Micromere Differentiation in the Sea Urchin Embryo: Expression of Primary Mesenchyme Cell Specific Antigen during Development

The temporal and spatial expression of antigen specific for primary mesenchyme cell (PMC) lineage cells during early development of the sea urchins Hemicentrotus pulcherrimus and Stronglyocentrotus nudus was studied with a monoclonal antibody (P4). P4 was produced by a hybridoma cell line prepared by fusion of myeloma cells and spleen cells from a mouse immunized with cultured spicule-forming cells. Immunofluorescence studies demonstrated that P4 antibody reacted strongly with the surfaces of PMC's and spicule-forming cells of both species. Immunoblot analysis showed that P4 antibody reacted with several proteins including those of 140–kDa, 120–kDa, 53-kDa, 43–kDa, and 41–kDa in H. pulcherrimus and with those of 130–kDa, 110–kDa, 51–kDa, and 43–kDa in S. nudus . These proteins appeared sequentially after the hatching blastula stage. Tunicamycin inhibited the expressions of these P4 antigens as well as spicule formation. Two of the P4-reactive antigens, the 140–kDa and 43–kDa proteins, in H. pulcherrimus were synthesized de novo and shown to be identical to micromere differentiation specific proteins. These results suggest that P4 binds to specific molecules that are important in spicule formation in developing sea urchin embryos.     

Micromere Differentiation in the Sea Urchin Embryo: Immunochemical Characterization of Primary Mesenchyme Cell-Specific Antigen and Its Biological Roles

Primary mesenchyme cell (PMC)-specific antigens in developing sea urchin embryos of five different species have been studied by using two different monoclonal antibodies, P4 and B2C2. Like B2C2 in Strongylocentrotus purpuratus (Anstrom et al. , 1987) P4 reacted with the N-linked carbohydrate in Strongylocentrotus intermedius embryo. Although both antibodies recognize the same group of glycoproteins in S. intermedius , P4 epitopes appeared earlier than B2C2 epitopes in Clypeaster japonicus embryo. PMCs of Anthocidaris crassispina blastulae raised in sulfate-deficient sea water were immuno-reactive with P4 but not with B2C2, although the embryos raised in normal sea water reacted with both antibodies at similar intensity. These results suggest that the epitopes of P4 and B2C2 are formed by glycosylation and sulfation, respectively. PMCs may display differential modification in their surface glycoprotein synthesis during differentiation. Furthermore, P4 inhibited cultured micromere descendant cells of Hemicentrotus pulcherrimus from attaching to the plastic dishes and forming spicules in vitro without detectable cytotoxic effect. P4-reactive glycoproteins may play important roles in cell-substrate interaction and spicule formation.     

Effect on Inhibition of Micromere Segregation on the Mitotic Pattern in the Sea Urchin Embryo

Detergent treatment of sea urchin eggs at the mid 4-cell stage results in prevention of micromere segregation at the fourth cleavage. In these embryos not only the formation of the primary mesenchyme is suppressed, but synchrony of cell division, which is the rule during the first four cleavage cycles, continues for several cycles after the 16-cell stage while the typical mitotic phase wave that sets in after micromere segregation is abolished.
These results support the hypothesis that micromeres act as coordinators of the mitotic activity of the embryo.     

Spore Germination and Rhizoid Differentiation in Onoclea sensibilis: A Two-Dimensional Electrophoretic Analysis of the Extant Soluble Proteins

Following a geometrically asymmetrical cell division during germination of spores of the fern Onoclea sensibilis L., the small cell differentiates into a rhizoid and the large cell divides to form the protonema. Using silver-staining of two-dimensional gels, we have examined the soluble proteins of spores during germination and of separated rhizoid protoplasts and protonemal cells. Of over 500 polypeptides followed, nearly 25% increased or decreased in prominence during spore germination and the initial phases of rhizoid elongation. Soluble proteins from purified protoplasts of young rhizoids were quantitatively different from those of protonemal cells and germinated spores. Nine polypeptides which appeared after cell division were substantially more prominent in rhizoid protoplasts than in whole germinated spores and have been putatively designated rhizoid-specific polypeptides. The differences in the soluble protein composition of young rhizoids and protonemal cells probably reflect the differential organelle distribution between the two cells as well as differential net protein synthesis in the cytoplasms of the two cells.     

Spicule Formation-Inducing Substance in Sea Urchin Embryo

Isolated micromeres of sea urchin produced spicules in sea water containing blastocoelic fluid (BCF) taken from embryos, or in a medium in which embryos had previously been dissociated (dissociated solution, DS). When isolated micromeres were cultured in vitro , their descendants initiated spicule formation only when BCF was added to the culture medium by the time when, in normal development, primary mesenchyme cells form two aggregates in the vegetal region. After the initiation of spicule formation, growth of spicules occurred under the continuous influence of DS. Spicule formation-inducing (SFI) activity in DS was first detected at the mesenchme blastula stage. The activity in BCF was heat-labile and was inactivated by trypsin.     

Chaperone-Assisted Folding of Newly Synthesized Proteins in the Cytosol

The way in which a newly synthesized polypeptide chain folds into its unique three-dimensional structure remains one of the fundamental questions in molecular biology. Protein folding in the cell is a problematic process and, in many cases, requires the assistance of a network of molecular chaperones to support productive protein folding in vivo. During protein biosynthesis, ribosome-associated chaperones guide the folding of the nascent polypeptide emerging from the ribosomal tunnel. In this review we summarize the basic principles of the protein-folding process and the involved chaperones, and focus on the role of ribosome-associated chaperones. Our discussion emphasizes the bacterial Trigger Factor, which is the best studied chaperone of this type. Recent advances have determined the atomic structure of the Trigger Factor, providing new, exciting insights into the role of ribosome-associated chaperones in co-translational protein folding.     

Distribution of Soluble Proteins Within Spinal Motoneurons: A Quantitative Two-Dimensional Electrophoretic Analysis

Soluble protein fractions obtained from bovine lumbar spinal motoneuron cell bodies, ventral gray matter, and ventral and dorsal roots were analyzed by two-dimensional gel electrophoresis. Each extract was separated into Coomassie blue-stained patterns of up to 350 polypeptides ranging in isoelectric point from pH 4 to 8 and in molecular weight from 10,000 to 200,000. Visual inspection of the protein pattern of the isolated cell bodies showed it to be substantially different from those of ventral gray matter and the spinal roots, while the patterns obtained from ventral and dorsal roots were indistinguishable. Computer-assisted densitometry of the major soluble proteins from spinal roots showed no quantitative difference between the predominant proteins in ventral and dorsal root extracts. Differences of 10-fold or more were common when the major proteins of the isolated perikarya were compared with those of the other fractions. Since most of the soluble proteins extracted from ventral and dorsal roots were probably derived from the axoplasm of motor and sensory nerves, respectively, these results are interpreted to mean that large differences exist in the distribution of individual soluble proteins between the cell body and axon of spinal motoneurons, while the major soluble proteins of spinal motor and sensory axons are highly similar.     

Changes in Ribosome-associated Proteins during Sea Urchin Development

Ribosomes isolated from unfertilised eggs of the sea urchin, Strongylocentrotus purpuratus , have a higher protein: RNA ratio than ribosomes extracted from blastula stage ribosomes. Approximately 64 additional protein equivalents are found per ribosome. Most of the proteins are of high molecular weight and are tightly bound, being resistant to high-salt and EDTA treatment. The majority of the proteins appear to be basic in nature and remain associated with the 40S subunit on dissociation of the ribosomes. The possible physiological significance of the additional proteins is discussed in terms of the activation of protein synthesis following fertilisation. Sea urchin ribosomes, isolated from various stages of development, showed differential protein-labelling patterns. The high molecular-weight proteins had preferentially higher specific activities and one ribosomal protein was particularly highly labelled, reaching a maximum at the gastrula stage of development. The functional role of this highly labelled protein during development is discussed.     

Spicule Formation by Isolated Micromeres of the Sea Urchin Embryo

Micromeres are isolated at the 16-cell stage from three speciesof Japanese sea urchins, Hemicentrotus pulcherrimus, Pseudocentrotusdepressus, and Anthocidaris crassispina, and are cultured insea water containing a small amount of horse serum. In all speciesused, isolated micromeres first divide unequally as they doin vivo. The pattern and number of the subsequent cleavagesare also the same as in vivo, although they are not necessarilyclear in all cases, since the border of the adjacent cells becomeinvisible at each resting stage in some batches of embryos. After cleavage, passing through the stage when the contoursof the individual cell are obscure, decendants of the isolatedmicromeres form cell aggregates similar to the group of primarymesenchyme cells in a blastula. Within such aggregates, a spicularrudiment appears which develops either into a triradiate spiculeas in normal gastrulae or into a rod. The triradiate spiculegrows into a three-dimensional skeleton which is very similarto the normal pluteus skeleton, not only in its final shapeand size including species-specific characters but in its developmentalcourse and crystallographic nature. The rod, on the other hand,develops into either a one-dimensional or two-dimensional skeleton.These skeletons probably correspond to a part of the completeskeleton.     

Cellular Origin and Biosynthesis of Rat Optic Nerve Proteins: A Two-Dimensional Gel Analysis

High resolution 2DGE (two-dimensional gel electrophoresis) was used to characterize neuronal and glial proteins of the rat optic nerve, to examine the phases of intraaxonal transport with which the neuronal proteins are associated, and to identify the ribosomal populations on which these proteins are synthesized. Neuronal proteins synthesized in the retinal ganglion cells were identified by injecting the eye with L-[35S]methionine, followed by 2DGE analysis of fast and slow axonally transported proteins in particulate and soluble fractions. Proteins synthesized by the glial cells were labeled by incubating isolated optic nerves in the presence of L-[35S]methionine and then analyzed by 2DGE. A number of differences were seen between filamentous proteins of neurons and glia. Most strikingly, proteins in the alpha- and beta-tubulin region of the 2D gels of glial proteins were distinctly different than was observed for axonal proteins. As expected, neurons but not glia expressed neurofilament proteins, which appeared among the slow axonally transported proteins in the particulate fraction; significant amounts of the glial filamentous protein, GFA, were also labeled under these conditions, which may have been due to transfer of amino acids from the axon to the glial compartment. The fast axonally transported proteins contained relatively large amounts of high-molecular-weight acidic proteins, two of which were shown to comigrate (on 2DGE) with proteins synthesized by rat CNS rough microsomes; this finding suggests that rough endoplasmic reticulum may be a major site of synthesis for fast transported proteins. In contrast, the free polysome population was shown to synthesize the principal components of slow axonal transport, including tubulin subunits, actin, and neurofilament proteins.     

Agarose Gel Electrophoretic Separation of Blood Serum Proteins in Cattle

The agarose gel electrophoresis described by Johansson (1972) was modified so that a buffer of pH 7.9 was used in the gel, whereas the buffer in the electrode vessels had a pH of 8.6. The cattle blood serum protein picture is described in detail. The β1-globulin zone shows a very distinct picture of the genetically polymorphic bovine transferrins. The region between the α- and β-globulins shows a number of faint and often very distinct bands. A faint background staining over the whole electrophoretogram may partly be caused by a rather strong lipoprotein in the α1-region, lipids thus having migrated all over the electrophoretogram. The modified method described is well suited as a “screen electrophoresis” for cattle serum and is also useful e.g. in studying bovine transferrin polymorphism.     

Genetic Variation in Proteins: Comparison of One-Dimensional and Two-Dimensional Gel Electrophoresis

Two proteins with known characteristics on one-dimensional gels were studied by two-dimensional electrophoresis to compare the sensitivities of the two methods in detecting genetic variation. Two-dimensional electrophoresis was found to be less sensitive than several types of one-dimensional gels in distinguishing variants of both proteins. Denaturation of proteins in urea in the two-dimensional method makes it possible to distinguish closely related proteins that differ from each other by units of charge. Many more types of variation in protein sequences can be distinguished on one-dimensional gels in the absence of denaturants. The estimates of heterozygosity based on two-dimensional gels are lower than those based on other methods, at least in part, because of the limited types of sequence differences that can be detected on two-dimensional gels. The application of two-dimensional electrophoresis to the measurement of genetic variation and to the detection of new mutations should be made carefully, in view of the limited sensitivity of the method in finding differences in sequence.     

Two-Dimensional Gel Electrophoretic Analysis of the Cerebella from Neuro-Pathologically-Mutant Weaver,Nervous and Staggerer Mice1

We have analysed the proteins of the cerebella from mutant and control mice by applying high resolution two-dimensional polyacrylamide gel electrophoresis. The tissue of each cerebellum and also the pallium cerebri were fractionated into water-soluble and particulate fractions, and these were used in gel electrophoresis. In order to augment the sensitivity for detection of protein spots, we applied silver staining. We used the cerebella from weaver (granule cell deficient), nervous (Purkinje cell deficient), and staggerer (poor dendritic arborization of Purkinje cells) mutant mice. The present technique revealed at least 700 to 800 protein spots. Among the spots detected we found 12 new significantly-changed proteins in the cerebella of the mutants. The possible significance of these proteins is discussed.