Showing papers in "The EMBO Journal in 2010"

ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import

Abstract: Mutations in fused in sarcoma (FUS) are a cause of familial amyotrophic lateral sclerosis (fALS). Patients carrying point mutations in the C-terminus of FUS show neuronal cytoplasmic FUS-positive inclusions, whereas in healthy controls, FUS is predominantly nuclear. Cytoplasmic FUS inclusions have also been identified in a subset of frontotemporal lobar degeneration (FTLD-FUS). We show that a non-classical PY nuclear localization signal (NLS) in the C-terminus of FUS is necessary for nuclear import. The majority of fALS-associated mutations occur within the NLS and impair nuclear import to a degree that correlates with the age of disease onset. This presents the first case of disease-causing mutations within a PY-NLS. Nuclear import of FUS is dependent on Transportin, and interference with this transport pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules. Moreover, proteins known to be stress granule markers co-deposit with inclusions in fALS and FTLD-FUS patients, implicating stress granule formation in the pathogenesis of these diseases. We propose that two pathological hits, namely nuclear import defects and cellular stress, are involved in the pathogenesis of FUS-opathies.

HDAC6 controls autophagosome maturation essential for ubiquitin-selective quality-control autophagy

Abstract: Autophagy is primarily considered a non-selective degradation process induced by starvation. Nutrient-independent basal autophagy, in contrast, imposes intracellular QC by selective disposal of aberrant protein aggregates and damaged organelles, a process critical for suppressing neurodegenerative diseases. The molecular mechanism that distinguishes these two fundamental autophagic responses, however, remains mysterious. Here, we identify the ubiquitin-binding deacetylase, histone deacetylase-6 (HDAC6), as a central component of basal autophagy that targets protein aggregates and damaged mitochondria. Surprisingly, HDAC6 is not required for autophagy activation; rather, it controls the fusion of autophagosomes to lysosomes. HDAC6 promotes autophagy by recruiting a cortactin-dependent, actin-remodelling machinery, which in turn assembles an F-actin network that stimulates autophagosome–lysosome fusion and substrate degradation. Indeed, HDAC6 deficiency leads to autophagosome maturation failure, protein aggregate build-up, and neurodegeneration. Remarkably, HDAC6 and F-actin assembly are completely dispensable for starvation-induced autophagy, uncovering the fundamental difference of these autophagic modes. Our study identifies HDAC6 and the actin cytoskeleton as critical components that define QC autophagy and uncovers a novel regulation of autophagy at the level of autophagosome–lysosome fusion.

LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis

Abstract: Autophagy, a critical process for bulk degradation of proteins and organelles, requires conjugation of Atg8 proteins to phosphatidylethanolamine on the autophagic membrane. At least eight different Atg8 orthologs belonging to two subfamilies (LC3 and GATE-16/GABARAP) occur in mammalian cells, but their individual roles and modes of action are largely unknown. In this study, we dissect the activity of each subfamily and show that both are indispensable for the autophagic process in mammalian cells. We further show that both subfamilies act differently at early stages of autophagosome biogenesis. Accordingly, our results indicate that LC3s are involved in elongation of the phagophore membrane whereas the GABARAP/GATE-16 subfamily is essential for a later stage in autophagosome maturation.

A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression.

Abstract: A growing number of long nuclear-retained non-coding RNAs (ncRNAs) have recently been described. However, few functions have been elucidated for these ncRNAs. Here, we have characterized the function of one such ncRNA, identified as metastasis-associated lung adenocarcinoma transcript 1 (Malat1). Malat1 RNA is expressed in numerous tissues and is highly abundant in neurons. It is enriched in nuclear speckles only when RNA polymerase II-dependent transcription is active. Knock-down studies revealed that Malat1 modulates the recruitment of SR family pre-mRNA-splicing factors to the transcription site of a transgene array. DNA microarray analysis in Malat1-depleted neuroblastoma cells indicates that Malat1 controls the expression of genes involved not only in nuclear processes, but also in synapse function. In cultured hippocampal neurons, knock-down of Malat1 decreases synaptic density, whereas its over-expression results in a cell-autonomous increase in synaptic density. Our results suggest that Malat1 regulates synapse formation by modulating the expression of genes involved in synapse formation and/or maintenance.

ADAM10 is the physiologically relevant, constitutive α-secretase of the amyloid precursor protein in primary neurons

Abstract: The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called α-secretase. This is considered an important mechanism preventing the generation of the Alzheimer's disease amyloid-β peptide (Aβ). α-Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel α-secretase-cleavage site-specific antibody, we found that RNAi-mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP α-secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of α-cleavage. This finding was further confirmed by mass-spectrometric detection of APP-cleavage fragments. Surprisingly, in different cell lines, the reduction of α-secretase cleavage was not paralleled by a corresponding increase in the Aβ-generating β-secretase cleavage, revealing that both proteases do not always compete for APP as a substrate. Instead, our data suggest a novel pathway for APP processing, in which ADAM10 can partially compete with γ-secretase for the cleavage of a C-terminal APP fragment generated by β-secretase. We conclude that ADAM10 is the physiologically relevant, constitutive α-secretase of APP.

The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation

Abstract: The transcription factor ATF4 regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses, and it is overexpressed in human solid tumours, suggesting that it has an important function in tumour progression. Here, we report that inhibition of ATF4 expression blocked proliferation and survival of transformed cells, despite an initial activation of cytoprotective macroautophagy. Knockdown of ATF4 significantly reduced the levels of asparagine synthetase (ASNS) and overexpression of ASNS or supplementation of asparagine in trans, reversed the proliferation block and increased survival in ATF4 knockdown cells. Both amino acid and glucose deprivation, stresses found in solid tumours, activated the upstream eukaryotic initiation factor 2α (eIF2α) kinase GCN2 to upregulate ATF4 target genes involved in amino acid synthesis and transport. GCN2 activation/overexpression and increased phospho-eIF2α were observed in human and mouse tumours compared with normal tissues and abrogation of ATF4 or GCN2 expression significantly inhibited tumour growth in vivo. We conclude that the GCN2-eIF2α-ATF4 pathway is critical for maintaining metabolic homeostasis in tumour cells, making it a novel and attractive target for anti-tumour approaches.

Mitochondrial fission and remodelling contributes to muscle atrophy

Abstract: Mitochondria are crucial organelles in the production of energy and in the control of signalling cascades. A machinery of pro-fusion and fission proteins regulates their morphology and subcellular localization. In muscle this results in an orderly pattern of intermyofibrillar and subsarcolemmal mitochondria. Muscular atrophy is a genetically controlled process involving the activation of the autophagy-lysosome and the ubiquitin–proteasome systems. Whether and how the mitochondria are involved in muscular atrophy is unknown. Here, we show that the mitochondria are removed through autophagy system and that changes in mitochondrial network occur in atrophying muscles. Expression of the fission machinery is per se sufficient to cause muscle wasting in adult animals, by triggering organelle dysfunction and AMPK activation. Conversely, inhibition of the mitochondrial fission inhibits muscle loss during fasting and after FoxO3 overexpression. Mitochondrial-dependent muscle atrophy requires AMPK activation as inhibition of AMPK restores muscle size in myofibres with altered mitochondria. Thus, disruption of the mitochondrial network is an essential amplificatory loop of the muscular atrophy programme.

Genome-Wide Analysis of ETS-Family DNA-Binding In Vitro and In Vivo

Abstract: Members of the large ETS family of transcription factors (TFs) have highly similar DNA-binding domains (DBDs)—yet they have diverse functions and activities in physiology and oncogenesis. Some differences in DNA-binding preferences within this family have been described, but they have not been analysed systematically, and their contributions to targeting remain largely uncharacterized. We report here the DNA-binding profiles for all human and mouse ETS factors, which we generated using two different methods: a high-throughput microwell-based TF DNA-binding specificity assay, and protein-binding microarrays (PBMs). Both approaches reveal that the ETS-binding profiles cluster into four distinct classes, and that all ETS factors linked to cancer, ERG, ETV1, ETV4 and FLI1, fall into just one of these classes. We identify amino-acid residues that are critical for the differences in specificity between all the classes, and confirm the specificities in vivo using chromatin immunoprecipitation followed by sequencing (ChIP-seq) for a member of each class. The results indicate that even relatively small differences in in vitro binding specificity of a TF contribute to site selectivity in vivo.

Neurotoxicity of Alzheimer's disease Aβ peptides is induced by small changes in the Aβ42 to Aβ40 ratio.

Abstract: The amyloid peptides Aβ(40) and Aβ(42) of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aβ(42) seems more pathogenic than Aβ(40), the reason for this is not well understood. We show here that small alterations in the Aβ(42):Aβ(40) ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ(42):Aβ(40) ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti-amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non-toxic intermediates.

Inhibition of mitochondrial fusion by α-synuclein is rescued by PINK1, Parkin and DJ-1.

Abstract: Aggregation of α-synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA-mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin Δ1–79 or by DJ-1 C106A.

High‐resolution profiling of γH2AX around DNA double strand breaks in the mammalian genome

Abstract: Chromatin acts as a key regulator of DNA-related processes such as DNA damage repair. Although ChIP-chip is a powerful technique to provide high-resolution maps of protein-genome interactions, its use to study DNA double strand break (DSB) repair has been hindered by the limitations of the available damage induction methods. We have developed a human cell line that permits induction of multiple DSBs randomly distributed and unambiguously positioned within the genome. Using this system, we have generated the first genome-wide mapping of gammaH2AX around DSBs. We found that all DSBs trigger large gammaH2AX domains, which spread out from the DSB in a bidirectional, discontinuous and not necessarily symmetrical manner. The distribution of gammaH2AX within domains is influenced by gene transcription, as parallel mappings of RNA Polymerase II and strand-specific expression showed that gammaH2AX does not propagate on active genes. In addition, we showed that transcription is accurately maintained within gammaH2AX domains, indicating that mechanisms may exist to protect gene transcription from gammaH2AX spreading and from the chromatin rearrangements induced by DSBs.

Architecture of the RNA polymerase II–TFIIF complex revealed by cross-linking and mass spectrometry

Abstract: Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes.

An ESRP‐regulated splicing programme is abrogated during the epithelial–mesenchymal transition

Abstract: Alternative splicing achieves coordinated changes in post-transcriptional gene expression programmes through the activities of diverse RNA-binding proteins. Epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) are cell-type-specific regulators of transcripts that switch splicing during the epithelial–mesenchymal transition (EMT). To define a comprehensive programme of alternative splicing that is regulated during the EMT, we identified an extensive ESRP-regulated splicing network of hundreds of alternative splicing events within numerous genes with functions in cell–cell adhesion, polarity, and migration. Loss of this global ESRP-regulated epithelial splicing programme induces the phenotypic changes in cell morphology that are observed during the EMT. Components of this splicing signature provide novel molecular markers that can be used to characterize the EMT. Bioinformatics and experimental approaches revealed a high-affinity ESRP-binding motif and a predictive RNA map that governs their activity. This work establishes the ESRPs as coordinators of a complex alternative splicing network that adds an important post-transcriptional layer to the changes in gene expression that underlie epithelial–mesenchymal transitions during development and disease.

Regulation of DNA‐damage responses and cell‐cycle progression by the chromatin remodelling factor CHD4

Abstract: The chromatin remodelling factor chromodomain helicase DNA-binding protein 4 (CHD4) is a catalytic subunit of the NuRD transcriptional repressor complex. Here, we reveal novel functions for CHD4 in the DNA-damage response (DDR) and cell-cycle control. We show that CHD4 mediates rapid poly(ADP-ribose)-dependent recruitment of the NuRD complex to DNA-damage sites, and we identify CHD4 as a phosphorylation target for the apical DDR kinase ataxia-telangiectasia mutated. Functionally, we show that CHD4 promotes repair of DNA double-strand breaks and cell survival after DNA damage. In addition, we show that CHD4 acts as an important regulator of the G1/S cell-cycle transition by controlling p53 deacetylation. These results provide new insights into how the chromatin remodelling complex NuRD contributes to maintaining genome stability.

Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients

Abstract: Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55–200 CGG repeats in the 5′-UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA-binding proteins by the expanded CGG repeats. Sam68 is an RNA-binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA-binding proteins sequentially, first Sam68, then hnRNP-G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing-regulatory function. Consequently, Sam68-responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain-of-function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.

c‐IAP1 and UbcH5 promote K11‐linked polyubiquitination of RIP1 in TNF signalling

Abstract: Ubiquitin ligases are critical components of the ubiquitination process that determine substrate specificity and, in collaboration with E2 ubiquitin-conjugating enzymes, regulate the nature of polyubiquitin chains assembled on their substrates. Cellular inhibitor of apoptosis (c-IAP1 and c-IAP2) proteins are recruited to TNFR1-associated signalling complexes where they regulate receptor-stimulated NF-κB activation through their RING domain ubiquitin ligase activity. Using a directed yeast two-hybrid screen, we found several novel and previously identified E2 partners of IAP RING domains. Among these, the UbcH5 family of E2 enzymes are critical regulators of the stability of c-IAP1 protein following destabilizing stimuli such as TWEAK or CD40 signalling or IAP antagonists. We demonstrate that c-IAP1 and UbcH5 family promote K11-linked polyubiquitination of receptor-interacting protein 1 (RIP1) in vitro and in vivo. We further show that TNFα-stimulated NF-κB activation involves endogenous K11-linked ubiquitination of RIP1 within the TNFR1 signalling complex that is c-IAP1 and UbcH5 dependent. Lastly, NF-κB essential modifier efficiently binds K11-linked ubiquitin chains, suggesting that this ubiquitin linkage may have a signalling role in the activation of proliferative cellular pathways.

Phosphorylation of Mcl‐1 by CDK1–cyclin B1 initiates its Cdc20‐dependent destruction during mitotic arrest

Abstract: The balance between cell cycle progression and apoptosis is important for both surveillance against genomic defects and responses to drugs that arrest the cell cycle. In this report, we show that the level of the human anti-apoptotic protein Mcl-1 is regulated during the cell cycle and peaks at mitosis. Mcl-1 is phosphorylated at two sites in mitosis, Ser64 and Thr92. Phosphorylation of Thr92 by cyclin-dependent kinase 1 (CDK1)–cyclin B1 initiates degradation of Mcl-1 in cells arrested in mitosis by microtubule poisons. Mcl-1 destruction during mitotic arrest requires proteasome activity and is dependent on Cdc20/Fizzy, which mediates recognition of mitotic substrates by the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Stabilisation of Mcl-1 during mitotic arrest by mutation of either Thr92 or a D-box destruction motif inhibits the induction of apoptosis by microtubule poisons. Thus, phosphorylation of Mcl-1 by CDK1–cyclin B1 and its APC/CCdc20-mediated destruction initiates apoptosis if a cell fails to resolve mitosis. Regulation of apoptosis, therefore, is linked intrinsically to progression through mitosis and is governed by a temporal mechanism that distinguishes between normal mitosis and prolonged mitotic arrest.

mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide

Abstract: The mechanisms that couple translation and protein processing are poorly understood in higher eukaryotes. Although mammalian target of rapamycin (mTOR) complex 1 (mTORC1) controls translation initiation, the function of mTORC2 in protein synthesis remains to be defined. In this study, we find that mTORC2 can colocalize with actively translating ribosomes and can stably interact with rpL23a, a large ribosomal subunit protein present at the tunnel exit. Exclusively during translation of Akt, mTORC2 mediates phosphorylation of the nascent polypeptide at the turn motif (TM) site, Thr450, to avoid cotranslational Akt ubiquitination. Constitutive TM phosphorylation occurs because the TM site is accessible, whereas the hydrophobic motif (Ser473) site is concealed in the ribosomal tunnel. Thus, mTORC2 can function cotranslationally by phosphorylating residues in nascent chains that are critical to attain proper conformation. Our findings reveal that mTOR links protein production with quality control.

Daughter cell separation is controlled by cytokinetic ring‐activated cell wall hydrolysis

Abstract: During bacterial cytokinesis, hydrolytic enzymes are used to split wall material shared by adjacent daughter cells to promote their separation. Precise control over these enzymes is critical to prevent breaches in wall integrity that can cause cell lysis. How these potentially lethal hydrolases are regulated has remained unknown. Here, we investigate the regulation of cell wall turnover at the Escherichia coli division site. We show that two components of the division machinery with LytM domains (EnvC and NlpD) are direct regulators of the cell wall hydrolases (amidases) responsible for cell separation (AmiA, AmiB and AmiC). Using in vitro cell wall cleavage assays, we show that EnvC activates AmiA and AmiB, whereas NlpD activates AmiC. Consistent with these findings, we show that an unregulated EnvC mutant requires functional AmiA or AmiB but not AmiC to induce cell lysis, and that the loss of NlpD phenocopies an AmiC− defect. Overall, our results suggest that cellular amidase activity is regulated spatially and temporally by coupling their activation to the assembly of the cytokinetic ring.

NANOG regulates glioma stem cells and is essential in vivo acting in a cross‐functional network with GLI1 and p53

Abstract: A cohort of genes associated with embryonic stem (ES) cell behaviour, including NANOG, are expressed in a number of human cancers. They form an ES-like signature we first described in glioblastoma multiforme (GBM), a highly invasive and incurable brain tumour. We have also shown that HEDGEHOG-GLI (HH-GLI) signalling is required for GBM growth, stem cell expansion and the expression of this (ES)-like stemness signature. Here, we address the function of NANOG in human GBMs and its relationship with HH-GLI activity. We find that NANOG modulates gliomasphere clonogenicity, CD133(+) stem cell cell behavior and proliferation, and is regulated by HH-GLI signalling. However, GLI1 also requires NANOG activity forming a positive loop, which is negatively controlled by p53 and vice versa. NANOG is essential for GBM tumourigenicity in orthotopic xenografts and it is epistatic to HH-GLI activity. Our data establish NANOG as a novel HH-GLI mediator essential for GBMs. We propose that this function is conserved and that tumour growth and stem cell behaviour rely on the status of a functional GLI1-NANOG-p53 network.

Wnt5a regulates distinct signalling pathways by binding to Frizzled2

Abstract: Wnt5a regulates multiple intracellular signalling cascades, but how Wnt5a determines the specificity of these pathways is not well understood. This study examined whether the internalization of Wnt receptors affects the ability of Wnt5a to regulate its signalling pathways. Wnt5a activated Rac in the β-catenin-independent pathway, and Frizzled2 (Fz2) and Ror1 or Ror2 were required for this action. Fz2 was internalized through a clathrin-mediated route in response to Wnt5a, and inhibition of clathrin-dependent internalization suppressed the ability of Wnt5a to activate Rac. As another action of Wnt5a, it inhibited Wnt3a-dependent lipoprotein receptor-related protein 6 (LRP6) phosphorylation and β-catenin accumulation. Wnt3a-dependent phosphorylation of LRP6 was enhanced in Wnt5a knockout embryonic fibroblasts. Fz2 was also required for the Wnt3a-dependent accumulation of β-catenin, and Wnt5a competed with Wnt3a for binding to Fz2 in vitro and in intact cells, thereby inhibiting the β-catenin pathway. This inhibitory action of Wnt5a was not affected by the impairment of clathrin-dependent internalization. These results suggest that Wnt5a regulates distinct pathways through receptor internalization-dependent and -independent mechanisms.

The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine‐specific demethylase 1

Abstract: Epithelial–mesenchymal transition (EMT) is a transdifferentiation programme. The mechanism underlying the epigenetic regulation of EMT remains unclear. In this study, we identified that Snail1 interacted with histone lysine-specific demethylase 1 (LSD1). We demonstrated that the SNAG domain of Snail1 and the amine oxidase domain of LSD1 were required for their mutual interaction. Interestingly, the sequence of the SNAG domain is similar to that of the histone H3 tail, and the interaction of Snail1 with LSD1 can be blocked by LSD1 enzymatic inhibitors and a histone H3 peptide. We found that the formation of a Snail1–LSD1–CoREST ternary complex was critical for the stability and function of these proteins. The co-expression of these molecules was found in cancer cell lines and breast tumour specimens. Furthermore, we showed that the SNAG domain of Snail1 was critical for recruiting LSD1 to its target gene promoters and resulted in suppression of cell migration and invasion. Our study suggests that the SNAG domain of Snail1 resembles a histone H3-like structure and functions as a molecular hook for recruiting LSD1 to repress gene expression in metastasis.

Ku prevents Exo1 and Sgs1‐dependent resection of DNA ends in the absence of a functional MRX complex or Sae2

Abstract: In this study, we investigate the interplay between Ku, a central non-homologous end-joining component, and the Mre11–Rad50–Xrs2 (MRX) complex and Sae2, end-processing factors crucial for initiating 5′-3′ resection of double-strand break (DSB) ends. We show that in the absence of end protection by Ku, the requirement for the MRX complex is bypassed and resection is executed by Exo1. In contrast, both the Exo1 and Sgs1 resection pathways contribute to DSB processing in the absence of Ku and Sae2 or when the MRX complex is intact, but functionally compromised by elimination of the Mre11 nuclease activity. The ionizing radiation sensitivity of a mutant defective for extensive resection (exo1Δ sgs1Δ) cannot be suppressed by the yku70Δ mutation, indicating that Ku suppression is specific to the initiation of resection. We provide evidence that replication-associated DSBs need to be processed by Sae2 for repair by homologous recombination unless Ku is absent. Finally, we show that the presence of Ku exacerbates DNA end-processing defects established in the sae2Δ sgs1Δ mutant, leading to its lethality.

The IKK complex contributes to the induction of autophagy

Abstract: In response to stress, cells start transcriptional and transcription-independent programs that can lead to adaptation or death. Here, we show that multiple inducers of autophagy, including nutrient depletion, trigger the activation of the IKK (IκB kinase) complex that is best known for its essential role in the activation of the transcription factor NF-κB by stress. Constitutively active IKK subunits stimulated autophagy and transduced multiple signals that operate in starvation-induced autophagy, including the phosphorylation of AMPK and JNK1. Genetic inhibition of the nuclear translocation of NF-κB or ablation of the p65/RelA NF-κB subunit failed to suppress IKK-induced autophagy, indicating that IKK can promote the autophagic pathway in an NF-κB-independent manner. In murine and human cells, knockout and/or knockdown of IKK subunits (but not that of p65) prevented the induction of autophagy in response to multiple stimuli. Moreover, the knockout of IKK-β suppressed the activation of autophagy by food deprivation or rapamycin injections in vivo, in mice. Altogether, these results indicate that IKK has a cardinal role in the stimulation of autophagy by physiological and pharmacological stimuli.

An intimate liaison: spatial organization of the endoplasmic reticulum–mitochondria relationship

Abstract: Organelle localization is often crucial to properly modulate cellular functions and signalling cascades. For example, the distribution of organelles in axons is crucial for their function and is dysregulated in several diseases. Similarly, relative positioning of two or more organelles is also important to perform certain specialized processes. Perhaps, the best-known form of interorganellar organization is that between endoplasmic reticulum (ER) and mitochondria. Close communication between these two compartments has been observed for a long time. Recent evidence suggests that this is the basis for a bidirectional communication regulating a number of physiological processes ranging from mitochondrial energy and lipid metabolism to Ca2+ signalling and cell death. The recent discovery of some of the molecular mediators of the tethering already allowed to extend the function of this paradigmatic spatial organization to previously unexpected functions, and will foster future research to explore it in cellular signalling cascades as well as in disease.

The march of the PINs: developmental plasticity by dynamic polar targeting in plant cells

Abstract: Development of plants and their adaptive capacity towards ever-changing environmental conditions largely depend on the spatial distribution of the plant hormone auxin. At the cellular level, various internal and external signals are translated into specific changes in the polar, subcellular localization of auxin transporters from the PIN family thereby directing and redirecting the intercellular fluxes of auxin. The current model of polar targeting of PIN proteins towards different plasma membrane domains encompasses apolar secretion of newly synthesized PINs followed by endocytosis and recycling back to the plasma membrane in a polarized manner. In this review, we follow the subcellular march of the PINs and highlight the cellular and molecular mechanisms behind polar foraging and subcellular trafficking pathways. Also, the entry points for different signals and regulations including by auxin itself will be discussed within the context of morphological and developmental consequences of polar targeting and subcellular trafficking.

Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival

Abstract: We have used mouse embryonic fibroblasts (MEFs) devoid of Ras proteins to illustrate that they are essential for proliferation and migration, but not for survival, at least in these cells. These properties are unique to the Ras subfamily of proteins because ectopic expression of other Ras-like small GTPases, even when constitutively active, could not compensate for the absence of Ras proteins. Only constitutive activation of components of the Raf/Mek/Erk pathway was sufficient to sustain normal proliferation and migration of MEFs devoid of Ras proteins. Activation of the phosphatidylinositol 3-kinase (PI3K)/PTEN/Akt and Ral guanine exchange factor (RalGEF)/Ral pathways, either alone or in combination, failed to induce proliferation or migration of Rasless cells, although they cooperated with Raf/Mek/Erk signalling to reproduce the full response mediated by Ras signalling. In contrast to current hypotheses, Ras signalling did not induce proliferation by inducing expression of D-type Cyclins. Rasless MEFs had normal levels of Cyclin D1/Cdk4 and Cyclin E/Cdk2. However, these complexes were inactive. Inactivation of the pocket proteins or knock down of pRb relieved MEFs from their dependence on Ras signalling to proliferate.

New insights into an old story: Agrobacterium- induced tumour formation in plants by plant transformation

Abstract: Agrobacterium tumefaciens causes tumour formation in plants. Plant signals induce in the bacteria the expression of a range of virulence (Vir) proteins and the formation of a type IV secretion system (T4SS). On attachment to plant cells, a transfer DNA (T-DNA) and Vir proteins are imported into the host cells through the bacterial T4SS. Through interaction with a number of host proteins, the Vir proteins suppress the host innate immune system and support the transfer, nuclear targeting, and integration of T-DNA into host cell chromosomes. Owing to extensive genetic analyses, the bacterial side of the plant–Agrobacterium interaction is well understood. However, progress on the plant side has only been achieved recently, revealing a highly complex molecular choreography under the direction of the Vir proteins that impinge on multiple processes including transport, transcription, and chromosome status of their host cells.

An in vivo RNAi assay identifies major genetic and cellular requirements for primary piRNA biogenesis in Drosophila

Abstract: In Drosophila, PIWI proteins and bound PIWI-interacting RNAs (piRNAs) form the core of a small RNA-mediated defense system against selfish genetic elements. Within germline cells, piRNAs are processed from piRNA clusters and transposons to be loaded into Piwi/Aubergine/AGO3 and a subset of piRNAs undergoes target-dependent amplification. In contrast, gonadal somatic support cells express only Piwi, lack signs of piRNA amplification and exhibit primary piRNA biogenesis from piRNA clusters. Neither piRNA processing/loading nor Piwi-mediated target silencing is understood at the genetic, cellular or molecular level. We developed an in vivo RNAi assay for the somatic piRNA pathway and identified the RNA helicase Armitage, the Tudor domain containing RNA helicase Yb and the putative nuclease Zucchini as essential factors for primary piRNA biogenesis. Lack of any of these proteins leads to transposon de-silencing, to a collapse in piRNA levels and to a failure in Piwi-nuclear accumulation. We show that Armitage and Yb interact physically and co-localize in cytoplasmic Yb bodies, which flank P bodies. Loss of Zucchini leads to an accumulation of Piwi and Armitage in Yb bodies, indicating that Yb bodies are sites of primary piRNA biogenesis.

Cytosolic pH is a second messenger for glucose and regulates the PKA pathway through V-ATPase.

Abstract: Glucose is the preferred carbon source for most cell types and a major determinant of cell growth. In yeast and certain mammalian cells, glucose activates the cAMP-dependent protein kinase A (PKA), but the mechanisms of PKA activation remain unknown. Here, we identify cytosolic pH as a second messenger for glucose that mediates activation of the PKA pathway in yeast. We find that cytosolic pH is rapidly and reversibly regulated by glucose metabolism and identify the vacuolar ATPase (V-ATPase), a proton pump required for the acidification of vacuoles, as a sensor of cytosolic pH. V-ATPase assembly is regulated by cytosolic pH and is required for full activation of the PKA pathway in response to glucose, suggesting that it mediates, at least in part, the pH signal to PKA. Finally, V-ATPase is also regulated by glucose in the Min6 β-cell line and contributes to PKA activation and insulin secretion. Thus, these data suggest a novel and potentially conserved glucose-sensing pathway and identify a mechanism how cytosolic pH can act as a signal to promote cell growth.