cleavage prematurely, so that presumptive somatic cells were left much larger than they normally would be at the end of cleavage, most cells differentiated as gonidia. This result was obtained both with wild-type embryos that had already divided asymmetrically (and should have segregated any cytoplasmic determinants involved in cell specification) and with embryos of a mutant that normally produces only somatic cells. (b) When individual wild-type blastomeres were isolated at the 16-cell stage, both the anterior blastomeres that normally produce two gonidia each and the posterior blastomeres that normally produce no gonidia underwent modified cleavage patterns and each produced an average of one large cell that developed as a gonidium. (c) When large cells were created microsurgically in a region of the embryo that normally makes only somatic cells, these large cells became gonidia. These data argue strongly for a central role of cell size in germ/soma specification in Volvox carteri, but leave open the question of how differences in cell size are actually transduced into differences in gene expression.     

Reproductive cell specification during Volvox obversus development

Asexual spheroids of the genus Volvox contain only two cell types: flagellated somatic cells and immotile asexual reproductive cells known as gonidia. During each round of embryogenesis in Volvox obversus, eight large gonidial precursors are produced at the anterior extremity of the embryo. These cells arise as a consequence of polarized, asymmetric divisions of the anteriormost blastomeres at the fourth through nine cleavage cycles, while all other blastomeres cleave symmetrically to yield somatic cell precursors. Blastomeres isolated from embryos at any point between the 2-cell and the 32-cell stage cleaved in the normal pattern and produced the same complement and spatial distribution of cell types as they would have in an intact embryo. This result indicates that intrinsic features control the cleavage patterns and developmental potentials of blastomeres, and rules out any significant role for cell-cell interactions in gonidial specification. When substantial quantities of anterolateral cytoplasm were deleted from uncleaved gonidia or 4-cell stage blastomeres, the cell fragments frequently regulated and embryos were produced with the expected number of asymmetrically cleaving cells and gonidial precursors at their anterior ends. However, when anterior cytoplasm was deleted from 8-cell stage blastomeres, the depleted cells frequently failed to cleave asymmetrically and produced no gonidial precursors. Furthermore, when compression was used to reorient cleavage planes at the fourth division cycle, so that anterior cytoplasm was transmitted to more than the normal number of cells, those cells receiving a significant amount of such cytoplasm cleaved asymmetrically to produce supernumerary gonidial precursors. Together, these last two experiments indicate that blastomeres in the V. obversus embryo acquire (at least by the end of the third cleavage cycle) a polarized organization in which anterior cytoplasm plays a causal role in the process of reproductive-cell specification.     

A posttranslationally regulated protease, VheA, is involved in the liberation of juveniles from parental spheroids in Volvox carteri

The lineage of volvocine algae includes unicellular Chlamydomonas and multicellular Volvox in addition to their colonial relatives intermediate in size and cell number. In an asexual life cycle, daughter cells of Chlamydomonas hatch from parental cell walls soon after cell division, while Volvox juveniles are released from parental spheroids after the completion of various developmental events required for the survival of multicellular juveniles. Thus, heterochronic change in the timing of hatching is considered to have played an important role in the evolution of multicellularity in volvocine algae. To study the hatching process in Volvox carteri, we purified a 125-kD Volvox hatching enzyme (VheA) from a culture medium with enzymatic activity to degrade the parental spheroids. The coding region of vheA contains a prodomain with a transmembrane segment, a subtilisin-like Ser protease domain, and a functionally unknown domain, although purified 125-kD VheA does not contain a prodomain. While 143-kD VheA with a prodomain is synthesized long before the hatching stage, 125-kD VheA is released into the culture medium during hatching due to cleavage processing at the site between the prodomain and the subtilisin-like Ser protease domain, indicating that posttranslational regulation is involved in the determination of the timing of hatching.