Isolation and characterization of a new class of acidic glycans implicated in sea urchin embryonal cell adhesion

Three major glycan fractions of 580 kDa (g580), 150 kDa (g150), and 2 kDa (g2) were isolated and purified from Lytechinus pictus sea urchin embryos at the mesenchyme blastula stage by gel filtration and high pressure liquid chromatography. Chemical analysis, by gas chromatography, revealed that g580 is highly sulfated and rich in N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, and fucose. The g150 fraction is less acidic than g580 and contains high amounts of amino sugars, xylose, and mannose. The g2 fraction is neutral, rich in N-acetylglucosamine, mannose, and galactose. The g580 and g150 fractions are resistant to glycosaminoglycan-degrading enzymes, indicating that they are distinct from the glycosaminoglycans. The g580 fraction resembles, with respect to chemical composition, a previously characterized 200 kDa sponge adhesion glycan (g200). The binding of the monoclonal antibody Block 2, which recognizes a repetitive epitope on g200, as well as of the anti-g580 polyclonal antibodies to both g580 and g200 indicated that these two glycans share similar antigenic determinants. The Fab fragments of the Block 2 antibody, which previously have been shown to inhibit cell adhesion in sponges, also blocked the reaggregation of dissociated sea urchin mesenchyme blastula cells. These results indicate that g580 carries a carbohydrate epitope, similar to the sponge adhesion epitope of g200, which is involved in sea urchin embryonal cell adhesion.     

Single cell human genomic analyses: a way to refine the knowledge of cellular heterogeneity origins in individual subject

Single cell genomics performed on individual human subjects' tumors, neural tissues, and sperm samples revealed the existence of genetic heterogeneity arising through either mutations in exomes, deletions, recombinations, and duplications of DNA sequences, as well as aneuploidy. These genetic changes happen during cell cycles followed by cell division. The aim of this review is to strictly focus on single cell human genomics and intends to deliver information that can help to refine fundamental knowledge relating to genetic causes of cellular heterogeneity origins in both healthy and disease states. Allogenic heterogeneity as well as heterogeneity origins of cells possessing the same genome with different gene expression patterns is not the subject of this review. Future research still requires: a) improvement for complete and errorless DNA acquisition and sequencing of not only selected parts of the genome, and b) analyses of more samples that contain millions of cells. These data will deliver a more precise comparative representation of genetic diversity among single cells in an individual human subject. Consequently, we will be able to better distinguish between the role of genetic, versus epigenetic, and stochastic factors in the cellular diversity of over 30 trillion cells present in a human body.     

Shape matters: Lifecycle of cooperative patches promotes cooperation in bulky populations

Natural cooperative systems take many forms, ranging from one‐dimensional cyanobacteria arrays to fractal‐like biofilms. We use in silico experimental systems to study a previously overlooked factor in the evolution of cooperation, physical shape of the population. We compare the emergence and maintenance of cooperation in populations of digital organisms that inhabit bulky (100 × 100 cells) or slender (4 × 2500) toroidal grids. Although more isolated subpopulations of secretors in a slender population could be expected to favor cooperation, we find the opposite: secretion evolves to higher levels in bulky populations. We identify the mechanistic explanation for the shape effect by analyzing the lifecycle and dynamics of cooperator patches, from their emergence and growth, to invasion by noncooperators and extinction. Because they are constrained by the population shape, the cooperator patches expand less in slender than in bulky populations, leading to fewer cooperators, less public good secretion, and generally lower cooperation. The patch dynamics and mechanisms of shape effect are robust across several digital cooperation systems and independent of the underlying basis for cooperation (public good secretion or a cooperation game). Our results urge for a greater consideration of population shape in the study of the evolution of cooperation across experimental and modeling systems.     

Molecular fingerprinting of carbohydrate structure phenotypes of three porifera proteoglycan-like glyconectins

Glyconectins (GNs) represent a new class of proteoglycan-like cell adhesion and recognition molecules found in several Porifera species. Physico-chemical properties of GN carbohydrate moieties, such as size, composition, and resistance to most glycosaminoglycan-degrading enzymes, distinguish them from any other type of known glycoproteins. The molecular mechanism of GN-mediated self/non-self discrimination function is based on highly species-specific and Ca(2+)-dependent GN to GN associations that approach the selectivity of the evolutionarily advanced immunoglobulin superfamily. Carbohydrates of glyconectins 1, 2, and 3 are essential for species-specific auto-aggregation properties in three respective Porifera species. To obtain a structural insight into the molecular mechanisms, we performed carbohydrate structural analyses of glyconectins isolated from the three sponge model systems, Microciona prolifera (GN1), Halichondria panicea (GN2), and Cliona celata (GN3). The glycan content of all three GNs ranged between 40 and 60% of their total mass. Our approach using sequential and selective chemical degradation of GN glycans and subsequent mass spectrometric and NMR analyses revealed that each glyconectin presents novel and highly species-specific carbohydrate sequences. All three GNs include distinct acid-resistant and acid-labile carbohydrate domains, the latter composed of novel repetitive units. We have sequenced four short sulfated and one pyruvilated unit in GN1, eight larger and branched pyruvilated oligosaccharides in GN2, which represent a heterogeneous but related family of structures, and four sulfated units in GN3.     

The species-specific cell-binding site of the aggregation factor from the sponge Microciona prolifera is a highly repetitive novel glycan containing glucuronic acid, fucose, and mannose

Species-specific adhesion of dissociated cells from the marine sponge Microciona prolifera is mediated by a Mr = 2 x 10(7) proteoglycan-like aggregation factor (MAF) via two highly polyvalent functional domains, a cell-binding and a self-interaction domain. Glycopeptide N-glycosidase F release of a major glycan of Mr = 6.3 gamma 10(3) (G-6) from the MAF protein core resulted in the loss of cell binding activity, indicating a role of this polysaccharide molecule in MAF-cell association. The G-6 glycan was isolated and purified after complete Pronase digestion of MAF using gel electrophoresis, gel filtration, and ion exchange chromatography. Quantification of the amount of carbohydrate recovered in G-6 showed that one MAF molecule has about 950 repeats of this glycan. In its monomeric state G-6 did not display any measurable binding to cells (K alpha less than or equal to 10(3) M-1). Intermolecular cross-linking of the G-6 glycan with glutaraldehyde resulted, however, in the concomitant recovery of polyvalency (about 2200 repeats of G-6 per polymer of Mr greater than or equal to 1.5 x 10(7) and species-specific high cell binding affinity (K alpha = 1.6 x 10(9) M-1) but not of the MAF-MAF self-interaction activity. Thus, the G-6 glycan is the multiple low affinity cell-binding site involved in cell-cell recognition and adhesion of sponge cells. The G-6 glycan has 7 glucuronic acids, 3 fucoses, 2 mannoses, 5 galactoses, 14 N-acetylglucosamines, 2 sulfates, and 1 asparagine. Such a unique chemical composition indicates a new type of structure which includes features of glycosaminolycans and N-linked polysaccharides.     

Involvement of carbohydrates as multiple low affinity interaction sites in the self-association of the aggregation factor from the marine sponge Microciona prolifera

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular weight aggregation factor (MAF) and is based on two functional properties, a Ca2+-independent cell binding activity and a Ca2+-dependent factor-factor self-interaction. Monoclonal antibodies were prepared against purified MAF, and one clone was characterized which selectively inhibited the MAF-MAF association activity. Binding of the blocking antibody (Block 1) to MAF demonstrated that this epitope structure was present in 1100 copies per one MAF molecule of Mr = 2 X 10(7). Such blocking antibodies precipitated a small molecular weight protein-free glycan fraction prepared from MAF by Pronase digestion, thus indicating that the highly repetitive epitope is located in the carbohydrate portion of the molecule. Since the inhibitory activity of the Block 1 antibody could only be achieved when most of the sites were occupied by Fab fragments of this antibody, the self-association of MAF seemed to be based on the polyvalency of the carbohydrate determinants. The affinity of the protein-free glycans was very low as shown by the fact that they did not display any measurable self or MAT binding activity in their monomeric form. After cross-linking them with glutaraldehyde into polymers of the size of MAF, however, the self-interaction could be reconstituted. Thus, the MAF-MAF association activity, which is needed for cell aggregation in sponges, seems to be based on multiple low affinity carbohydrate-carbohydrate interactions, which is different from most interactions mediated by adhesion molecules characterized so far.     

Natural selection fails to optimize mutation rates for long-term adaptation on rugged fitness landscapes

The rate of mutation is central to evolution. Mutations are required for adaptation, yet most mutations with phenotypic effects are deleterious. As a consequence, the mutation rate that maximizes adaptation will be some intermediate value. Here, we used digital organisms to investigate the ability of natural selection to adjust and optimize mutation rates. We assessed the optimal mutation rate by empirically determining what mutation rate produced the highest rate of adaptation. Then, we allowed mutation rates to evolve, and we evaluated the proximity to the optimum. Although we chose conditions favorable for mutation rate optimization, the evolved rates were invariably far below the optimum across a wide range of experimental parameter settings. We hypothesized that the reason that mutation rates evolved to be suboptimal was the ruggedness of fitness landscapes. To test this hypothesis, we created a simplified landscape without any fitness valleys and found that, in such conditions, populations evolved near-optimal mutation rates. In contrast, when fitness valleys were added to this simple landscape, the ability of evolving populations to find the optimal mutation rate was lost. We conclude that rugged fitness landscapes can prevent the evolution of mutation rates that are optimal for long-term adaptation. This finding has important implications for applied evolutionary research in both biological and computational realms.     

Sulfate restriction induces hyposecretion of the adhesion proteoglycan and cell hypomotility associated with increased 35SO42− uptake and expression of a band 3 like protein in the marine sponge,Microciona prolifera

Sulfate is an important component relating to normal proteoglycan secretion and normal motility in the marine sponge, Microciona prolifera. The following alterations were observed in sponge cells when sulfate free artificial sea water was used as the suspension medium: (1) impairment of aggregation, (2) loss of cell movements, (3) a marked reduction in the secretion of the adhesion proteoglycan (AP). Reversal of this effect occurred if sulfate depleted cells were again rotated in sulfate containing artificial sea water. Motility and reaggregation of sulfate deprived cells could be completely restored by purified AP, but only if cells were first pre-conditioned in normal sea water. Comparisons of ³⁵SO₄^2? uptake between normal and sulfate deprived cells which had been treated to reduce preformed secretions showed a marked increase in ³⁵SO₄^2? uptake and incorporation which could be greatly augmented in the presence of Ca²⁺/Mg²⁺. Excessive retention of AP in sulfate starved cells demonstrated by immunostaining suggested that AP secretion and cellular motility may be controlled by a sulfate dependent secretogogue or that undersulfated AP itself had developed a secretory defect. SDS-PAGE of Triton treated cellular extracts demonstrated a 116 kDa ³⁵SO₄^2? sulfated band which co-migrated with AP, but only in extracts derived from sulfate starved cells. Western blots prepared from such extracts incubated in the presence of a monoclonal anti-band 3 antibody demonstrated labelling of a single 97 kDa band only in material from sulfate deprived cells. The absence of this component in normal cell extracts indicated that this protein may be involved in facilitated sulfate transport. This study lends support to a heretofore unrecognized role for sulfate in cell motility and secretion.