Planar polarity: out of joint?

Epithelial structures, such as the wing hairs and ommatidia in Drosophila, are aligned in the plane of the epithelium. This planar polarity requires the transmembrane receptor Frizzled. Recent studies have shed new light on mechanisms that could be involved in generating or transducing the polarity signal.     

Amphisomes: out of the autophagosome shadow?

EMBO J (2013) 32: 3130–3144 doi: 10.1038/emboj.2013.233; published online November012013Amphisomes are intermediate organelles, formed during autophagy through the fusion between autophagosomes and endosomes. Complex multivesicular vacuoles that resemble amphisomes have been observed in various cell types, but whether they have cellular roles other than being a precursor structure is still enigmatic. While autophagy-related (ATG) proteins interact with the endocytic pathways in other processes different from autophagy, Patel and colleagues now report that these factors come together to generate amphisome-like compartments that regulate mucin secretion in goblet cells.ATG and endosomal proteins have been linked to secretion, and the specific loss of them impairs the function of different secretory cell types (Jung et al, 2008; DeSelm et al, 2011; Ushio et al, 2011; Sasidharan et al, 2012). ATG proteins have also been shown to interact with the endocytic pathway in few situations that do not involve autophagy. For example in phagocytic cells, the surface of bacteria-containing phagosomes acquires LC3/Atg8 through the concerted action of a subpopulation of ATG proteins. This process, which has been termed LC3-associated phagocytosis (LAP), promotes the fusion of phagosomes with lysosomes (Sanjuan et al, 2007). Something similar occurs during entotic cell death, an engulfment programme leading to the elimination of cells into lysosomes. The entotic vacuole membranes surrounding the internalized cells also recruit LC3 through a mechanism that depends on several ATG proteins, but not on autophagosome formation (Florey et al, 2011).In their work aimed to understand the function of ATG proteins in goblet cells, Patel et al (2013) show that the autophagy and endocytic machinery converge at the amphisomes to promote the secretion of mucins. In the gastrointestinal tract, secretory cells have a crucial role in providing the mucus barrier that protects against intestinal pathogens. Mucins, the main components of the mucus, are produced in goblet cells where large polymers of these highly glycosylated proteins are packed into secretory granules that accumulate at the apical surface. The release of these mucin granules relies on a series of cellular events that are tightly coordinated. Patel et al (2013) show that knockout mice lacking ATG5 in the intestinal epithelium, that is, Atg5VC mice, exhibit both a dramatic accumulation of mucin granules in goblet cells and a diminished mucus secretion. Taking advantage of a newly developed in vitro system to culture and differentiate intestinal epithelial stem cells into secretory goblet cells, the authors also demonstrate that the ablation of other ATG proteins causes the same phenotype showing that the autophagy machinery is required for mucin secretion in these specialized cells (Patel et al, 2013). Interestingly, ATG proteins affect the functionality of another gastrointestinal secretory lineage, the Paneth cells. Paneth cells homozygous for the atg16L1 risk allele, associated with Crohn disease, produce less secretory granules than in controls (Cadwell et al, 2008). This suggests that although ATG proteins regulate secretion in the two most abundant secretory lineages in the intestinal tract, two different mechanisms are probably involved.A microarray analysis of mRNA from Atg5VC mouse colonic epithelial cells revealed a possible alteration in the endocytic pathway. Indeed, blocking endocytosis also provoked an accumulation of mucin granules. While LC3B has been previously found on the surface of secretory granules (Ushio et al, 2011; Ishibashi et al, 2012), immuno-electron microscopy of wild-type mouse intestinal tissue revealed a distribution of LC3B not on mucin granules, but on multivesicular vacuoles positive for several endosomal proteins (Patel et al, 2013). Because of the morphological and molecular characteristics of these compartments, it appears that the ATG proteins together with the endocytic pathway regulate secretion in goblet cells by converging in what could be a new amphisome-like organelle (Figure 1).Open in a separate windowFigure 1Schematic representation for the regulated secretion of mucin granules by amphisome-like structures in goblet cells. ROS generated by NADPH oxidases promote the fusion of LC3-positive vesicles with endosomes marked by Rab5 and containing the NADPH oxidase subunit p22phox. The resulting amphisomes-like organelles are decorated with LC3, endosomal proteins (Rab5, Rab7 and EEA1) and p22phox and localize near the mucin granules. The formation of these copartments probably prolong and/or enhance the production of ROS by the NADPH oxidase, which in turn increases the levels of cytoplasmic calcium through an unknown mechanism leading to the release of the mucin granules.NADPH oxidases are known to be present in endosomes, and NADPH oxidase-generated reactive oxygen species (ROS) are necessary for LC3 recruitment to phagosomes.(Huang et al, 2009). Patel et al (2013) thus explored whether these enzymes played a role in mucin granule secretion in goblet cells. Indeed, expression of a mutant form of p22phox, a transmembrane subunit of several NADPH oxidase complexes, altered the exocytosis of these carriers. Moreover, p22phox was found to localize to Rab5-positive endosomes and also with the observed amphisome-like structures (Figure 1). Because a mutant form of p22phox also caused a misslocalization of both LC3 and the early-endosomal marker protein EEA1, the obvious conclusion was that ROS production by endosomes is necessary to trigger the formation of the amphisome-like organelles via the acquisition of the ATG machinery (Figure 1). Interestingly, addition of H₂O₂ that mimics ROS generation was able to induce mucin granule exocytosis in the p22phox mutant cells, showing that ROS was also required to regulate secretion in goblet cells (Patel et al, 2013). Furthermore, H₂O₂ bypassed as well the mucin granule secretion defect in autophagy and endocytosis-deficient goblet cells through an increase of cytosolic calcium levels (Patel et al, 2013). This, together with the observation that the loss of ATG5 and the block of the endocytic pathway impair the production of ROS has led Patel et al (2013) to propose that amphisome-like organelles are a signalling platform, where NADPH oxidase-driven ROS production promotes the release of the mucin granules.Amphisomes have been characterized and defined as autophagic vacuoles formed upon fusion between autophagosomes and endosomes. Given that ATG and endosomal proteins converge in multivesicular and/or vacuolar compartments resembling amphisomes in cellular processes independent of autophagy, one could consider to use the term amphisomes to describe a more heterogenous and ampler population of unnamed compartments where part of the autophagy and endosomal machineries co-localize. Based on this consideration, the study by Patel et al (2013) has identified an amphisome-like structure where molecular events interconnect to trigger granule secretion. While their work adds to the still limited number of non-degradative roles of the autophagic pathway, which include unconventional secretion (Subramani and Malhotra, 2013), it is one of the first reports highlighting that amphisomes (or any autophagosomal intermediate structure) could be more than just a transport intermediate, and at least in goblet cells, they could act as a platform where signals integrating some aspects of the cell physiology are elicited.Though it remains to be establish whether the organelles described by Patel et al (2013) are indeed amphisomes, especially as they are formed by fusion of endosomes with LC3-positive single-membrane vesicles rather than LC3-positive double-membrane autophagosomes, their study raises some intriguing questions. Are these compartments persistent or will they eventually fuse with lysosomes? Why has the cell opted to signal from amphisomes and not from endosomes, where the NADPH oxidases are normally present? Maybe the answer to these questions is hidden in the transient life of amphisomes. In the most classical signalling pathways, the transduction cascade amplifies the initial cue but it also turn it off subsequently through negative feedback loops. This permits to precisely modulate the signal output temporally (and locally). The amphisome-like structures observed in goblet cells could also act as the molecular switch for the signal-stimulating mucin granule secretion. The ROS generated initially from endosomes would trigger the recruitment of LC3 through vesicle fusion events, and the production of this second messenger will be prolonged and/or enhanced in the resulting amphisomes-like structure, leading to a stimulation of mucin granule exocytosis (Figure 1). The subsequent fusion of the amphisomes with lysosomes could lead to the termination of the signal. Other scenarios, however, cannot be excluded like, for example, the delivery of a protein enhancing the NADPH oxidase activity to the endosomes by the LC3-positive vesicles.While these are just hypotheses, it is clear that Patel et al (2013) have opened a window on a new and unexplored area of the autophagy field. Future investigations will tell us whether what observed in goblet cells is a unique situation or the intermediate organelles characterizing autophagy can carry out cellular functions different from the one delivering unwanted structures into the lysosome interior for degradation, including to serve as signalling platforms.     

Stress-induced morphogenic responses: growing out of trouble?

Plants exposed to sub-lethal abiotic stress conditions exhibit a broad range of morphogenic responses. Despite the diversity of phenotypes, a generic 'stress-induced morphogenic response' can be recognized that appears to be carefully orchestrated and comprises three components: (a) inhibition of cell elongation, (b) localized stimulation of cell division and (c) alterations in cell differentiation status. It is hypothesized that the similarities in the morphogenic responses induced by distinct stresses, reflect common molecular processes such as increased ROS-production and altered phytohormone transport and/or metabolism. The stress-induced morphogenic response (SIMR) is postulated to be part of a general acclimation strategy, whereby plant growth is redirected to diminish stress exposure.     

Review: transport of tRNA out of the nucleus-direct channeling to the ribosome?

Although tRNA was the first substrate whose export from the nuclei of eukaryotic cells had been shown to be carrier-mediated and active, it has only been in the last 2 years that the first mechanistic details of this nucleocytoplasmic transport pathway have begun to emerge. A member of the importin/karyopherin beta superfamily, Los1p in yeast and Xpo-t in vertebrates, has been shown to export tRNA in cooperation with the small GTPase Ran (Gsp1p) from the nucleus into the cytoplasm, where tRNA becomes available for translation. However, Los1p is not essential for viability in yeast cells, suggesting that alternative tRNA export pathways exist. Recent results show that aminoacylation and a translation factor are also required for efficient nuclear tRNA export. Thus, protein translation and nuclear export of tRNA appear to be coupled processes.     

Penetrability of the inner mucus layer: who is out there?

Large numbers of microorganisms colonise the skin and mucous membranes of animals, with their highest density in the lower gastrointestinal tract. The impact of these microbes on the host can be demonstrated by comparing animals (usually mice) housed under germ‐free conditions, or colonised with different compositions of microbes. Inbreeding and embryo manipulation programs have generated a wide variety of mouse strains with a fixed germ‐line (isogenic) and hygiene comparisons robustly show remarkably strong interactions between the microbiota and the host, which can be summarised in three axioms. (I) Live microbes are largely confined to their spaces at body surfaces, provided the animal is not suffering from an infection. (II) There is promiscuous molecular exchange throughout the host and its microbiota in both directions 1 . (III) Every host organ system is profoundly shaped by the presence of body surface microbes. It follows that one must draw a line between live microbial and host “spaces” (I) to understand the crosstalk (II and III) at this interesting interface of the host‐microbial superorganism. Of course, since microbes can adapt to very different niches, there has to be more than one line. In this issue of EMBO Reports, Johansson and colleagues have studied mucus, which is the main physical frontier for most microbes in the intestinal tract: they report how different non‐pathogenic microbiota compositions affect its permeability and the functional protection of the epithelial surface 2 .     

The genomics of local adaptation in trees: are we out of the woods yet?

There is substantial interest in uncovering the genetic basis of the traits underlying adaptive responses in tree species, as this information will ultimately aid conservation and industrial endeavors across populations, generations, and environments. Fundamentally, the characterization of such genetic bases is within the context of a genetic architecture, which describes the mutlidimensional relationship between genotype and phenotype through the identification of causative variants, their relative location within a genome, expression, pleiotropic effect, environmental influence, and degree of dominance, epistasis, and additivity. Here, we review theory related to polygenic local adaptation and contextualize these expectations with methods often used to uncover the genetic basis of traits important to tree conservation and industry. A broad literature survey suggests that most tree traits generally exhibit considerable heritability, that underlying quantitative genetic variation (Q_ST) is structured more so across populations than neutral expectations (F_ST) in 69% of comparisons across the literature, and that single-locus associations often exhibit small estimated per-locus effects. Together, these results suggest differential selection across populations often acts on tree phenotypes underlain by polygenic architectures consisting of numerous small to moderate effect loci. Using this synthesis, we highlight the limits of using solely single-locus approaches to describe underlying genetic architectures and close by addressing hurdles and promising alternatives towards such goals, remark upon the current state of tree genomics, and identify future directions for this field. Importantly, we argue, the success of future endeavors should not be predicated on the shortcomings of past studies and will instead be dependent upon the application of theory to empiricism, standardized reporting, centralized open-access databases, and continual input and review of the community’s research.     

Rare events out of equilibrium: mobility through the liquid–liquid interface

Transition state theory provides a well established means to compute the rate at which rare events occur; however, this is strictly an equilibrium approach. Here we consider a nonequilibrium problem of this nature in the form of transport through a liquid–liquid interface. When two immiscible liquids are coexisting in equilibrium, there will be a certain amount of mixing between the two phases, resulting in a finite linear mobility across the liquid–liquid interface. We derive an exact relationship between the mobility and the local diffusion in the direction perpendicular to the interface. We compute the mobility using both nonequilibrium molecular dynamics and a variety of linear response type approaches, with accurate agreement being obtained for the best of these. Our analysis makes it clear how the local diffusion is influenced by the inhomogeneities of the interface, even when at a distance from it. This nonlocal character to the mobility has not been appreciated before and results in a strong variation in the local diffusion, which is formally coupled to the variation in the potential of mean force. The nonlocal aspect of the diffusion requires the velocity autocorrelation function to be integrated out to far longer times than is the case for homogeneous liquids, and requires special care with regard to the choice of numerical approach.     

ERAD substrates: Which way out?

Global folding of polypeptides entering the endoplasmic reticulum (ER) starts as soon as they emerge from the narrow Sec61 translocon. Attainment of the native structure can take from several minutes to hours, depending on the gene product. Until then, non-native folding intermediates must be protected from molecular chaperones that recognize misfolded determinants and could prematurely interrupt folding programs by re-directing them to disposal pathways. On the other hand, futile folding attempts must actively be stopped to prevent intraluminal accumulation of defective cargo. This review describes recent advances in understanding how terminally misfolded polypeptides are extracted from the folding environment and directed to specific dislocons within the ER membrane for transfer to the cytoplasm for proteasome-mediated degradation.     

Fading out of vole and predator cycles?

Northern voles and lemmings are famous for their spectacular multiannual population cycles with high amplitudes. Such cyclic vole populations in Scandinavia have shown an unexpected and marked long-term decline in density since the early 1970s, particularly with a marked shift to lower spring densities in the early 1980s. The vole decline, mainly characterized by a strongly decreased rate of change in numbers over winter, is associated with an increased occurrence of mild and wet winters brought about by a recent change in the North Atlantic Oscillation. This has led to a decrease in winter stability and has shortened the period with protective snow cover, the latter considered as an important prerequisite for the occurrence of multiannual, high-amplitude cycles in vole populations. Although the vole decline is predicted to be negative for predators' reproduction and abundance, empirical data showing this are rare. Here we show that the dynamics of a predator-prey system (Tengmalm's owl, Aegolius funereus, and voles), have in recent years gradually changed from 3-4 yr, high-amplitude cycles towards more or less annual fluctuations only.     

Lipid modifications of proteins – slipping in and out of membranes

Protein lipid modification, once thought to act as a stable membrane anchor for soluble proteins, is now attracting more widespread attention for its emerging role in diverse signaling pathways and regulatory mechanisms. Most multicellular organisms have recruited specific types of lipids and a suite of unique enzymes to catalyze the modification of a select number of proteins, many of which are evolutionarily conserved in plants, animals and fungi. Each of the three known types of lipid modification – palmitoylation, myristylation and prenylation – allows cells to target proteins to the plasma membrane, as well as to other subcellular compartments. Among the lipid modifications, protein prenylation might also function as a relay between cytoplasmic isoprene biosynthesis and regulatory pathways that control cell cycle and growth. Molecular and genetic studies of an Arabidopsis mutant that lacks farnesyl transferase suggest that the enzyme has a role in abscisic acid signaling during seed germination and in the stomata. It is becoming clear that lipid modifications are not just fat for the protein, but part of a highly conserved intricate network that plays a role in coordinating complex cellular functions.     

Review: Transport of tRNA out of the Nucleus—Direct Channeling to the Ribosome?

Although tRNA was the first substrate whose export from the nuclei of eukaryotic cells had been shown to be carrier-mediated and active, it has only been in the last 2 years that the first mechanistic details of this nucleocytoplasmic transport pathway have begun to emerge. A member of the importin/karyopherin β superfamily, Los1p in yeast and Xpo-t in vertebrates, has been shown to export tRNA in cooperation with the small GTPase Ran (Gsp1p) from the nucleus into the cytoplasm, where tRNA becomes available for translation. However, Los1p is not essential for viability in yeast cells, suggesting that alternative tRNA export pathways exist. Recent results show that aminoacylation and a translation factor are also required for efficient nuclear tRNA export. Thus, protein translation and nuclear export of tRNA appear to be coupled processes.