Tissues Of Multicellular Organisms Research Articles

Single-cell cDNA high-density oligonucleotide microarray analysis: detection of individual cell types and properties in complex biological processes

Gene expression analyses at the single-cell level have been identifying genes that are expressed and play key roles in distinct populations of cells constituting a wide variety of tissues in multicellular organisms. With the development of high-density oligonucleotide microarray platforms covering whole genome transcript information from many organisms, the demand has grown for methods of quantitative global mRNA amplification from a small number of cells, especially single cells. This article reviews briefly some of the pioneering studies in the detection of genes expressed in single cells of interest, with emphasis on the quantitative performance of the amplification methodologies employed. Based on these studies, a highly quantitative global single-cell cDNA amplification procedure has recently been developed that is immediately applicable to high-density oligonucleotide microarray analysis. Indeed, the method has revealed that the inner cell mass cells of mouse early blastocysts show distinctive gene expression indicative of their differentiation towards either primitive endoderm or pluripotent epiblast as early as embryonic day 3.5, 1 day before these cells are morphologically distinguishable. Thus, it has now become more practical to uncover the genome-wide expression properties of any given cell type in desired biological contexts at single-cell resolution, creating unprecedented analytical opportunities in the biomedical sciences.

Tolerance of dividing cells to replication stress in UVB-irradiated Arabidopsis roots: Requirements for DNA translesion polymerases η and ζ

In tissues of multicellular organisms, DNA lesions that block replication can disrupt division of the transiently amplifying (TA) cells and stem cells that drive growth. To study how tissue growth is maintained despite DNA damage, stem cells and other cell types must be clearly identifiable. In plants, root growth depends directly on cell divisions in the root meristem. In Arabidopsis thaliana, cell identities in root meristems are unambiguously defined by position relative to the quiescent center and are readily visualized by microscopy. We evaluated roles of two DNA translesion polymerases, AtPolη (Eta) and AtPolζ (Zeta), in resistance of dividing root cells to a model genotoxin, UVB-radiation. The major UV photoproducts in DNA, cyclobutane pyrimidine dimers (CPDs), were induced to roughly 0.03 CPD/kb by a threshold dose (0.28 kJ m −2) that minimally affected wild-type roots. In roots lacking AtPolη and/or AtPolζ, this dose inhibited cell division and tissue growth and specifically killed stem cells; severities of all three phenotypes increased in the order η − < ζ − < η −ζ −. One to 2 days after CPDs had disappeared from η −ζ − roots, TA cell pools were depleted and there were novel cell divisions in the quiescent center. This delayed “secondary” response to genotoxic stress may reflect changes in the balance of proliferation and differentiation signals. In η −ζ − roots, death of stem cells was substantial even in the absence of irradiation. The lethality of Polζ ablation in mice had confined most previous analyses of Polζ (and concomitant Polη) function to unicellular (yeast) and chicken-cell culture models, so these studies illustrate the advantages afforded by the Arabidopsis-root model system in studies of growth and development of multicellular tissues.

Increased cell-to-cell variation in gene expression in ageing mouse heart

The accumulation of somatic DNA damage has been implicated as a cause of ageing in metazoa. One possible mechanism by which increased DNA damage could lead to cellular degeneration and death is by stochastic deregulation of gene expression. Here we directly test for increased transcriptional noise in aged tissue by dissociating single cardiomyocytes from fresh heart samples of both young and old mice, followed by global mRNA amplification and quantification of mRNA levels in a panel of housekeeping and heart-specific genes. Although gene expression levels already varied among cardiomyocytes from young heart, this heterogeneity was significantly elevated at old age. We had demonstrated previously an increased load of genome rearrangements and other mutations in the heart of aged mice. To confirm that increased stochasticity of gene expression could be a result of increased genome damage, we treated mouse embryonic fibroblasts in culture with hydrogen peroxide. Such treatment resulted in a significant increase in cell-to-cell variation in gene expression, which was found to parallel the induction and persistence of genome rearrangement mutations at a lacZ reporter locus. These results underscore the stochastic nature of the ageing process, and could provide a mechanism for age-related cellular degeneration and death in tissues of multicellular organisms.

Mapping Functional Domains of Chloride Intracellular Channel (CLIC) Proteins in Vivo

Chloride intracellular channel (CLIC) proteins are small proteins distantly related to the omega family of glutathione S-transferases (GSTs). CLIC proteins are expressed in a wide variety of tissues in multicellular organisms and are targeted to specific cellular membranes. Members of this family are capable in vitro of changing conformation from a globular, soluble state to a membrane-inserted state in which they provide chloride conductance. The structural basis for in vivo CLIC protein function, however, is not well understood. We have mapped the functional domains of CLIC family members using an in vivo assay for membrane localization and function of CLIC proteins in the nematode Caenorhabditis elegans. A < 70 amino acid N-terminal domain is a key determinant of membrane localization and function of invertebrate CLIC proteins. This domain, which we term the ‘‘PTM‘‘ domain, named after an amphipathic putative trans membrane helix contained within it, directs distinct C. elegans CLIC homologs to distinct subcellular membranes. We find that within the PTM region, the cysteine residues required for GST-type activity are unnecessary for invertebrate CLIC function, but that specific residues within the proposed transmembrane helix are necessary for correct targeting and protein function. We find that among all tested invertebrate CLIC proteins, function appears to be completely conserved despite striking differences in the charged residues contained within the amphipathic helix. This indicates that these residues do not contribute to anion selectivity as previously suggested. We find that outside the PTM region, the remaining three-quarters of CLIC protein sequence is functionally equivalent not only among vertebrate and invertebrate CLIC proteins, but also among the more distantly related GST-omega and GST-sigma proteins. The PTM region thus provides both targeting information and CLIC functional specificity, possibly adapting GST-type proteins to function as ion channels.

Nuclear Actin-Related Proteins as Epigenetic Regulators of Development

Complex regulatory networks control cell fate and the development of organs and tissues in multicellular organisms. But what mechanisms initiate the necessary global changes in patterns of gene expression? What regulates the regulators of organismal development? The nuclear actin-related proteins (

Evaluation of Chondroitin Sulfate Bioactivity in Hippocampal Neurones and the Astrocyte Cell Line U373: Influence of Position of Sulfate Groups and Charge Density

Chondroitin sulfates are linear polysaccharides of alternating glucuronic acid and N-acetylgalactosamine, sulfated in varying positions. They form the extracellular framework providing the information for the structural establishment of tissues in multicellular organisms. Growth cones of neurones modulate their outgrowth according to signals received from proteoglycans. The exact molecular structures behind these functions are not fully understood, but structural details of the carbohydrate backbone are crucial. In this report we have employed quantitative cytometry on hippocampal neurite outgrowth in the presence of chondroitin sulfate added in solution to determine the influence of the position and density of the sulfate groups of the N-acetyl-D-galactosamine-residues of chondroitin sulfates. It is of profound interest whether externally added chondroitin sulfates can compete with core protein bound chondroitin sulfate to modulate the effects of tissue-synthesized matrix. In series of microscopic images 3 parameters of neuritic outgrowth activity, neurite length, number of neurites and fasciculation (thickness of neurites) are analyzed at concentrations occurring in intact tissues. Fasciculation increased and number of neurites decreased with high di-sulfation. No significant differences on process length reduction were found between the isotypes. Specificity of effects found is emphasized, as no influence on cell proliferation with U373 human astrocyte cell line is detectable, while neurones clearly are inhibited. The IC30 and IC50 values of chondroitin sulfates isoforms are presented for neurones. The data indicate that the soluble fragments from chondroitin sulfate are actively modulating cell development. Besides dosage, sulfation density and position are relevant for effects of chondroitin sulfate in neuronal regenerative activity.

The linear process of somatic evolution.

Cancer is the consequence of an unwanted evolutionary process. Cells receive mutations that alter their phenotype. Especially dangerous are those mutations that increase the net reproductive rate of cells, thereby leading to neoplasia and later to cancer. The standard models of evolutionary dynamics consider well mixed populations of individuals in symmetric positions. Here we introduce a spatially explicit, asymmetric stochastic process that captures the essential architecture of evolutionary dynamics operating within tissues of multicellular organisms. The "linear process" has the property of cancelling out selective differences among cells yet retaining the protective function of apoptosis. This design can slow down the rate of somatic evolution dramatically and therefore delay the onset of cancer.

Fas antigen (CD95) expression and apoptosis in hepatocytes of patients with chronic viral hepatitis.

Apoptosis may be defined as programmed cell death. It is involved in the normal development and homeostasis of tissues in multicellular organisms. An increased or decreased rate of apoptosis may lead to a range of diseases. Fas antigen is a cell-surface receptor that induces apoptotic pathways when treated with Fas ligand or anti-Fas antibody. There is increasing evidence that apoptosis plays an important role in the immunopathogenesis of chronic viral hepatitis, in which the Fas antigen-Fas ligand pathway is particularly involved. Fas antigen expression and apoptosis (apoptotic index) were assayed using flow cytometry in the hepatocytes of 27 patients with chronic viral hepatitis. Histopathological activity, scored by Knodell's histological activity index, other histopathological parameters, serum transaminase values and patient age were then compared with apoptotic index and Fas antigen expression. Apoptosis and Fas antigen expression in hepatocytes were correlated closely with histological activity (grade) of chronic viral hepatitis, but there were no correlations with histological stage, patient age or serum transaminase levels. Apoptosis and its triggering molecule, Fas antigen, induce mechanisms that appear to be associated with the pathogenesis of chronic viral hepatitis.

Cell volume control and signal transduction in apoptosis.

Apoptosis is a physiological form of death in which cells turn-on an intrinsic genetic program that eventually leads to their destruction in a highly regulated manner. This process renders elimination of "unwanted cells" in the body, and accounts for cellular turnover and homeostasis of tissues in multicellular organisms. Consequently, an imbalance in the apoptotic rate in a particular tissue can lead to profound effects in the whole organism. Exposure of cells to apoptotic stimuli induces a rapid loss of cell volume (apoptotic volume decrease) that plays a pivotal role in the decision of a cell to undergo apoptosis. Interestingly, the apoptotic volume decrease is driven by changes in ionic fluxes across the plasma membrane that promote a decrease in the intracellular ions that ultimately also leads to a reduction in intracellular ionic strength. Despite an intensive research effort however, the cellular and molecular mechanisms that trigger changes in cell volume during apoptosis remain poorly understood. Nevertheless, this apoptotic volume decrease has been shown to be a necessary component of the apoptotic cascade and an important point of modulation for the entire cell death process. In this review, we will focus on the importance of the apoptotic volume decrease in the context of signaling and modulation of programmed cell death.

The p85 subunit of phosphoinositide 3-kinase is associated with beta-catenin in the cadherin-based adhesion complex.

Cell adhesion is fundamental to establishing and maintaining the discrete tissues in multicellular organisms. Adhesion must be sufficiently strong to preserve tissue architecture, whilst having the capacity to readily dissociate to permit fundamental processes, such as wound repair, to occur. However, very little is known about the signalling mechanisms involved in temporary down-regulation of cell adhesion to facilitate such processes. Cadherins are the principal mediators of cell-cell adhesion in a wide variety of tissues and species and form multi-protein complexes with cytosolic and cytoskeletal proteins to express their full adhesive capacity. In the present study we report that the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) is associated with the cadherin-based adhesion complex in human epithelial cells. The interaction of p85 with the complex is via beta-catenin. We also show that the interaction of p85 and beta-catenin is direct, involves the N-terminal Src homology domain 2 of p85 and is regulated by tyrosine phosphorylation. These data suggest that PI 3-kinase may play a role in the functional regulation of the cadherin-based adhesion complex.

The p85 subunit of phosphoinositide 3-kinase is associated with β-catenin in the cadherin-based adhesion complex

Cell adhesion is fundamental to establishing and maintaining the discrete tissues in multicellular organisms. Adhesion must be sufficiently strong to preserve tissue architecture, whilst having the capacity to readily dissociate to permit fundamental processes, such as wound repair, to occur. However, very little is known about the signalling mechanisms involved in temporary down-regulation of cell adhesion to facilitate such processes. Cadherins are the principal mediators of cell–cell adhesion in a wide variety of tissues and species and form multi-protein complexes with cytosolic and cytoskeletal proteins to express their full adhesive capacity. In the present study we report that the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) is associated with the cadherin-based adhesion complex in human epithelial cells. The interaction of p85 with the complex is via β-catenin. We also show that the interaction of p85 and β-catenin is direct, involves the N-terminal Src homology domain 2 of p85 and is regulated by tyrosine phosphorylation. These data suggest that PI 3-kinase may play a role in the functional regulation of the cadherin-based adhesion complex.

Autophagy in yeast: mechanistic insights and physiological function.

Unicellular eukaryotic organisms must be capable of rapid adaptation to changing environments. While such changes do not normally occur in the tissues of multicellular organisms, developmental and pathological changes in the environment of cells often require adaptation mechanisms not dissimilar from those found in simpler cells. Autophagy is a catabolic membrane-trafficking phenomenon that occurs in response to dramatic changes in the nutrients available to yeast cells, for example during starvation or after challenge with rapamycin, a macrolide antibiotic whose effects mimic starvation. Autophagy also occurs in animal cells that are serum starved or challenged with specific hormonal stimuli. In macroautophagy, the form of autophagy commonly observed, cytoplasmic material is sequestered in double-membrane vesicles called autophagosomes and is then delivered to a lytic compartment such as the yeast vacuole or mammalian lysosome. In this fashion, autophagy allows the degradation and recycling of a wide spectrum of biological macromolecules. While autophagy is induced only under specific conditions, salient mechanistic aspects of autophagy are functional in a constitutive fashion. In Saccharomyces cerevisiae, induction of autophagy subverts a constitutive membrane-trafficking mechanism called the cytoplasm-to-vacuole targeting pathway from a specific mode, in which it carries the resident vacuolar hydrolase, aminopeptidase I, to a nonspecific bulk mode in which significant amounts of cytoplasmic material are also sequestered and recycled in the vacuole. The general aim of this review is to focus on insights gained into the mechanism of autophagy in yeast and also to review our understanding of the physiological significance of autophagy in both yeast and higher organisms.

Proteases

AbstractThe processes of growth and remodeling of cells and tissues in multicellular organisms require the breakdown of old protein molecules, in concert with the synthesis of new ones. For example, many newly‐synthesized molecules require proteolytic processing to convert them to biologically active forms. Proteolysis can terminate the activity of a protein—e.g., capsases mediate apoptosis, which is a vital step in the life cycle of the cell. Proteolysis contributes to defense systems too, as the recognition of peptide fragments of foreign proteins triggers the immune response. Proteases are the class of enzymes involved in these important reactions. This unit discusses the general categories of proteases, and sets the stage for addition of overview units on cysteine proteases, aspartic proteases, and metalloproteases, as well as protocol units featuring techniques for analyzing mammalian and yeast proteasomes and protease inhibitors, among other topics.

Cadherin-catenin complex: protein interactions and their implications for cadherin function.

Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. In epithelial cells, E-cadherin represents a key molecule in the establishment and stabilization of cellular junctions. On the cellular level, E-cadherin is concentrated at the adherens junction and interacts homophilically with E-cadherin molecules of adjacent cells. Significant progress has been made in understanding the extra- and intracellular interactions of E-cadherin. Recent success in solving the three-dimensional structure of an extracellular cadherin domain provides a structural basis for understanding the homophilic interaction mechanism and the calcium requirement of cadherins. According to the crystal structure, individual cadherin molecules cooperate to form a linear cell adhesion zipper. The intracellular anchorage of cadherins is regulated by the dynamic association with cytoplasmic proteins, termed catenins. The cytoplasmic domain of E-cadherin is complexed with either beta-catenin or plakoglobin (gamma-catenin). Beta-catenin and plakoglobin bind directly to alpha-catenin, giving rise to two distinct cadherin-catenin complexes (CCC). Alpha-catenin is thought to link both CCC's to actin filaments. The anchorage of cadherins to the cytoskeleton appears to be regulated by tyrosine phosphorylation. Phosphorylation-induced junctional disassembly targets the catenins, indicating that catenins are components of signal transduction pathways. The unexpected association of catenins with the product of the tumor suppressor gene APC has led to the discovery of a second, cadherin-independent catenin complex. Two separate catenin complexes are therefore involved in the cross-talk between cell adhesion and signal transduction. In this review we focus on protein interactions regulating the molecular architecture and function of the CCC. In the light of a fundamental role of the CCC during mammalian development and tissue morphogenesis, we also discuss the phenotypes of embryos lacking E-cadherin or beta-catenin.

Extraction of intraerythrocytic malarial parasites by phagocytic cells

Phagocytosis is an intricate process adopted by some unicellular organisms as a feeding behaviour. It has developed in the tissues of multicellular organisms, both vertebrates and invertebrates, as a defence system to confine and eliminate foreign matter and, in this manner, protect the host against infection. During evolutionary development, phagocytic cells have evolved to show greater specificity. Lakshmi Kumaratilake, Antonio Ferrante, Jaliya Kumaratilake and Anthony Allison here describe a unique mechanism used by phogocytic leukocytes to engulf intra-erythrocytic malarial parasites.