Multicellular Morphology Research Articles

Experimental evolution of multicellularity via cuboidal cell packing in fission yeast

Abstract The evolution of multicellularity represents a major transition in life’s history, enabling the rise of complex organisms. Multicellular groups can evolve through multiple developmental modes, but a common step is the formation of permanent cell–cell attachments after division. The characteristics of the multicellular morphology that emerges have profound consequences for the subsequent evolution of a nascent multicellular lineage, but little prior work has investigated these dynamics directly. Here, we examine a widespread yet understudied emergent multicellular morphology: cuboidal packing. Extinct and extant multicellular organisms across the tree of life have evolved to form groups in which spherical cells divide but remain attached, forming approximately cubic subunits. To experimentally investigate the evolution of cuboidal cell packing, we used settling selection to favor the evolution of simple multicellularity in unicellular, spherical Schizosaccharomyces pombe yeast. Multicellular clusters with cuboidal organization rapidly evolved, displacing the unicellular ancestor. These clusters displayed key hallmarks of an evolutionary transition in individuality: groups possess an emergent life cycle driven by physical fracture, group size is heritable, and they respond to group-level selection via multicellular adaptation. In 2 out of 5 lineages, group formation was driven by mutations in the ace2 gene, preventing daughter cell separation after division. Remarkably, ace2 mutations also underlie the transition to multicellularity in Saccharomyces cerevisiae and Candida glabrata, lineages that last shared a common ancestor >300 million years ago. Our results provide insight into the evolution of cuboidal cell packing, an understudied multicellular morphology, and highlight the deeply convergent potential for a transition to multicellular individuality within fungi.

Rapid evolution of an adaptive multicellular morphology of Candida auris during systemic infection

Candida auris has become a serious threat to public health. The mechanisms of how this fungal pathogen adapts to the mammalian host are poorly understood. Here we report the rapid evolution of an adaptive C. auris multicellular aggregative morphology in the murine host during systemic infection. C. auris aggregative cells accumulate in the brain and exhibit obvious advantages over the single-celled yeast-form cells during systemic infection. Genetic mutations, specifically de novo point mutations in genes associated with cell division or budding processes, underlie the rapid evolution of this aggregative phenotype. Most mutated C. auris genes are associated with the regulation of cell wall integrity, cytokinesis, cytoskeletal properties, and cellular polarization. Moreover, the multicellular aggregates are notably more recalcitrant to the host antimicrobial peptides LL-37 and PACAP relative to the single-celled yeast-form cells. Overall, to survive in the host, C. auris can rapidly evolve a multicellular aggregative morphology via genetic mutations.

Gene loss during a transition to multicellularity

Multicellular evolution is a major transition associated with momentous diversification of multiple lineages and increased developmental complexity. The volvocine algae comprise a valuable system for the study of this transition, as they span from unicellular to undifferentiated and differentiated multicellular morphologies despite their genomes being similar, suggesting multicellular evolution requires few genetic changes to undergo dramatic shifts in developmental complexity. Here, the evolutionary dynamics of six volvocine genomes were examined, where a gradual loss of genes was observed in parallel to the co-option of a few key genes. Protein complexes in the six species exhibited novel interactions, suggesting that gene loss could play a role in evolutionary novelty. This finding was supported by gene network modeling, where gene loss outpaces gene gain in generating novel stable network states. These results suggest gene loss, in addition to gene gain and co-option, may be important for the evolution developmental complexity.

MorphoSim: an efficient and scalable phase-field framework for accurately simulating multicellular morphologies

The phase field model can accurately simulate the evolution of microstructures with complex morphologies, and it has been widely used for cell modeling in the last two decades. However, compared to other cellular models such as the coarse-grained model and the vertex model, its high computational cost caused by three-dimensional spatial discretization hampered its application and scalability, especially for multicellular organisms. Recently, we built a phase field model coupled with in vivo imaging data to accurately reconstruct the embryonic morphogenesis of Caenorhabditis elegans from 1- to 8-cell stages. In this work, we propose an improved phase field model by using the stabilized numerical scheme and modified volume constriction. Then we present a scalable phase-field framework, MorphoSim, which is 100 times more efficient than the previous one and can simulate over 100 mechanically interacting cells. Finally, we demonstrate how MorphoSim can be successfully applied to reproduce the assembly, self-repairing, and dissociation of a synthetic artificial multicellular system - the synNotch system.

An environmentally induced multicellular life cycle of a unicellular cyanobacterium

Understanding the evolutionary transition to multicellularity is a key problem in biology.1,2,3,4 Nevertheless, the ecological conditions driving such transitions are not well understood. The first known transition to multicellularity occurred 2.5 billion years ago in cyanobacteria,5,6,7 and today's cyanobacteria are characterized by enormous morphological diversity. They range from unicellular species; unicellular cyanobacteria with packet-like phenotypes, e.g., tetrads; and simple filamentous species to highly differentiated filamentous species.8,9,10 The cyanobacterium Cyanothece sp. ATCC 51142, an isolate from the intertidal zone of the U.S. Gulf Coast,11 was classified as a unicellular species.12 We report a facultative life cycle of Cyanothece sp. in which multicellular filaments alternate with unicellular stages. In a series of experiments, we identified salinity and population density as environmental factors triggering the phenotypic switch between the two morphologies. Then, we used numerical models to test hypotheses regarding the nature of the environmental cues and the mechanisms underlying filament dissolution. While the results predict that the observed response is likely caused by an excreted compound in the medium, we cannot fully exclude changes in nutrient availability (as in Tuomi etal.13 and Matz and Jürgens14). The best-fit modeling results show a nonlinear effect of the compound, which is characteristic of density-dependent sensing systems.15,16 Furthermore, filament fragmentation is predicted to occur by connection cleavage rather than cell death of each alternating cell, which is supported by fluorescent and scanning electron microscopy results. The switch between unicellular and multicellular morphology constitutes an environmentally dependent life cycle that is likely an important step en route to permanent multicellularity.

The extra-embryonic space and the local contour are crucial geometric constraints regulating cell arrangement.

ABSTRACTIn multicellular systems, cells communicate with adjacent cells to determine their positions and fates, an arrangement important for cellular development. Orientation of cell division, cell-cell interactions (i.e. attraction and repulsion) and geometric constraints are three major factors that define cell arrangement. In particular, geometric constraints are difficult to reveal in experiments, and the contribution of the local contour of the boundary has remained elusive. In this study, we developed a multicellular morphology model based on the phase-field method so that precise geometric constraints can be incorporated. Our application of the model to nematode embryos predicted that the amount of extra-embryonic space, the empty space within the eggshell that is not occupied by embryonic cells, affects cell arrangement in a manner dependent on the local contour and other factors. The prediction was validated experimentally by increasing the extra-embryonic space in the Caenorhabditis elegans embryo. Overall, our analyses characterized the roles of geometrical contributors, specifically the amount of extra-embryonic space and the local contour, on cell arrangements. These factors should be considered for multicellular systems.

Deciphering Molecular Mechanism Underlying Self-Flocculation of Zymomonas mobilis for Robust Production.

Zymomonas mobilis metabolizes sugar anaerobically through the Entner-Doudoroff pathway with less ATP generated for lower biomass accumulation to direct more sugar for product formation with improved yield, making it a suitable host to be engineered as microbial cell factories for producing bulk commodities with major costs from feedstock consumption. Self-flocculation of the bacterial cells presents many advantages, such as enhanced tolerance to environmental stresses, a prerequisite for achieving high product titers by using concentrated substrates. ZM401, a self-flocculating mutant developed from ZM4, the unicellular model strain of Z. mobilis, was employed in this work to explore the molecular mechanism underlying this self-flocculating phenotype. Comparative studies between ZM401 and ZM4 indicate that a frameshift caused by a single nucleotide deletion in the poly-T tract of ZMO1082 fused the putative gene with the open reading frame of ZMO1083, encoding the catalytic subunit BcsA of the bacterial cellulose synthase to catalyze cellulose biosynthesis. Furthermore, the single nucleotide polymorphism mutation in the open reading frame of ZMO1055, encoding a bifunctional GGDEF-EAL protein with apparent diguanylate cyclase/phosphodiesterase activities, resulted in the Ala526Val substitution, which consequently compromised in vivo specific phosphodiesterase activity for the degradation of cyclic diguanylic acid, leading to intracellular accumulation of the signaling molecule to activate cellulose biosynthesis. These discoveries are significant for engineering other unicellular strains from Z. mobilis with the self-flocculating phenotype for robust production. IMPORTANCE Stress tolerance is a prerequisite for microbial cell factories to be robust in production, particularly for biorefinery of lignocellulosic biomass to produce biofuels, bioenergy, and bio-based chemicals for sustainable socioeconomic development, since various inhibitors are released during the pretreatment to destroy the recalcitrant lignin-carbohydrate complex for sugar production through enzymatic hydrolysis of the cellulose component, and their detoxification is too costly for producing bulk commodities. Although tolerance to individual stress has been intensively studied, the progress seems less significant since microbial cells are inevitably suffering from multiple stresses simultaneously under production conditions. When self-flocculating, microbial cells are more tolerant to multiple stresses through the general stress response due to enhanced quorum sensing associated with the morphological change for physiological and metabolic advantages. Therefore, elucidation of the molecular mechanism underlying such a self-flocculating phenotype is significant for engineering microbial cells with the unique multicellular morphology through rational design to boost their production performance.

Fundamental control of grade-specific colorectal cancer morphology by Src regulation of ezrin-centrosome engagement.

The phenotypic spectrum of colorectal cancer (CRC) is remarkably diverse, with seemingly endless variations in cell shape, mitotic figures and multicellular configurations. Despite this morphological complexity, histological grading of collective phenotype patterns provides robust prognostic stratification in CRC. Although mechanistic understanding is incomplete, previous studies have shown that the cortical protein ezrin controls diversification of cell shape, mitotic figure geometry and multicellular architecture, in 3D organotypic CRC cultures. Because ezrin is a substrate of Src tyrosine kinase that is frequently overexpressed in CRC, we investigated Src regulation of ezrin and morphogenic growth in 3D CRC cultures. Here we show that Src perturbations disrupt CRC epithelial spatial organisation. Aberrant Src activity suppresses formation of the cortical ezrin cap that anchors interphase centrosomes. In CRC cells with a normal centrosome number, these events lead to mitotic spindle misorientation, perturbation of cell cleavage, abnormal epithelial stratification, apical membrane misalignment, multilumen formation and evolution of cribriform multicellular morphology, a feature of low-grade cancer. In isogenic CRC cells with centrosome amplification, aberrant Src signalling promotes multipolar mitotic spindle formation, pleomorphism and morphological features of high-grade cancer. Translational studies in archival human CRC revealed associations between Src intensity, multipolar mitotic spindle frequency and high-grade cancer morphology. Collectively, our study reveals Src regulation of CRC morphogenic growth via ezrin-centrosome engagement and uncovers combined perturbations underlying transition to high-grade CRC morphology. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The complex multicellular morphology of the food spoilage bacteria Brochothrix thermosphacta strains isolated from ground chicken.

Herein we describe a highly structured, filamentous growth phenotype displayed by an isolate of the food spoilage microorganism Brochothrix thermosphacta. The growth morphology of this B. thermosphacta strain (strain BII) was dependent on environmental factors such as the growth media, incubation temperatures, and the inoculum concentration. Inoculation of cultures in highly dilute suspensions resulted in the formation of isolated, tight aggregates resembling fungal growth in liquid media. This same strain also formed stable, mesh-like structures in 6-well tissue culture plates under specific growth conditions. The complex growth phenotype does not appear to be unique to strain BII but was common among B. thermosphacta strains isolated from chicken. Light and electron micrographs showed that the filaments of multiple BII cells can organize into complex, tertiary structures resembling multistranded cables. Time-lapse microscopy was employed to monitor the development of such aggregates over 18 h and revealed growth originating from short filaments into compact ball-like clusters that appeared fuzzy due to protruding filaments or cables. This report is the first to document this complex filamentous growth phenotype in a wild-type bacterial isolate of B. thermosphacta.

Adhesion of freshwater sponge cells mediated by carbohydrate-carbohydrate interactions requires low environmental calcium.

Marine ancestors of freshwater sponges had to undergo a series of physiological adaptations to colonize harsh and heterogeneous limnic environments. Besides reduced salinity, river-lake systems also have calcium concentrations far lower than seawater. Cell adhesion in sponges is mediated by calcium-dependent multivalent self-interactions of sulfated polysaccharide components of membrane-bound proteoglycans named aggregation factors. Cells of marine sponges require seawater average calcium concentration (10mM) to sustain adhesion promoted by aggregation factors. We demonstrate here that the freshwater sponge Spongilla alba can thrive in a calcium-poor aquatic environment and that their cells are able to aggregate and form primmorphs with calcium concentrations 40-fold lower than that required by marine sponges cells. We also find that their gemmules need calcium and other micronutrients to hatch and generate new sponges. The sulfated polysaccharide purified from S. alba has sulfate content and molecular size notably lower than those from marine sponges. Nuclear magnetic resonance analyses indicated that it is composed of a central backbone of non- and 2-sulfated α- and β-glucose units decorated with branches of α-glucose. Assessments with atomic force microscopy/single-molecule force spectroscopy show that S. alba glucan requires 10-fold less calcium than sulfated polysaccharides from marine sponges to self-interact efficiently. Such an ability to retain multicellular morphology with low environmental calcium must have been a crucial evolutionary step for freshwater sponges to successfully colonize inland waters.

A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns

Synthetic multicellular systems hold promise as models for understanding natural development of biofilms and higher organisms and as tools for engineering complex multi-component metabolic pathways and materials. However, such efforts require tools to adhere cells into defined morphologies and patterns, and these tools are currently lacking. Here, we report a100% genetically encoded synthetic platform for modular cell-cell adhesion in Escherichia coli, which provides control over multicellular self-assembly. Adhesive selectivity is provided by a library of outer membrane-displayed nanobodies and antigens with orthogonal intra-library specificities, while affinity iscontrolled by intrinsic adhesin affinity, competitive inhibition, and inducible expression. We demonstrate the resulting capabilities for quantitative rational design of well-defined morphologies and patterns through homophilic and heterophilic interactions, lattice-like self-assembly, phase separation, differential adhesion, and sequential layering. Compatible with synthetic biology standards, this adhesion toolbox will enable construction of high-level multicellular designs and shed light on the evolutionary transition to multicellularity.

Three Substrains of the Cyanobacterium Anabaena sp. Strain PCC 7120 Display Divergence in Genomic Sequences and hetC Function.

Anabaena sp. strain PCC 7120 is a model strain for molecular studies of cell differentiation and patterning in heterocyst-forming cyanobacteria. Subtle differences in heterocyst development have been noticed in different laboratories working on the same organism. In this study, 360 mutations, including single nucleotide polymorphisms (SNPs), small insertion/deletions (indels; 1 to 3 bp), fragment deletions, and transpositions, were identified in the genomes of three substrains. Heterogeneous/heterozygous bases were also identified due to the polyploidy nature of the genome and the multicellular morphology but could be completely segregated when plated after filament fragmentation by sonication. hetC is a gene upregulated in developing cells during heterocyst formation in Anabaena sp. strain PCC 7120 and found in approximately half of other heterocyst-forming cyanobacteria. Inactivation of hetC in 3 substrains of Anabaena sp. PCC 7120 led to different phenotypes: the formation of heterocysts, differentiating cells that keep dividing, or the presence of both heterocysts and dividing differentiating cells. The expression of P hetZ -gfp in these hetC mutants also showed different patterns of green fluorescent protein (GFP) fluorescence. Thus, the function of hetC is influenced by the genomic background and epistasis and constitutes an example of evolution under way.IMPORTANCE Our knowledge about the molecular genetics of heterocyst formation, an important cell differentiation process for global N2 fixation, is mostly based on studies with Anabaena sp. strain PCC 7120. Here, we show that rapid microevolution is under way in this strain, leading to phenotypic variations for certain genes related to heterocyst development, such as hetC This study provides an example for ongoing microevolution, marked by multiple heterogeneous/heterozygous single nucleotide polymorphisms (SNPs), in a multicellular multicopy-genome microorganism.

Protein kinase C zeta suppresses low- or high-grade colorectal cancer (CRC) phenotypes by interphase centrosome anchoring.

Histological grading provides prognostic stratification of colorectal cancer (CRC) by scoring heterogeneous phenotypes. Features of aggressiveness include aberrant mitotic spindle configurations, chromosomal breakage, and bizarre multicellular morphology, but pathobiology is poorly understood. Protein kinase C zeta (PKCz) controls mitotic spindle dynamics, chromosome segregation, and multicellular patterns, but its role in CRC phenotype evolution remains unclear. Here, we show that PKCz couples genome segregation to multicellular morphology through control of interphase centrosome anchoring. PKCz regulates interdependent processes that control centrosome positioning. Among these, interaction between the cytoskeletal linker protein ezrin and its binding partner NHERF1 promotes the formation of a localized cue for anchoring interphase centrosomes to the cell cortex. Perturbation of these phenomena induced different outcomes in cells with single or extra centrosomes. Defective anchoring of a single centrosome promoted bipolar spindle misorientation, multi‐lumen formation, and aberrant epithelial stratification. Collectively, these disturbances induce cribriform multicellular morphology that is typical of some categories of low‐grade CRC. By contrast, defective anchoring of extra centrosomes promoted multipolar spindle formation, chromosomal instability (CIN), disruption of glandular morphology, and cell outgrowth across the extracellular matrix interface characteristic of aggressive, high‐grade CRC. Because PKCz enhances apical NHERF1 intensity in 3D epithelial cultures, we used an immunohistochemical (IHC) assay of apical NHERF1 intensity as an indirect readout of PKCz activity in translational studies. We show that apical NHERF1 IHC intensity is inversely associated with multipolar spindle frequency and high‐grade morphology in formalin‐fixed human CRC samples. To conclude, defective PKCz control of interphase centrosome anchoring may underlie distinct categories of mitotic slippage that shape the development of low‐ or high‐grade CRC phenotypes. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A high-content image analysis approach for quantitative measurements of chemosensitivity in patient-derived tumor microtissues

Organotypic, three-dimensional (3D) cancer models have enabled investigations of complex microtissues in increasingly realistic conditions. However, a drawback of these advanced models remains the poor biological relevance of cancer cell lines, while higher clinical significance would be obtainable with patient-derived cell cultures. Here, we describe the generation and data analysis of 3D microtissue models from patient-derived xenografts (PDX) of non-small cell lung carcinoma (NSCLC). Standard of care anti-cancer drugs were applied and the altered multicellular morphologies were captured by confocal microscopy, followed by automated image analyses to quantitatively measure phenotypic features for high-content chemosensitivity tests. The obtained image data were thresholded using a local entropy filter after which the image foreground was split into local regions, for a supervised classification into tumor or fibroblast cell types. Robust statistical methods were applied to evaluate treatment effects on growth and morphology. Both novel and existing computational approaches were compared at each step, while prioritizing high experimental throughput. Docetaxel was found to be the most effective drug that blocked both tumor growth and invasion. These effects were also validated in PDX tumors in vivo. Our research opens new avenues for high-content drug screening based on patient-derived cell cultures, and for personalized chemosensitivity testing.

Abstract 4473: SRRM4 drives treatment-induced neuroendocrine transdifferentiation of prostate adenocarcinoma under androgen receptor pathway inhibition

Abstract Treatment-induced neuroendocrine prostate cancer (t-NEPC) is an aggressive subtype of castration-resistant prostate cancer that typically does not respond to AR pathway inhibition (ARPI) and its diagnosis is increasing. To understand its development, we analyzed transcriptomes of prostate tumors to identify t-NEPC-specific RNA splicing. Interestingly, more than 66% of the splice events are predicted to be regulated by the RNA splicing factor, Serine/Arginine Repetitive Matrix 4 (SRRM4). We confirmed in vitro and in vivo that one SRRM4 target gene is the RE1-Silencer Factor (REST), a master regulator of neurogenesis. SRRM4 strongly stimulates adenocarcinoma cells to express t-NEPC biomarkers and this is exacerbated by ARPI. ARPI combined with gain of SRRM4 results in adenocarcinoma cells to assume multicellular spheroid morphology and establishes progressive NEPC xenografts. These SRRM4 actions are further enhanced by loss-of-function of TP53. We conclude that SRRM4 drives t-NEPC progression. This knowledge may guide the development of novel therapeutics aimed at t-NEPC. Citation Format: Yinan Li. SRRM4 drives treatment-induced neuroendocrine transdifferentiation of prostate adenocarcinoma under androgen receptor pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4473. doi:10.1158/1538-7445.AM2017-4473

Quantitative Phenotypic Image Analysis of Three-Dimensional Organotypic Cultures.

Glandular epithelial cells differentiate into three-dimensional (3D) multicellular or acinar structures, particularly when embedded in laminin-rich extracellular matrix (ECM). The spectrum of different multicellular morphologies formed in 3D is a reliable indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. Motile cancer cells may actively invade the matrix, utilizing epithelial, mesenchymal, or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are also very sensitive to small-molecule inhibitors that, e.g., target the actin cytoskeleton. Our strategy is to recapitulate the formation and the histology of complex solid cancer tissues in vitro, based on cell culture technologies that promote the intrinsic differentiation potential of normal and transformed epithelial cells, and also including stromal fibroblasts and other key components of the tumor microenvironment. We have developed a streamlined stand-alone software solution that supports the detailed quantitative phenotypic analysis of organotypic 3D cultures. This approach utilizes the power of automated image analysis as a phenotypic readout in cell-based assays. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of a large number of multicellular structures, which can form a multitude of different organoid shapes, sizes, and textures according to their capacity to engage in epithelial differentiation programs or not. At the far end of this spectrum of tumor-relevant differentiation properties, there are highly invasive tumor cells or multicellular structures that may rapidly invade the surrounding ECM, but fail to form higher-order epithelial tissue structures. Furthermore, this system allows us to monitor dynamic changes that can result from the extraordinary plasticity of tumor cells, e.g., epithelial-to-mesenchymal transition in live cell settings. Furthermore, AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate cell-based organotypic 3D assays in basic research, drug discovery, and target validation.

Evolutionary engineering reveals divergent paths when yeast is adapted to different acidic environments

Tolerance of yeast to acid stress is important for many industrial processes including organic acid production. Therefore, elucidating the molecular basis of long term adaptation to acidic environments will be beneficial for engineering production strains to thrive under such harsh conditions. Previous studies using gene expression analysis have suggested that both organic and inorganic acids display similar responses during short term exposure to acidic conditions. However, biological mechanisms that will lead to long term adaptation of yeast to acidic conditions remains unknown and whether these mechanisms will be similar for tolerance to both organic and inorganic acids is yet to be explored. We therefore evolved Saccharomyces cerevisiae to acquire tolerance to HCl (inorganic acid) and to 0.3M L-lactic acid (organic acid) at pH 2.8 and then isolated several low pH tolerant strains. Whole genome sequencing and RNA-seq analysis of the evolved strains revealed different sets of genome alterations suggesting a divergence in adaptation to these two acids. An altered sterol composition and impaired iron uptake contributed to HCl tolerance whereas the formation of a multicellular morphology and rapid lactate degradation was crucial for tolerance to high concentrations of lactic acid. Our findings highlight the contribution of both the selection pressure and nature of the acid as a driver for directing the evolutionary path towards tolerance to low pH. The choice of carbon source was also an important factor in the evolutionary process since cells evolved on two different carbon sources (raffinose and glucose) generated a different set of mutations in response to the presence of lactic acid. Therefore, different strategies are required for a rational design of low pH tolerant strains depending on the acid of interest.

ChemInform Abstract: Isolation and Synthesis of a Bacterially Produced Inhibitor of Rosette Development in Choanoflagellates.

AbstractAn atypical sulfonolipid (I) with rare syn‐diol configuration that inhibits the conversion from unicellular to a multicellular morphology in choanoflagellates is isolated.

Carbohydrate-Carbohydrate Interactions Mediated by Sulfate Esters and Calcium Provide the Cell Adhesion Required for the Emergence of Early Metazoans

Early metazoans had to evolve the first cell adhesion mechanism addressed to maintain a distinctive multicellular morphology. As the oldest extant animals, sponges are good candidates for possessing remnants of the molecules responsible for this crucial evolutionary innovation. Cell adhesion in sponges is mediated by the calcium-dependent multivalent self-interactions of sulfated polysaccharides components of extracellular membrane-bound proteoglycans, namely aggregation factors. Here, we used atomic force microscopy to demonstrate that the aggregation factor of the sponge Desmapsamma anchorata has a circular supramolecular structure and that it thus belongs to the spongican family. Its sulfated polysaccharide units, which were characterized via nuclear magnetic resonance analysis, consist preponderantly of a central backbone composed of 3-α-Glc1 units partially sulfated at 2- and 4-positions and branches of Pyr(4,6)α-Gal1→3-α-Fuc2(SO3)1→3-α-Glc4(SO3)1→3-α-Glc→4-linked to the central α-Glc units. Single-molecule force measurements of self-binding forces of this sulfated polysaccharide and their chemically desulfated and carboxyl-reduced derivatives revealed that the sulfate epitopes and extracellular calcium are essential for providing the strength and stability necessary to sustain cell adhesion in sponges. We further discuss these findings within the framework of the role of molecular structures in the early evolution of metazoans.

Bio-inspired configurable multiscale extracellular matrix-like structures for functional alignment and guided orientation of cells

Inspired by the hierarchically organized protein fibers in extracellular matrix (ECM) as well as the physiological importance of multiscale topography, we developed a simple but robust method for the design and manipulation of precisely controllable multiscale hierarchical structures using capillary force lithography in combination with an original wrinkling technique. In this study, based on our proposed fabrication technology, we approached a conceptual platform that can mimic the hierarchically multiscale topographical and orientation cues of the ECM for controlling cell structure and function. We patterned the polyurethane acrylate-based nanotopography with various orientations on the microgrooves, which could provide multiscale topography signals of ECM to control single and multicellular morphology and orientation with precision. Using our platforms, we found that the structures and orientations of fibroblast cells were greatly influenced by the nanotopography, rather than the microtopography. We also proposed a new approach that enables the generation of native ECM having nanofibers in specific three-dimensional (3D) configurations by culturing fibroblast cells on the multiscale substrata. We suggest that our methodology could be used as efficient strategies for the design and manipulation of various functional platforms, including well-defined 3D tissue structures for advanced regenerative medicine applications.