Transcriptional activation by the sexual pheromone and wounding: a new gene family from Volvox encoding modular proteins with (hydroxy)proline-rich and metalloproteinase homology domains

The green alga Volvox represents the simplest kind of multicellular organism: it is composed of only two cell types, somatic and reproductive, making it suitable as a model system. The sexual development of males and females of Volvox carteri is triggered by a sex-inducing pheromone at a concentration of < 10-16 M. Early biochemical responses to the pheromone involve structural modifications within the extracellular matrix (ECM). By differential screenings of cDNA libraries made from mRNAs of pheromone-treated Volvox, four novel genes were identified that encode four closely related Volvox metalloproteinases that we use to define a new protein family, the VMPs. The existence of several features common to matrix glycoproteins, such as signal peptides, a (hydroxy)proline content of 12-25%, and Ser(Pro)2-4 repeats, suggest an extracellular localization of the VMPs within the ECM. Synthesis of VMP cDNAs is triggered not only by the sex-inducing pheromone, but also by wounding, and is restricted to the somatic cell type. Sequence comparisons suggest that the VMPs are members of the MB clan of zinc-dependent matrix metalloproteinases, although the putative zinc binding site of all VMPs is QEXXHXXGXXH rather than HEXXHXXGXXH. The presence of glutamine instead of histidine in the zinc binding motif suggests a novel family, or even clan, of peptidases. Like the matrixin family of human collagenases, Volvox VMPs exhibit a modular structure: they possess a metalloproteinase homology domain and a (hydroxy)proline-rich domain, and one of them, VMP4, also has two additional domains. Metalloproteinases seem to be crucial for biochemical modifications of the ECM during development or after wounding in the lower eukaryote Volvox with only two cell types, just as in higher organisms.     

Targeted migration of pherophorin‐S indicates extensive extracellular matrix dynamics in Volvox carteri

Hydroxyproline‐rich glycoproteins (HRGPs) constitute a major group of proteins of the extracellular matrix (ECM). The multicellular green alga Volvox carteri is a suitable model organism in which to study the evolutionary transition to multicellularity, including the basic principles and characteristics of an ECM. In Volvox, the ECM is dominated by a single HRGP family: the pherophorins. Our inventory amounts to 117 pherophorin‐related genes in V. carteri. We focused on a pherophorin with an unexpected characteristic: pherophorin‐S is a soluble, non‐cross‐linked ECM protein. Using transformants expressing a YFP‐tagged pherophorin‐S we observed the synthesis and secretion of pherophorin‐S by somatic cells in vivo, and we then traced the protein during its conspicuous migration to the ECM around prehatching juveniles and its localized concentration there. Our results provide insights into how an ECM zone surrounding the progeny is remotely affected by distantly located parental somatic cells. In view of the properties and migration of pherophorin‐S, we conclude that pherophorin‐S is likely to act as an ECM plasticizer to allow for dynamic ECM remodeling.     

Protein synthesis in a new system for the study of senescence

In asexual individuals of the green alga Volvox carteri, more than 99% of the cells are somatic cells which undergo synchronous programmed senescence and cell death every generation. Only a small number of reproductive cells survive to produce the next generation. The specific activity of pulse-labelled somatic cell protein preparations declines sharply during senescence, but no decline is seen in the nonageing reproductive cells. Two-dimensional polyacrylamide gel electrophoresis reveals that somatic and reproductive cells synthesize very different patterns of polypeptides. During the period when observable senescent changes are first evident in somatic cells, there is a change in the pattern of polypeptides being synthesized. Our results suggest that senescence in Volvox somatic cells is triggered by a change in the pattern of gene expression and are consistent with theories of programmed cell senescence.     

Volvox carteri as a model for studying the genetic and cytological control of morphogenesis

The green alga Volvox carteri has a very simple and regular adult form that arises through a short sequence of well-defined morphogenetic steps. A mature gonidium (asexual reproductive cell) initiates a stereotyped sequence of rapid cleavage divisions that will produce all of the cells found later in an adult. A predictable subset of these divisions are asymmetric and result in production of a small set of germ cells in a precise spatial pattern. Throughout cleavage, all intracellular components are held in predictable spatial relationships by a cytoskeleton of unusually regular structure, while neighboring cells are also held in fixed spatial relationships by an extensive network of cytoplasmic bridges that form as a result of incomplete cytokinesis. As a result of these two orienting mechanisms combined, dividing cells are arranged around the anterior-posterior axis of the embryo with precise rotational symmetry. These relationships are maintained by the cytoplasmic bridge system when the embryo that was inside out at the end of cleavage turns right-side out in the gastrulation-like process of inversion. Inversion is driven by a cytoskeleton-mediated sequence of cell shape changes, cellular movements and coordinated contraction. Then, by the time the cytoplasmic bridges begin to break down shortly after inversion, a preliminary framework of extracellular matrix (ECM) has been formed. The ECM traps the cells and holds them in the rotational relationships that were established during cleavage, and that must be maintained in order for the adult to be able to swim. Transposon tagging is now being used to clone and characterize the genes regulating these morphogenetic processes.     

Evidence for autocatalytic cross-linking of hydroxyproline-rich glycoproteins during extracellular matrix assembly in Volvox

The alga Volvox carteri is one of the simplest multicellular organisms, yet it has a surprisingly complex extracellular matrix (ECM), making Volvox suitable as a model system in which to study ECM self-assembly. Here, we analyze the primary structures and post-translational modifications of two main ECM components synthesized in response to sexual induction as well as wounding. These proteins are members of the pherophorin family with as yet unknown properties. They contain polyhydroxyproline spacers as long as 500 and 2750 residues. Even the highly purified proteins retain the capacity to self-assemble and cross-link, producing an insoluble fibrous network in an apparently autocatalytic reaction. This pherophorin-based network is located within the deep zone of the ECM. A molecular genetic search for additional members of the pherophorin family indicates that at least nine different pherophorin species can be expected to serve as precursors for ECM substructures. Therefore, the highly diversified members of the pherophorin family represent region-specific morphological building blocks for ECM assembly and cross-linking.     

Cyclic AMP as an intraspheroidal differentiation signal in Volvox carteri

The action of the macromolecular inducer glycoprotein on sexual reproduction in the green alga Volvox carteri can be modified by altering the external (intraspheroidal) cAMP concentration. Direct proof for the presence of cAMP in the spheroids is given. Protein binding assay and HPLC-fluorimetric analysis independently demonstrate the existence of cAMP in the matrix, cells, and culture medium. Its concentration is higher in sexual cultures, pointing to a transmitting function in sex induction. The presence in the matrix of other members of a protein phosphorylation system suggests an induction-specific signal cascade in this plant.     

Self-assembly and cross-linking of Volvox extracellular matrix glycoproteins are specifically inhibited by Ellman's reagent.

A major impediment to the biochemical characterization of extracellular matrices from algae (as well as higher plants) is the extensive covalent cross-linking that exists in the matrix, rendering most components insoluble and resistant to conventional extraction procedures. In the multicellular green alga Volvox, biogenesis of the extracellular matrix (ECM) is initiated immediately after the process of embryonic inversion. At this stage of development, the sulfhydryl reagent 5, 5'-dithio-bis(2-nitrobenzoic acid), known as Ellman's reagent, interferes in a highly specific manner with ECM biogenesis. Treated post-inversion embryos are no longer able to assemble an intact ECM and consequently dissociate into a suspension of single cells. Dissociated cells remain viable and continue to secrete ECM proteins into the growth medium, as documented by the identification of several members of the pherophorin family. Cross-linked ECM polymers such as sulfated surface glycoprotein 185 remain in a soluble state. Thus, treatment with Ellman's reagent opens a simple approach for the isolation and characterization of otherwise inaccessible monomeric precursors.     

Two light-activated conductances in the eye of the green alga Volvox carteri

Photoreceptor currents of the multicellular green alga Volvox carteri were analyzed using a dissolver mutant. The photocurrents are restricted to the eyespot region of somatic cells. Photocurrents are detectable from intact cells and excised eyes. The rhodopsin action spectrum suggests that the currents are induced by Volvox rhodopsin. Flash-induced photocurrents are a composition of a fast Ca2+-carried current (PF) and a slower current (PS), which is carried by H+. PF is a high-intensity response that appears with a delay of less than 50 micros after flash. The stimulus-response curve of its initial rise is fit by a single exponential and parallels the rhodopsin bleaching. These two observations suggest that the responsible channel is closely connected to the rhodopsin, both forming a tight complex. At low flash energies PS is dominating. The current delay increases up to 10 ms, and the PS amplitude saturates when only a few percent of the rhodopsin is bleached. The data are in favor of a second signaling system, which includes a signal transducer mediating between rhodopsin and the channel. We present a model of how different modes of signal transduction are accomplished in this alga under different light conditions.     

Genes specifically expressed in sexually differentiated female spheroids of Volvox carteri

Volvox carteri is a multicellular green alga with only two cell types, somatic cells and reproductive cells. Phylogenetic analysis suggests that this organism has evolved from a Chlamydomonas-like unicellular ancestor along with multicellularity, cellular differentiation, and a change in the mode of sexual reproduction from isogamy to oogamy. To examine the mechanism of sexual differentiation and the evolution of oogamy, we isolated 6 different cDNA sequences specifically expressed in sexually differentiated female spheroids. The genes for the cDNAs were designated SEF1 to SEF6. The time course of accumulation of each mRNA was shown to be distinct. The expression of some of these genes was not significantly affected when the sexual inducer was removed after the induction of sexual development. Sequence analysis indicates that SEF5 and SEF6 encode pherophorin-related proteins. Of these, SEF5 has the unique structural feature of a polyproline stretch in the C-terminal domain in addition to the one found in the central region.     

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.     

Characterization of a heat-shock-inducible hsp70 gene of the green alga Volvox carteri

The green alga Volvox carteri possesses several thousand cells, but just two cell types: large reproductive cells called gonidia, and small, biflagellate somatic cells. Gonidia are derived from large precursor cells that are created during embryogenesis by asymmetric cell divisions. The J domain protein GlsA (Gonidialess A) is required for these asymmetric divisions and is believed to function with an Hsp70 partner. As a first step toward identifying this partner, we cloned and characterized V. carteri hsp70A, which is orthologous to HSP70A of the related alga Chlamydomonas reinhardtii. Like HSP70A, V. carteri hsp70A contains multiple heat shock elements (HSEs) and is highly inducible by heat shock. Consistent with these properties, Volvox transformants that harbor a glsA antisense transgene that is driven by an hsp70A promoter fragment express Gls phenotypes that are temperature-dependent. hsp70A appears to be the only gene in the genome that encodes a cytoplasmic Hsp70, so we conclude that Hsp70A is clearly the best candidate to be the chaperone that participates with GlsA in asymmetric cell division.     

Environmentally induced responses co-opted for reproductive altruism

Reproductive altruism is an extreme form of altruism best typified by sterile castes in social insects and somatic cells in multicellular organisms. Although reproductive altruism is central to the evolution of multicellularity and eusociality, the mechanistic basis for the evolution of this behaviour is yet to be deciphered. Here, we report that the gene responsible for the permanent suppression of reproduction in the somatic cells of the multicellular green alga, Volvox carteri, evolved from a gene that in its unicellular relative, Chlamydomonas reinhardtii, is part of the general acclimation response to various environmental stress factors, which includes the temporary suppression of reproduction. Furthermore, we propose a model for the evolution of soma, in which by simulating the acclimation signal (i.e. a change in cellular redox status) in a developmental rather than environmental context, responses beneficial to a unicellular individual can be co-opted into an altruistic behaviour at the group level. The co-option of environmentally induced responses for reproductive altruism can contribute to the stability of this behaviour, as the loss of such responses would be costly for the individual. This hypothesis also predicts that temporally varying environments, which will select for more efficient acclimation responses, are likely to be more conducive to the evolution of reproductive altruism.     

The pherophorins: common, versatile building blocks in the evolution of extracellular matrix architecture in Volvocales

Green algae of the order Volvocales provide an unrivalled opportunity for exploring the transition from unicellularity to multicellularity. They range from unicells, like Chlamydomonas, through homocytic colonial forms with increasing cooperation of individual cells, like Gonium or Pandorina, to heterocytic multicellular forms with different cell types and a complete division of labour, like Volvox. A fundamental requirement for the evolution of multicellularity is the development of a complex, multifunctional extracellular matrix (ECM). The ECM has many functions, which can change under developmental control or as a result of environmental factors. Here molecular data from 15 novel proteins are presented. These proteins have been identified in Chlamydomonas reinhardtii, Gonium pectorale, Pandorina morum and Volvox carteri, and all belong to a single protein family, the pherophorins. Pherophorin-V1 is shown to be a glycoprotein localized to the 'cellular zone' of the V. carteri ECM. Pherophorin-V1 and -V2 mRNAs are strongly induced not only by the sex inducer, which triggers sexual development at extremely low concentrations, but also by mechanical wounding. Like the extensins of higher plants, which are also developmentally controlled or sometimes inducible by wounding, the pherophorins contain a (hydroxy-)proline-rich (HR) rod-like domain and are abundant within the extracellular compartment. In contrast to most extensins, pherophorins have additional globular A and B domains on both ends of the HR domains. Therefore pherophorins most closely resemble a particular class of higher plant extensin, the solanaceous lectins (e.g. potato lectin), suggesting multivalent carbohydrate-binding functions are present within the A and B domains and are responsible for cross-linking. Our results suggest that pherophorins are used as the building blocks for the extracellular scaffold throughout the Volvocales, with the characteristic mesh sizes in different ECM structures being a result of the highly diverse extensions of the HR domains. Pherophorins have therefore been a versatile element during the evolution of ECM architecture in these green algae.     

Sex as a response to oxidative stress: stress genes co-opted for sex

Despite a great deal of interest, the evolutionary origins and roles of sex remain unclear. Recently, we showed that in the multicellular green alga, Volvox carteri, sex is a response to increased levels of reactive oxygen species (ROS), which could be indicative of the ancestral role of sex as an adaptive response to stress-induced ROS. To provide additional support for the suggestion that sex evolved as a response to oxidative stress, this study addresses the hypothesis that genes involved in sexual induction are evolutionarily related to genes associated with various stress responses. In particular, this study investigates the evolutionary history of genes specific to the sexual induction process in V. carteri--including those encoding the sexual inducer (SI) and several SI-induced extracellular matrix (ECM) proteins. Surprisingly, (i) a highly diversified multigene family with similarity to the V. carteri SI and SI-induced pherophorin family is present in its unicellular relative, Chlamydomonas reinhardtii (which lacks both a SI and an ECM) and (ii) at least half of the 12 identified gene members are induced (as inferred from reported expressed sequence tags) under various stress conditions. These findings suggest an evolutionary connection between sex and stress at the gene level, via duplication and/or co-option.     

Germ-soma differentiation in volvox.

Volvox carteri is a spherical green alga with a predominantly asexual mode of reproduction and a complete germ-soma division of labor. Its somatic cells are specialized for motility, incapable of dividing, and programmed to die when only a few days old, whereas its gonidia (asexual reproductive cells) are nonmotile, specialized for growth and reproduction, and potentially immortal. When a gonidium is less than 2 days old it divides to produce a juvenile spheroid containing all of the somatic cells and gonidia that will be present in an adult of the next generation. The first visible step in germ-soma differentiation is a set of asymmetric cleavage divisions in the embryo that set apart small somatic initials from their large gonidial-initial sister cells. Three types of genes have been found to play key roles in germ-soma specification. First a set of gls genes act in the embryos to shift cell-division planes, resulting in the asymmetric divisions that set apart the large-small sister-cell pairs. Then a set of lag genes act in the large cells to prevent somatic differentiation, while the regA gene acts in the small cells to prevent reproductive development. An inducible transposon was used to tag and recover some of these and other developmentally important genes. The glsA gene encodes a chaperone-like protein that, like another chaperone that is one of its putative binding partners, is associated with the cell division apparatus, although how this leads to asymmetric division remains to be elucidated. The regA gene encodes a somatic-cell-specific nuclear protein that appears to function by repressing genes required for chloroplast biogenesis, thereby preventing somatic cells from growing enough to reproduce. Somatic-cell-specific expression of regA is controlled by three intronic enhancers.     

Differential targeting of closely related ECM glycoproteins: the pherophorin family from Volvox.

The alga Volvox carteri represents one of the simplest multicellular organisms. Its extracellular matrix (ECM) is modified under developmental control, e.g. under the influence of the sex-inducing pheromone that triggers development of males and females at a concentration below 10(-16) M. A novel ECM glycoprotein (pherophorin-S) synthesized in response to this pheromone was identified and characterized. Although being a typical member of the pherophorins, which are identified by a C-terminal domain with sequence homology to the sex-inducing pheromone, pherophorin-S exhibits a completely novel set of properties. In contrast to the other members of the family, which are found as part of the insoluble ECM structures of the cellular zone, pherophorin-S is targeted to the cell-free interior of the spherical organism and remains in a soluble state. A main structural difference is the presence of a polyhydroxyproline spacer in pherophorin-S that is linked to a saccharide containing a phosphodiester bridge between two arabinose residues. Sequence comparisons indicate that the self-assembling proteins that create the main parts of the complex Volvox ECM have evolved from a common ancestral gene.     

Role of the extracellular matrix in cell-cell signalling: paracrine paradigms

The plant extracellular matrix (ECM) is complex and diverse, and is involved in cell-cell communication in a wide range of developmental, reproductive and pathogenic processes. Characterisation of integral ECM components is leading to improved understanding of their roles in signalling. Interactions between the extracellular domains of plant plasma membrane receptor kinases and their ligands are potentially regulated by the properties of the ECM. Several of these interactions, for example those involving the S-locus receptor kinase, are being characterised in some detail. Non-protein constituents are also implicated in regulating the movement of signalling molecules in the ECM, which is associated with developmental patterning. In contrast to the situation in animal cells, cytoskeleton-integrin-ECM signalling complexes appear not to be dominant features of signal transduction in plant cells. Nevertheless, structural adhesions between the plasma membrane and cell wall are important for a variety of functions.