Stimulating cROSstalk between commensal bacteria and intestinal stem cells

EMBO J (2013) 32 23, 3017–3028 10.1038/emboj.2013.224; published online October182013Commensal gut bacteria benefit their host in many ways, for instance by aiding digestion and producing vitamins. In a new study in The EMBO Journal, Jones et al (2013) report that commensal bacteria can also promote intestinal epithelial renewal in both flies and mice. Interestingly, among commensals this effect is most specific to Lactobacilli, the friendly bacteria we use to produce cheese and yogurt. Lactobacilli stimulate NADPH oxidase (dNox/Nox1)-dependent ROS production by intestinal enterocytes and thereby activate intestinal stem cells.The human gut contains huge numbers of bacteria (∼10¹⁴/person) that play beneficial roles for our health, including digestion, building our immune system and competing with harmful microbes (Sommer and Backhed, 2013). Both commensal and pathogenic bacteria can elicit antimicrobial responses in the intestinal epithelium and also stimulate epithelial turnover (Buchon et al, 2013; Sommer and Backhed, 2013). In contrast to gut pathogens, relatively little is known about how commensal bacteria influence intestinal turnover. In a simple yet elegant study reported recently in The EMBO Journal, Jones et al (2013) show that among several different commensal bacteria tested, only Lactobacilli promoted much intestinal stem cell (ISC) proliferation, and it did so by stimulating reactive oxygen species (ROS) production. Interestingly, the specific effect of Lactobacilli was similar in both Drosophila and mice. In addition to distinguishing functional differences between species of commensals, this work suggests how the ingestion of Lactobacillus-containing probiotic supplements or food (e.g., yogurt) might support epithelial turnover and health.In both mammals and insects, ISCs give rise to intestinal enterocytes, which not only absorb nutrients from the diet but must also interact with the gut microbiota (Jiang and Edgar, 2012). The metazoan intestinal epithelium has developed conserved responses to enteric bacteria, for instance the expression of antimicrobial peptides (AMPs; Gallo and Hooper, 2012; Buchon et al, 2013), presumably to kill harmful bacteria while allowing symbiotic commensals to flourish. In addition to AMPs, intestinal epithelial cells use NADPH family oxidases to generate ROS that are used as microbicides (Lambeth and Neish, 2013). High ROS levels during enteric infections likely act non-discriminately against both commensals and pathogens, but controlled, low-level ROS can act as signalling molecules that regulate various cellular processes including proliferation (Lambeth and Neish, 2013). In flies, exposure to pathogenic Gram-negative bacteria has been reported to result in ROS (H₂O₂) production by an enzyme called dual oxidase (Duox; Ha et al, 2005). Duox activity in the fly intestine (and likely also the mammalian one) has recently been discovered to be stimulated by uracil secretion by pathogenic bacteria (Lee et al, 2013). In the mammalian intestine another enzyme, NADPH oxidase (Nox), has also been shown to produce ROS in the form of superoxide (O₂⁻), in this case in response to formylated bacterial peptides (Lambeth and Neish, 2013). A conserved role for Nox in the Drosophila intestinal epithelium had not until now been explored.Jones et al (2013) checked seven different commensal bacterial to see which would stimulate ROS production by the fly''s intestinal epithelium, and found that only one species, a Gram-positive Lactobacillus, could stimulate significant production of ROS in intestinal enterocytes. Five bacterial species were checked in mice or cultured intestinal cells, and again it was a Lactobacillus that generated the strongest ROS response. Although not all of the most prevalent enteric bacteria were assayed, those others that were—such as E. coli—induced only mild, barely detectable levels of ROS in enterocytes. Surprisingly, although bacteria pathogenic to Drosophila, like Erwinia caratovora, were expected to stimulate ROS production via Duox, Jones et al (2013) did not observe this using the ROS detecting dye hydrocyanine-Cy3, or a ROS-sensitive transgene reporter, Glutatione S-transferase-GFP, in flies. Further, Jones et al (2013) found that genetically suppressing Nox in either Drosophila or mice decreased ROS production after Lactobacillus ingestion. Consistent with the important role of Nox, Duox appeared not to be required for ROS production after Lactobacillus ingestion. In addition, Jones et al (2013) found that Lactobacilli also promoted DNA replication—a metric of cell proliferation and epithelial renewal—in the fly''s intestine, and that this was also ROS- and Nox-dependent. Again, the same relationship was found in the mouse small intestine. Together, these results suggest a conserved mechanism by which Lactobacilli can stimulate Nox-dependent ROS production in intestinal enterocytes and thereby promote ISC proliferation and enhance gut epithelial renewal.In the fly midgut, uracil produced by pathogenic bacteria can stimulate Duox-dependent ROS production, which is thought to act as a microbicide (Lee et al, 2013), and can also promote ISC proliferation (Buchon et al, 2009). However, Duox-produced ROS may also damage the intestinal epithelium itself and thereby promote epithelial regeneration indirectly through stress responses. In this disease scenario, ROS appears to be sensed by the stress-activated Jun N-terminal Kinase (JNK; Figure 1A), which can induce pro-proliferative cytokines of the Leptin/IL-6 family (Unpaireds, Upd1–3) (Buchon et al, 2009; Jiang et al, 2009). These cytokines activate JAK/STAT signalling in the ISCs, promoting their growth and proliferation, and accelerating regenerative repair of the gut epithelium (Buchon et al, 2009; Jiang et al, 2009). It is also possible, however, that low-level ROS, or specific types of ROS (e.g., H₂O₂) might induce ISC proliferation directly by acting as a signal between enterocytes and ISCs. Since commensal Lactobacillus stimulates ROS production via Nox rather than Duox, this might be a case in which a non-damaging ROS signal promotes intestinal epithelial renewal without stress signalling or a microbicidal effect (Figure 1B). However, Jones et al (2013) stopped short of ruling out a role for oxidative damage, cell death or stress signalling in the intestinal epithelium following colonization by Lactobacilli, and so these parameters must be checked in future studies. Perhaps even the friendliest symbiotes cause a bit of ‘healthy'' damage to the gut lining, stimulating it to refresh and renew. Whether damage-dependent or not, the stimulation of Drosophila ISC proliferation by commensals and pathogens alike appears to involve the same cytokine (Upd3; Buchon et al, 2009), and so some of the differences between truly pathogenic and ‘friendly'' gut microbes might be ascribed more to matters of degree than qualitative distinctions. Future studies exploring exactly how different types of ROS signals stimulate JNK activity, gut cytokine expression and epithelial renewal should be able to sort this out, and perhaps help us learn how to better manage the ecosystems in our own bellies. From the lovely examples reported by Jones et al (2013), an experimental back-and-forth between the Drosophila and mouse intestine seems an informative way to go.Open in a separate windowFigure 1Metazoan intestinal epithelial responses to commensal and pathogenic bacteria. (A) High reactive oxygen species (ROS) levels generated by dual oxidase (Duox) in response to uracil secretion by pathogenic bacteria. (B) Low ROS levels generated by NADPH oxidase (Nox) in response to commensal bacteria. In addition to acting as a microbiocide, ROS in flies may stimulate JNK signaling and cytokine (Upd 1–3) expression in enterocytes, thereby stimulating ISC proliferation and epithelial turnover or regeneration. Whether this stimulation required damage to or loss of enterocytes has yet to be explored.     

A structural road map to unveil basal body composition and assembly

EMBO J 31 3, 552–562 (2012); published online December132011The Basal Body (BB) acts as the template for the axoneme, the microtubule-based structure of cilia and flagella. Although several proteins were recently implicated in both centriole and BB assembly and function, their molecular mechanisms are still poorly characterized. In this issue of The EMBO journal, Li and coworkers describe for the first time the near-native structure of the BB at 33 Å resolution obtained by Cryo-Electron Microscopy analysis of wild-type (WT) isolated Chlamydomonas BBs. They identified several uncharacterized non-tubulin structures and variations along the length of the BB, which likely reflect the binding and function of numerous macromolecular complexes. These complexes are expected to define BB intrinsic properties, such as its characteristic structure and stability. Similarly to the high-resolution structures of ribosome and nuclear pore complexes, this study will undoubtedly contribute towards the future analysis of centriole and BB biogenesis, maintenance and function.The microtubule (MT)-based structure of the cilium/flagellum grows from the distal part of the Basal Body (BB), which in many animal cells develops from the mature centriole in the centrosome. Electron microscopic (EM) images of chemically fixed resin-embedded centrioles and basal bodies (CBBs) suggest that their ultrastructure is similar, and that their key components are MTs. The mechanisms underlying the organization of CBB MTs, comprising highly stable closed and open MTs, are likely to hold many surprises as they are remarkably different from other microtubular structures in the cell. Additionally, non-MT-based structures are also part of the CBB, including a cartwheel in the proximal lumen region that reinforces CBB symmetry (reviewed in Azimzadeh and Marshall, 2010 and Carvalho-Santos et al, 2011).Several centriole components and BB proteins were identified by comparative and/or functional genomics and proteomics studies of purified CBBs (reviewed in Azimzadeh and Marshall, 2010 and Carvalho-Santos et al, 2011). Advances in our understanding of the molecular mechanisms of CBB assembly depend on high-resolution comparative studies of wild-type (WT) and mutant structures, as well as characterization of the localization of molecular complexes within the small CBB structure. Despite the existence of beautiful ultrastructure data acquired from chemically fixed specimens (Geimer and Melkonian, 2004; Ibrahim et al, 2009), high-resolution structures of native CBBs were missing. Using electron cryo-tomography and 3D subtomogram averaging, Li et al (2012) solved the structure of the near-native BB triplet at 33 Å resolution. A pseudo-atomic model of the tubulin protofilaments at the core of the triplets was built by fitting the atomic structure of α/β-tubulin monomers into the BB tomograms.The 3D density map reveals several additional densities that represent non-tubulin proteins attached, both internally and externally, to all triplet MTs, some linking MTs inside the triplets and/or MTs in consecutive triplets (Li et al, 2012; for a summary, see Li et al, 2012; Geimer and Melkonian, 2004; Ibrahim et al, 2009), but with less detail and complexity. The authors speculate that some of the additional densities present at the A- and B-tubule inner wall might correspond to proteins of the tektin family, probably conferring rigidity to the BB triplet (Amos, 2008).

Table 1

Characteristics of the non-α/β-tubulin structures reported in Li et al (2012) in this issue of The EMBO journal

Protection by natural IgG: a sweet partnership with soluble lectins does the trick!

EMBO J 32: 2905–2919 10.1038/emboj.2013.199; published online September032013Some B cells of the adaptive immune system secrete polyreactive immunoglobulin G (IgG) in the absence of immunization or infection. Owing to its limited affinity and specificity, this natural IgG is thought to play a modest protective role. In this issue, a report reveals that natural IgG binds to microbes following their opsonization by ficolin and mannan-binding lectin (MBL), two carbohydrate receptors of the innate immune system. The interaction of natural IgG with ficolins and MBL protects against pathogenic bacteria via a complement-independent mechanism that involves IgG receptor FcγRI expressing macrophages. Thus, natural IgG enhances immunity by adopting a defensive strategy that crossovers the conventional boundaries between innate and adaptive microbial recognition systems.The adaptive immune system generates protective somatically recombined antibodies through a T cell-dependent (TD) pathway that involves follicular B cells. After recognizing antigen through the B-cell receptor (BCR), follicular B cells establish a cognate interaction with CD4⁺ T follicular helper (T_FH) cells and thereafter either rapidly differentiate into short-lived IgM-secreting plasmablasts or enter the germinal centre (GC) of lymphoid follicles to complete class switch recombination (CSR) and somatic hypermutation (SHM) (Victora and Nussenzweig, 2012). CSR from IgM to IgG, IgA and IgE generates antibodies with novel effector functions, whereas SHM provides the structural correlate for the induction of affinity maturation (Victora and Nussenzweig, 2012). Eventually, this canonical TD pathway generates long-lived bone marrow plasma cells and circulating memory B cells that produce protective class-switched antibodies capable to recognize specific antigens with high affinity (Victora and Nussenzweig, 2012).In addition to post-immune monoreactive antibodies, B cells produce pre-immune polyreactive antibodies in the absence of conventional antigenic stimulation (Ehrenstein and Notley, 2010). These natural antibodies form a vast and stable repertoire that recognizes both non-protein and protein antigens with low affinity (Ehrenstein and Notley, 2010). Natural antibodies usually emerge from a T cell-independent (TI) pathway that involves innate-like B-1 and marginal zone (MZ) B cells. These are extrafollicular B-cell subsets that rapidly differentiate into short-lived antibody-secreting plasmablasts after detecting highly conserved microbial and autologus antigens through polyreactive BCRs and nonspecific germline-encoded pattern recognition receptors (Pone et al, 2012; Cerutti et al, 2013).The most studied natural antibody is IgM, a pentameric complement-activating molecule with high avidity but low affinity for antigen (Ehrenstein and Notley, 2010). In addition to promoting the initial clearance of intruding microbes, natural IgM regulates tissue homeostasis, immunological tolerance and tumour surveillance (Ochsenbein et al, 1999; Zhou et al, 2007; Ehrenstein and Notley, 2010). Besides secreting IgM, B-1 and MZ B cells produce IgG and IgA after receiving CSR-inducing signals from dendritic cells (DCs), macrophages and neutrophils of the innate immune system (Cohen and Norins, 1966; Cerutti et al, 2013). In humans, certain natural IgG and IgA are moderately mutated and show some specificity, which may reflect the ability of human MZ B cells to undergo SHM (Cerutti et al, 2013). Yet, natural IgG and IgA are generally perceived as functionally quiescent.In this issue, Panda et al show that natural IgG bound to a broad spectrum of bacteria with high affinity by cooperating with ficolin and MBL (Panda et al, 2013), two ancestral soluble lectins of the innate immune system (Holmskov et al, 2003). This binding involved some degree of specificity, because it required the presence of ficolin or MBL on the microbial surface as well as lower pH and decreased calcium concentration in the extracellular environment as a result of infection or inflammation (see Figure 1).Open in a separate windowFigure 1Ficolins and MBL are produced by hepatocytes and various cells of the innate immune system and opsonize bacteria after recognizing conserved carbohydrates. Low pH and calcium concentrations present under infection-inflammation conditions promote the interaction of ficolin or MBL with natural IgG on the surface of bacteria. The resulting immunocomplex is efficiently phagocytosed by macrophages through FcγR1 independently of the complement protein C3, leading to the clearance of bacteria.Ficolins and MBL are soluble pattern recognition receptors that opsonize microbes after binding to glycoconjugates through distinct carbohydrate recognition domain (CRD) structures (Holmskov et al, 2003). While ficolins use a fibrinogen domain, MBL and other members of the collectin family use a C-type lectin domain attached to a collagen-like region (Holmskov et al, 2003). Similar to pentraxins, ficolins and MBL are released by innate effector cells and hepatocytes, and thus may have served as ancestral antibody-like molecules prior to the inception of the adaptive immune system (Holmskov et al, 2003; Bottazzi et al, 2010). Of note, MBL and the MBL-like complement protein C1q are recruited by natural IgM to mediate complement-dependent clearance of autologous apoptotic cells and microbes (Holmskov et al, 2003; Ehrenstein and Notley, 2010). Panda et al found that a similar lectin-dependent co-optation strategy enhances the protective properties of natural IgG (Panda et al, 2013).By using bacteria and the bacterial glycan N-acetylglicosamine, Panda et al show that natural IgG isolated from human serum or T cell-deficient mice interacted with the fibrinogen domain of microbe-associated ficolins (Panda et al, 2013). The resulting immunocomplex was phagocytosed by macrophages via the IgG receptor FcγRI in a complement-independent manner (Panda et al, 2013). The additional involvement of MBL was demonstrated by experiments showing that natural IgG retained some bacteria-binding activity in the absence of ficolins (Panda et al, 2013).Surface plasmon resonance provided some clues regarding the molecular requirements of the ficolin–IgG interaction (Panda et al, 2013), but the conformational changes required by ficolin to interact with natural IgG remain to be addressed. In particular, it is unclear what segment of the effector Fc domain of natural IgG binds to ficolins and whether Fc-associated glycans are involved in this binding. Specific glycans have been recently shown to mitigate the inflammatory properties of IgG emerging from TI responses (Hess et al, 2013) and this process could implicate ficolins and MBL. Moreover, it would be important to elucidate whether and how the antigen-binding Fab portion of natural IgG regulates its interaction with ficolins and MBL.The in vivo protective role of natural IgG was elegantly demonstrated by showing that reconstitution of IgG-deficient mice lacking the CSR-enzyme activation-induced cytidine deaminase with natural IgG from T cell-insufficient animals enhanced resistance to pathogenic Pseudomonas aeruginosa (Panda et al, 2013). This protective effect was associated with reduced production of proinflammatory cytokines, occurred independently of the complement protein C3 and was impaired by peptides capable to inhibit the binding of natural IgG to ficolin (Panda et al, 2013). Additional in vivo studies will be needed to determine whether natural IgG exerts protective activity in mice lacking ficolin, MBL or FcγRI, and to ascertain whether these molecules also enhance the protective properties of canonical or natural IgG and IgA released by bone marrow plasma cells and mucosal plasma cells, respectively.In conclusion, the findings by Panda et al show that natural IgG adopts ‘crossover'' defensive strategies that blur the conventional boundaries between the innate and adaptive immune systems. The sophisticated integration of somatically recombined and germline-encoded antigen recognition systems described in this new study shall stimulate immunologists to further explore the often underestimated protective virtues of our vast natural antibody repertoire. This effort may lead to the development of novel therapies against infections.     

Autophagy plays a WASHing game

EMBO J (2013) 32: 2685–2696 doi:10.1038/emboj.2013.189; published online August232013Beclin 1 is a crucial regulator of autophagy. It forms a complex with ATG14L, VPS34 or the class III phosphatidylinositol 3-kinase AMBRA1 to control autophagosome formation. A study in this issue of The EMBO Journal (Xia et al, 2013) reports that WASH, a protein known to regulate endosomal sorting, hampers autophagy by inhibiting the AMBRA1-dependent polyubiquitylation of Beclin 1. This modification is required to promote VPS34 activity and to initiate autophagy during starvation.Macroautophagy (hereafter called autophagy) is a lysosomal degradation pathway for cytoplasmic components. Autophagy is initiated by the formation of a double-membrane bound vacuole, the autophagosome that sequesters the autophagic cargo and eventually fuses with the lysosomal compartment, leading to cargo degradation. Autophagy plays a major role in cytoplasm homeostasis by removing protein aggregates and controlling organelle quality, and it is stimulated to promote cell survival during stressful situations, such as nutrient starvation and microbial infection. Autophagosome formation is dependent on evolutionarily conserved Atg (Autophagy-related) proteins initially identified in yeast (Mizushima et al, 2011). They function in complexes or functional modules on a membrane known as the phagophore that matures into the autophagosome via several stages (initiation, elongation and sealing). Phosphatidylinositol 3-phosphate kinase complex I (PI3K complex I) plays a key role in the initiation step. In this complex, Beclin 1 (the mammalian homologue of yeast Atg6) interacts with ATG14L, AMBRA1, and class III PI3K or VPS34 (Cecconi and Levine, 2008; Figure 1). The activity of this complex, which produces the lipid phosphatidylinositol 3-phosphate (PI3P) to recruit Atg18 homologues (WIPIs), has to be tightly regulated to keep autophagy under control.Open in a separate windowFigure 1Role of WASH in autophagosome formation. WASH interacts with Beclin 1 via a sequence (121–221) that is not involved in endosomal sorting. In the absence of WASH, when autophagy is stimulated by nutrient deprivation, Beclin 1 interacts with VPS34 (and its adaptor VPS15), ATG14L and AMBRA1. In this complex, Beclin 1 is polyubiquitylated by AMBRA1, which acts as an E3 ligase. The activation of VPS34 produces PI3P at the phagophore to recruit WIPI proteins, and then triggers the machinery to elongate and seal the membrane, thus forming an autophagosome. The VAC domain of WASH involved in endosomal sorting is not required for autophagy regulation.In this issue of The EMBO Journal, Xia et al (2013) demonstrate that WASH (Wiskott-Aldrich syndrome protein (WASP) and SCAR homologue) regulates autophagosome formation in response to nutrient starvation by influencing the ubiquitylation of Beclin 1. WASH forms a complex with four other proteins and plays an essential role in endosomal sorting by promoting actin polymerization to facilitate segregation of endosomal proteins (Seaman et al, 2013). Using WASH−/− mice, Xia et al (2013) observe that WASH deficiency causes embryonic lethality (E7.5−E9.5) with massive apoptosis-independent cell death and the accumulation of autophagic structures. However, whether autophagy is instrumental in the cell death observed in WASH−/− embryos remains to be investigated. From a series of classical readouts, the authors conclude that WASH downregulates starvation-induced autophagy. Interestingly, WASH is detected on the autophagosomal membrane before and after closure of the autophagosome, but not on the autolysosomes (organelles formed when the autophagosome fuses with the lysosome). The authors first show that WASH has distinct roles in autophagy and in endosomal sorting, because deletion of the VCA domain of WASH that is required for its endosomal function and the knockdown of FAM21 (a protein that directs WASH to the endosomal membrane) do not influence autophagy. They then show that WASH interacts with the coil-coiled domain of Beclin 1. WASH depletion induces more VPS34 to interact with Beclin 1, leading to augmented formation of PtdIns3P and increased recruitment of WIPI-1 to the autophagosomal membrane. However, WASH does not influence the stability of Beclin 1, although it does regulate its K63-linked polyubiquitylation at position K437 during starvation (K63-linked polyubiquitylation suggests a regulatory role, whereas K48-linked polyubiquitylation is frequently associated with protein degradation). Overexpression of WASH reduces the ubiquitylation of Beclin 1, and the K437R Beclin 1 mutant that cannot be ubiquitylated has a low level of interaction with VPS34, which prevents the stimulation of autophagy by starvation (Figure 1). Recently, Beclin 1 has been shown to be ubiquitylated at position K117 during TLR4-induced autophagy (Shi and Kehrl, 2010). Interestingly, Xia et al (2013) show that the K117R Beclin 1 mutant is still ubiquitylated in response to nutrient starvation. Finally, the authors identify the substrate-specific E3 ubiquitin ligase for Beclin 1 in this context. They exclude TRAF6 and NEDD4, two E3 ligases reported to ubiquitylate Beclin 1 (Kuang et al, 2013). AMBRA1, which regulates autophagy in the PI3K complex I, is also known as DCAF3 and interacts with the Cullin 4–DDB1 E3 ligase complex (Jin et al, 2006). Following in vitro E3 ligase assays, Xia et al (2013) conclude that AMBRA1 is the substrate receptor for Beclin 1 ubiquitylation at position K437, adding Cullin4/DDB1 and AMBRA1 to the list of E3 ligases involved in autophagy (Kuang et al, 2013). The finding that WASH and AMBRA1 compete to regulate starvation-induced autophagy raises several questions: how do WASH and AMBRA1 influence one another''s binding to Beclin 1? WASH is evolutionarily conserved, although it is not present in yeast (Linardopoulou et al, 2007)—does it also reduce starvation-induced autophagy in non-mammalian cells? WASH has recently been reported to be required for the lysosomal digestion of autophagy cargo in Dictyostelium during starvation (King et al, 2013). Does this mean that the function of WASH in autophagy has evolved from controlling cargo degradation to forming autophagosomes? Xia et al (2013) do not report any association between WASH and the lysosomal compartment during autophagy; the role of WASH in autophagosome maturation calls for further investigation. Finally, a recent report demonstrates that AMBRA1 interacts with the E3 ligase TRAF6 to ubiquitylate ULK1 (the mammalian orthologue of the yeast Atg1) and to ensure its subsequent stabilization and function (Nazio et al, 2013). ULK1 is part of a complex that acts with PI3K complex 1 to regulate the initiation of autophagy (Mizushima et al, 2011). How are these two AMBRA1-dependent ubiquitylation processes coordinated to control autophagy? In conclusion, both the study reported here (Xia et al, 2013) and the recent study of Nazio et al (2013) clearly demonstrate that AMBRA1 plays a central role in regulating the initiation of autophagy.     

A bacterial actin unites to divide bacterial cells

EMBO J 31 10, 2249–2260 (2012); published online March302012Once thought to exist only in eukaryotic cells, the highly conserved bacterial cytoskeleton is now known to function analogously to its eukaryotic counterparts, particularly in cell shape and division. For instance, the actin-like MreB protein and its homologs are important to maintain cell shape in many rod-shaped bacteria, probably by organizing how peptidoglycan is synthesized. FtsZ, a tubulin homolog, forms a scaffold for the cytokinetic ring, or divisome, by GTP-dependent polymerization into protofilaments. In this issue of The EMBO Journal, Szwedziak et al (2012) reveal the first crystal structures of cell division protein FtsA polymerizing into actin-like filaments, along with in vivo evidence that this self-interaction is crucial for proper cell division.FtsA is an actin homolog required for cytokinesis in many bacterial species and has several key roles in cell division, including helping to tether FtsZ to the cytoplasmic membrane via a membrane-targeting sequence (MTS), recruiting other essential proteins to the divisome, and perhaps promoting divisome constriction (de Boer, 2010). Szwedziak et al (2012) recapitulate the FtsZ-FtsA-membrane association in vitro using liposomes with FtsZ and FtsA proteins from Thermotoga maritima. To get a closer look at the FtsA-FtsZ interface, the authors co-crystallize FtsA with the carboxy-terminal tail of FtsZ, which is known to interact with FtsA. Intriguingly, the crystal reveals an FtsA homodimer. Contrary to the previous bioinformatics model of FtsA self-interaction that proposed a 180° rotation between the two subunits (Carettoni et al, 2003), the FtsA-FtsA interface in the crystal structure shows no rotation, similar to F-actin. Szwedziak et al (2012) also show that FtsA can form longer, actin-like polymers in the presence of non-hydrolysable ATP or on lipid monolayers. These results are surprising because FtsA has a divergent subdomain architecture compared to other actin-family proteins (van den Ent and Löwe).A critical question now is whether FtsA needs to form polymers in vivo to function properly. Purified Streptococcus pneumoniae FtsA assembles into large polymers that are not like F-actin, and it remains unclear if these structures are relevant in vivo (Krupka et al, 2012). Wild-type FtsA proteins do not form detectable filaments in cells, but C-terminal truncations of FtsA that remove the MTS form polymers quite readily in cells when overproduced, although they are not functional (Pichoff and Lutkenhaus, 2007). Even so, starting with an MTS truncation derivative of FtsA to visualize in vivo polymers, Szwedziak et al (2012) design site-directed mutants of Bacillus subtilis FtsA based on the FtsA-FtsA interface of their crystals; these fail to assemble into polymers in vivo. Using a similar MTS truncation derivative, Pichoff et al (2012) created random mutations in Escherichia coli FtsA, and found that those mapping to the same interface found by Szwedziak et al (2012) also disrupted polymer formation. Together, these data suggest that these residues are needed for FtsA self-interaction. Perplexingly, when these mutants were subsequently tested for functionality in the context of full-length FtsA, the results were mixed. Pichoff et al (2012) showed that FtsA mutants deficient for self-interaction in E. coli have a gain-of-function phenotype, whereas Szwedziak et al (2012) report that analogous mutants in B. subtilis FtsA suffer a loss of function. These results support the idea that FtsA self-association is related to its activity (Shiomi and Margolin, 2007), yet understanding how self-interaction regulates FtsA function clearly requires further study.The ability of eukaryotic cytoskeletal proteins to form long polymers is essential to their function, but the physiological relevance of long polymer formation by bacterial cytoskeletal proteins is now a topic of debate (Figure 1). For example, it has been hypothesized that FtsZ protofilaments wrap around the entire circumference of the cell to form the cytokinetic ring. However, recent studies using photoactivated localization microscopy (PALM) and electron cryotomography reveal a different model in which FtsZ forms a series of very short polymers that overlap to encompass the diameter of the cell (Li et al, 2007; Fu et al, 2010). MreB was also originally thought to form long-range helical polymers extending the length of the cell, but recent data obtained with more sophisticated microscopic techniques suggest that MreB is distributed in patches that move circumferentially and independently (White and Gober, 2012). It is not yet clear which of these models represents the true cellular architecture of MreB, although it is likely that some degree of MreB polymerization is still needed for function. It is notable that other bacterial homologs of actin and tubulin used for generating scaffolds or partitioning plasmid DNA, but not for essential cellular processes such as cell division and growth, tend to form long polymers that extend throughout the cell (Pogliano, 2008). The continued combined use of microscopic, biochemical, and genetic methods, as demonstrated by Szwedziak et al (2012) will enhance future understanding of ancestral tubulin and actin proteins in prokaryotes.Open in a separate windowFigure 1Bacterial actin and tubulin filaments involved in cell growth and division. (A) MreB (purple) has long been thought of as a spiral filament twisting along the cell length to control cell shape. Likewise, FtsZ protofilaments (blue) were once thought to wrap around the cell midpoint to organize the divisome. (B) Recent work using high-resolution microscopy has revealed that long cytoskeletal filaments are more likely to be short patches of polymers. The present work by Szwedziak et al (2012) has added FtsA actin-like filaments (green) to the model of possible divisome architecture.     

Up for grabs; trashing peroxisomes

EMBO J 31 13, 2852–2868 (2012); published online May292012Together with the proteasome, autophagy is one of the major catabolic pathways of the cell. In response to cellular needs or environmental cues, this transport route targets specific structures for degradation into the mammalian lysosomes or the yeast and plant vacuoles. The mechanisms allowing exclusive autophagic elimination of unwanted structures are currently the object of intensive investigations. The emerging picture is that there is a series of autophagy receptors that determines the specificity of the different selective types of autophagy. How cargo binding and recognition is regulated by these receptors, however, is largely unknown. In their study, Motley et al (2012) have shed light into the molecular principles underlying the turnover of excess peroxisomes in the budding yeast Saccharomyces cerevisiae.Peroxisomes perform a series of crucial functions and their number is regulated in response to the metabolic demands of the cell. After proliferation and when no more required, a selective type of autophagy called pexophagy degrades superfluous peroxisomes (Manjithaya et al, 2010). This turnover allows the cell to save the energy required for the maintenance of excess organelles and to generate metabolites that can be used to carry out other functions. Like all selective types of autophagy, pexophagy relies on the conserved core of the autophagy-related (Atg) machinery, but also requires additional proteins that confer specificity of the pathway such as cargo selection and membrane dynamics (Manjithaya et al, 2010). It is still unclear, however, which peroxisomal protein allows the recognition of peroxisomes by the autophagosomes. Although Pex3 and Pex14 have previously been indicated as possible suspects (Bellu et al, 2001, 2002; Farre et al, 2008), their specific contribution to pexophagy was difficult to establish. Deletion of either PEX3 or PEX14, as well as most other PEX genes, leads to defects in peroxisome biogenesis, which makes the dissection of their contribution to peroxisome degradation very difficult to assess. Motley et al (2012) have elegantly exploited S. cerevisiae genetics to isolate pex3 alleles specifically impaired in pexophagy and could thus demonstrate that Pex3 (and not Pex14) mediates the selective engulfment of peroxisomes by autophagosomes. In support to this result, the authors have also identified a new protein, Atg36, which binds Pex3 (Figure 1). Importantly, Atg36 interacts with Atg11, an autophagy adaptor involved in numerous selective types of autophagy in yeast, thereby bringing peroxisomes to the site where autophagosomes will be generated and coordinating the activation of the Atg machinery at this location (Kim et al, 2001; Reggiori et al, 2005; Monastyrska et al, 2008). Atg36, however, is only present in S. cerevisiae and related yeasts. Methylotrophic yeasts, in contrast, appear to have a different protein with the same properties, Atg30 (Farre et al, 2008). It is unclear, however, whether Atg30 is the functional counterpart of Atg36 because these two proteins do not display similarities in their amino-acid sequence.Open in a separate windowFigure 1Schematic representation for a putative Pex3 checkpoint. The peroxisomal integral membrane protein Pex3 acts as a master regulator to determine peroxisome fate. Organelle abundance is regulated by formation of new organelles, and their subsequent segregation (inheritance) and degradation. A new paradigm has been uncovered, whereby Pex3 controls peroxisome abundance through the regulated binding to specific co-factors. At the endoplasmic reticulum (ER), together with Pex19, it initiates biogenesis of new peroxisomes. At the peroxisomal membrane, it ensures that both mother and daughter cells obtain the correct number of peroxisomes, whereas when the organelles become dispensable, Pex3 can initiate their selective degradation. To keep peroxisomes in the mother cell during cell division, Pex3 associates with Inp1 and tether peroxisomes to cortical actin patches. Under pexophagy-inducing conditions, Pex3 binds the newly identified pexophagy factor Atg36 and delivers peroxisomes to the site of autophagosome formation for subsequent degradation into the vacuole.While it is unmistakable that Atg36 (and Atg30) is essential for pexophagy, it remains unclear whether this protein is an autophagy receptor. This class of molecules has four characteristics (Kraft et al, 2010). First, each autophagy receptor binds a specific cargo. Second, they often interact with adaptor proteins, which function as scaffolds that bring the cargo–receptor complex in contact with the core Atg machinery to allow the specific sequestration of the cargo. Third, they possess at least one LC3-interacting region (LIR) motif that enables them to interact with the LC3/Atg8 pool present in the interior autophagomes and assures the hermetic enwrapping of the cargo into these vesicles. Fourth, autophagy receptors are degraded in the lysosome/vacuole together with the cargo that they bind to. While Atg36 (and Atg30) binds both the cargo and the adaptor protein Atg11, this protein does not appear to be turned over in the vacuole during pexophagy and a LIR motif has not been pinpointed yet. Consequently, it is unclear whether Atg36 is a new type of autophagy receptor or acts together with a not yet identified autophagy receptor involved in pexophagy.A very interesting concept emerging from the work of Motley et al (2012) is that a single protein, that is, Pex3, could be the central regulator of peroxisome homoeostasis (Figure 1). Pex3 is involved in peroxisome biogenesis, segregation and degradation (Bellu et al, 2002; Hoepfner et al, 2005; Farre et al, 2008; Munck et al, 2009; Ma et al, 2011). As a result, the cell could regulate peroxisome abundance by modulating Pex3 function and/or its array of interactions. In this context, it would be particularly interesting to determine whether Pex3 is also the decision maker of a quality control mechanism that eliminates peroxisomes when not correctly assembled and thus dysfunctional, or when not accurately distributed during cell division. Clearly, additional experiments are needed to understand how Pex3 regulates peroxisome homoeostasis, but this protein and this organelle could represent a convenient system to unveil the principles that regulate the steady-state level of other subcellular compartments.     

Autophagy--alias self-eating--appetite and ageing

Two articles—one published online in January and in the March issue EMBO reports—implicate autophagy in the control of appetite by regulating neuropeptide production in hypothalamic neurons. Autophagy decline with age in POMC neurons induces obesity and metabolic syndrome.Kaushik et al. EMBO reports, this issue doi:10.1038/embor.2011.260Macroautophagy, which I will call autophagy, is a critical process that degrades bulk cytoplasm, including organelles, protein oligomers and a range of selective substrates. It has been linked with diverse physiological and disease-associated functions, including the removal of certain bacteria, protein oligomers associated with neurodegenerative diseases and dysfunctional mitochondria [1]. However, the primordial role of autophagy—conserved from yeast to mammals—appears to be its ability to provide nutrients to starving cells by releasing building blocks, such as amino acids and free fatty acids, obtained from macromolecular degradation. In yeast, autophagy deficiency enhances death in starvation conditions [2], and in mice it causes death from starvation in the early neonatal period [3,4]. Two recent articles from the Singh group—one of them in this issue of EMBO reports—also implicate autophagy in central appetite regulation [5,6].Autophagy seems to decline with age in the liver [7], and it has thus been assumed that autophagy declines with age in all tissues, but this has not been tested rigorously in organs such as the brain. Conversely, specific autophagy upregulation in Caenorhabditis elegans and Drosophila extends lifespan, and drugs that induce autophagy—but also perturb unrelated processes, such as rapamycin—promote longevity in rodents [8].Autophagy literally means self-eating, and it is therefore interesting to see that this cellular ‘self-eating'' has systemic roles in mammalian appetite control. The control of appetite is influenced by central regulators, including various hormones and neurotransmitters, and peripheral regulators, including hormones, glucose and free fatty acids [9]. Autophagy probably has peripheral roles in appetite and energy balance, as it regulates lipolysis and free fatty acid release [10]. Furthermore, Singh and colleagues have recently implicated autophagy in central appetite regulation [5,6].The arcuate nucleus in the hypothalamus has received extensive attention as an integrator and regulator of energy homeostasis and appetite. Through its proximity to the median eminence, which is characterized by an incomplete blood–brain barrier, these neurons rapidly sense metabolic fluctuations in the blood. There are two different neuronal populations in the arcuate nucleus, which appear to have complementary effects on appetite (Fig 1). The proopiomelanocortin (POMC) neurons produce the neuropeptide precursor POMC, which is cleaved to form α-melanocyte stimulating hormone (α-MSH), among several other products. The α-MSH secreted from these neurons activates melanocortin 4 receptors on target neurons in the paraventricular nucleus of the hypothalamus, which ultimately reduce food intake. The second group of neurons contain neuropeptide Y (NPY) and Agouti-related peptide (AgRP). Secreted NPY binds to downstream neuronal receptors and stimulates appetite. AgRP blocks the ability of α-MSH to activate melanocortin 4 receptors [11]. Furthermore, AgRP neurons inhibit POMC neurons [9].Open in a separate windowFigure 1Schematic diagram illustrating the complementary roles of POMC and NPY/AgRP neurons in appetite control. AgRP, Agouti-related peptide; MC4R, melanocortin 4 receptor; α-MSH, α-melanocyte stimulating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin.The first study from Singh''s group started by showing that starvation induces autophagy in the hypothalamus [5]. This finding alone merits some comment. Autophagy is frequently assessed by using phosphatidylethanolamine-conjugated Atg8/LC3 (LC3-II), which is specifically associated with autophagosomes and autolysosomes. LC3-II levels on western blot and the number of LC3-positive vesicles strongly correlate with the number of autophagosomes [1]. To assess whether LC3-II formation is altered by a perturbation, its level can be assessed in the presence of lysosomal inhibitors, which inhibit LC3-II degradation by blocking autophagosome–lysosome fusion [12]. Therefore, differences in LC3-II levels in response to a particular perturbation in the presence of lysosomal inhibitors reflect changes in autophagosome synthesis. An earlier study using GFP-LC3 suggested that autophagy was not upregulated in the brains of starved mice, compared with other tissues where this did occur [13]. However, this study only measured steady state levels of autophagosomes and was performed before the need for lysosomal inhibitors was appreciated. Subsequent work has shown rapid flux of autophagosomes to lysosomes in primary neurons, which might confound analyses without lysosomal inhibitors [14]. Thus, the data of the Singh group—showing that autophagy is upregulated in the brain by a range of methods including lysosomal inhibitors [5]—address an important issue in the field and corroborate another recent study that examined this question by using sophisticated imaging methods [15].“…decreasing autophagy with ageing in POMC neurons could contribute to the metabolic problems associated with age”Singh and colleagues then analysed mice that have a specific knockout of the autophagy gene Atg7 in AgRP neurons [5]. Although fasting increases AgRP mRNA and protein levels in normal mice, these changes were not seen in the knockout mice. AgRP neurons provide inhibitory signals to POMC neurons, and Kaushik and colleagues found that the AgRP-specific Atg7 knockout mice had higher levels of POMC and α-MSH, compared with the normal mice. This indicated that starvation regulates appetite in a manner that is partly dependent on autophagy. The authors suggested that the peripheral free fatty acids released during starvation induce autophagy by activating AMP-activated protein kinase (AMPK), a known positive regulator of autophagy. This, in turn, enhances degradation of hypothalamic lipids and increases endogenous intracellular free fatty acid concentrations. The increased intracellular free fatty acids upregulate AgRP mRNA and protein expression. As AgRP normally inhibits POMC/α-MSH production in target neurons, a defect in AgRP responses in the autophagy-null AgRP neurons results in higher α-MSH levels, which could account for the decreased mouse bodyweight.In follow-up work, Singh''s group have now studied the effects of inhibiting autophagy in POMC neurons, again using Atg7 deletion [6]. These mice, in contrast to the AgRP autophagy knockouts, are obese. This might be accounted for, in part, by an increase in POMC preprotein levels and its cleavage product adrenocorticotropic hormone in the knockout POMC neurons, which is associated with a failure to generate α-MSH. Interestingly, these POMC autophagy knockout mice have impaired peripheral lipolysis in response to starvation, which the authors suggest might be due to reduced central sympathetic tone to the periphery from the POMC neurons. In addition, POMC-neuron-specific Atg7 knockout mice have impaired glucose tolerance.This new study raises several interesting issues. How does the autophagy defect in the POMC neurons alter the cleavage pattern of POMC? Is this modulated within the physiological range of autophagy activity fluctuations in response to diet and starvation? Importantly, in vivo, autophagy might fluctuate similarly (or possibly differently) in POMC and AgRP neurons in response to diet and/or starvation. Given the tight interrelation of these neurons, how does this affect their overall response to appetite regulation in wild-type animals?Finally, the study also shows that hypothalamic autophagosome formation is decreased in older mice. To my knowledge, this is the first such demonstration of this phenomenon in the brain. The older mice phenocopied aspects of the POMC-neuron autophagy null mice—increased hypothalamic POMC preprotein and ACTH and decreased α-MSH, along with similar adiposity and lipolytic defects, compared with young mice. These data are provocative from several perspectives. In the context of metabolism, it is tantalizing to consider that decreasing autophagy with ageing in POMC neurons could contribute to the metabolic problems associated with ageing. Again, this model considers the POMC neurons in isolation, and it would be important to understand how reduced autophagy in aged AgRP neurons counterbalances this situation. In a more general sense, the data strongly support the concept that neuronal autophagy might decline with age.Autophagy is a major clearance route for many mutant, aggregate-prone intracytoplasmic proteins that cause neurodegenerative disease, such as tau (Alzheimer disease), α-synuclein (Parkinson disease), and huntingtin (Huntington disease), and the risk of these diseases is age-dependent [1]. Thus, it is tempting to suggest that the dramatic age-related risks for these diseases could be largely due to decreased neuronal capacity of degrading these toxic proteins. Neurodegenerative pathology and age-related metabolic abonormalities might be related—some of the metabolic disturbances that occur in humans with age could be due to the accumulation of such toxic proteins. High levels of these proteins are seen in many people who do not have, or who have not yet developed, neurodegenerative diseases, as many of them start to accumulate decades before any sign of disease. These proteins might alter metabolism and appetite either directly by affecting target neurons, or by influencing hormonal and neurotransmitter inputs into such neurons.     

Telomere dysfunction puts the brakes on oncogene-induced cancers

EMBO J 31 13, 2839–2851 (2012); published online May082012Senescence represents a major tumour suppressor checkpoint activated by telomere dysfunction or cellular stress factors such as oncogene activation. In this issue of The EMBO Journal, Suram et al (2012) reveal a surprising interconnection between oncogene activation and telomere dysfunction induced senescence. The study supports an alternative model of tumour suppression, indicating that oncogene-induced accumulation of telomeric DNA damage contributes to the induction of senescence in telomerase-negative tumours.Telomere shortening limits the proliferative capacity of primary human cells after 50–70 cell divisions by induction of replicative senescence activated by critically short, dysfunctional telomeres. Different mechanisms were thought to initiate senescence in response to oncogene activation, which occurs abruptly within a few cell doublings (Serrano et al, 1997). Oncogene-induced senescence (OIS) involves an activation of DNA damage signals at stalled replication forks induced by DNA replication stress (Bartkova et al, 2006; Di Micco et al, 2006). Replication fork stalling in response to oncogene activation preferentially affects common fragile sites of the DNA (Tsantoulis et al, 2008). The ends of eukaryotic chromosomes—the telomeres–represent common fragile sites that are sensitive to replication fork stalling (Sfeir et al, 2009). These data made it tempting to speculate whether replication fork stalling at telomeres was causatively involved in OIS. Studies on replicative senescence in human fibroblast also supported this possibility showing that mitogenic signals amplify DNA damage responses in senescent cells (Satyanarayana et al, 2004).Multiple studies revealed experimental evidences that senescence suppresses tumour progression in mouse models and early human tumours (for review see Collado and Serrano, 2010). The relative contribution of OIS and telomere dysfunction induced senescence (TDIS) to tumour suppression and possible interconnections between the two pathways at the level of checkpoint induction were not investigated in previous studies. In this issue of The EMBO Journal, Suram et al (2012) describe the presence of TDIS in human precursor lesions but not in the corresponding malignant tumours. Mechanistically, the study shows that oncogenic signals cause replication fork stalling, resulting in telomeric DNA damage accumulation and activation of DNA damage checkpoints reminiscent to TDIS. Telomerase expression does not rescue replication fork stalling but prevents the accumulation of DNA damage at telomeres allowing a bypass of OIS.The study has several important implications for molecular pathways and therapeutic approaches in cancer that need to be further explored (Figure 1):Open in a separate windowFigure 1Traditional and new models of senescence in tumour suppression. (A) Traditional model of replicative senescence: Telomerase-negative tumour cell clones experience telomere shortening as a consequence of cell division. After a lack period depending on the initial telomere length, tumour cells accumulate telomere dysfunction and activation of senescence impairs tumour growth. Telomerase activation represents a late event allowing tumour progression. (B) New model of oncogene induced, telomere-dependent senescence: Oncogene activation leads to abrupt accumulation of DNA damage at telomeres resulting in senescence and tumour suppression. Telomerase-positive stem cells could be resistant to OIS and may be selected as the cell type of origin of tumour development.(i) Telomere length independent roles of telomeres in tumour suppressionThe classical model of telomere-dependent tumour suppression indicates that proliferation-dependent telomere shortening leads to telomere dysfunction, activation of DNA damage checkpoints, and induction of senescence suppressing the growth of telomerase-negative tumour clones. Studies on mouse models supported this concept showing that telomere shortening impairs the progression of initiated tumours in a telomere length-dependent manner (Feldser and Greider, 2007). The new data from Suram et al (2012) indicate that oncogene-induced replication fork stalling activates a telomere-dependent senescence checkpoint, which is independent of telomere length. The study shows that replication forks stall in response to oncogene activation throughout the genome. However, stalled replication forks are resolved in non-telomeric regions, whereas fork stalling inside telomeres leads to un-repairable DNA damage in telomerase-negative cells. These findings are in line with recent publication showing accumulation of un-repairable DNA damage in telomeric DNA in response to aging and stress-induced DNA damage (Fumagalli et al, 2012).(ii) Telomere length independent roles of telomerase in tumour progressionFollowing the classical model telomeres in tumour suppression (Figure 1A), telomerase re-activation is required for tumour progression by limiting telomere dysfunction and the induction of DNA damage checkpoints in response to telomere shortening. The new data from Suram et al (2012) indicate that telomerase has an additional telomere length independent role in tumour progression. The study shows that catalytically active telomerase prevents the activation of DNA damage signals originating from stalled replication forks inside telomeres in response to oncogene activation (Figure 1B). The exact mechanisms of telomerase-dependent healing of stalled replication forks at telomeres remain to be elucidated. It is also unclear whether telomerase activity can prevent any type of DNA damage at telomeres as an over-expression of TERT could not suppress irradiation-induced cellular senescence or the persistence of telomeric DDR following irradiation, H₂O₂, or chemotherapy induced DNA damage (Hewitt et al, 2012).The data could provide a plausible explanation for the increased tumorigenesis in telomerase transgenic mice—a finding which is difficult to explain by telomere length dependent effects of telomerase given the long telomere reserves in mouse tissues (Gonzalez-Suarez et al, 2001). According to the findings of Suram et al (2012), anti-telomerase therapies could have immediate anti-cancer effects in tumours depending on telomerase-mediated healing of stalled replication forks at telomeres. Specific markers for this dependency could be of clinical value. In addition, the data support the concept that somatic stem cells could represent the cell type of origin of cancers. In contrast to differentiated somatic cells, tissues stem cells are often telomerase-positive, indicating that stem cells might be less sensitive to OIS.     

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.     

A POTluck of peptide transporters

EMBO J (2012) 31 16, 3411–3421 doi:10.1038/emboj.2012.157EMBO J (2010) 30 2, 417–426 doi:10.1038/emboj.2010.309The uptake of diet-derived peptides is mediated by the conserved proton-dependent oligopeptide transporter (POT) family. Crystal structures of bacterial transporters in the inward-open conformation and in an occluded conformation published in The EMBO Journal provide structural insight into the proton-driven peptide transport cycle.Lipid bilayers are generally not permeable to amino acids, peptides and their derivatives. Cells therefore rely on active transporters or facilitators for the uptake and redistribution of these valuable compounds. In the late 1970s, many transporter activities were identified and characterized from brain synaptic or kidney/intestinal brush border membranes, including for example transporters for serotonin, glutamate and alanine (Fass et al, 1977; Rudnick, 1977; Kanner and Sharon, 1978). Their activities were shown to be dependent on the Na⁺ gradient maintained by the Na⁺, K⁺-ATPase, and for a while, an attractive model was that active, secondary transporters of the plasma membrane in animals are driven by the Na⁺ gradient whereas those in plants, fungi and bacteria are coupled to the proton gradient.This distinction changed when it was shown that transport of a glycyl-L-proline dipetide across rabbit kidney brush border membranes was in fact dependent on a proton gradient (Ganapathy and Leibach, 1983). In 1994, the rabbit PEPT1 protein was successfully cloned, expressed and characterized as the proton-dependent peptide transporter (Fei et al, 1994) responsible of this activity, and it became a founding member of the large proton-dependent oligopeptide transporter (POT) family.Human PEPT1 is recognized for its key function in the uptake of peptide nutrients from the intestinal tract. However, along with the PEPT2 isoform, it exerts also a range of other important peptide transport functions in various tissues such as kidney, brain, glandular and endocrine tissues and placenta. For a general overview of PEPT1 tissue distribution, see for example http://www.proteinatlas.org/ENSG00000088386.POT proteins are found in eukaryotes and eubacteria, while being surprisingly absent in archaea. In 1989, Konings and coworkers identified an H⁺-dependent transporter for di- and tripeptides in Lactococcus lactis (Smid et al, 1989), and many bacterial members of the family have since then been characterized and recognized as members of the POT family, such as the E. coli yjdL transporter (Jensen et al, 2011).For many years, the mechanism of POT proteins, which selectively transport di- or tripeptides of any kind, remained puzzling. Quite clearly, recognition of the N- and C-termini of the peptide, rather than interactions with the side chains, would be expected to determine optimal cargo recognition. Furthermore, the question remained of how transport would be coupled to an H⁺ gradient.Two reports from the Newstead/Iwata group published in The EMBO Journal provide critical new insight into the structure and transport mechanism of the POT family (Newstead et al, 2011; Solcan et al, 2012). Based on a wide-spread approach in membrane protein crystallography of using bacterial homologues of mammalian membrane proteins, two different bacterial species of POT transporters—the PEPT_So from Shewanella oneidensis and the PEPT_St from Streptococcus thermophilus were crystallized and their structures determined at medium resolution. Overall, the POT structure is similar to other proteins of the Major Facilitator Superfamily (MFS) such as the sugar permeases (see Figure 1). A simple mechanistic model has been proposed for MFS proteins, the rocking bundle model. In this model, two related ‘halves'' of the transporter (transmembrane spanning segments 1–6 and 7–12, respectively) form a V-shaped structure with the substrate site in the middle that is exposed to either the outside (uptake) or the inside (release) of the cell via an occluded intermediate state. The proton gradient is thought to stimulate the switch between these two conformations. In real life, the picture is not so simple as seen here for the POT transporters: the first structure, PEPT_So, revealed an asymmetric, occluded state with an undefined substrate in a buried cavity. Transition to the inward-open conformation, as revealed form the latest structure and further supported by e.g. molecular dynamics simulations and mutational studies, shows both rigid-body motions and more flexible changes. A markedly polar environment with charged residues along the transport pathway is the basis for the proton-coupled mechanism, which involves the formation and dissociation of salt bridges to open and close gates to the intracellular and extracellular environment. In the occluded state, substrate binding is mediated by conserved tyrosines and a glutamate in a manner homologous to what has been predicted for PEPT1 and yjdL (Meredith et al, 2000; Jensen et al, 2011).Open in a separate windowFigure 1Alternating access mechanism of the proton-dependent oligopeptide transporter (POT) family. To the right, the PEPT_St transporter is shown in the inward-open conformation (Solcan et al, 2012, PDB 4APS), in the middle, the PEPT_So in the inward-occluded conformation (Newstead et al, 2011, PDB 2XUT). To the left, another protein of the Major Facilitator Family, the Fucose transporter is shown in the outward-facing conformation (Dang et al, 2010, PDB 3O7Q).Besides their critical function as peptide transporters, PEPT1 and 2 are also responsible for the uptake of drugs with peptide-like moieties, such as penicillin antibiotics and PEPT1 and 2 are widely recognized as drug uptake vehicles in drug development (Nielsen and Brodin, 2003). Clearly, we need more detailed information of how specific compounds are recognized and the kinetics of active transport is determined. Furthermore, in agricultural livestock production, the efficiency of protein nutrient uptake is extremely important and relies on the optimal match between uptake kinetics and the available resources of feed (D''Inca et al, 2011). There is no doubt that we wish to see the POTluck increasing from many sides.     

Cargo carriers from the Golgi to the cell surface

EMBO J (2012) 31 20, 3976–3990 doi:10.1038/emboj.2012.235; published online August212012In this issue, Malhotra and colleagues use biochemical approaches to identify a new class of secretory cargo carriers (CARTS) that do not contain the larger cargoes, collagen or Vesicular stomatitis virus (VSV)-G glycoprotein. CARTS appear to be basolateral membrane-directed carriers that use myosin for their motility but not for their formation.Protein secretion involves the collection of proteins into transport carriers that form at the exit (or ‘trans'') face of the Golgi apparatus for delivery to the cell surface. Multiple classes of secretory carriers form at the trans Golgi (Anitei and Hoflack, 2011). Some deliver cargo continuously to the cell surface; others release cargo in response to a signal. Regulated and constitutive secretory cargoes traverse the Golgi complex together and are sorted just before their exit. Proteins destined for different domains of the plasma membrane are also packaged into different carriers that bud from the Golgi and are delivered to either the apical or basolateral surface, respectively. Also departing the Golgi are clathrin-coated vesicles that carry newly synthesized lysosomal enzymes to endocytic compartments.Despite the importance of protein secretion, the carriers that transport cargo from the Golgi to the cell surface have not yet been isolated or characterized. When visualized in live cells expressing GFP-tagged cargo, Golgi-to-cell surface carriers appear as variably sized vesicles and tubules of 1–8 μm in length (Hirschberg et al, 1998; Toomre et al, 1999; Polishchuk et al, 2003; Anitei and Hoflack, 2011). Both actin- and microtubule-based motors participate in their formation, along with phosphatidylinositol 4-phosphate that is needed to recruit components that participate in membrane budding and scission.In this issue, Wakana et al (2012) report the identification of transport carriers (CARriers from the trans Golgi network to the cell surface or CARTS) that mediate the Golgi-to-cell surface transport of a select set of cargo proteins. Unexpectedly, the authors report that collagen and VSV-G glycoprotein use a different carrier for their transport to the cell surface; CARTS also use myosin II for motility but not for vesicle scission (see Figure 1).Open in a separate windowFigure 1PAUF and collagen export from the Golgi require protein kinase D, which distinguishes these export events from the transport of proteins to the apical surface. Small cargoes like PAUF use myosin II for vesicle motility after carrier formation; large cargoes like collagen and VSV-G may use myosin for both carrier formation and motility.Wakana et al (2012) first characterize the vesicle formation process by monitoring TGN46. TGN46 is a protein of unknown function that localizes to the trans Golgi at steady state but cycles between the Golgi and the cell surface. Thus, TGN46 should be present in the Golgi and to a lesser extent, in secretory transport vesicles and endocytic and recycling vesicles. The authors use digitonin to permeabilize HeLa cells and monitor vesicle budding that occurs upon addition of ATP and rat liver cytosol. They use differential centrifugation to remove large membranes and identify a population of putative carriers that only sediment upon centrifugation at high speed and form in the presence of ATP and cytosol. TGN46-vesicle formation requires protein kinase D, a kinase needed for secretory carrier formation in cells (Liljedahl et al, 2001). Next, the authors use antibodies that recognize the cytoplasmic domain of TGN46 to immuno-isolate intact vesicles; controls show that the isolated membranes do not represent lysosomes, endosomes or the Golgi itself. Satisfyingly, the isolated vesicles include a secretory cargo: exogenously expressed, signal sequence containing, horseradish peroxidase. This is good evidence that the isolated carriers represent exocytic vesicles.Mass spectrometry was used to identify candidate transport vesicle proteins; low yields precluded the authors from carrying out a rigorous analysis. Nevertheless, pancreatic adenocarcinoma upregulated factor (PAUF or ZG16B) and lysozyme were identified and confirmed as endogenous, soluble cargo proteins, together with synaptotagmin II, Rab6A, Rab8A and myosin II. Expression of a protein kinase D mutant enabled the authors to accumulate PAUF in trans Golgi tubules; in cells, PAUF carriers were distinct from those coated with COPI, COPII and clathrin. By EM, the carriers were round to elongated, 100–250 nm diameter structures. The identification of an endogenous, constitutively secreted protein will be valuable to those studying secretion.Myosin II has been reported to play a role in the formation of vesicles containing VSV-G glycoprotein (cf. Miserey-Lenkei et al, 2010). Wakana et al (2012) showed that PAUF secretion was inhibited in the presence of blebbistatin, a myosin II inhibitor. However, in the presence of blebbistatin, PAUF-containing punctate structures detected by light microscopy were unchanged in total number or distribution, suggesting that CARTS formation is myosin II independent.Many studies of protein secretion have monitored the trafficking of VSV-G glycoprotein (Hirschberg et al, 1998; Toomre et al, 1999; Polishchuk et al, 2003). G protein is convenient and well studied but an important property that is often overlooked is the tendency of viral glycoproteins to form crystalline arrays within the secretory pathway, especially if proteins are accumulated in the trans Golgi by incubation of cells at 20°C (Griffiths et al, 1985). Under these conditions, cryoelectron microscopy has documented the oligomerization of viral glycoproteins. Large protein assemblies like these and like collagen may require modification of the vesicle formation process to accommodate the larger proteins (Malhotra and Erlmann, 2011; Jin et al, 2012). Thus, it was especially interesting that collagen and VSV-G protein are not detected in PAUF-containing vesicles en route to the cell surface. This may explain why PAUF carriers were not dependent upon myosin II (Wakana et al, 2012) while VSV-G carriers were (Miserey-Lenkei et al, 2010)—perhaps the larger carriers of VSV-G and collagen have a greater need for myosin II in their formation.Several models can explain the formation of the two transport vesicle classes detected. A trivial explanation would be that the carriers are distinct because they are destined for different plasma membrane domains—apical versus basolateral. However, only basolateral transport requires protein kinase D (Yeaman et al, 2004) and protein kinase D is important for all the cargoes studied here—suggesting that both carrier types are basolaterally directed. Simply by default, collection of large assemblies into a nascent vesicle may physically exclude soluble PAUF protein. Alternatively, larger cargoes may use a molecularly distinct class of transport carrier. Yet to be identified are the protein constituents that define CARTS—proteins that collect cargoes together with the vesicle targeting and fusion machinery that must be included in all functional, newly formed transport vesicles. Once these markers are identified, it will become possible to distinguish between these two models and to isolate CARTS in larger quantities for full mass spec analysis. For now, the findings confirm the segregation of small and large cargoes into different vesicles that traverse the path from the Golgi to the cell surface and clarify the role of myosin in transporting these vesicles, but not necessarily pinching them off from the trans Golgi.