Glyconectin Cell Adhesion Epitope, ß-d-GlcpNAc3S-(1→3)-α-l-Fucp, Is Involved in Blastulation of Lytechinus pictus Sea Urchin Embryos.

Glycans, as the most peripheral cell surface components, are the primary candidates to mediate the initial steps of cell recognition and adhesion via glycan-glycan binding. This molecular mechanism was quantitatively demonstrated by biochemical and biophysical measurements at the cellular and molecular level for the glyconectin 1 ß-d-GlcpNAc3S-(1→3)-α-l-Fucp glycan structure (GN1). The use of adhesion blocking monoclonal antibody Block 2 that specifically recognize this epitope showed that, besides Porifera, human colon carcinoma also express this structure in the apical glycocalyx. Here we report that Block 2 selectively immune-precipitate a Mr 580 × 103 (g580) acidic non-glycosaminoglycan glycan from the total protein-free glycans of Lytechinus pictus sea urchin hatched blastula embryos. Immuno-fluorescence confocal light microscopy and immunogold electron microscopy localized the GN1 structure in the apical lamina glycocalyx attachments of ectodermal cells microvilli, and in the Golgi complex. Biochemical and immune-chemical analyses showed that the g580 glycan is carrying about 200 copies of the GN1 epitope. This highly polyvalent g580 glycan is one of the major components of the glycocalyx structure, maximally expressed at hatched blastula and gastrula. The involvement of g580 GN1 epitope in hatched blastula cell adhesion was demonstrated by: (1) enhancement of cell aggregation by g580 and sponge g200 glycans, (2) inhibition of cell reaggregation by Block 2, (3) dissociation of microvilli from the apical lamina matrix by the loss of its gel-like structure resulting in a change of the blastula embryonal form and consequent inhibition of gastrulation at saturating concentration of Block 2, and (4) aggregation of beads coated with the immune-purified g580 protein-free glycan. These results, together with the previous atomic force microscopy measurements of GN1 binding strength, indicated that this highly polyvalent and calcium ion dependent glycan-glycan binding can provide the force of 40 nanonewtons per single ectodermal cell association of microvilli with the apical lamina, and conservation of glycocalyx gel-like structure. This force can hold the weight of 160,000 cells in sea water, thus it is sufficient to establish, maintain and preserve blastula form after hatching, and prior to the complete formation of further stabilizing basal lamina.

Single-cell omics analyses with single molecular detection: challenges and perspectives.

The ultimate goal of single-cell analyses is to obtain the biomolecular content for each cell in unicellular and multicellular organisms at different points of their life cycle under variable environmental conditions. These require an assessment of: a) the total number of cells, b) the total number of cell types, and c) the complete and quantitative single molecular detection and identification for all classes of biopolymers, and organic and inorganic compounds, in each individual cell. For proteins, glycans, lipids, and metabolites, whose sequences cannot be amplified by copying as in the case of nucleic acids, the detection limit by mass spectrometry is about 105 molecules. Therefore, proteomic, glycomic, lipidomic, and metabolomic analyses do not yet permit the assembly of the complete single-cell omes. The construction of novel nanoelectrophoretic arrays and nano in microarrays on a single 1-cm-diameter chip has shown proof of concept for a high throughput platform for parallel processing of thousands of individual cells. Combined with dynamic secondary ion mass spectrometry, with 3D scanning capability and lateral resolution of 50 nm, the sensitivity of single molecular quantification and identification for all classes of biomolecules could be reached. Further development and routine application of such technological and instrumentation solution would allow assembly of complete omes with a quantitative assessment of structural and functional cellular diversity at the molecular level.

Glycan-to-Glycan Binding: Molecular Recognition through Polyvalent Interactions Mediates Specific Cell Adhesion.

Glycan-to-glycan binding was shown by biochemical and biophysical measurements to mediate xenogeneic self-recognition and adhesion in sponges, stage-specific cell compaction in mice embryos, and in vitro tumor cell adhesion in mammals. This intermolecular recognition process is accepted as the new paradigm accompanying high-affinity and low valent protein-to-protein and protein-to-glycan binding in cellular interactions. Glycan structures in sponges have novel species-specific sequences. Their common features are the large size >100 kD, polyvalency >100 repeats of the specific self-binding oligosaccharide, the presence of fucose, and sulfated and/or pyruvylated hexoses. These structural and functional properties, different from glycosaminoglycans, inspired their classification under the glyconectin name. The molecular mechanism underlying homophilic glyconectin-to-glyconectin binding relies on highly polyvalent, strong, and structure-specific interactions of small oligosaccharide motifs, possessing ultra-weak self-binding strength and affinity. Glyconectin localization at the glycocalyx outermost cell surface layer suggests their role in the initial recognition and adhesion event during the complex and multistep process. In mammals, Lex-to-Lex homophilic binding is structure-specific and has ultra-weak affinity. Cell adhesion is achieved through highly polyvalent interactions, enabled by clustering of small low valent structure in plasma membranes.

Electrical properties of human skin as aging biomarkers.

A non-invasive bioimpedance spectroscopy (BIS) and Cole-Cole impedance model parameters (R0, R∞, τ and α) were used to analyze electrical properties of intact and stripped human skin for both gender subjects divided into younger and older age groups. R0, R∞ and τ significantly increased while α significantly decreased with age in stripped skin for both genders (p<0.031). Using pooled data with respect to age, gender and skin stripping, R0, R∞ and τ values were shown to increase with age (p<0.0034), R0, τ and α were different between genders (p<0.024) and R0, R∞ and τ decreased with skin stripping (p<0.000008). All of four Cole-Cole parameters were age dependent with specific differences observed for genders and intact and stripped skin layers. Therefore, Cole-Cole parameters, obtained by non-invasive BIS measurements, are a new type of age dependent biomarkers.

Fractional calculus model of electrical impedance applied to human skin.

Fractional calculus is a mathematical approach dealing with derivatives and integrals of arbitrary and complex orders. Therefore, it adds a new dimension to understand and describe basic nature and behavior of complex systems in an improved way. Here we use the fractional calculus for modeling electrical properties of biological systems. We derived a new class of generalized models for electrical impedance and applied them to human skin by experimental data fitting. The primary model introduces new generalizations of: 1) Weyl fractional derivative operator, 2) Cole equation, and 3) Constant Phase Element (CPE). These generalizations were described by the novel equation which presented parameter [Formula: see text] related to remnant memory and corrected four essential parameters [Formula: see text] We further generalized single generalized element by introducing specific partial sum of Maclaurin series determined by parameters [Formula: see text] We defined individual primary model elements and their serial combination models by the appropriate equations and electrical schemes. Cole equation is a special case of our generalized class of models for[Formula: see text] Previous bioimpedance data analyses of living systems using basic Cole and serial Cole models show significant imprecisions. Our new class of models considerably improves the quality of fitting, evaluated by mean square errors, for bioimpedance data obtained from human skin. Our models with new parameters presented in specific partial sum of Maclaurin series also extend representation, understanding and description of complex systems electrical properties in terms of remnant memory effects.

Glyconectin glycans as the self-assembling nano-molecular-velcrosystem mediating self-nonself recognition and adhesion implicated in evolution of multicellularity.

The goal of this chapter is to make a specific contribution about glyconectin glycan as the self-assembling nano-molecular-velcro system mediating initial steps of self-nonself recognition and cell adhesion in Porifera, the first descendants of the most simple primordial multicellular organisms. Two original findings will be described: (i) Velcro like concept based on highly polyvalent and specific intermolecular glycan to glycan associations with extremely low affinity of the single binding site and (ii) novel structures of the large and newly emerging family of glyconectin like glycan molecules. The emphasis will be put on the interdisciplinary approach for studying structure to function relationship at the different size scale levels by combining the knowledge and technologies (instrumentation and methods) of physics, chemistry, biology and mathematics. Applying such strategy which is crossing the boundaries of different science disciplines enabled us to develop a new Atomic Force Microscopy (AFM) based nano-bio-technology and perform the first quantitative measurements of intermolecular binding forces at the single molecular level under physiological conditions. We propose that nano-velcro systems of the glyconectin glycans, which are the constituents on the cell surface that are the most exposed to the environment, were responsible for the molecular self-nonself recognition and adhesion processes that underpinned the emergence of multicellular life forms.

Atomic force microscopy measurements of intermolecular binding forces.

Atomic force microscopy (AFM) measurements of intermolecular binding strength between a single pair of complementary cell adhesion molecules in physiological solutions provided the first quantitative evidence for their cohesive function. This novel AFM-based nanobiotechnology opens a molecular mechanic approach for studying structure- to function-related properties of any type of individual biological macromolecules. The presented example of Porifera cell adhesion glyconectin proteoglycans showed that homotypic carbohydrate to carbohydrate interactions between two primordial proteoglycans can hold the weight of 1,600 cells. Thus, glyconectin type carbohydrates, as the most peripheral cell surface molecules of sponges (today's simplest living Metazoa), are proposed to be the primary cell adhesive molecules essential for the evolution of the multicellularity.

Chemical microscopy of biological samples by dynamic mode secondary ion mass spectrometry.

3D chemical microscopy is one of the emerging applications of secondary ion mass spectrometry (SIMS) in biology. Tissues, cells, extracellular matrices, and polymer films can be imaged at present with a lateral resolution of 50 nm and depth resolution of 1 nm using the latest generation of CAMECA magnetic sector NanoSIMS 50 or with a lower lateral resolution (above 100 nm) using IMS 4f Cameca SIMS equipped with cold stage. Dynamic mode SIMS analysis is performed in ultrahigh vacuum and thus requires specific and careful preparation of biological samples aimed at preserving and minimizing destruction of the original structural and chemical properties of the samples. Here we describe a methodology based on the ultrafast plunge-freezing of biological tissues, preparation of the sample for SIMS analyses and transfer to the SIMS cold stage without interruption of the cold chain during the mounting procedure and subsequent SIMS analyses. Using this strategy, SIMS chemical microscopy can be performed on biological tissue in which unwanted molecular and/or structural reorganization, loss of constituents and chemical modifications are minimized and in which structures are therefore optimally preserved.

Toward a hyperstructure taxonomy.

Bacterial cells contain many large, spatially extended assemblies of ions, molecules, and macromolecules, called hyperstructures, that are implicated in functions that range from DNA replication and cell division to chemotaxis and secretion. Interactions between these hyperstructures would create a level of organization intermediate between macromolecules and the cell itself. To explore this level, a taxonomy is needed. Here, we describe classification criteria based on the form of the hyperstructure and on the processes responsible for this form. These processes include those dependent on coupled transcription-translation, protein-protein affinities, chromosome site-binding by protein, and membrane structures. Various combinations of processes determine the formation, maturation, and demise of many hyperstructures that therefore follow a trajectory within the space of classification by form/process. Hence a taxonomy by trajectory may be desirable. Finally, we suggest that working toward a taxonomy based on speculative interactions between hyperstructures promises most insight into life at this level.

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.

Molecular recognition between glyconectins as an adhesion self-assembly pathway to multicellularity.

The appearance of multicellular forms of life has been tightly coupled to the ability of an organism to retain its own anatomical integrity and to distinguish self from non-self. Large glycoconjugates, which make up the outermost cell surface layer of all Metazoans, are the primary candidates for the primordial adhesion and recognition functions in biological self-assembly systems. Atomic force microscopy experiments demonstrated that the binding strength between a single pair of Porifera cell surface glyconectin 1 glycoconjugates from Microciona prolifera can hold the weight of 1600 cells, proving their adhesion functions. Here, measurement of molecular self-recognition of glyconectins (GNs) purified from three Porifera species was used as an experimental model for primordial xenogeneic self/non-self discrimination. Physicochemical and biochemical characterization of the three glyconectins, their glycans, and peptides using gel electrophoresis, ultracentrifugation, NMR, mass spectrometry, glycosaminoglycan-degrading enzyme treatment, amino acid and carbohydrate analyses, and peptide mapping showed that GNs define a new family of proteoglycan-like molecules exhibiting species-specific structures with complex and repetitive acidic carbohydrate motives different from the classical proteoglycans and mucins. In functional self-assembly color-coded bead, cell, and blotting assays, glyconectins displayed species-specific recognition and adhesion. Affinity-purified monospecific polyclonal antibodies prepared against GN1, -2, and -3 glycans selectively inhibited cell adhesion of the respective sponge species. These results together with species-specific coaggregation of GN carbohydrate-coated beads with cells showed that GN glycans are functional in cell recognition and adhesion. The specificity of carbohydrate-mediated homophilic GN interactions in Porifera approaches the binding selectivity of the evolutionarily advanced immunoglobulin superfamily. Xenoselectivity of primordial glyconectin to glyconectin recognition may be a new paradigm in the self-assembly and non-self discrimination pathway of cellular adhesion leading to multicellularity.

Hypothesis: a phospholipid translocase couples lateral and transverse bilayer asymmetries in dividing bacteria.

Cell division in bacteria such as Escherichia coli entails changes in the radii of curvature of the invaginating cytoplasmic membrane which culminate in rearrangements of its monolayers. Division therefore risks perturbing transverse and lateral asymmetries and compromising membrane integrity. This leads us to propose that a strong selective pressure exists for a phospholipid translocator that would transfer phospholipids across the cytoplasmic membrane so as to both demarcate the division site and mediate lipid composition during division. This translocase has an affinity for phospholipids with small headgroups and unsaturated acyl chains which it translocates so as to (1) generate changes in the radius of curvature, (2) facilitate septum formation, (3) minimise bilayer disruption during fusion and (4) prevent septum formation at old or inappropriate division sites. We discuss briefly possible candidates for this translocase including ABC transporters and proteins localised to the division site.