The trafficking protein Tmed2/p24β1 is required for morphogenesis of the mouse embryo and placenta

During vesicular transport between the endoplasmic reticulum and the Golgi, members of the TMED/p24 protein family form hetero-oligomeric complexes that facilitate protein-cargo recognition as well as vesicle budding. In addition, they regulate each other's level of expression. Despite analyses of TMED/p24 protein distribution in mammalian cells, yeast, and C. elegans, little is known about the role of this family in vertebrate embryogenesis. We report the presence of a single point mutation in Tmed2/p24β₁ in a mutant mouse line, 99J, identified in an ENU mutagenesis screen for recessive developmental abnormalities. This mutation does not affect Tmed2/p24β₁ mRNA levels but results in loss of TMED2/p24β₁ protein. Prior to death at mid-gestation, 99J homozygous mutant embryos exhibit developmental delay, abnormal rostral-caudal elongation, randomized heart looping, and absence of the labyrinth layer of the placenta. We find that Tmed2/p24β₁ is normally expressed in tissues showing morphological defects in 99J mutant embryos and that these affected tissues lack the TMED2/p24β₁ oligomerization partners, TMED7/p24γ₃ and TMED10/p24δ₁. Our data reveal a requirement for TMED2/p24β₁ protein in the morphogenesis of the mouse embryo and placenta.     

Heat shock protein 70 is required for tabtoxinine-β-lactam-induced cell death in Nicotiana benthamiana

Tabtoxinine-β-lactam (TβL), a non-specific bacterial toxin, is produced by Pseudomonas syringae pv. tabaci, the causal agent of tobacco wildfire disease. TβL causes death of plant cells through the inhibition of glutamine synthetase, which leads to an abnormal accumulation of ammonium ions and the characteristic necrotic wildfire lesions. To better understand the mechanisms involved in TβL-induced cell death, we studied its regulation in Nicotiana benthamiana. TβL-induced lesions, similar to those in controls, could be observed in SGT1-, RAR1- and Hsp90-silenced plants. In contrast, Hsp70-silenced plants showed suppression of lesion formation. Expression of hin1, a marker gene for the hypersensitive response (HR), which is a characteristic of programmed cell death in plants, was strongly induced in controls by TβL treatment but only slightly in Hsp70-silenced plants. However, in these TβL-treated Hsp70-silenced plants, the amount of ammonium ions was considerably increased. Furthermore, the silencing of Hsp70 also suppressed l-methionine sulfoximine-induced cell death and hin1 expression and caused the over-accumulation of ammonium ions. When inoculated directly with P. syringae pv. tabaci, Hsp70-silenced plants showed only reduced symptoms. Our results suggest that the TβL-induced pathway to cell death in N. benthamiana is at least partially similar to HR response, and that Hsp70 might play an essential role in these events.     

IFITM3/STAT3 axis promotes glioma cells invasion and is modulated by TGF-β

Molecular Biology Reports - Glioma is the most aggressive primary brain tumor. We have previously provided evidence that IFITM3 promoted glioma cells migration. However, the mechanism of how IFITM3...     

A cell polarity protein aPKCλ is required for eye lens formation and growth

The organisation of individual cells into a functional three-dimensional tissue is still a major question in developmental biology. Modulation of epithelial cell shape is a critical driving force in forming tissues. This is well illustrated in the eye lens where epithelial cells elongate extensively during their differentiation into fibre cells. It is at the lens equator that epithelial cells elongate along their apical-basal axis. During this process the elongating epithelial cells and their earliest fibre cell derivatives remain anchored at their apical tips, forming a discrete region or modiolus, which we term the lens fulcrum. How this is achieved has received scant attention and is little understood. Here, we show that conditional depletion of aPKCλ, a central effector of the PAR polarity complex, disrupts the apical junctions in elongating epithelial cells so that the lens fulcrum fails to form. This results in disorganised fibre cell alignment that then causes cataract. Interestingly, aPKCλ depletion also promotes epithelial-mesenchymal transition of the lens epithelial cells, reducing their proliferation, leading ultimately to a small lens and microphthalmia. These observations indicate that aPKCλ, a regulator of polarity and apical junctions, is required for development of a lens that is the correct size and shape.     

LmIntegrinβ-PS is required for wing morphogenesis and development in Locusta migratoria

Wings are an important flight organ of insects and their morphogenesis depends on a series of cell-to-cell and cell-to-extracellular matrix interactions. Integrin as a transmembrane protein receptor mediates cell-to-cell adhesion, cell-to-extracellular matrix interactions and signal transduction. In the present study, we characterized an integrin gene that encodes integrinβ-PS protein in Locusta migratoria. LmIntegrinβ-PS is highly expressed in the wing pads and the middle stages of 5th instar nymphs. Immunohistochemical analysis revealed that the LmIntegrinβ-PS protein was localized at the cell base of the two layers of wings. After suppression of LmIntegrinβ-PS by RNA interference, the wing pads or wings were unable to form normally, with a blister wing appearance during nymph to nymph transition and nymph to adult transition. We further found that the dorsal and ventral epidermis of the wings after dsLmIntegrinβ-PS injection were improperly connected and formed huge cavities revealed by hematoxylin and eosin staining. Furthermore, the morphology and structure of the wing cuticle was significantly disturbed which affected the stable arrangement and attachments of the wing epidermis. Moreover, the expression of related cell adhesion genes was significantly decreased in LmIntegrinβ-PS-suppressed L. migratoria, suggesting that LmIntegrinβ-PS is required for the morphogenesis and development of wings during molting by stabilizing cell adhesion and maintaining the cytoskeleton of these cells.     

Wnt/β-catenin signaling is required for development of the exocrine pancreas

Background  

β-catenin is an essential mediator of canonical Wnt signaling and a central component of the cadherin-catenin epithelial adhesion complex. Dysregulation of β-catenin expression has been described in pancreatic neoplasia. Newly published studies have suggested that β-catenin is critical for normal pancreatic development although these reports reached somewhat different conclusions. In addition, the molecular mechanisms by which loss of β-catenin affects pancreas development are not well understood. The goals of this study then were; 1] to further investigate the role of β-catenin in pancreatic development using a conditional knockout approach and 2] to identify possible mechanisms by which loss of β-catenin disrupts pancreatic development. A Pdx1-cre mouse line was used to delete a floxed β-catenin allele specifically in the developing pancreas, and embryonic pancreata were studied by immunohistochemistry and microarray analysis.     

N-terminal region of rice polycomb group protein OsEZ1 is required for OsEZ1–OsFIE2 protein interaction

Polycomb group (PcG) proteins form Polycomb Repressive Complex 2 (PRC2), which regulates seed development by the epigenetic control of gene expression. Interaction assay among Arabidopsis Fertilization-independent-seed2 (FIS) class PcG proteins showed that Fertilization-independent endosperm (FIE) interacts with Medea (MEA), a SET-domain polycomb protein, of which N-terminal region is crucial for the interaction. In this study, rice SET-domain PcG protein OsEZ1, also known as OsiEZ1 in indica rice, was analyzed to identify an interacting domain of OsEZ1 required for OsEZ1–OsFIE2 protein interaction. A series of OsEZ1 deletions were generated and used to determine an interacting domain of OsEZ1 with OsFIE2 using the yeast two-hybrid system. Among OsEZ1 deletions, only OsEZ1?2 and OsEZ1?3 interacted with OsFIE2, indicating that the 155K–169R or N-proximal region of OsEZ1 is crucial for OsFIE2–OsEZ1 interaction. To examine the physiological roles of OsEZ1, 35S:OsEZ1 Arabidopsis lines were generated. OsEZ1 overexpressors exhibited altered seedling growth and seed size, implying that OsEZ1 may play important roles in seedling and seed development.     

Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened

Hedgehog (Hh) signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo), but how vertebrate Smo is activated remains poorly understood. In Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo) and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo) is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that graded Hh signals induce increasing levels of mSmo phosphorylation that fine-tune its ciliary localization, conformation, and activity. We show that mSmo phosphorylation is induced by its agonists and oncogenic mutations but is blocked by its antagonist cyclopamine, and efficient mSmo phosphorylation depends on the kinesin-II ciliary motor. Furthermore, we provide evidence that Hh signaling recruits CK1α to initiate mSmo phosphorylation, and phosphorylation further increases the binding of CK1α and GRK2 to mSmo, forming a positive feedback loop that amplifies and/or sustains mSmo phosphorylation. Hence, despite divergence in their primary sequences and their subcellular trafficking, mSmo and dSmo employ analogous mechanisms for their activation.     

The β subunit gate loop is required for RNA polymerase modification by RfaH and NusG

In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the β gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the β' clamp helices. We hypothesize that upon binding to the clamp helices and the gate loop RfaH bridges the gap across the DNA channel, stabilizing RNAP contacts with nucleic acid and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity.     

Translationally controlled tumor protein (TCTP) is required for TGF-β1 induced epithelial to mesenchymal transition and influences cytoskeletal reorganization

Epithelial-mesenchymal transition (EMT) is a programed course of developmental changes resulting in the acquisition of invasiveness and mobility in cells. In cancer, this course is used by epithelial cells to attain movability. Translationally controlled tumor protein (TCTP) has been extensively characterized following the observation on tumor reversion ensuing its depletion. However, the role of TCTP in cancer progression is still elusive. Here, we demonstrate for the first time that TCTP is a target of transforming growth factor-β1 (TGF-β1), a key regulator of EMT in A549 cells. We here present changes in expression patterns of intermediate filament markers (vimentin and cytokeratin 18a) of EMT following TCTP knockdown or over expression. The TCTP over-expression in cancer cells is associated with mesenchymal characters, while downregulation promotes the epithelial markers in the cells. Interaction of TCTP with β-catenin seems to stabilize β-catenin, preparative to its nuclear localization highlighting a role for β-catenin signaling in EMT. Moreover, the induction of urokinase plasminogen activator (uPA) following ectopic expression of TCTP leads to destabilization of ECM. The cells knocked down for TCTP show diminished invasiveness and migration under TGF-β1 treatment. The present results for the first time demonstrate that TGF-β1 dependent TCTP expression is required for EMT in cells.     

NFkappaB activation is essential for miR-21 induction by TGFβ1 in high glucose conditions

Transforming growth factor beta1 (TGFβ1) is a pleiotropic growth factor with a very broad spectrum of effects on wound healing. Chronic non-healing wounds such as diabetic foot ulcers express reduced levels of TGFβ1. On the other hand, our previous studies have shown that the microRNA miR-21 is differentially regulated in diabetic wounds and that it promotes migration of fibroblast cells. Although interplay between TGFβ1 and miR-21 are studied in relation to cancer, their interaction in the context of chronic wounds has not yet been investigated. In this study, we examined if TGFβ1 could stimulate miR-21 in fibroblasts that are subjected to high glucose environment. MiR-21 was, in fact, induced by TGFβ1 in high glucose conditions. The induction by TGFβ1 was dependent on NFκB activation and subsequent ROS generation. TGFβ1 was instrumental in degrading the NFκB inhibitor IκBα and facilitating the nuclear translocation of NFκB p65 subunit. EMSA studies showed enhanced DNA binding activity of NFκB in the presence of TGFβ1. ChIP assay revealed binding of p65 to miR-21 promoter. NFκB activation was also required for the nuclear translocation of Smad 4 protein and subsequent direct interaction of Smad proteins with primary miR-21 as revealed by RNA-IP studies. Our results show that manipulation of TGFβ1–NFκB–miR-21 pathway could serve as an innovative approach towards therapeutics to heal diabetic ulcers.