Distinct Cellular Mechanisms Research Articles

Combinatorial Control of Th17 and Th1 Cell Functions by Genetic Variations in Genes Associated With the Interleukin‐23 Signaling Pathway in Spondyloarthritis

Recent genome-wide association studies have revealed numerous genetic associations between specific single-nucleotide polymorphisms (SNPs) and immune-mediated inflammatory diseases. The current challenge is to identify associations of the genetic variants with effector mechanisms implicated in pathogenesis. This study was undertaken to investigate the link between genetic variation at loci associated with spondyloarthritis (SpA) and the effector function of CD4+ T lymphocyte subsets involved in chronic inflammatory disease. Expression of Th17 and Th1 cytokines and transcription factors was measured in CD4+ T cells isolated from patients with SpA. Correlation analyses were performed to assess potential associations of these expression levels with the patient's genotype at loci genetically linked to SpA. The effector functions of Th17 and Th1 cells in patients with SpA were found to be under combinatorial control by multiple SNPs at genes associated with the interleukin-23 (IL-23)/Th17 pathway. Patients with SpA carrying risk-associated alleles of genes in the IL-23/Th17 pathway expressed the highest levels of genes involved in the differentiation and function of Th17 and Th1 cells, whereas the presence of protective alleles was associated with low-level expression of these genes. In contrast, variation at loci that were genetically linked to SpA, but not associated with the IL-23 pathway, did not affect the expression of Th17- and Th1-specific genes, suggesting that these SNPs may contribute to the pathogenesis of SpA through distinct cellular mechanisms. These results show that genetic variations at genes associated with the IL-23 signaling pathway may influence the effector functions of Th17 and Th1 cells in patients with SpA. These findings provide a framework to delineate the mechanisms by which genetic variants contribute to pathology.

Cellular and molecular mechanisms underlying muscular dystrophy

The muscular dystrophies are a group of heterogeneous genetic diseases characterized by progressive degeneration and weakness of skeletal muscle. Since the discovery of the first muscular dystrophy gene encoding dystrophin, a large number of genes have been identified that are involved in various muscle-wasting and neuromuscular disorders. Human genetic studies complemented by animal model systems have substantially contributed to our understanding of the molecular pathomechanisms underlying muscle degeneration. Moreover, these studies have revealed distinct molecular and cellular mechanisms that link genetic mutations to diverse muscle wasting phenotypes.

Cellular mechanisms of developmental and sex differences in the rapid hormonal modulation of a social communication signal

Some gymnotiform electric fish species rapidly modify their electric signal waveforms by altering the action potential (AP) waveforms of their electrocytes, the excitable cells that produce the electric organ discharge (EOD). The fish Brachyhypopomus gauderio alters EOD amplitude and pulse duration as a social signal in accordance with the prevailing social conditions, under the dual regulation of melanocortins and androgens. We show here that B. gauderio uses two distinct cellular mechanisms to change signal amplitude, and its use of these two mechanisms varies with age and sex of the signaler. EOD amplitude and waveform are regulated by the coordinated timing and shaping of two APs generated from two opposing excitable membranes in each electrocyte. The two membranes fire in sequence within 100μs of each other with the second AP being broader than the first. We have shown previously that mature males increase EOD amplitude and duration when melanocortin peptide hormones act directly on electrocytes to selectively broaden the second AP and increase the delay between the two APs by approximately 25μs. Here we show that females selectively broaden only the second AP as males do, but increase amplitude of both APs with no change in delay between them, a previously unreported second mechanism of EOD amplitude change in B. gauderio. Juvenile fish broaden both APs and increase the delay between the APs. Cellular mechanisms of EOD plasticity are therefore shaped during development, presumably by sex steroids, becoming sexually dimorphic at maturity.

Long Non-Coding RNAs in Infection Biology.

Long non-coding RNA have emerged as an increasingly well studied subset of non-coding RNAs (ncRNAs) following their recent discovery in a number of organisms including humans and characterization of their functional and regulatory roles in variety of distinct cellular mechanisms. The recent annotations of long ncRNAs in humans peg their numbers as similar to protein-coding genes. However, despite the rapid advancements in the field the functional characterization and biological roles of most of the long ncRNAs still remain unidentified, although some candidate long ncRNAs have been extensively studied for their roles in cancers and biological phenomena such as X-inactivation and epigenetic regulation of genes. A number of recent reports suggest an exciting possibility of long ncRNAs mediating host response and immune function, suggesting an elaborate network of regulatory interactions mediated through ncRNAs in infection. The present role of long ncRNAs in host-pathogen cross talk is limited to a handful of mechanistically distinct examples. The current commentary chronicles the findings of these reports on the role of long ncRNAs in infection biology and further highlights the bottlenecks and future directions toward understanding the biological significance of the role of long ncRNAs in infection biology.

Actomyosin-based self-organization of cell internalization during C. elegans gastrulation.

BackgroundGastrulation is a key transition in embryogenesis; it requires self-organized cellular coordination, which has to be both robust to allow efficient development and plastic to provide adaptability. Despite the conservation of gastrulation as a key event in Metazoan embryogenesis, the morphogenetic mechanisms of self-organization (how global order or coordination can arise from local interactions) are poorly understood.ResultsWe report a modular structure of cell internalization in Caenorhabditis elegans gastrulation that reveals mechanisms of self-organization. Cells that internalize during gastrulation show apical contractile flows, which are correlated with centripetal extensions from surrounding cells. These extensions converge to seal over the internalizing cells in the form of rosettes. This process represents a distinct mode of monolayer remodeling, with gradual extrusion of the internalizing cells and simultaneous tissue closure without an actin purse-string. We further report that this self-organizing module can adapt to severe topological alterations, providing evidence of scalability and plasticity of actomyosin-based patterning. Finally, we show that globally, the surface cell layer undergoes coplanar division to thin out and spread over the internalizing mass, which resembles epiboly.ConclusionsThe combination of coplanar division-based spreading and recurrent local modules for piecemeal internalization constitutes a system-level solution of gradual volume rearrangement under spatial constraint. Our results suggest that the mode of C. elegans gastrulation can be unified with the general notions of monolayer remodeling and with distinct cellular mechanisms of actomyosin-based morphogenesis.

Canonical Transient Receptor Channel 5 (TRPC5) and TRPC1/4 Contribute to Seizure and Excitotoxicity by Distinct Cellular Mechanisms

Seizures are the manifestation of highly synchronized burst firing of a large population of cortical neurons. Epileptiform bursts with an underlying plateau potential in neurons are a cellular correlate of seizures. Emerging evidence suggests that the plateau potential is mediated by neuronal canonical transient receptor potential (TRPC) channels composed of members of the TRPC1/4/5 subgroup. We previously showed that TRPC1/4 double-knockout (DKO) mice lack epileptiform bursting in lateral septal neurons and exhibit reduced seizure-induced neuronal cell death, but surprisingly have unaltered pilocarpine-induced seizures. Here, we report that TRPC5 knockout (KO) mice exhibit both significantly reduced seizures and minimal seizure-induced neuronal cell death in the hippocampus. Interestingly, epileptiform bursting induced by agonists for metabotropic glutamate receptors in the hippocampal CA1 area is unaltered in TRPC5 KO mice, but is abolished in TRPC1 KO and TRPC1/4 DKO mice. In contrast, long-term potentiation is greatly reduced in TRPC5 KO mice, but is normal in TRPC1 KO and TRPC1/4 DKO mice. The distinct changes from these knockouts suggest that TRPC5 and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms. Furthermore, the reduced seizure and excitotoxicity and normal spatial learning exhibited in TRPC5 KO mice suggest that TRPC5 is a promising novel molecular target for new therapy.

Many ways to make a novel structure: a new mode of sex comb development in Drosophilidae

On macroevolutionary time scales, the same genes can regulate the development of homologous structures through strikingly different cellular processes. Comparing the development of similar morphological traits in closely related species may help elucidate the evolutionary dissociation between pattern formation and morphogenesis. We address this question by focusing on the interspecific differences in sex comb development in Drosophilids. The sex comb is a recently evolved, male-specific structure composed of modified bristles. Previous work in the obscura and melanogaster species groups (Old World Sophophora) has identified two distinct cellular mechanisms that give rise to nearly identical adult morphologies. Here, we describe sex comb development in a species from a more distantly related lineage, the genus Lordiphosa. Although the expression of key regulatory genes is largely conserved in both clades, the cell behaviors responsible for sex comb formation show major differences between Old World Sophophora and Lordiphosa. We suggest that the many-to-one mapping between development and adult phenotype increases the potential for evolutionary innovations.

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis

Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes. Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy. Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization. Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways.

Editorial: Molecular Endocrinology Articles in the Spotlight for May 2012

This month’s issue of Molecular Endocrinology features many excellent articles. We have picked some of the most compelling new research to spotlight: In “Mutations in MCT8 in Patients With AllanHerndon-Dudley-Syndrome Affecting Its Cellular Distribution,” Kersseboom et al (freely available at http://mend.endojournals.org/) observes cell-type dependent effects of mutations within the monocarboxylate transporter 8 (MCT8) thyroid hormone transporter, suggesting tissue-specific effects on thyroid hormone transport in patients with MCT8 mutations. Patients with Allan-Herndon-Dudley-syndrome exhibit severe psychomotor retardation illustrating the importance of MCT8 in maintaining thyroid hormone homeostasis in the brain, particularly during development. The authors identify 2 distinct cellular pathogenic mechanisms of MCT8 mutations: some mutations result in partial or complete loss of thyroid hormone transport activity without affecting cellular MCT8 protein distribution, whereas other mutations mainly disturb protein expression and trafficking. The severity of the phenotype of patients with MCT8 mutations is independent of the underlying pathogenic mechanism, suggesting that even minor perturbations in thyroid hormone transport via MCT8 could have severe consequences on brain development. The report by Katano-Toki et al, “THRAP3 Interacts With HELZ2 and Plays a Novel Role in Adipocyte Differentiation,” used coimmunoprecipitation coupled with liquid chromatography and tandem mass spectrometry to identify Thrap3 (thyroid hormone receptor-associated protein 3), a putative component of the Mediator/tartrate-resistant acid phosphatase complex, as a Helz2 associating protein. The authors also showed that endogenous Helz2, Thrap3, and peroxisome proliferator-activating receptor(PPAR) associate with each other in mature 3T3-L1 adipocytes. Furthermore, Thrap3 and Helz2 are corecruited to PPAR-response elements within the Fabp4/aP2 and Adipoqenhancers in 3T3-L1 cells in response to PPARligands andcooperativelyenhancePPAR-mediatedgenetranscription in vitro. Thus, Thrap3 is a novel PPAR cofactor that plays an important role in the terminal differentiation of adipocytes. “An Essential Role for Insulin and IGF1 Receptors in Regulating Sertoli Cell Proliferation, Testis Size, and FSH Action in Mice” by Pitetti et al provides in vivo evidence that IGFs are the major regulators of Sertoli cell number and testis size in mammals. Both the insulin receptor and the type I IGF1 receptor are required to obtain an optimal number of adult Sertoli cells and testis size. It was also found that the pituitary hormone FSH requires the insulin/IGF1 signaling pathway to mediate its proliferative effects on immature Sertoli cells and thus testis size. These resultssuggestthatgermcellsthemselvesmayregulatethe number of Sertoli cells and thus testis size and daily sperm outputthroughthesecretionofgrowthfactorspromoting Sertoli cell survival and maturation during postnatal testicular development. Congratulations to these researchers as well as those who produced all of the outstanding science in the May issue.

Formation of Axon‐dendrite polarity in situ: Initiation of axons from polarized and non‐polarized cells

Neurons are polarized cells that extend a single axon and several dendrites. Historically, how neurons establish their axon-dendrite polarity has been extensively studied using dissociated hippocampal cells in culture. Although such studies have identified the cellular and molecular mechanisms underlying axon-dendrite polarization, the conclusions have been limited to in vitro conditions. Recent progress using live imaging has enabled us to directly observe axon formation in situ, revealing distinct cellular mechanisms that regulate axon-dendrite polarization in vivo. In this review, we compare the cellular events during axon formation studied in various systems both in vivo and in vitro and discuss possible common mechanisms underlying the axon-dendrite polarization.

Cannabinoid CB1 receptors transactivate multiple receptor tyrosine kinases and regulate serine/threonine kinases to activate ERK in neuronal cells

Signalling networks that regulate the progression of cannabinoid CB(1) receptor-mediated extracellular signal-regulated kinase (ERK) activation in neurons are poorly understood. We investigated the cellular mechanisms involved in CB(1) receptor-stimulated ERK phosphorylation in a neuronal cell model. Murine N18TG2 neuronal cells were used to analyse the effect of specific protein kinase and phosphatase inhibitors on CB(1) receptor-stimulated ERK phosphorylation. The LI-COR In Cell Western assay and immunoblotting were used to measure ERK phosphorylation. The time-course of CB(1) receptor-stimulated ERK activation occurs in three phases that are regulated by distinct cellular mechanisms in N18TG2 cells. Phase I (0-5 min) maximal ERK phosphorylation is mediated by CB(1) receptor-stimulated ligand-independent transactivation of multiple receptor tyrosine kinases (RTKs). Phase I requires G(i/o) βγ subunit-stimulated phosphatidylinositol 3-kinase activation and Src kinase activation and is modulated by inhibition of cAMP-activated protein kinase A (PKA) levels. Src kinase activation is regulated by the protein tyrosine phosphatases 1B and Shp1. The Phase II (5-10 min) rapid decline in ERK phosphorylation involves PKA inhibition and serine/threonine phosphatase PP1/PP2A activation. The Phase III (>10 min) plateau in ERK phosphorylation is mediated by CB(1) receptor-stimulated, ligand-independent, transactivation of multiple RTKs. The complex expression of CB(1) receptor-stimulated ERK activation provides cellular selectivity, modulation of sensitivity to agonists, and coincidence detection with RTK signalling. RTK and PKA pathways may provide routes to novel CB(1) -based therapeutic interventions in the treatment of addictive disorders or neurodegenerative diseases. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Augmentation of Kv4.2-encoded Currents by Accessory Dipeptidyl Peptidase 6 and 10 Subunits Reflects Selective Cell Surface Kv4.2 Protein Stabilization

Rapidly activating and inactivating somatodendritic voltage-gated K(+) (Kv) currents, I(A), play critical roles in the regulation of neuronal excitability. Considerable evidence suggests that native neuronal I(A) channels function in macromolecular protein complexes comprising pore-forming (α) subunits of the Kv4 subfamily together with cytosolic, K(+) channel interacting proteins (KChIPs) and transmembrane, dipeptidyl peptidase 6 and 10 (DPP6/10) accessory subunits, as well as other accessory and regulatory proteins. Several recent studies have demonstrated a critical role for the KChIP subunits in the generation of native Kv4.2-encoded channels and that Kv4.2-KChIP complex formation results in mutual (Kv4.2-KChIP) protein stabilization. The results of the experiments here, however, demonstrate that expression of DPP6 in the mouse cortex is unaffected by the targeted deletion of Kv4.2 and/or Kv4.3. Further experiments revealed that heterologously expressed DPP6 and DPP10 localize to the cell surface in the absence of Kv4.2, and that co-expression with Kv4.2 does not affect total or cell surface DPP6 or DPP10 protein levels. In the presence of DPP6 or DPP10, however, cell surface Kv4.2 protein expression is selectively increased. Further addition of KChIP3 in the presence of DPP10 markedly increases total and cell surface Kv4.2 protein levels, compared with cells expressing only Kv4.2 and DPP10. Taken together, the results presented here demonstrate that the expression and localization of the DPP accessory subunits are independent of Kv4 α subunits and further that the DPP6/10 and KChIP accessory subunits independently stabilize the surface expression of Kv4.2.

How Can Satellite DNA Divergence Cause Reproductive Isolation? Let Us Count the Chromosomal Ways

Satellites are one of the most enigmatic parts of the eukaryotic genome. These highly repetitive, noncoding sequences make up as much as half or more of the genomic content and are known to play essential roles in chromosome segregation during meiosis and mitosis, yet they evolve rapidly between closely related species. Research over the last several decades has revealed that satellite divergence can serve as a formidable reproductive barrier between sibling species. Here we highlight several key studies on Drosophila and other model organisms demonstrating deleterious effects of satellites and their rapid evolution on the structure and function of chromosomes in interspecies hybrids. These studies demonstrate that satellites can impact chromosomes at a number of different developmental stages and through distinct cellular mechanisms, including heterochromatin formation. These findings have important implications for how loci that cause postzygotic reproductive isolation are viewed.

Optimizing Megakaryocyte Polyploidization in Culture by Targeting Multiple Pathways of Cytokinesis

Abstract 4820 Introduction:The large-scale laboratory production of platelets for transfusion purposes using cord blood stem cells is one of goals of stem cell research. One step toward this goal will be to produce and expand megakaryocytes with high ploidy which are capable of releasing higher amounts of platelets than low ploidy cells. Megakaryocyte polyploidization requires a combination of distinct cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. Normal bone marrow megakaryocytes reach a DNA content of 128N, with a modal ploidy of 16N. In contrast, cultured human megakaryocytes have typically reached a maximum ploidy of 16N with the majority of cells at 2N/4N. In this study we combined the inhibition of each of the principle mechanisms driving cytokinesis with the goal of driving polyploidization of cultured CD34+ umbilical cord blood cells. The effects of inhibiting each cytokinesis process was characterized by its effects on cell expansion and ploidy, and the additive or inhibitory effects of combining inhibition of multiple processes was determined. The results of this study provide a strategy to produce high ploidy megakaryocytes and provide new insight into the mechanisms underlying megakaryocyte polyploidization. Methods:Human cord blood derived megakaryocytes were cultured with thrombopoietin (TPO) and stem cell factor (SCF) along with reagents that inhibit different mechanisms of the cytokinesis process: Rho-Rock inhibitor, Y27632 (RRI); Src-inhibitor, SU6656 (SI); Nicotinamide (NIC); Aurora-B inhibitor, ZM447439 (ABI); and Myosin Light Chain Kinase Inhibitor (MLCKI). Reagents were added in one single day (day 8) or on three consecutive days (days 8–10) and ploidy was analyzed on day 11 of culture with PI/RNase. Combinations of reagents were used in order to determine their additive or inhibitory interactions and to maximize megakaryocyte ploidy. Results:Treatment with RRI resulted in the highest megakaryocyte ploidy. Up to 48% of megakaryocytes were high ploidy (≥ 8N), with highest ploidy ≥ 64N (p = 0.0007 vs. control). Treatment with NIC resulted in up to 24% of cells reaching high ploidy, with a maximum ploidy of ≥ 32N (p = 0.003 vs. control). Treatment with SI resulted in up to 24% megakaryocytes reaching high ploidy, with a maximum ploidy of ≥ 32N (p = 0.026 vs. control). Treatment with ABI resulted in up to 28% of megakaryocytes reaching high ploidy, with a maximum ploidy of ≥ 32N (p = 0.018 vs. control). The MLCKI had no effect in final ploidy. The combinations of reagents all significantly increased both percentage of cells reaching high ploidy and the highest ploidy compared to control. However, none of the combinations achieved a more robust effect in final ploidy than the RRI alone. However, the combination of MLCKI with SI, NIC and ABI increased the high ploidy cells up to 51.3% with a maximum ploidy ≥ 64N (p < 0.0001 vs. control) (Table 1). [Display omitted] Conclusion:The RRI proved to be the most effective agent in driving umbilical cord-derived megakaryocyte polyploidization. All other reagents tested, except MLCKI, also moderately increased megakaryocyte ploidy. RRI and MLCKI, both reagents that inhibit the late stages of cytokinesis and furrow formation, resulted in opposite ploidy outcomes, suggesting that RRI acts on ploidy through pathways other than inhibition of myosin activation. While MLCK is active primarily in the late stages of cytokinesis, the Rho/Rock pathway overlaps with other signaling pathways involved in cytokinesis, including src activation in actin polymerization and Aurora kinase activity in microtubule formation. Although inhibition of myosin light chain activation by itself did not appear to drive polyploidization, the combination of MLCKI with other cytokinesis inhibitors increased polyploidization to the same extent as RRI. Our results indicate that induction of high ploidy in cord blood derived megakaryocytes involves a combination of distinct cytokinesis pathways. Disclosures:No relevant conflicts of interest to declare.

Disparate changes in the expression of transient receptor potential vanilloid type 1 receptor mRNA and protein in dorsal root ganglion neurons following local capsaicin treatment of the sciatic nerve in the rat

In situ hybridization, quantitative reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis were applied to study the changes in expression of the major nociceptive ion channel transient receptor potential vanilloid type 1 receptor (TRPV1) after the perineural application of capsaicin or nerve transection. In control rats, quantitative morphometric and statistical analyses of TRPV1 protein and mRNA expression in L5 dorsal root ganglion cells revealed distinct populations of small (type C) and small-to-medium (type B) neurons, which showed very high and moderate levels of TRPV1, whereas larger (type A) neurons mostly did not express this receptor. After either transection or capsaicin treatment of the sciatic nerve, immunohistochemistry and Western blotting demonstrated a massive (up to 80%) decrease in the proportion of TRPV1-immunoreactive neurons and TRPV1 protein at all postoperative survival times. In situ hybridization indicated marked decreases (up to 85%) in the proportion of neurons that expressed TRPV1 mRNA after sciatic nerve transection. In contrast, although perineural treatment with capsaicin resulted in similar substantial decreases in the proportions of type B and C neurons of the L5 dorsal root ganglia 3 days postoperatively, a clear-cut tendency to recovery was observed thereafter. Hence, the proportions of both type B and C neurons expressing TRPV1 mRNA reached up to 70% of the control levels at 30 days postoperatively. In accord with these findings, quantitative RT-PCR revealed a marked and significant recovery in TRPV1 mRNA after perineural capsaicin but not after nerve transection. These observations suggest the involvement of distinct cellular mechanisms in the regulation of the TRPV1 mRNA expression of damaged neurons, specifically triggered by the nature of the injury. The present findings imply that the antinociceptive and anti-inflammatory effects of perineurally applied capsaicin involve distinct changes in neuronal TRPV1 mRNA expression and long-lasting alterations in (post)translational regulation.

Distinct Cellular Mechanisms of Blood Vessel Fusion in the Zebrafish Embryo

Although many of the cellular and molecular mechanisms of angiogenesis have been intensely studied [1], little is known about the processes that underlie vascular anastomosis. We have generated transgenic fish lines expressing an EGFP-tagged version of the junctional protein zona occludens 1 (ZO1) to visualize individual cell behaviors that occur during vessel fusion and lumen formation in vivo. These life observations show that endothelial cells (ECs) use two distinct morphogenetic mechanisms, cell membrane invagination and cord hollowing to generate different types of vascular tubes. During initial steps of anastomosis, cell junctions that have formed at the initial site of cell contacts expand into rings, generating a cellular interface of apical membrane compartments, as defined by the localization of the apical marker podocalyxin-2 (Pdxl2). During the cord hollowing process, these apical membrane compartments are brought together via cell rearrangements and extensive junctional remodeling, resulting in lumen coalescence and formation of a multicellular tube. Vessel fusion by membrane invagination occurs adjacent to a preexisting lumen in a proximal to distal direction and is blood-flow dependent. Here, the invaginating inner cell membrane undergoes concomitant apicobasal polarization and the vascular lumen is formed by the extension of a transcellular lumen through the EC, which forms a unicellular or seamless tube.

Associative, bidirectional changes in neural signaling utilizing NMDA receptor- and endocannabinoid-dependent mechanisms

Persistent, bidirectional changes in synaptic signaling (that is, potentiation and depression of the synapse) can be induced by the precise timing of individual pre- and postsynaptic action potentials. However, far less attention has been paid to the ability of paired trains of action potentials to elicit persistent potentiation or depression. We examined plasticity following the pairing of spike trains in the touch mechanosensory neuron (T cell) and S interneuron (S cell) in the medicinal leech. Long-term potentiation (LTP) of T to S signaling was elicited when the T-cell spike train preceded the S-cell train. An interval 0 to +1 sec between the T- and S-cell spike trains was required to elicit long-term potentiation (LTP), and this potentiation was NMDA receptor (NMDAR)-dependent. Long-term depression (LTD) was elicited when S-cell activity preceded T-cell activity and the interval between the two spike trains was -0.2 sec to -10 sec. This surprisingly broad temporal window involved two distinct cellular mechanisms; an NMDAR-mediated LTD (NMDAR-LTD) when the pairing interval was relatively brief (<-1 sec) and an endocannabinoid-mediated LTD (eCB-LTD) when longer pairing intervals were used (-1 to -10 sec). This eCB-LTD also required activation of a presynaptic transient receptor potential vanilloid (TRPV)-like receptor, presynaptic Ca(2+) release from intracellular stores and activation of voltage-gated Ca(2+) channels (VGCCs). These findings demonstrate that the pairing of spike trains elicits timing-dependent forms of LTP and LTD that are supported by a complex set of cellular mechanisms involving NMDARs and endocannabinoid activation of TRPV-like receptors.

Toll-like receptors

Summary Beginning with the physical barrier presented by the epithelium, infectious agents such as viruses and bacteria encounter an array of cellular and molecular countermeasures that evolved within the host to resist them. Host immune responses are of two types, termed innate and adaptive. Immediate defensive responses, which include inflammation, phagocytosis of pathogens, and recruitment of a variety of immune cells, are employed against all classes of microbe, irrespective of prior exposure, and are collectively termed the innate immune response. Innate immunity is evolutionarily ancient, and selected mechanisms are known to be conserved from plants to humans. In contrast, the adaptive immune response is mobilized over a more protracted timescale, is influenced by prior exposure, and, by virtue of antigen-specific receptors generated through somatic DNA recombination within lymphoid clones, is highly specific at the molecular level, often to the point of specificity for a particular microbial species. Adaptive immunity evolved relatively recently and exists only in vertebrates. In mammals, the immune response encompasses the innate and adaptive responses, and, although cross-talk occurs between them, each can be carried out independently by distinct cellular and molecular mechanisms.

Heterogeneity in the processing of CLCN5 mutants related to Dent disease

Mutations in the electrogenic Cl(-)/H(+) exchanger ClC-5 gene CLCN5 are frequently associated with Dent disease, an X-linked recessive disorder affecting the proximal tubules. Here, we investigate the consequences in Xenopus laevis oocytes and in HEK293 cells of nine previously reported, pathogenic, missense mutations of ClC-5, most of them which are located in regions forming the subunit interface. Two mutants trafficked normally to the cell surface and to early endosomes, and displayed complex glycosylation at the cell surface like wild-type ClC-5, but exhibited reduced currents. Three mutants displayed improper N-glycosylation, and were nonfunctional due to being retained and degraded at the endoplasmic reticulum. Functional characterization of four mutants allowed us to identify a novel mechanism leading to ClC-5 dysfunction in Dent disease. We report that these mutant proteins were delayed in their processing, and that the stability of their complex glycosylated form was reduced, causing lower cell surface expression. The early endosome distribution of these mutants was normal. Half of these mutants displayed reduced currents, whereas the other half showed abolished currents. Our study revealed distinct cellular mechanisms accounting for ClC-5 loss of function in Dent disease.

Glutaminyl cyclase contributes to the formation of focal and diffuse pyroglutamate (pGlu)-Aβ deposits in hippocampus via distinct cellular mechanisms

In the hippocampal formation of Alzheimer’s disease (AD) patients, both focal and diffuse deposits of Aβ peptides appear in a subregion- and layer-specific manner. Recently, pyroglutamate (pGlu or pE)-modified Aβ peptides were identified as a highly pathogenic and seeding Aβ peptide species. Since the pE modification is catalyzed by glutaminyl cyclase (QC) this enzyme emerged as a novel pharmacological target for AD therapy. Here, we reveal the role of QC in the formation of different types of hippocampal pE-Aβ aggregates. First, we demonstrate that both, focal and diffuse pE-Aβ deposits are present in defined layers of the AD hippocampus. While the focal type of pE-Aβ aggregates was found to be associated with the somata of QC-expressing interneurons, the diffuse type was not. To address this discrepancy, the hippocampus of amyloid precursor protein transgenic mice was analysed. Similar to observations made in AD, focal (i.e. core-containing) pE-Aβ deposits originating from QC-positive neurons and diffuse pE-Aβ deposits not associated with QC were detected in Tg2576 mouse hippocampus. The hippocampal layers harbouring diffuse pE-Aβ deposits receive multiple afferents from QC-rich neuronal populations of the entorhinal cortex and locus coeruleus. This might point towards a mechanism in which pE-Aβ and/or QC are being released from projection neurons at hippocampal synapses. Indeed, there are a number of reports demonstrating the reduction of diffuse, but not of focal, Aβ deposits in hippocampus after deafferentation experiments. Moreover, we demonstrate in neurons by live cell imaging and by enzymatic activity assays that QC is secreted in a constitutive and regulated manner. Thus, it is concluded that hippocampal pE-Aβ plaques may develop through at least two different mechanisms: intracellularly at sites of somatic QC activity as well as extracellularly through seeding at terminal fields of QC expressing projection neurons.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-011-0806-2) contains supplementary material, which is available to authorized users.