The Wnt Pathway Controls Cell Death Engulfment,Spindle Orientation,and Migration through CED-10/Rac

Wnt signalling pathways have extremely diverse functions in animals, including induction of cell fates or tumours, guidance of cell movements during gastrulation, and the induction of cell polarity. Wnt can induce polar changes in cellular morphology by a remodelling of the cytoskeleton. However, how activation of the Frizzled receptor induces cytoskeleton rearrangement is not well understood. We show, by an in depth 4-D microscopy analysis, that the Caenorhabditis elegans Wnt pathway signals to CED-10/Rac via two separate branches to regulate modulation of the cytoskeleton in different cellular situations. Apoptotic cell clearance and migration of the distal tip cell require the MOM-5/Fz receptor, GSK-3 kinase, and APC/APR-1, which activate the CED-2/5/12 branch of the engulfment machinery. MOM-5 (Frizzled) thus can function as an engulfment receptor in C. elegans. Our epistatic analyses also suggest that the two partially redundant signalling pathways defined earlier for engulfment may act in a single pathway in early embryos. By contrast, rearrangement of mitotic spindles requires the MOM-5/Fz receptor, GSK-3 kinase, and β-catenins, but not the downstream factors LIT-1/NLK or POP-1/Tcf. Taken together, our results indicate that in multiple developmental processes, CED-10/Rac can link polar signals mediated by the Wnt pathway to rearrangements of the cytoskeleton.     

Nutritional exploitation of dead trunks: another function of cypress knees (Taxodium distichum)?

Summary In one cypress dome in the Everglades National Park, cypress knees seem to develop from vertical root loops that grow along and ramify into dead cypress stumps. Nearly all young root loops emerge close to a decaying stump. The proportion of these associations decreases as the diameter and presumed age of the loops increase. Loop density correlates with the density of dead but not of live trunks. These preliminary findings suggest that the root loops emerge primarily near dead stumps and exploit their nutrients until the stumps rot away and the loops develop into mature knees.     

The effect of some micronutrients and heavy metals on phosphate absorption by maize root cortex segments

The effect of salts (nitrates, chlorides, and sulfates) of microelements, Cd²⁺, Ni²⁺, and Co²⁺ and the effect of boric acid and ammonium molybdate on phosphate uptake by maize root cortex segments were tested. Higher concentration (0.1 mM) of Cu²⁺ salts caused enhancement of phosphate efflux to the extent that efflux was higher than influx. Inhibitory action on phosphate uptake by maize root cortex segments was exerted by following salts: 0.01 mM Cu²⁺ salts (20–30% inhibition), 0.5 mM ZnSO₄ (9.7%), 0.5 and 0.05 mM ZnCl₂ (34.3% and 20.8%), 0.1 mM salts of Cd²⁺, Ni²⁺, Co²⁺ (35–78%). 1 mM FeS_O4 had significant stimulatory effect (92%) on phosphate uptake. Much weaker stimulatory effect was exerted by 1 mM FeCl₃ (14%), 0.05 mM ZnS_O4 (9.6%), 0.005 mM ZnCla and ZnS_O4 (8.4 and 18.5%) and 0.001 mM CdCl2 and CdS_O4 (20.8 and 12.4%). All other tested salts-salts of Mn²⁺ (0.1 and 0.01 mM), 0.01 and 0.001 mM salts of Co²⁺ and Ni²⁺, 0.001 mM salts of Cu²⁺, 0.001–10 mM boric acid, and 0.001–0.1 mM ammonium molybdate left phosphate uptake unaffected.     

Proteolytic and N-Glycan Processing of Human α1-Antitrypsin Expressed in Nicotiana benthamiana

Plants are increasingly being used as an expression system for complex recombinant proteins. However, our limited knowledge of the intrinsic factors that act along the secretory pathway, which may compromise product integrity, renders process design difficult in some cases. Here, we pursued the recombinant expression of the human protease inhibitor α1-antitrypsin (A1AT) in Nicotiana benthamiana. This serum protein undergoes intensive posttranslational modifications. Unusually high levels of recombinant A1AT were expressed in leaves (up to 6 mg g⁻¹ of leaf material) in two forms: full-length A1AT located in the endoplasmic reticulum displaying inhibitory activity, and secreted A1AT processed in the reactive center loop, thus rendering it unable to interact with target proteinases. We found that the terminal protein processing is most likely a consequence of the intrinsic function of A1AT (i.e. its interaction with proteases [most likely serine proteases] along the secretory pathway). Secreted A1AT carried vacuolar-type paucimannosidic N-glycans generated by the activity of hexosaminidases located in the apoplast/plasma membrane. Notwithstanding, an intensive glycoengineering approach led to secreted A1AT carrying sialylated N-glycan structures largely resembling its serum-derived counterpart. In summary, we elucidate unique insights in plant glycosylation processes and show important aspects of postendoplasmic reticulum protein processing in plants.Recombinant protein-based drugs are among the fastest growing areas of development in the pharmaceutical industry. Consequently, there is a demand for exploring new production systems. Plants are increasingly being used for the expression of recombinant proteins, primarily because of their remarkable production speed and yield (for review, see Gleba et al., 2014). The highly conserved secretory pathway between human and plant cells allows similar, if not identical, protein folding, assembly, and posttranslational modifications. Importantly, plants are able to synthesize complex N-glycans, a prerequisite for the in vivo activity of many therapeutically interesting proteins. Despite substantial differences in N-glycan diversity, we and others have shown that plants are highly amendable to glycan engineering and allow proteins with controlled human-type N-glycosylation profiles to be generated (Castilho and Steinkellner, 2012). Moreover, it has even been possible to reconstruct entire human glycosylation pathways, which was shown by the introduction of the human sialylation and O-glycosylation processes in Nicotiana benthamiana (Castilho et al., 2010, 2012). These accomplishments render plants suitable for the production of human proteins that require a complex glycosylation profile.Notwithstanding, to use plants as a versatile expression host for complex human proteins, it is important to fully understand intracellular processes. Particularly detailed knowledge about constraints along the plant cell secretory pathway, including proteolytic processing, is required, because these constraints may compromise protein integrity and quality. Despite major achievements in controlling protein-bound oligosaccharide formation, some plant glycosylation peculiarities are not entirely understood. For example, plant cells synthesize so-called paucimannosidic N-glycans, a type of truncated glycans usually absent in mammals (Lerouge et al., 1998). The biosynthesis and physiological significance of this N-glycan formation has yet to be completely explained (Strasser et al., 2007; Liebminger et al., 2011). Another process not fully understood in plants is subcellular localization of proteins. Aberrant intracellular deposition and as a consequence, incorrect glycosylation of recombinant proteins are often reported. For example, recombinant proteins designed for secretion are frequently also located in the endoplasmic reticulum (ER) and as a consequence, carry oligomannosidic carbohydrates instead of the desired complex-type glycans (Loos et al., 2011; Schneider et al., 2014a). By contrast, KDEL-tagged proteins designed for ER retention are sometimes partially secreted (Van Droogenbroeck et al., 2007; Niemer et al., 2014). How and at which biosynthetic stage these plant-specific peculiarities arise are largely unpredictable, which makes controlled expression of recombinant proteins with features authentically to their natural counterparts a difficult task.One human protein that is pharmaceutically interesting, and thus needed in large amounts at high quality, is α1-antitrypsin (A1AT). This highly glycosylated protease inhibitor from the serpin superfamily interacts with a wide variety of proteases (Gettins, 2002). Like other serpins, A1AT is characterized by an exposed and mobile reactive center loop (RCL) with a Met (³⁵⁸M) residue acting as bait for specific target proteinases (Travis and Salvesen, 1983). The main biological role of plasma A1AT is to prevent excessive action of leukocyte-derived Ser proteinases, especially neutrophil elastase, in the circulatory system (Blank and Brantly, 1994). Therapeutic A1AT used in augmentation therapies is currently purified from pooled human serum, and the treatment can cost up to $100,000 per year per patient (Alkins and O’Malley, 2000). Concerns over the supply and safety of the products have urged searches for alternative recombinant sources of A1AT. Recombinant A1AT has been produced in human and nonhuman cell production systems with limited success (Blanchard et al., 2011; Brinkman et al., 2012; Ross et al., 2012; Lee et al., 2013). The production suffers from two major drawbacks: low expression levels and/or incorrect glycosylation (Garver et al., 1987; Chang et al., 2003; McDonald et al., 2005; Hasannia et al., 2006; Karnaukhova et al., 2006; Plesha et al., 2007; Agarwal et al., 2008; Nadai et al., 2009; Huang et al., 2010; Arjmand et al., 2011; Jha et al., 2012). The mature plasma-derived 52-kD protein has three N-linked glycosylation sites that are mainly decorated with disialylated structures (Kolarich et al., 2006). Sialylated N-glycans are a well-known requisite for the plasma half-life of A1AT (Mast et al., 1991; Lindhout et al., 2011; Lusch et al., 2013); the difficulties associated with obtaining them hamper the generation of biologically active A1AT in many expression systems.Here, we pursued the expression of recombinant human A1AT in glycoengineered N. benthamiana and investigated the system’s ability to generate active sialylated variants. Unusually high amounts of A1AT were obtained using a plant viral-based transient expression system. The inhibitor was efficiently secreted to the intercellular space (IF); however, peptide mapping showed that the secreted A1AT was truncated at both the N and C termini. Mass spectrometry (MS) -based N-glycan analysis of IF-derived A1AT showed that vacuolar typical paucimannosidic N-glycans were present. By expressing A1AT in Arabidopsis (Arabidopsis thaliana) knockout plants lacking β-N-acetylhexosaminidase (HEXO) activity (Liebminger et al., 2011), we showed that paucimannosidic structures are generated by the action of HEXO3 located at the plasma membrane.Coexpression with the mammalian genes necessary for in planta sialylation allowed the synthesis of disialylated A1AT, and sialylation levels could be increased by the synthesis of multiantennary glycans. By contrast, full-length A1AT purified from total soluble extracts exhibited ER-typical oligomannosidic carbohydrates. Using live-cell imaging, a GFP-tagged A1AT fusion did, indeed, exhibit aberrant ER-associated deposition of full-length A1AT. Elastase inhibition assays showed that ER-retained A1AT exhibits inhibitory activity, whereas the IF-derived truncated form was rendered inactive by cleavage within its RCL.     

Effect of FGFR inhibitors on chicken limb development

Fibroblast growth factor (FGF) signalling appears essential for the regulation of limb development, but a full complexity of this regulation remains unclear. Here, we addressed the effect of three different chemical inhibitors of FGF receptor tyrosine kinases (FGFR) on growth and patterning of the chicken wings. The inhibitor PD173074 caused shorter and thinner wing when using lower concentration. Microinjection of higher PD173074 concentrations (25 and 50 mmol/L) into the wing bud at stage 20 resulted in the development of small wing rudiment or the total absence of the wing. Skeletal analysis revealed the absence of the radius but not ulna, deformation of metacarpal bones and/or a reduction of digits. Treatment with PD161570 resembled the effects of PD173074. NF449 induced shortening and deformation of the developing wing with reduced autopodium. These malformed embryos mostly died at the stage HH25–29. PD173074 reduced chondrogenesis also in the limb micromass cultures together with early inhibition of cartilaginous nodule formation, evidenced by lack of sulphated proteoglycan and peanut agglutinin expression. The effect of FGFR inhibition on limb development observed here was unlikely mediated by excessive cell death as none of the inhibitors caused massive apoptosis at low concentrations. More probably, FGFR inhibition decreased both the proliferation and adhesion of mesenchymal chondroprogenitors. We conclude that FGFR signalling contributes to the regulation of the anterior‐posterior patterning of zeugopod during chicken limb development.     

Layered water in crystal interfaces as source for bone viscoelasticity: arguments from a multiscale approach

Extracellular bone material can be characterised as a nanocomposite where, in a liquid environment, nanometre-sized hydroxyapatite crystals precipitate within as well as between long fibre-like collagen fibrils (with diameters in the 100 nm range), as evidenced from neutron diffraction and transmission electron microscopy. Accordingly, these crystals are referred to as ‘interfibrillar mineral’ and ‘extrafibrillar mineral’, respectively. From a topological viewpoint, it is probable that the mineralisations start on the surfaces of the collagen fibrils (‘mineral-encrusted fibrils’), from where the crystals grow both into the fibril and into the extrafibrillar space. Since the mineral concentration depends on the pore spaces within the fibrils and between the fibrils (there is more space between them), the majority of the crystals (but clearly not all of them) typically lie in the extrafibrillar space. There, larger crystal agglomerations or clusters, spanning tens to hundreds of nanometers, develop in the course of mineralisation, and the micromechanics community has identified the pivotal role, which this extrafibrillar mineral plays for tissue elasticity. In such extrafibrillar crystal agglomerates, single crystals are stuck together, their surfaces being covered with very thin water layers. Recently, the latter have caught our interest regarding strength properties (Fritsch et al. 2009 J Theor Biol. 260(2): 230–252) – we have identified these water layers as weak interfaces in the extrafibrillar mineral of bone. Rate-independent gliding effects of crystals along the aforementioned interfaces, once an elastic threshold is surpassed, can be related to overall elastoplastic material behaviour of the hierarchical material ‘bone’. Extending this idea, the present paper is devoted to viscous gliding along these interfaces, expressing itself, at the macroscale, in the well-known experimentally evidenced phenomenon of bone viscoelasticity. In this context, a multiscale homogenisation scheme is extended to viscoelasticity, mineral-cluster-specific creep parameters are identified from three-point bending tests on hydrated bone samples, and the model is validated by statistically and physically independent experiments on partially dried samples. We expect this model to be relevant when it comes to prediction of time-dependent phenomena, e.g. in the context of bone remodelling.