Tissues Of Multicellular Organisms Research Articles

The role of apical cell-cell junctions and associated cytoskeleton in mechanotransduction.

Tissues of multicellular organisms are characterised by several types of specialised cell-cell junctions. In vertebrate epithelia and endothelia, tight and adherens junctions (AJ) play critical roles in barrier and adhesion functions, and are connected to the actin and microtubule cytoskeletons. The interaction between junctions and the cytoskeleton is crucial for tissue development and physiology, and is involved in the molecular mechanisms governing cell shape, motility, growth and signalling. The machineries which functionally connect tight and AJ to the cytoskeleton comprise proteins which either bind directly to cytoskeletal filaments, or function as adaptors for regulators of the assembly and function of the cytoskeleton. In the last two decades, specific cytoskeleton-associated junctional molecules have been implicated in mechanotransduction, revealing the existence of multimolecular complexes that can sense mechanical cues and translate them into adaptation to tensile forces and biochemical signals. Here, we summarise the current knowledge about the machineries that link tight and AJ to actin filaments and microtubules, and the molecular basis for mechanotransduction at epithelial and endothelial AJ.

BAMORF: A Novel Computational Method for Predicting the Extracellular Matrix Proteins

Extracellular matrix (ECM) proteins play a major role in the tissues of multicellular organisms. The ECM presents structural support for cells inside a tumor. Meanwhile, it also works homeostatically to mediate the interaction between cells. However, the current bioinformatics tools to predict the ECM proteins seem often fail. This paper introduces a method for predicting the ECM proteins from the protein sequence as well as the molecular characteristics. We report a novel hybrid animal migration optimization and random forest method to predict the ECM protein sequences adapting four various features design methods. Binary animal migration optimization (AMORF) is used to select a near-optimal subset of informative features that are most relevant for the classification. AMORF experiments on a data set, including 145 ECM and 3887 non-ECM proteins. Our algorithm performs 86.4700% accuracy, a sensitive of 84.9655%, a specificity of 86.5261%, a Matthew’s correlation coefficient of 0.3627, and an area under receiver operating characteristic of 0.877804. The results confirm that the proposed method is promising. From the results, we can summarize that it can choose small subsets of features and still increase the classification efficiency.

Abstract A51: Notch4 acts as an oncogenic signal in pancreatic tumorigenesis

Abstract The Notch signaling network is an evolutionarily conserved intercellular signaling pathway which regulates interactions between adjacent cells. The Notch family receptor and its signaling pathways are involved in the development of numerous tissues in multicellular organisms. Recent studies from our Acvr1bflox/flox;LSL-KrasG12D;Pdx1-Cre mice suggest that Notch4 plays an oncogenic role in dictating cell fate and contributes to pancreatic tumorigenesis, particularly in the development of pancreatic intraductal papillary mucious neoplasms (IPMNs). The perinuclear localization of the activated Notch4 (but not other Notch members) intracellular domain (ICD) in the apical cytoplasm of neoplastic cells appeared to be associated with the expansion of the IPMN lesions. The unique role of Notch4 signaling pathway in IPMN development was also confirmed by unbiased RNA-Seq analyses. The deletion of Notch4 in the p16flox/flox;LSL-KrasG12D;Pdx1-Cre mice prolonged survival, further supporting its oncogenic role in pancreatic tumorigenesis. Citation Format: Wanglong Qiu, David D. Suh, Natasha M. Tsay, Simona Chadi, Adam R. Chambers, Peter A. Sims, Carrie J. Shawber, Jan Kitajewski, Helen E. Remotti, Gloria H. Su.{Authors}. Notch4 acts as an oncogenic signal in pancreatic tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A51.

Lateral Interactions Affect Cadherin Binding Kinetics and Function

Adhesion protein interactions between cell membranes (2D) are indispensable for the formation and maintenance of tissues in multicellular organisms. However, binding and organization of membrane proteins within these interfaces is not well understood at the molecular level. To address this question, we use cell-cell adhesion proteins Cadherins as our model. Cadherins form adhesive interactions by binding to identical proteins on opposite cells, but are also hypothesized to form clusters on a single membrane. The protein-protein bonds proposed to stabilize these “lateral” interactions were not detected in any solution binding studies (3D), and their existence as well as their relevance for establishing cell-cell junctions and transducing signals are not clearly known. We investigated the relevance of these lateral interactions using quantitative micropipette measurements of cadherin-mediated cell-cell binding kinetics. We found that classical E-cadherin exhibits kinetics that exhibit two distinct kinetic processes. This ‘biphasic’ kinetics is not consistent with kinetic models based on solution binding data, and suggested that one of the kinetic steps might be due to clustering of the confined proteins. In support of this interpretation, mutating the proposed lateral interaction sites eliminated the kinetic step that we attributed to clustering. We also demonstrated the functional significance of this putative lateral clustering step, by quantifying the leakiness of cell-cell junctions to macromolecules. Cell junctions formed by the cadherin mutants were leakier than WT E-cadherin. Wound healing assays also showed that cells expressing the mutants migrated faster than cells with WT protein. Together, these data reveal that cadherins in “2D” environments undergo additional interactions than in solution, and these alter the assembly of intercellular junctions. These findings might also be relevant to similar class of adhesion proteins, like nectins and CAMs.

Direct roles of SPEECHLESS in the specification of stomatal self-renewing cells.

Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In Arabidopsis, the initiation and proliferation of stomatal lineage cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). SPCH-driven asymmetric and self-renewing divisions allow flexibility in stomatal production and overall organ growth. How SPCH directs stomatal lineage cell behaviors, however, is unclear. Here, we improved the chromatin immunoprecipitation (ChIP) assay and profiled the genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH controls key regulators of cell fate and asymmetric cell divisions and modulates responsiveness to peptide and phytohormone-mediated intercellular communication. Our results delineate the molecular pathways that regulate an essential adult stem cell lineage in plants.

PECM: Prediction of extracellular matrix proteins using the concept of Chou’s pseudo amino acid composition

The extracellular matrix proteins (ECMs) are widely found in the tissues of multicellular organisms. They consist of various secreted proteins, mainly polysaccharides and glycoproteins. The ECMs involve the exchange of materials and information between resident cells and the external environment. Accurate identification of ECMs is a significant step in understanding the evolution of cancer as well as promises wide range of potential applications in therapeutic targets or diagnostic markers. In this paper, an accurate computational method named PECM is proposed for identifying ECMs. Here, we explore various sequence-derived discriminative features including evolutionary information, predicted secondary structure, and physicochemical properties. Rather than simply combining the features which may bring information redundancy and unwanted noises, we use Fisher–Markov selector and incremental feature selection approach to search the optimal feature subsets. Then, we train our model by the technique of support vector machine (SVM). PECM achieves good prediction performance with the ACC scores about 86% and 90% on testing and independent datasets, which are competitive with the state-of-the-art ECMs prediction tools. A web-server named PECM which implements the proposed approach is freely available at http://59.73.198.144:8088/PECM/.

Kolmogorov complexity of epithelial pattern formation: The role of regulatory network configuration

The tissues of multicellular organisms are made of differentiated cells arranged in organized patterns. This organization emerges during development from the coupling of dynamic intra- and intercellular regulatory networks. This work applies the methods of information theory to understand how regulatory network structure both within and between cells relates to the complexity of spatial patterns that emerge as a consequence of network operation. A computational study was performed in which undifferentiated cells were arranged in a two dimensional lattice, with gene expression in each cell regulated by identical intracellular randomly generated Boolean networks. Cell–cell contact signalling between embryonic cells is modeled as coupling among intracellular networks so that gene expression in one cell can influence the expression of genes in adjacent cells. In this system, the initially identical cells differentiate and form patterns of different cell types. The complexity of network structure, temporal dynamics and spatial organization is quantified through the Kolmogorov-based measures of normalized compression distance and set complexity. Results over sets of random networks that operate in the ordered, critical and chaotic domains demonstrate that: (1) ordered and critical networks tend to create the most information-rich patterns; (2) signalling configurations in which cell-to-cell communication is non-directional mostly produce simple patterns irrespective of the internal network domain; and (3) directional signalling configurations, similar to those that function in planar cell polarity, produce the most complex patterns, but only when the intracellular networks function in non-chaotic domains.

Resolving Cadherin Catch Bond Formation at the Single Molecule Level

Cadherins are Ca2+-dependent cell-cell adhesion proteins which play key roles in the formation and maintenance of tissues in multi-cellular organisms. We recently showed that cadherins withstand tensile force by forming catch bonds where their bond lifetimes increase with tensile force. Here we resolve the molecular mechanism of cadherin catch bond formation. Using Steered Molecular Dynamics (SMD) simulations, we show that catch bonds are formed because tensile force re-orients the cadherins such that they lock into a tighter contact. Our simulations also predict that cadherins are unable to lock into a tighter binding conformation if their extracellular regions are made more floppy by titrating Ca2+ ions from solution. Using single molecule force measurements with an Atomic Force Microscope (AFM), we confirm that catch bond formation is abolished as Ca2+ concentration is reduced. Based on these results we propose a molecular model by which cadherins withstand mechanical stress and strengthen cell-cell junctions.

The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C

Author SummaryCentromeres are regions of eukaryotic chromosomes that recruit the kinetochores and are essential for faithful segregation of DNA during all cell divisions. The centromere-specific histone H3 variant CENP-A accumulates at the centromere, defining this region, and is maintained throughout cellular generations by epigenetic mechanisms in most eukaryotes. Previous studies have discovered many factors regulating both the maintenance and assembly of CENP-A at centromeres during mitosis in cultured cells, but the mode of regulation of CENP-A assembly during meiosis and mitosis in animal tissues is unknown. In this study, we use Drosophila melanogaster as an organismal model to investigate the timing and requirements for assembly of CID, the fly CENP-A homolog. We find that that CID is loaded at centromeres during telophase/G1 phase in brain stem and nonstem cells. In male meiosis, CID is loaded in two phases, during the first stages of meiosis I and after the second meiotic division. Meiosis I loading time is also conserved in females. We also report an unprecedented drop in CID levels after meiosis I and before meiosis II, which correlates with the timing of kinetochore reorientation. Additionally, we find that two essential centromere proteins (CAL1 and CENP-C) are necessary for CID assembly and chromosome segregation during meiosis. Our data demonstrate novel differential timing for CENP-A assembly during mitosis and meiosis in the whole organism.

Characterizing the mechanics of cultured cell monolayers

One-cell-thick monolayers are the simplest tissues in multicellular organisms, yet they fulfill critical roles in development and normal physiology. In early development, embryonic morphogenesis results largely from monolayer rearrangement and deformation due to internally generated forces. Later, monolayers act as physical barriers separating the internal environment from the exterior and must withstand externally applied forces. Though resisting and generating mechanical forces is an essential part of monolayer function, simple experimental methods to characterize monolayer mechanical properties are lacking. Here, we describe a system for tensile testing of freely suspended cultured monolayers that enables the examination of their mechanical behavior at multi-, uni-, and subcellular scales. Using this system, we provide measurements of monolayer elasticity and show that this is two orders of magnitude larger than the elasticity of their isolated cellular components. Monolayers could withstand more than a doubling in length before failing through rupture of intercellular junctions. Measurement of stress at fracture enabled a first estimation of the average force needed to separate cells within truly mature monolayers, approximately ninefold larger than measured in pairs of isolated cells. As in single cells, monolayer mechanical properties were strongly dependent on the integrity of the actin cytoskeleton, myosin, and intercellular adhesions interfacing adjacent cells. High magnification imaging revealed that keratin filaments became progressively stretched during extension, suggesting they participate in monolayer mechanics. This multiscale study of monolayer response to deformation enabled by our device provides the first quantitative investigation of the link between monolayer biology and mechanics.

Dynamics of Mutant Cells in Hierarchical Organized Tissues

Most tissues in multicellular organisms are maintained by continuous cell renewal processes. However, high turnover of many cells implies a large number of error-prone cell divisions. Hierarchical organized tissue structures with stem cell driven cell differentiation provide one way to prevent the accumulation of mutations, because only few stem cells are long lived. We investigate the deterministic dynamics of cells in such a hierarchical multi compartment model, where each compartment represents a certain stage of cell differentiation. The dynamics of the interacting system is described by ordinary differential equations coupled across compartments. We present analytical solutions for these equations, calculate the corresponding extinction times and compare our results to individual based stochastic simulations. Our general compartment structure can be applied to different tissues, as for example hematopoiesis, the epidermis, or colonic crypts. The solutions provide a description of the average time development of stem cell and non stem cell driven mutants and can be used to illustrate general and specific features of the dynamics of mutant cells in such hierarchically structured populations. We illustrate one possible application of this approach by discussing the origin and dynamics of PIG-A mutant clones that are found in the bloodstream of virtually every healthy adult human. From this it is apparent, that not only the occurrence of a mutant but also the compartment of origin is of importance.

Tonight, the same old, deadly programme: BH3-only proteins, mitochondria and yeast

Tonight, the same old, deadly programme: BH3-only proteins, mitochondria and yeast

Polycomb purification by in vivo biotinylation tagging reveals cohesin and Trithorax group proteins as interaction partners

The maintenance of specific gene expression patterns during cellular proliferation is crucial for the identity of every cell type and the development of tissues in multicellular organisms. Such a cellular memory function is conveyed by the complex interplay of the Polycomb and Trithorax groups of proteins (PcG/TrxG). These proteins exert their function at the level of chromatin by establishing and maintaining repressed (PcG) and active (TrxG) chromatin domains. Past studies indicated that a core PcG protein complex is potentially associated with cell type or even cell stage-specific sets of accessory proteins. In order to better understand the dynamic aspects underlying PcG composition and function we have established an inducible version of the biotinylation tagging approach to purify Polycomb and associated factors from Drosophila embryos. This system enabled fast and efficient isolation of Polycomb containing complexes under near physiological conditions, thereby preserving substoichiometric interactions. Novel interacting proteins were identified by highly sensitive mass spectrometric analysis. We found many TrxG related proteins, suggesting a previously unrecognized extent of molecular interaction of the two counteracting chromatin regulatory protein groups. Furthermore, our analysis revealed an association of PcG protein complexes with the cohesin complex and showed that Polycomb-dependent silencing of a transgenic reporter depends on cohesin function.

Resolving Desmosomal Cadherin Interactions at the Single Molecule Level

Desmocollin and Desmoglein are essential Ca2+ dependent cell adhesion proteins that mediate the integrity and functional organization of tissues in multi-cellular organisms; however, the molecular interactions that mediate their binding are not understood. It is currently believed that desmosomal cadherin cis-dimers are required to mediate adhesion, evidence for the role of cis-dimers is lacking. Furthermore, it is unclear if desmosomal cadherins interact via homophilic or heterophilic binding and the molecular mechanism by which desmosomal cadherins enhance their bond-strength. To resolve these questions we used Dynamic Force Spectroscopy with an Atomic Force Microscope to measure the homophilic and heterophilic binding of desmosomal cadherin monomers and cis-dimers at the single molecule level. Our measurements showed that desmosomal cadherin monomers alone mediate Ca2+ dependent adhesion, cis-dimers are not essential for adhesion. Furthermore, while desmocollin and desmoglein participate in Ca2+ dependent heterophilic binding, we could not measure Ca2+ dependent homophilic interactions between these cadherins. Dynamic Force Spectroscopy revealed that desmosomal cadherin interactions occur via a double barrier interaction potential; at low loading rates the outer barrier serves as the dominant impedance to unbinding while at higher loading rates the inner barrier serves as the primary kinetic barrier. These experiments resolve the molecular details of desmosomal cadherin binding and suggest a mechanism by which these essential cell adhesion molecules enhance bond strength and lifetime in the presence of force.

Morphogenesis by coupled regulatory networks: Reliable control of positional information and proportion regulation

Based on a non-equilibrium mechanism for spatial pattern formation we study how position information can be controlled by locally coupled discrete dynamical networks, similar to gene regulation networks of cells in a developing multicellular organism. As an example we study the developmental problems of domain formation and proportion regulation in the presence of noise, as well as in the presence of cell flow. We find that networks that solve this task exhibit a hierarchical structure of information processing and are of similar complexity as developmental circuits of living cells. Proportion regulation is scalable with system size and leads to sharp, precisely localized boundaries of gene expression domains, even for large numbers of cells. A detailed analysis of noise-induced dynamics, using a mean-field approximation, shows that noise in gene expression states stabilizes (rather than disrupts) the spatial pattern in the presence of cell movements, both for stationary as well as growing systems. Finally, we discuss how this mechanism could be realized in the highly dynamic environment of growing tissues in multicellular organisms.

Bistable switches control memory and plasticity in cellular differentiation

Development of stem and progenitor cells into specialized tissues in multicellular organisms involves a series of cell fate decisions. Cellular differentiation in higher organisms is generally considered irreversible, and the idea of developmental plasticity in postnatal tissues is controversial. Here, we show that inhibition of mitogen-activated protein kinase (MAPK) in a human bone marrow stromal cell-derived myogenic subclone suppresses their myogenic ability and converts them into satellite cell-like precursors that respond to osteogenic stimulation. Clonal analysis of the induced osteogenic response reveals ultrasensitivity and an "all-or-none" behavior, hallmarks of a bistable switch mechanism with stochastic noise. The response demonstrates cellular memory, which is contingent on the accumulation of an intracellular factor and can be erased by factor dilution through cell divisions or inhibition of protein synthesis. The effect of MAPK inhibition also exhibits memory and appears to be controlled by another bistable switch further upstream that determines cell fate. Once the memory associated with osteogenic differentiation is erased, the cells regain their myogenic ability. These results support a model of cell fate decision in which a network of bistable switches controls inducible production of lineage-specific differentiation factors. A competitive balance between these factors determines cell fate. Our work underscores the dynamic nature of cellular differentiation and explains mechanistically the dual properties of stability and plasticity associated with the process.

Resolving Cadherin Interactions at the Single Molecule Level

The cadherin family of Calcium-dependent cell adhesion proteins are critical for the morphogenesis and functional organization of tissues in multicellular organisms, but the molecular interactions that are at the core of cadherin binding are poorly understood. The generally accepted model is that cadherins adhere in three stages. First, the functional unit of cadherin adhesion is a cis-dimer formed by the binding of the extracellular regions of two cadherins on the same cell surface. Second, formation of low affinity trans interactions between cadherin cis-dimers on opposing cell surfaces initiates cell-cell adhesion, and third lateral clustering of cadherins cooperatively strengthens intercellular adhesion. Direct molecular proof of these cadherin binding states during adhesion is, however, contradictory, and evidence for cooperativity is lacking. We used single molecule structural (Fluorescence Resonance Energy Transfer) and functional (Atomic Force Microscopy) assays to demonstrate directly that cadherin monomers interact via their outermost domain to form trans adhesive complexes. We could not detect the formation of cadherin cis-dimers, but found that increasing the density of cadherin monomers cooperatively increased the probability of trans-adhesive binding. We also resolved the role of Tryptophan-2 a key amino acid in cadherin trans-binding. These results resolve conflicting data on trans- and cis-cadherin binding states, and provide quantitative evidence for cooperativity in trans-cadherin adhesion.

Resolving cadherin interactions and binding cooperativity at the single-molecule level

The cadherin family of Ca(2+)-dependent cell adhesion proteins are critical for the morphogenesis and functional organization of tissues in multicellular organisms, but the molecular interactions between cadherins that are at the core of cell-cell adhesion are a matter of considerable debate. A widely-accepted model is that cadherins adhere in 3 stages. First, the functional unit of cadherin adhesion is a cis dimer formed by the binding of the extracellular regions of 2 cadherins on the same cell surface. Second, formation of low-affinity trans interactions between cadherin cis dimers on opposing cell surfaces initiates cell-cell adhesion. Third, lateral clustering of cadherins cooperatively strengthens intercellular adhesion. Evidence of these cadherin binding states during adhesion is, however, contradictory, and evidence for cooperativity is lacking. We used single-molecule structural (fluorescence resonance energy transfer) and functional (atomic force microscopy) assays to demonstrate directly that cadherin monomers interact via their N-terminal EC1 domain to form trans adhesive complexes. We could not detect the formation of cadherin cis dimers, but found that increasing the density of cadherin monomers cooperatively increased the probability of trans adhesive binding.

Nox enzymes in immune cells

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of phagocytes is a multi-component electron transferase that uses cytoplasmic NADPH to convert molecular oxygen to superoxide anion, consequently delivering reactive oxygen species to the site of invading microorganisms. Together with soluble factors and other phagocyte-derived agents, the resultant toxic species kill and degrade the ingested microbe. Flavocytochrome b (558), a heterodimeric protein composed of gp91 phox and p22 phox, is the membrane component of the NADPH oxidase and was previously thought to be uniquely expressed in phagocytes. Based on structural homology with gp91 phox, recent studies have defined a family of NADPH oxidase proteins (Nox) that is widely distributed throughout the plant and animal kingdoms and in many tissues in multicellular organisms. The goals of this review are to review features of the phagocyte NADPH oxidase that serve as a paradigm for exploiting oxidants for host defense, and to discuss contributions of other Nox proteins to innate immunity.

Cell contact-inhibition signaling as part of wound-healing processes in brain

Cell contact-dependent signaling is a major regulatory mechanism in the organization of developing tissues and in the reorganization (post-injury responses) of specialized tissues in multicellular organisms. In this review we contribute to the further understanding of post-injury recovery processes in adult nervous tissue. We emphasize evidence that supports the involvement of cell contact-inhibition signaling in the cell proliferation, growth and differentiation that occurs during healing and neural reorganization after brain damage.