The syntaxins SYP31 and SYP81 control ER-Golgi trafficking in the plant secretory pathway

Overexpression of the Golgi and endoplasmic reticulum (ER) syntaxins SYP31 and SYP81 strongly inhibits constitutive secretion. By comparing the secreted reporter alpha-amylase with the ER-retained reporter alpha-amylase-HDEL, it was concluded that SYP81 overexpression inhibits both retrograde and anterograde transport, while SYP31 overexpression mainly affected anterograde transport. Of the other interacting SNAREs investigated, only the overexpression of MEMB11 led to an inhibition of protein secretion. Although the position of a fluorescent tag does not influence the correct localization of the fusion protein, only N-terminal-tagged SYP31 retained the ability of the untagged SNARE to inhibit transport. C-terminal-tagged SYP31 failed to exhibit this effect. Overexpression of both wild-type and N-terminal-tagged syntaxins caused standard Golgi marker proteins to redistribute into the ER. Nevertheless, green fluorescent protein (GFP)-SYP31 was still visible as fluorescent punctae, which, unlike SYP31-GFP, were resistant to brefeldin A treatment. Immunogold electron microscopy showed that endogenous SYP81 is not only present at the ER but also in the cis Golgi, indicating that this syntaxin cycles between these two organelles. However, when expressed at non-inhibitory levels, YFP-SYP81 was seen to locate principally to subdomains of the ER. These punctate structures were physically separated from the Golgi, suggesting that they might possibly reflect the position of ER import sites.     

Transitions between epithelial and mesenchymal states and the morphogenesis of the early mouse embryo

Multicellular organisms arise from the generation of different cell types and the organization of cells into tissues and organs. Cells of metazoa display two main phenotypes, the ancestral epithelial state and the recent mesenchymal derivative. Epithelial cells are usually stationary and reside in twodimensional sheets. By contrast mesenchymal cells are loosely packed and can move to new positions, thereby providing a vehicle for cell rearrangement, dispersal and novel cell-cell interactions. Transitions between epithelial and mesenchymal states drive key morphogenetic events in the early vertebrate embryo, including gastrulation, germ layer formation and somitogenesis. The cell behaviors and molecular mechanisms promoting transitions between these two states in the early mouse embryo are discussed in this review.Key words: mouse embryo, EMT, MET, morphogenesis, gastrulation, somitogenesis, epiblast, mesoderm, endoderm, primitive streak, paraxial mesoderm     

Afp::mCherry, a red fluorescent transgenic reporter of the mouse visceral endoderm

Live imaging of genetically encoded fluorescent protein reporters is increasingly being used to investigate details of the cellular behaviors that underlie the large-scale tissue rearrangements that shape the embryo. However, the majority of mouse fluorescent reporter strains are based on the green fluorescent protein (GFP). Mouse reporter strains expressing fluorescent colors other than GFP are therefore valuable for co-visualization studies with GFP, where relative positioning and relationship between two different tissues or compartments within cells are being investigated. Here, we report the generation and characterization of a transgenic Afp::mCherry mouse strain in which cis-regulatory elements from the Alpha-fetoprotein (Afp) locus were used to drive expression of the monomeric Cherry red fluorescent protein. The Afp::mCherry transgene is based on and recapitulates reporter expression of a previously described Afp::GFP strain. However, we note that perdurance of mCherry protein is not as prolonged as GFP, making the Afp::mCherry line a more faithful reporter of endogenous Afp expression. Afp::mCherry transgenic mice expressed mCherry specifically in the visceral endoderm and its derivatives, including the visceral yolk sac, gut endoderm, fetal liver, and pancreas of the embryo. The Afp::mCherry reporter was also noted to be expressed in other documented sites of Afp expression including hepatocytes as well as in pancreas, digestive tract, and brain of postnatal mice.     

Sorting nexins 1 and 2a locate mainly to the TGN

The subcellular localization of the sorting nexins (SNXs) in higher plants is a matter of controversy. Previous confocal laser scanning microscopy (CLSM studies on root cells from a transgenic Arabidopsis line expressing SNX1-GFP have suggested that this SNX is present on an endosome having characteristics of both the trans-Golgi network (TGN) and the multivesicular body (MVB). In contrast, SNX2a locates exclusively to the TGN when transiently expressed in tobacco mesophyll protoplasts. By performing immunogold electron microscopy on cryofixed Arabidopsis roots, we have tried to clarify the situation. Both SNX1-GFP and endogenous SNX2a locate principally to the TGN. Labeling of MVBs could not be confirmed with any certainty.     

Arabidopsis Stromal-derived Factor2 (SDF2) Is a Crucial Target of the Unfolded Protein Response in the Endoplasmic Reticulum

Stresses increasing the load of unfolded proteins that enter the endoplasmic reticulum (ER) trigger a protective response termed the unfolded protein response (UPR). Stromal cell-derived factor2 (SDF2)-type proteins are highly conserved throughout the plant and animal kingdoms. In this study we have characterized AtSDF2 as crucial component of the UPR in Arabidopsis thaliana. Using a combination of biochemical and cell biological methods, we demonstrate that SDF2 is induced in response to ER stress conditions causing the accumulation of unfolded proteins. Transgenic reporter plants confirmed induction of SDF2 during ER stress. Under normal growth conditions SDF2 is highly expressed in fast growing, differentiating cells and meristematic tissues. The increased production of SDF2 due to ER stress and in tissues that require enhanced protein biosynthesis and secretion, and its association with the ER membrane qualifies SDF2 as a downstream target of the UPR. Determination of the SDF2 three-dimensional crystal structure at 1.95 Å resolution revealed the typical β-trefoil fold with potential carbohydrate binding sites. Hence, SDF2 might be involved in the quality control of glycoproteins. Arabidopsis sdf2 mutants display strong defects and morphological phenotypes during seedling development specifically under ER stress conditions, thus establishing that SDF2-type proteins play a key role in the UPR.     

Decreased level of calpain inhibitor activity in kidney from Milan hypertensive rats

Rat kidney contains two different calpain isozymes distinguishable on the basis of their Ca2+ requirement and of their activation mechanisms. The two calpain isozymes are present in comparable amounts in kidney of normotensive and hypertensive rats of the Milan strain. Conversely, the level of the natural inhibitor of calpain is significantly decreased in kidney of hypertensive rats as compared to control normotensive rats. This deficiency is more pronounced in the cortical region than in other kidney fractions. These results taken together with previous observations indicating the existence of an identical defect in red cells from the same hypertensive rat strain, (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Viotti, P., Michetti, M., Duzzi, L., and Bianchi, G. (1986) Biochem. Biophys. Res. Commun. 138, 1370-1375) emphasize the possible role of an unbalanced intracellular proteolytic system in the development of genetically determined hypertension.     

Adaptation of the tRNA population of maize endosperm for zein synthesis.

Maize endosperm, 30 days after pollination is actively synthesizing zein, a storage protein containing high amounts of glutamine. leucine and alanine. Endosperm tRNAs have a higher accepting activity than embryo tRNAs for these three amino acids, but not for some other (control) amino acids. This increase in accepting activity is accompanied by a change in the distribution of the isoaccepting tRNA species corresponding to these three amino acids, but not of the isoacceptors corresponding to some other (control) amino acids. These results are in favor of the theory of functional adaptation of tRNA population.