Discovering the Molecular Components of Intercellular Junctions��A Historical View

The organization of metazoa is based on the formation of tissues and on tissue-typical functions and these in turn are based on cell–cell connecting structures. In vertebrates, four major forms of cell junctions have been classified and the molecular composition of which has been elucidated in the past three decades: Desmosomes, which connect epithelial and some other cell types, and the almost ubiquitous adherens junctions are based on closely cis-packed glycoproteins, cadherins, which are associated head-to-head with those of the hemi-junction domain of an adjacent cell, whereas their cytoplasmic regions assemble sizable plaques of special proteins anchoring cytoskeletal filaments. In contrast, the tight junctions (TJs) and gap junctions (GJs) are formed by tetraspan proteins (claudins and occludins, or connexins) arranged head-to-head as TJ seal bands or as paracrystalline connexin channels, allowing intercellular exchange of small molecules. The by and large parallel discoveries of the junction protein families are reported.In the year of the bicenturial jubilee of Charles Darwin (born 1809) and his 1859 publication of the concept of natural selection as the decisive driving force of evolution, it is perhaps appropriate to begin this review with the notion that the four major kinds of cell–cell junctions are among the oldest and most important structures contributing to the formation and functional diversification of multilayered metazoan organisms. From mere associations of individual cells, whether protozoan or parazoan, it was the cooperation of the molecular ensembles of these junctions to provide the basis for eumetazoan life. Note, however, that some protocadherin glycoproteins and armadillo-type proteins already occur in certain nonmetazoa (e.g., King et al. 2003; Nichols et al. 2006; for refs. Halbleib and Nelson 2006). In particular, diverse cell–cell junction molecules were—and are—needed to assemble and organize metazoan architecture, notably that of the Bilateria, to allow the formation of the epithelial layers of ectoderm and endoderm, mesoderm-derived tissues, and the segregation of diverse kinds of interstitial cells and the organs derived therefrom. So, it is not so surprising that major kinds of cell junctional structures already exist in the lowest divisions of eumetazoa (e.g., Hobmayer et al. 1996, 2000).In general, metazoan animals possess three intercellular junction systems of the adhering type formed by characteristic transmembrane molecules and proteins that assemble into specific submembranous plaques (

Desmosomes (maculae adherentes) are by far the most abundant junctions in stratified epithelia and have been studied with special impetus by researchers analyzing the cytoskeleton and tissue architecture, and by dermatologists.

Adherens junctions, including the zonulae and fasciae adherentes, have first attracted the special interest of developmental biologists because of their importance in mammalian embryogenesis.

Tight junctions (zonulae occludentes), and in particular the transmembrane molecules involved, had long been sought as this structure was important in controlling the paracellular transport of molecules and particles. Finally, the centrally important tetraspan proteins, desired by a generation of physiologists and membranologists, have been found as the result of careful cell particle fractionation work and immunoelectron microscopy.

Gap junctions (nexus) had always fascinated electron microscopists and crystallographers because of their esthetic paracrystalline substructural order, as well as physiologists studying lateral direct molecule exchange from cell to cell. Here, the stability of the structure itself helped in its isolation and reconstitution in vitro.

Table 1.

Constitutive molecular components of the major types of symmetrical (homotypic) junctions

	Occurrence	Associated filaments	Transmembrane proteins and glycoproteins	Specific plaque proteins
DesmosomesMaculae adherentes	Epithelial cells, various types of cardiomyocytes, meningothelial cells, dendritic reticulum cells of the thymus and lymph follicles	Intermediate-sized filaments (keratins, vimentin, desmin)	Desmogleins 1–4a desmocollins 1–3a	Plakoglobinb desmoplakin I/II plakophilins 1–3a
Adherens junctionsZonulae adherentes Fasciae adherentes Puncta adhaerentia	Epithelial cells, endothelial cells, various types of cardiomyocytes, mesenchymal and neural cells	Microfilaments (actin)	Typical cadherinsa (e.g., E-cadherin, N-cadherin, P-cadherin, VE-cadherin, cadherin-11)	α- and β-Catenin, plakoglobin, protein p120, protein ARVCF, protein p0071, neurojungin (δ-catenin)a, (plakophilin-2c) proteins ZO-1, ZO-2, ZO-3
Tight junctionsZonulae occludentes Fasciae occludentes Puncta adhaerentia	Epithelial cells, endothelial cells	–d	Occludin, claudins 1–24a tricellulin(s)e proteins of the JAMA-group, CARa, ESAMa	Proteins ZO-1, ZO-2, ZO-3, cingulin
Gap junctions (nexus)	All kinds of tissue-forming cells	–	Connexins 1–21a	Proteins ZO-1, ZO-2, ZO-3

Open in a separate windowOnly established, i.e., repeatedly confirmed, constituent structural molecules are mentioned here; some further regulatory or peripherally associated molecules are not listed here but are discussed in the text.^aOne or combinations of a few representatives, with cell type and cell layer specificities.^bProteins of the so-called armadillo family are in italics.^cOnly in specific proliferatively active cells (Rickelt et al. 2009).^dActin microfilaments are seen near some tight junctions but their specific association is not clear.^eThere are at least two mRNA splice products but only one protein has so far been localized.Thus, at about the same time in the late 1970s, when the ultrastructural organization and specificities of the diverse kinds of these junctions had been well determined (for reviews, see Farquhar and Palade 1963; Staehelin 1974), the race by cell biologists to elucidate the molecular compositions of these junctions began. Although this analytical research period is not quite over and a few new junction diamonds may still just lie around the corner, the prime interest of the community of cell biological researchers has already moved on to the next research arena, studying the mechanisms of junction formation and the functions of the junctions. Over the last two decades, these major structural elements of our bodies have also become objects of intense medical research, already with some startling results.In the following, the by and large parallel searches for constituent molecules of the four major categories of cell–cell junctions in vertebrate cells is described. However, only constituents localized and confirmed by different groups and with various methods are mentioned as generally accepted components. This, of course, does not exclude the presence of others for which the available evidence does not yet seem sufficient. Furthermore, certain special types of junctions that do not fit one of the four major categories will be reviewed elsewhere (Franke et al. 2009).     

Bio-sintering processes in hexactinellid sponges: Fusion of bio-silica in giant basal spicules from Monorhaphis chuni

The two sponge classes, Hexactinellida and Demospongiae, comprise a skeleton that is composed of siliceous skeletal elements (spicules). Spicule growth proceeds by appositional layering of lamellae that consist of silica nanoparticles, which are synthesized via the sponge-specific enzyme silicatein. While in demosponges during maturation the lamellae consolidate to a solid rod, the lamellar organization of hexactinellid spicules largely persists. However, the innermost lamellae, near the spicule core, can also fuse to a solid axial cylinder. Similar to the fusion of siliceous nanoparticles and lamella, in several hexactinellid species individual spicules unify during sintering-like processes. Here, we study the different stages of a process that we termed bio-sintering, within the giant basal spicule (GBS) of Monorhaphis chuni. During this study, a major GBS protein component (27 kDa) was isolated and analyzed by MALDI-TOF-MS. The sequences were used to isolate and clone the encoding cDNA via degenerate primer PCR. Bioinformatic analyses revealed a significant sequence homology to silicatein. In addition, the native GBS protein was able to mediate bio-silica synthesis in vitro. We conclude that the syntheses of bio-silica in M. chuni, and the subsequent fusion of nanoparticles to lamellae, and finally to spicules, are enzymatically-driven by a silicatein-like protein. In addition, evidence is now presented that in hexactinellids those fusions involve sintering-like processes.     

Dynein light chain regulates axonal trafficking and synaptic levels of Bassoon

Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. In this study, we report the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. We demonstrate that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon–DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material.     

Sequential estimation for prescribed statistical accuracy in stochastic simulation of biological systems

Stochastic simulation of biological systems proceeds by repeatedly generating sample paths or trajectories of the underlying stochastic process, from which many relevant and important system properties can be obtained. While a great deal of research is targeted towards accelerated trajectory generation, issues concerned with the variability across trajectories are often neglected. Advanced methods for properly quantifying the statistical accuracy and determining a reasonable number of trajectories are hardly addressed formally in the context of biological system simulation, though mathematical statistics provides a large body of powerful theory. We invoke this theory and show how mathematically well-founded sequential estimation approaches serve for systematically generating enough but not too many trajectories for achieving a certain prescribed accuracy. The practical applicability is demonstrated and illustrated by numerical examples through simulation studies of an immigration-death process and a gene regulatory network.     

Chromosomal evolution of the PKD1 gene family in primates

Correction to Kirsch S, Pasantes J, Wolf A, Bogdanova N, Münch C, Pennekamp P, Krawczak M, Dworniczak B, Schempp W: Chromosomal evolution of the PKD1 gene family in primates. BMC Evolutionary Biology 2008, 8 :263 (doi:10.1186/1471-2148-8-263)     

Development and bin mapping of a Rosaceae Conserved Ortholog Set (COS) of markers

Background

Detailed comparative genome analyses within the economically important Rosaceae family have not been conducted. This is largely due to the lack of conserved gene-based molecular markers that are transferable among the important crop genera within the family [e.g. Malus (apple), Fragaria (strawberry), and Prunus (peach, cherry, apricot and almond)]. The lack of molecular markers and comparative whole genome sequence analysis for this family severely hampers crop improvement efforts as well as QTL confirmation and validation studies.

Results

We identified a set of 3,818 rosaceaous unigenes comprised of two or more ESTs that correspond to single copy Arabidopsis genes. From this Rosaceae Conserved Orthologous Set (RosCOS), 1039 were selected from which 857 were used for the development of intron-flanking primers and allele amplification. This led to successful amplification and subsequent mapping of 613 RosCOS onto the Prunus TxE reference map resulting in a genome-wide coverage of 0.67 to 1.06 gene-based markers per cM per linkage group. Furthermore, the RosCOS primers showed amplification success rates from 23 to 100% across the family indicating that a substantial part of the RosCOS primers can be directly employed in other less studied rosaceaous crops. Comparisons of the genetic map positions of the RosCOS with the physical locations of the orthologs in the Populus trichocarpa genome identified regions of colinearity between the genomes of Prunus-Rosaceae and Populus-Salicaceae.

Conclusion

Conserved orthologous genes are extremely useful for the analysis of genome evolution among closely and distantly related species. The results presented in this study demonstrate the considerable potential of the mapped Prunus RosCOS for genome-wide marker employment and comparative whole genome studies within the Rosaceae family. Moreover, these markers will also function as useful anchor points for the genome sequencing efforts currently ongoing in this family as well as for comparative QTL analyses.

     

Pathogenesis of tendinopathies: inflammation or degeneration?

The intrinsic pathogenetic mechanisms of tendinopathies are largely unknown and whether inflammation or degeneration has the prominent role is still a matter of debate. Assuming that there is a continuum from physiology to pathology, overuse may be considered as the initial disease factor; in this context, microruptures of tendon fibers occur and several molecules are expressed, some of which promote the healing process, while others, including inflammatory cytokines, act as disease mediators. Neural in-growth that accompanies the neovessels explains the occurrence of pain and triggers neurogenic-mediated inflammation. It is conceivable that inflammation and degeneration are not mutually exclusive, but work together in the pathogenesis of tendinopathies.     

Complex Evolution of a Y-Chromosomal Double Homeobox 4 (DUX4)-Related Gene Family in Hominoids

The human Y chromosome carries four human Y-chromosomal euchromatin/heterochromatin transition regions, all of which are characterized by the presence of interchromosomal segmental duplications. The Yq11.1/Yq11.21 transition region harbours a peculiar segment composed of an imperfectly organized tandem-repeat structure encoding four members of the double homeobox (DUX) gene family. By comparative fluorescence in situ hybridization (FISH) analysis we have documented the primary appearance of Y-chromosomal DUX genes (DUXY) on the gibbon Y chromosome. The major amplification and dispersal of DUXY paralogs occurred after the gibbon and hominid lineages had diverged. Orthologous DUXY loci of human and chimpanzee show a highly similar structural organization. Sequence alignment survey, phylogenetic reconstruction and recombination detection analyses of human and chimpanzee DUXY genes revealed the existence of all copies in a common ancestor. Comparative analysis of the circumjacent beta-satellites indicated that DUXY genes and beta-satellites evolved in concert. However, evolutionary forces acting on DUXY genes may have induced amino acid sequence differences in the orthologous chimpanzee and human DUXY open reading frames (ORFs). The acquisition of complete ORFs in human copies might relate to evolutionary advantageous functions indicating neo-functionalization. We propose an evolutionary scenario in which an ancestral tandem array DUX gene cassette transposed to the hominoid Y chromosome followed by lineage-specific chromosomal rearrangements paved the way for a species-specific evolution of the Y-chromosomal members of a large highly diverged homeobox gene family.     

Chlamydia pneumoniae Hides inside Apoptotic Neutrophils to Silently Infect and Propagate in Macrophages

Background

Intracellular pathogens have developed elaborate strategies for silent infection of preferred host cells. Chlamydia pneumoniae is a common pathogen in acute infections of the respiratory tract (e.g. pneumonia) and associated with chronic lung sequelae in adults and children. Within the lung, alveolar macrophages and polymorph nuclear neutrophils (PMN) are the first line of defense against bacteria, but also preferred host phagocytes of chlamydiae.

Methodology/Principal Findings

We could show that C. pneumoniae easily infect and hide inside neutrophil granulocytes until these cells become apoptotic and are subsequently taken up by macrophages. C. pneumoniae infection of macrophages via apoptotic PMN results in enhanced replicative activity of chlamydiae when compared to direct infection of macrophages, which results in persistence of the pathogen. Inhibition of the apoptotic recognition of C. pneumoniae infected PMN using PS- masking Annexin A5 significantly lowered the transmission of chlamydial infection to macrophages. Transfer of apoptotic C. pneumoniae infected PMN to macrophages resulted in an increased TGF-ß production, whereas direct infection of macrophages with chlamydiae was characterized by an enhanced TNF-α response.

Conclusions/Significance

Taken together, our data suggest that C. pneumoniae uses neutrophil granulocytes to be silently taken up by long-lived macrophages, which allows for efficient propagation and immune protection within the human host.