Association of centrioles with the marginal band of a molluscan erythrocyte

Continuous circumferential bundles of microtubules, or marginal bands (MBs), are best known as a prominent structural feature of all nonmammalian vertebrate erythrocytes and mammalian blood platelets. Since their discovery in the late 19th century, MBs have been thought to play a cellular morphogenetic role, but no cytological clues to the mechanism of MB biogenesis have been reported. In previous work we have established the presence of MBs in serveral invertebrate blood cell types, including amebocytes and coelomocytes of certain Arthropod species and erythrocytes of a Sipunculan. We report here the occurrence of MBs in erythrocytes of the ark Anadara transversa (Mollusca) and four closely related species. The MBs of these arks have a striking structural feature; each is physically associated with a pair of centrioles. The centrioles are identified as such on the basis of morphological criteria: size, cylindrical shape, right-angle orientation, pairing, and 9-triplet ultrastructure. This intimate association between centrioles and MBs suggests that centrioles may be MB-organizing centers and invites comparative investigation of their possible role in vertebrate erythrocyte and platelet morphogenesis.     

The cytoskeletal system of mammalian primitive erythrocytes: studies in developing marsupials

Seeking to resolve conflicting literature on cytoskeletal structure in mammalian "primitive" generation erythrocytes, we have utilized the circulating blood of developing marsupials. In young of the Tammar Wallaby (Macropus eugenii) and the Gray Short-tailed Opossum (Monodelphis domestica), relatively large, nucleated primitive erythrocytes constituted nearly 100% of the circulating population at birth (= day 0) and in fetuses (Tammar) several days before birth. These cells were discoidal or elliptical, and flattened except for a nuclear bulge. Their cytoskeletal system, consisting of a marginal band of microtubules enclosed within a cell surface-associated network (membrane skeleton), closely resembled that of non-mammalian vertebrate erythrocytes. By day 2 or 3, much smaller anucleate erythrocytes of "definitive" morphology, lacking marginal bands, appeared in abundance. These accounted for greater than 90% of the circulating population of both species by day 6-8. Non-nucleated erythrocytes of a different type, constituting 1-6% of the cells in most blood samples up to day 7, were identified as anucleate primitives on the basis of size, shape, and presence of a marginal band. Thus, loss of erythrocyte nuclei in mammals appears to begin earlier than generally recognized, i.e., in the primitive generation. Counts of these anucleate primitives in young of various ages implicated nucleated primitives as their probable source. Pointed erythrocytes, occasionally found in younger neonates of both species, occurred in greatest number in fetuses (Tammar) prior to birth. This is in accord with previous work on non-mammalian vertebrates suggesting that such cells are morphogenetic intermediates. The results confirm the long-suspected similarity between mammalian primitive erythrocytes and the nucleated erythrocytes of all non-mammalian vertebrates.     

Lethal and non-lethal responses of spermatozoa from a wide variety of vertebrates and invertebrates to lysenin, a protein from the coelomic fluid of the earthworm Eisenia foetida

Lysenin, a novel protein that we isolated from the coelomic fluid of the earthworm Eisenia foetida, binds specifically to sphingomyelin (SM) among various phospholipids found in cell membranes, and causes cytolysis. The plasma membrane of mammalian spermatozoa is known to contain SM at relatively high levels and we therefore examined the effects of lysenin on the spermatozoa of various animals. Lysenin had lethal effects on spermatozoa of 5 of 33 species of invertebrates tested and on spermatozoa of 30 of 39 species of vertebrates. We postulated that plasma membranes of the spermatozoa of most invertebrates might not contain SM whereas those of most vertebrate species might contain SM. These possibilities were supported by our failure to detect SM chemically in the testes of three species of invertebrates, in none of which spermatozoa responded to lysenin. In contrast, we detected SM in the testes of all 25 vertebrate species examined, irrespective of a negative or positive response of spermatozoa to lysenin. None of the six species of Protista examined was affected by lysenin. Our survey suggests that, in general, the spermatozoa of animals can be grouped into two categories, invertebrate and vertebrate, depending on the absence or presence of SM in their plasma membrane. The incorporation of SM into spermatozoa seems first to have occurred in protochordates during the course of evolution. Discussions about the exceptional responses to lysenin observed in the spermatozoa of five species of invertebrates and of nine species of vertebrates are made from phylogenetic and reproductive viewpoints. J. Exp. Zool. 286:538-549, 2000.     

On erythrocyte morphology.

The significance of the distinctive morphological difference between mammalian and non-mammalian vertebrate erythrocytes (disc versus ellipse) is discussed in the light of mammalian erythrocyte rheology.     

Rosette formation by insect macrophages inhibition by cytochalasin B.

Macrophages from the insect Spodoptera eridania possess membrane receptors for unmodified avian and mammalian erythrocytes, with which they form spontaneous rosettes. Rosette formation occurs in the absence of serum proteins and divalent cations. Individual macrophages bear receptors for several types of red cells. The level of naturally-occurring hemagglutinins against a particular test erythrocyte is not correlated with macrophage reactivity against that red cell. In contrast with mammalian macrophages, neuraminidase treatment of either hemocytes or erythrocytes does not cause a marked enhancement of binding. Pretreatment of macrophages or erythrocytes with cytochalasin B causes reversible inhibition of resetting probably by interfering with normal microfilament function, suggesting that optimal binding occurs when membranes are functioning normally on both macrophages and red cells. Colchicine and vinblastine do not influence resetting; therefore, microtubules are probably not involved in erythrocyte binding.     

Invertebrate aquaporins: a review

Aquaporins (AQPs) or water channels render the lipid bilayer of cell membranes permeable to water. The numerous AQP subtypes present in any given species, the transport properties of each subtype and the variety of methods of their regulation allows different cell types to be transiently or permanently permeable to water or other solutes that AQPs are capable of transporting (e.g. urea or glycerol). AQPs have been well characterized in all vertebrate classes, other than reptilia. Here we review the current state of knowledge of invertebrate AQPs set in the context of the much more thoroughly studied vertebrate AQPs. By phylogenetic analysis of the total AQP complement of several completed insect genomes, we propose a classification system of insect AQPs including three sub-families (DRIP, BIB and PRIP) that have one representative from all the complete insect genomes. The physiological role of AQPs in invertebrates (insects, ticks and nematodes) is discussed, including their function in common invertebrate phenomena such as high-volume liquid diets, cryoprotection and anhydrobiosis.     

Exploited marine invertebrates: genetics and fisheries

The application of genetic techniques to invertebrate fisheries is in many ways essentially similar to that in vertebrate (i.e. finfish) fisheries, for which there is already an extensive body of published data. However, there are also relative differences which lead to particular problems in the use of genetic data to study commercially important invertebrate species. The main role for genetics of both vertebrates and invertebrates has been, and is likely to continue to be, the identification of groups of interbreeding individuals as the basis for a fishery. It is in the identification of the breeding unit that the genetic differences between vertebrates and invertebrates can be of practical significance. The genetic breeding unit, usually called a 'stock' in fisheries biology, generally shows a certain uniformity of size in most marine fish which have been studied. Smaller or less mobile fish (e.g. flatfish) may only range a few tens of kilometres to their breeding grounds, whilst in more mobile, particularly migratory pelagic species (e.g. Scombridae), the area occupied by a stock is likely to be far greater and for a few (e.g. large pelagic elasmobranchs), a single unit of stock may be almost circumglobal. However, marine fish generally, particularly those large or plentiful enough to be of commercial interest, are likely to be fairly mobile and in many cases the order of mobility is likely to be in the region we might predict from our knowledge of the biology and habits of the species. In the genetic assessment of `stocks' for invertebrate fisheries, we face a number of additional problems, mostly related to the large evolutionary range of invertebrates exploited and their widely different biology. Although in Europe and North America marine invertebrate fisheries may be thought of as being mainly for decapod crustaceans and bivalve molluscs, globally commercially important marine invertebrate fisheries range from sponges to squid and include such diverse groups as sea cucumbers, barnacles, krill, octopuses, cuttlefish, sea anemones, ascidians, polychaetes, sea urchins, gastropods and jellyfish. An obvious feature of many of these invertebrates is that the adult (i.e. commercial) stage of the life cycle is sessile (e.g. barnacles, sponges, ascidians) or of very limited mobility (e.g. sea anemones, sea urchins, bivalves, gastropods), with the result that the dispersive phase of the life cycle is the larva. Other groups (e.g. krill, jellyfish) are planktonic or nektonic and may cover very large distances, but, unlike fish, have little control over the distance or direction of travel, whilst some of the open ocean pelagic squid are more mobile than most fish and may migrate thousands or kilometres to spawning grounds. The very low mobility of both larva and adult in some invertebrates indicates that dispersal, and hence stock size, is likely to be low and that, therefore, stocks are far more vulnerable to overfishing than in most fish species. An additional difficulty is that genetic studies to date indicate a remarkably high incidence of cryptic speciation in marine invertebrates, sometimes even in comparatively well studied commercially important species. Thus, although to date marine invertebrate fisheries have not received the same level of attention from geneticist as finfish fisheries, it is clear that for invertebrate fisheries genetic data are relatively far more important if a fishery is to be exploited without being endangered.     

Cephalochordate melanopsin: evolutionary linkage between invertebrate visual cells and vertebrate photosensitive retinal ganglion cells

Animal photoreceptor cells can be classified into two distinct types, depending on whether the photopigment is borne on the membrane of a modified cilium (ciliary type) or apical microvilli (rhabdomeric type) [1]. Ciliary photoreceptors are well known as vertebrate rods and cones and are also found in several invertebrates. The rhabdomeric photoreceptor, in contrast, is a predominant type of invertebrate visual cell, but morphologically identifiable rhabdomeric photoreceptors have never been found in vertebrates. It is hypothesized that the rhabdomeric photoreceptor cell had evolved to be the photosensitive retinal ganglion cell for the vertebrate circadian photoentrainment [2, 3 and 4] owing to the fact that some molecules involved in cell differentiation are common among them [5]. We focused on the cephalochordate amphioxus because it is the closest living invertebrate to the vertebrates, and interestingly, it has rhabdomeric photoreceptor cells for putative nonvisual functions [6]. Here, we show that the amphioxus homolog of melanopsin [7, 8 and 9], the circadian photopigment in the photosensitive retinal ganglion cells of vertebrates, is expressed in the rhabdomeric photoreceptor cells of the amphioxus and that its biochemical and photochemical properties, not just its primary structure, are considerably similar to those of the visual rhodopsins in the rhabdomeric photoreceptor cells of higher invertebrates. The cephalochordate rhabdomeric photoreceptor represents an evolutionary link between the invertebrate visual photoreceptor and the vertebrate circadian photoreceptor.     

Presence of invertebrate dystrophin-like products in obliquely striated muscle of the leech, Pontobdella muricata (Annelida, Hirudinea)

Dystrophin is a 427-kDa cytoskeletal protein, which occurs in scant amounts in vertebrate muscle and nerve cells. No previous references to dystrophin or associated proteins in invertebrates at the protein level have been found, while two recent studies investigated the presence of genes encoding proteins homologous to dystrophin in sea urchin and other invertebrates such as Drosophila melanogaster. In this study, the possible presence and distribution of dystrophin-like proteins were studied in different invertebrate muscle cell types and species through Western blot analysis and light and electron microscope immunohistochemistry using a panel of antibodies whose specificities have been determined in vertebrates. Crude protein extracts of leech Pontobdella muricata were analysed by Western blotting. The revealed protein band, with 140kDa molecular weight, was related to dystrophin, utrophin or dystrophin-related protein-2 (DRP2) according to the specificities of the antibodies used to detect them. The immunofluorescence study showed positive immunoreactions in obliquely striated muscle of this hyrudinean. The immunoelectron microscopy study confirmed specific immunogold labelling beneath the sarcolemma of muscle cells. We thus assume that this protein is an invertebrate dystrophin-like product that is referred to as IDLp140. The potential functions of this invertebrate dystrophin-like protein in invertebrate muscles are discussed relative to previous data in vertebrate tissues.     

The cytoskeletal system of nucleated erythrocytes. III. Marginal band function in mature cells

Marginal bands (MBs) of microtubules are believed to function during morphogenesis of nonmammalian vertebrate erythrocytes, but there has been little evidence favoring a continuing role in mature cells. To test MB function, we prepared dogfish erythrocytes with and without MBs at the same temperature by (a) stabilization of the normally cold- labile MB at 0 degree C by taxol, and (b) inhibition of MB reassembly at room temperature by nocodazole or colchicine. We then compared the responses of these cells to mechanical stress by fluxing them through capillary tubes. Before fluxing , cells with or without MBs had normal flattened elliptical shape. After fluxing , deformation was consistently observed in a much greater percentage of cells lacking MBs. The difference in percent deformation between the two cell types was highly significant. That the MB is an effector of cell shape was further documented in studies of the formation of singly or doubly pointed dogfish erythrocytes that appear during long-term incubation of normal cells at room temperature. On-slide perfusion experiments revealed that the pointed cells contain MBs of corresponding pointed morphology. Incubation of cells with and without MBs showed that they become pointed only when they contain MBs, indicating that the MB acts as a flexible frame which can deform and support the cell surface from within. To test this idea further, cells with and without MBs were exposed to hyperosmotic conditions. Many of the cells without MBs collapsed and shriveled , whereas those with MBs did not. The results support the view that the MB has a continuing function in mature erythrocytes, resisting deformation and/or rapidly returning deformed cells to an efficient equilibrium shape in the circulation.     

Properties of the C-terminal domain of 4.1 proteins.

At the C-terminus of all known 4.1 proteins is a sequence domain unique to these proteins, known as the C-terminal domain (CTD). Mammalian CTDs are associated with a growing number of protein-protein interactions, although such activities have yet to be associated with invertebrate CTDs. Mammalian CTDs are generally defined by sequence alignment as encoded by exons 18-21. Comparison of known vertebrate 4.1 proteins with invertebrate (Caenorhabditis elegans and Drosophila melanogaster) 4.1 proteins indicates that mammalian 4.1 exon 19 represents a vertebrate adaptation that extends the sequence of the CTD with a Ser/Thr-rich sequence. The CTD was first described as a 22/24-kDa domain by chymotryptic digestion of erythrocyte 4.1 (4.1R) [Leto, T.L. & Marchesi, V.T. (1984) J. Biol. Chem. 259, 4603-4608]. Here we show that in 4.1R the 22/24-kDa fragment is not stable but rapidly processed to a 15-kDa fragment by chymotrypsin. The 15-kDa fragment is extremely stable, being resistant to overnight digestion in chymotrypsin on ice. Analysis of this fragment indicates that it is derived from residues 709-858 (SwissProt accession no. P48193), and represents the CTD of 4.1R. The fragment behaves as a globular monomer in solution. Secondary-structure predictions indicate that this domain is composed of five or six beta strands with an alpha helix before the most C-terminal of these. Together these data indicate that the CTD probably represents an independent folding structure which has gained function since the divergence of vertebrates from invertebrates.     

Fatty-acid-binding protein from the flight muscle of Locusta migratoria: evolutionary variations in fatty acid binding

Intracellular lipid-binding proteins have evolved from a common ancestral gene with the appearance of mitochondrial oxidation, to guarantee, for example, transport of fatty acids through the aqueous cytosol to their site of utilization. The mammalian forms of these lipid carriers are structurally well-characterized and have been categorized, on the basis of sequence similarities and several typical ligand-binding features, into four subfamilies. Only a single complex structure of an invertebrate fatty-acid-binding protein (FABP) has been reported to date, which reveals a unique ligand-binding arrangement yet unknown in vertebrate FABPs. In the present study, the structure of a second invertebrate FABP (locust muscle) complexed with a fatty acid has been determined on the basis of intermolecular NOE connectivities between the protein and the uniformly (13)C-enriched oleate ligand. The resulting ligand conformation, although resembling the closely related mammalian heart- and adipocyte-type FABPs, is characterized by certain binding features that differ significantly from the typical hairpin-turn ligand shapes of the latter forms. This is primarily due to an alanine-to-leucine substitution in locust FABPs that produces a steric hindrance for ligand binding. A comparison with an FABP from tobacco hornworm larvae furthermore demonstrates that certain amino acid substitutions that appear to be specific for invertebrates decidedly influence the binding arrangement inside the protein cavity. Hence, as a result of these evolutionary variations, invertebrate FABPs may display a much greater diversity in intracellular lipid binding than observed for the mammalian transport proteins, thus possibly providing new insights for the design of modified lipid carriers.     

Role of a single amino acid in the evolution of glycans of invertebrates and vertebrates

Structures of glycoconjugate N-glycans and glycolipids of invertebrates show significant differences from those of vertebrates. These differences are due largely to the vertebrate beta1,4-galactosyltransferase-1 (beta4Gal-T1), which is found as a beta1,4-N-acetylgalactosaminyltransferase (beta4GalNAc-T1) in invertebrates. Mutation of Tyr285 to Ile or Leu in human beta4Gal-T1 converts the enzyme into an equally efficient beta4GalNAc-T1. A comparison of all the human beta4Gal-T1 ortholog enzymes shows that this Tyr285 residue in human beta4Gal-T1 is conserved either as Tyr or Phe in all vertebrate enzymes, while in all invertebrate enzymes it is conserved as an Ile or Leu. We find that mutation of the corresponding Ile residue to Tyr in Drosophila beta4GalNAc-T1 converts the enzyme to a beta4Gal-T1 by reducing its N-acetylgalactosaminyltransferase activity by nearly 1000-fold, while enhancing its galactosyltransferase activity by 80-fold. Furthermore, we find that, similar to the vertebrate/mammalian beta4Gal-T1 enzymes, the wild-type Drosophila beta4GalNAc-T1 enzyme binds to a mammary gland-specific protein, alpha-lactalbumin (alpha-LA). Thus, it would seem that, during the evolution of vertebrates from invertebrates over 500 million years ago, beta4Gal-T1 appeared as a result of the single amino acid substitution of Tyr or Phe for Leu or Ile in the invertebrate beta4GalNAc-T1. Subsequently, the pre-existing alpha-LA-binding site was utilized during mammalian evolution to synthesize lactose in the mammary gland during lactation.     

Immunocytogenetics. V. A highly conserved NOR antigen (He) is facultatively associated with nucleolar or nucleoplasmic granules

A new protein antigen of the nucleolus organizer region (NOR), designated He, was recognized by human autoantibodies obtained from a patient with Raynaud phenomenon. In mitotic cells of all vertebrate species tested. He serum selectively immunostained the chromosomal NORs. A completely unexpected characteristic of the He antigen was its location during interphase. In mammalian cell substrates, it was concentrated in numerous nucleoplasmic granules, with minor amounts of the antigen uniformly distributed throughout the entire nucleus. In interphase nuclei of lower vertebrate cells, however, the antigen was preferentially located in the nucleolus. The antigenicity of He is not dependent on RNA or DNA; its cytochemical properties operationally classify it as a nonhistone component of the chromosome scaffold. The He antigen was present in the residual nucleolar structures of cells that were not at all active in rRNA synthesis, such as mammalian late spermatids and amphibian erythrocytes.     

Sexual size dimorphism in parasitic oxyurid nematodes

As in many invertebrates, female oxyurids are larger than male. Sexual size dimorphism (SSD) of oxyurid nematodes (the hosts of which are both invertebrate and vertebrate), is investigated regarding body size of both host and parasite. SSD of parasites appeared to be weakly, but not significandy, correlated with invertebrate and vertebrate host body size. However, this study reveals a different pattern for SSD with respect to either type of host. SSD does not increase in tandem with body size in vertebrate parasites either at the level of species or genus. SSD is much more pronounced in Syphaciidae than in Heteroxynematidae, two families of vertebrate parasites exhibiting different modes of transmission (members of the Syphaciidae are transmitted through perianal contamination). SSD is investigated in one monophyletic group of parasites of primates, for which a phylogeny is known. Independent comparisons method is used and we find that the body size of female parasite is strongly correlated with that of the male. The hypoallometry (slope<1) of the relationship suggests that the SSD is not linked to an increase of parasite body size. Moreover, there is no influence of host body size on parasite SSD. The pattern in parasites of invertebrates is different. First, SSD has been found to increase with parasite body size in two groups of invertebrate parasites: the oxyurids of Dictyoptera and Coleoptera. Second, female body size of invertebrate parasites is not correlated with male body size either at genus or species level. Finally, the evolution of SSD is discussed in relation to the demographic patterns of invertebrate parasites and the haplodiploid mode of reproduction of these parasitic nematodes.     

Alkylacyl glycerophosphoinositol in human and bovine erythrocytes. Molecular species composition and comparison with glycosyl-inositolphospholipid anchors of erythrocyte acetylcholinesterases.

Glycosyl-inositolphospholipid (glycosyl-PtdIns) anchors of proteins in mammalian cells which have been analyzed so far are exclusively of the alkylacyl type. However, little is known about the putative precursor of glycosyl-PtdIns, the alkylacyl derivative of glycerophosphoinositol (GroPIns), in these cells since it is generally believed that cellular GroPIns consists of diacyl-type molecular species only. In this report, we describe the isolation and identification of alkylacyl GroPIns molecular species in both human and bovine erythrocytes, and compare it with the molecular species compositions of the glycosyl-PtdIns anchors of human and bovine erythrocyte acetylcholinesterase. Diradyl GroPIns was isolated from lipid extracts of ghost membranes and treated with phospholipase C. Diradylglycerols of the glycosyl-PtdIns anchors of affinity-purified human and bovine erythrocyte acetylcholinesterase were generated by sequential treatment with glycoprotein phospholipase D and acidic phosphatase and by PtdIns-specific phospholipase C, respectively. Diradylglycerols were subsequently converted into benzoate derivatives and separated into diacyl, alkylacyl, and alkenylacylglycerol subclasses. The molecular species compositions were quantitated and determined by combined HPLC/mass spectrometry. We found that human and bovine erythrocyte membrane diradyl GroPIns consist of 1.5-4.8% alkylacyl GroPIns. Molecular species analysis showed a heterogeneous species composition for both human and bovine erythrocyte alkylacyl GroPIns. Their compositions are distinctly different from those of human and bovine erythrocyte acetylcholinesterase glycosyl-PtdIns anchors. The number of alkylacyl GroPIns molecules/cell is roughly equal with the number of glycosyl-PtdIns-anchored proteins in human erythrocytes.     

The phylogenetic odyssey of the erythrocyte. III. Fish, the lower vertebrate experience.

The piscine erythrocyte can be considered the prototype of the red cells that are distributed among inframmalian vertebrates. It is a permanently nucleated, hemoglobin-ladened, oval, flattened, biconvex disc. Ultrastructurally it demonstrates a cytoskeleton comprised of a marginal band and a membrane skeleton which are responsible for the erythrocyte's conversion to an ellipsoid during morphogenesis and endow it with resilience to physical trauma. Erythropoiesis initiates in the yolk sac, followed in many fishes, by the intermediate cell mass. These sites are the sources of the transitory, primitive generation red cells which apparently make their first phylogenetic appearance in fishes and which are subsequently represented in all classes of vertebrates including mammals. Production of definitive generation erythrocytes is centered in evolutionary "pre-splenic" tissue of the gastrointestinal tract or in the spleen in cyclostomes, dipnoi, and chondrichthyes while in teleosts it is typically located in the kidneys with or without splenic participation. The blood is a major site of erythrocyte maturation in the lower fishes and exhibits significant numbers of immature erythroid cells plus occasional mitotic figures. Some teleosts also circulate developing erythroid cells. Certain fishes have occasional circulating erythroplastids, conceptually a portent of phylogenetic changes in higher vertebrates. Remarkably, some bristlemouths have denucleated erythrocytes exclusively in the circulation. The largest piscine erythrocytes are found in the dipnoi, myxines, and chondrichthyes. Primitive fish with the exception of the endothermic sharks tend to have lower hemoglobin concentrations than the modern teleosteans. The very highest hemoglobin concentrations are attained by the endothermic scombrids. Erythrocyte-based data have a broad extent and are variably affected by age, sex, season and environment. This report includes a substantial selection of illustrations (fish species and rbc micrographs).     

The occurrence of muscle spindles in extraocular muscles of various vertebrates

Extraocular muscles from representative species of vertebrate groups ranging from amphibians to the higher mammals were examined in serial histological sections for the presence of muscle spindles. These observations and data from the literature indicate that extraocular muscles of the pig, calf, sheep and other even-toed ungulates are richly supplied with well-defined spindles having a generous complement of intrafusal fibers distinguishable as nuclear bag and chain fibers. Spindles in human eye muscles are also numerous. In macaque and chimpanzee muscles a few poorly developed spindles were present in some, but not all, muscles. No encapsulated receptors were found in 20 other mammalian and submammalian species examined in this study. When present, spindles tended to be located in the zone of small muscle fibers found along the orbital surface of the muscle. Rectus and oblique muscles in all species had such a zone, so that its existence did not determine whether spindles would occur.