The lysosomal-autophagic pathway is activated by starvation and plays an important role in both cellular clearance and lipid catabolism. However, the transcriptional regulation of this pathway in response to metabolic cues is currently uncharacterized. Here we show that the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is induced by starvation through an autoregulatory feedback loop and exerts a global transcriptional control on lipid catabolism via PGC1α and PPARα. Thus, during starvation a transcriptional mechanism links the autophagic pathway to cellular energy metabolism. The conservation of this mechanism in Caenorhabditis elegans suggests a fundamental role for TFEB in the evolution of the adaptive response to food deprivation. Viral delivery of TFEB to the liver prevented weight gain and metabolic syndrome in both diet-induced and genetic mouse models of obesity, suggesting a novel therapeutic strategy for disorders of lipid metabolism.
Fat is stored or mobilized according to food availability. Malfunction of the mechanisms that ensure this coordination underlie metabolic diseases in humans. In mammals, lysosomal and autophagic function is required for normal fat storage and mobilization in the presence or absence of food. Autophagy is tightly linked to nutrients. However, if and how lysosomal lipolysis is coupled to nutritional status remains to be determined. Here we identify MXL-3 and HLH-30 as transcriptional switches coupling lysosomal lipolysis and autophagy to nutrient availability and controlling fat storage and ageing in Caenorhabditis elegans. Transcriptional coupling of lysosomal lipolysis and autophagy to nutrients is also observed in mammals. Thus, MXL-3 and HLH-30 orchestrate an adaptive and conserved cellular response to nutritional status and regulate lifespan.
Cellular decision-making is mediated by a complex interplay of external stimuli with the intracellular environment, in particular transcription factor regulatory networks. Here we have determined the expression of a network of 18 key haematopoietic transcription factors (TFs) in 597 single primary blood stem and progenitor cells isolated from mouse bone marrow. We demonstrate that different stem/progenitor populations are characterised by distinctive TF expression states, and through comprehensive bioinformatic analysis reveal positively and negatively correlated TF pairings, including previously unrecognised relationships between Gata2, Gfi1 and Gfi1b. Validation using transcriptional and transgenic assays confirmed direct regulatory interactions consistent with a regulatory triad in immature blood stem cells, where Gata2 may function to modulate cross-inhibition between Gfi1 and Gfi1b. Single cell expression profiling therefore identifies network states and allows reconstruction of network hierarchies involved in controlling stem cell fate choices, and provides a blueprint for studying both normal development and human disease.
Increased stromal collagen deposition in human breast tumours correlates with metastases. We show that activation of the collagen I receptor DDR2 regulates Snail1 protein stability by stimulating ERK2 activity, in a Src-dependent manner. Activated ERK2 directly phosphorylates Snail1, leading to Snail1 nuclear accumulation, reduced ubiquitination, and increased protein half-life. DDR2-mediated stabilization of Snail1 promotes breast cancer cell invasion and migration in vitro, and metastasis in vivo. DDR2 expression was observed in the majority of human invasive ductal breast carcinomas studied, and was associated with nuclear Snail1 and absence of E-cadherin expression. We propose that DDR2 maintains Snail1 protein level and activity in tumor cells that have undergone EMT, thereby facilitating continued tumor cell invasion through collagen I-rich ECM by sustaining the EMT phenotype. As such, DDR2 could be an RTK target for the treatment of breast cancer metastasis.
Discoidin Domain Receptor (DDR); Snail; Epithelial Mesenchymal Transition (EMT); Collagen
Shelterin binds and protects mammalian telomeres. Here, we generated cells and mice conditionally deleted for the shelterin component RAP1. We find that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. Mice with Rap1 deletion in stratified epithelia are viable but have shorter telomeres and develop skin hyperpigmentation at aduldhood. By performing chromatin immunoprecipitation coupled with ultra-highthroughput sequencing, we find that RAP1 binds to telomeres and to extra-telomeric sites through the (TTAGGG)2 consensus motif. Extra-telomeric RAP1 binding sites are enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. More than 70% of extra-telomeric RAP1 binding sites are at the vicinity of genes and 31% of the genes deregulated in Rap1-null cells contain RAP1 binding sites, suggesting a role of RAP1 in transcriptional control. These findings place a shelterin component at the interface between telomere function and transcriptional regulation.
telomeres; RAP1; TERT; TPE; subtelomeres; DNA damage; transcription; ChIP-seq; metabolism; conditional knock out mice
Lgr5 stem cells reside at small intestinal crypt bottoms, generating both the enterocyte and secretory lineage. Entry into the latter epithelial lineage requires silencing of Notch signaling. The Notch ligand Dll1 is strongly up-regulated in a small subset of immediate stem cell daughters. Lineage tracing utilizing a novel Dll1GFP-ires-CreERT2 knock-in mouse reveals that single Dll1high cells generate small, short-lived clones of all four secretory cell types. In culture, sorted Dll1high cells can form long-lived organoids when briefly exposed to Wnt3A. When Dll1 cells are genetically marked prior to tissue damage, significant numbers of stem cell tracing events occur. Lineage specification therefore occurs already in the earliest stem cell daughters through Notch lateral inhibition. Yet, specified secretory progenitors display plasticity and can regain stemness upon tissue damage.
Angiogenesis, the formation of new blood vessels, is fundamental to development and post-injury tissue repair. Vascular endothelial growth factor (VEGF)-A guides and enhances endothelial cell (EC) migration to initiate angiogenesis. Profilin-1 (Pfn-1) is an actin-binding protein that enhances actin filament formation and cell migration, but stimulus-dependent regulation of Pfn-1 has not been observed. Here, we show VEGF-A-inducible phosphorylation of Pfn-1 at Tyr129 is critical for EC migration and angiogenesis. Chemotactic activation of VEGF receptor kinase-2 (VEGFR2) and Src induce Pfn-1 phosphorylation in the cell leading edge, promoting Pfn-1 binding to actin and actin polymerization. Furthermore, Pfn-1 phosphorylation is robustly and preferentially elevated in blood vessels during tissue repair after myocardial infarction in humans. Conditional endothelial knock-in of phosphorylation-deficient Pfn-1Y129F in mice reveals that Pfn-1 phosphorylation is critical for angiogenesis in response to wounding and ischemic injury, but not for developmental angiogenesis. Thus, VEGFR2/Src-mediated phosphorylation of Pfn-1 bypasses canonical, multistep intracellular signaling events to initiate EC migration and angiogenesis, and might serve as a selective therapeutic target for anti-angiogenic therapy.
In blood, transcription factor C/EBPa is essential for myeloid differentiation and has been implicated in regulating self-renewal of fetal liver (FL) hematopoietic stem cells (HSCs). However, its function in adult HSCs has remained unknown. Here, using an inducible knockout model we found that C/EBPa deficient adult HSCs underwent a pronounced expansion with enhanced proliferation, characteristics resembling FL HSCs. Consistently, transcription profiling of C/EBPa deficient HSCs revealed a gene expression programme similar to FL HSCs. Moreover we observed that age-specific C/EBPa expression correlated with its inhibitory effect on HSC cell cycle. Mechanistically we identified N-Myc as C/EBPa downstream target, and loss of C/EBPa resulted in de-repression of N-Myc. Our data establish C/EBPa as a central determinant in the switch from fetal to adult HSCs.
Transcription factor; C/EBPa; hematopoietic stem cell; quiescence; proliferation; N-Myc
Generation of research quality, clinically relevant cell types in vitro from human pluripotent stem cells (hPSCs) requires detailed understanding of the equivalent human cell types. Here we analyzed 134 human embryonic and fetal samples from 6–20 developmental weeks and identified the stages in which cKIT+ primordial germ cells (PGCs), the precursors of gametes, undergo whole genome epigenetic reprogramming with global depletion of 5mC, H3K27me3, H2A.Z and the time where imprint erasure is initiated and 5hmC is present. Using five alternate in vitro differentiation strategies combined with single-cell microfluidic analysis and a bona fide human cKIT+ PGC signature, we show the stage of cKIT+ PGC formation in the first 16 days of differentiation. Taken together, our study creates a resource of human germ line ontogeny that is essential for future studies aimed at in vitro differentiation and unveiling mechanisms necessary to pass human DNA from one generation to the next.
Intracellular transport is vital for the function, survival and architecture of every eukaryotic cell. Long range transport in animal cells is thought to depend exclusively on microtubule tracks. This study reveals an unexpected actin-dependent but microtubule-independent mechanism for long range transport of vesicles. Vesicles organize their own actin tracks by recruiting the actin nucleation factors Spire1, Spire2 and Formin-2, which assemble an extensive actin network from the vesicles’ surfaces. The network connects the vesicles with each other and with the plasma membrane. Vesicles move directionally along these connections in a Myosin 5b-dependent manner to converge and to reach the cell surface. The overall outward directed movement of the vesicle-actin-network is driven by recruitment of vesicles to the plasma membrane in the periphery of the oocyte. Being organized in a dynamic vesicle-actin-network allows vesicles to move in a local random manner and a global directed manner at the same time: they can reach any position in the cytoplasm, but also move directionally to the cell surface as a collective. Thus, collective movement within a network is a powerful and flexible mode of vesicle transport.
The actin cortex both facilitates and hinders exocytosis of secretory granules. How cells consolidate these two opposing roles was not well understood. Here we show that antigen activation of mast cells induces oscillations in Ca2+ and PI(4,5)P2 lipids that in turn drive cyclic recruitment of N-WASP and cortical actin oscillations. Experimental and computational analysis argues that vesicle fusion correlates with the observed actin and Ca2+ oscillations. A vesicle secretion cycle starts with the capture of vesicles by actin when cortical F-actin is high, followed by vesicle passage through the cortex when F-actin levels are low, and vesicle fusion with the plasma membrane when Ca2+ levels subsequently increase. Thus, cells employ oscillating levels of Ca2+, PI(4,5)P2 and cortical F-actin to increase secretion efficiency, explaining how the actin cortex can function as a carrier as well as barrier for vesicle secretion.
The systems that refine actomyosin forces during motility remain poorly understood. Septins assemble on the T cell cortex and are enriched at the mid-zone in filaments. Septin knockdown causes membrane blebbing, excess leading edge protrusions, and lengthening of the trailingedge uropod. The associated loss of rigidity permits motility, but cells become uncoordinated and poorly persistent. This also relieves a previously unrecognized restriction to migration through small pores. Pharmacologically rigidifying cells counteracts this effect, and relieving cytoskeletal rigidity synergizes with septin-depletion. These data suggest that septins tune actomyosin forces during motility, and likely regulate lymphocyte trafficking in confined tissues.
Micro (mi)RNAs form a class of short RNAs (~21 nt) that post-transcriptionally regulate partially complementary messenger (m)RNAs. Each miRNA may target tens-to-hundreds of transcripts to control key biological processes. While the biochemical reactions underpinning miRNA biogenesis and activity are relatively well-defined1,2, and the importance of their homeostasis increasingly evident, the processes underlying regulation of miRNA pathways in vivo are still largely elusive3. Autophagy, a degradative process in which cytoplasmic material is targeted into double-membrane vacuoles, is recognized to critically contribute to cellular homeostasis. Here, we show that the miRNA-processing enzyme, DICER, and the major miRNA effector, AGO2, are targeted for degradation as miRNA-free entities by the selective autophagy receptor NDP52. Autophagy establishes a checkpoint required for continued loading of miRNA into AGO2; accordingly, NDP52 and autophagy are required for homeostasis and activity of tested miRNAs. Autophagy also engages post-transcriptional regulation of the DICER mRNA, underscoring the importance of fine-tuned regulation of the miRNA pathway. These findings have implications for human diseases linked to misregulated autophagy, DICER- and miRNA-levels, including cancer.
Autophagy; NDP52; DICER; Argonaute; microRNA; post-transcriptional
Epithelial cells have an apical–basolateral axis of polarity, which is required for epithelial functions including barrier formation, vectorial ion transport and sensory perception. Here we review what is known about the sorting signals, machineries and pathways that maintain this asymmetry, and how polarity proteins interface with membrane-trafficking pathways to generate membrane domains de novo. It is becoming apparent that membrane traffic does not simply reinforce polarity, but is critical for the generation of cortical epithelial cell asymmetry.
Epithelial-mesenchymal transitions (EMT) are essential for organogenesis and triggered in carcinoma progression into an invasive state1. Transforming growth factor-β (TGF-β) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT2-5, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT6, 7. The SNAIL1-SMAD3/4 complex was targeted to the gene promoters of CAR, a tight junction protein, and E-cadherin during TGF-β-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited the repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1-SMAD3/4 transcriptional complex represents a novel mechanism of gene repression during EMT.
As the main microtubule-organizing center in animal cells, the centrosome has a fundamental role in cell function. Surrounding the centrioles, the Pericentriolar material (PCM) provides a dynamic platform for nucleating microtubules. While the PCM’s importance is established, its amorphous electron-dense nature has made it refractory to structural investigation. By using SIM and STORM sub-diffraction resolution microscopies to visualize proteins critical for centrosome maturation, we demonstrate that the PCM is organized into two major structural domains: a layer juxtaposed to the centriole wall, and proteins extending further away from the centriole organized in a matrix. Analysis of Pericentrin-like protein (Plp) reveals that its C-terminus is positioned at the centriole wall, it radiates outward into the matrix and is organized in clusters having quasi-nine-fold symmetry. By RNAi we show that Plp fibrils are required for interphase recruitment and proper mitotic assembly of the PCM matrix.
Mature adipocytes are generated through the proliferation and differentiation of precursor cells. Our prior studies identified adipocyte progenitors in white adipose tissue (WAT) as Lin−:CD29+:CD34+:Sca-1+:CD24+ (CD24+) cells that are capable of generating functional WAT1. Here, we employ several Cre recombinase mouse models to identify the adipocyte cellular lineage in vivo. While it has been proposed that white adipocytes are derived from endothelial2 and hematopoietic3, 4 lineages, we find that neither of these lineages label white adipocytes. However, platelet-derived growth factor receptor α (PdgfRα)-Cre trace labels all white adipocytes. Analysis of WAT from PdgfRα-Cre reporter mice identifies CD24+ and Lin−:CD29+:CD34+:Sca-1+:CD24− (CD24−) cells as adipocyte precursors. We show that CD24+ cells generate the CD24− population in vivo and the CD24− cells express late markers of adipogenesis. From these data we propose a model where the CD24+ adipocyte progenitors become further committed to the adipocyte lineage as CD24 expression is lost, generating CD24− preadipocytes. This characterization of the adipocyte cellular lineage will facilitate study of the mechanisms that regulate WAT formation in vivo and WAT mass expansion in obesity.
A key issue in cancer biology is whether oncogenic transformation of different cell types of origin within an adult tissue gives rise to distinct tumor subtypes that differ in their prognosis and/or treatment response. We now show that initiation of prostate tumors in basal or luminal epithelial cells in mouse models results in tumors with distinct molecular signatures that are predictive of human patient outcomes. Furthermore, our analysis of untransformed basal cells reveals an unexpected assay-dependence of their stem cell properties in sphere formation and transplantation assays versus genetic lineage-tracing during prostate regeneration and adult tissue homeostasis. Although oncogenic transformation of basal cells gives rise to tumors with luminal phenotypes, cross-species bioinformatic analyses indicate that luminal origin tumors are more aggressive than basal origin tumors, and identify a molecular signature associated with patient outcome. Our results reveal the inherent plasticity of basal cells, and support a model in which different cells of origin generate distinct molecular subtypes of prostate cancer.
The specification of tissue size during development involves the coordinated action of many signalling pathways responding to organ-intrinsic signals, such as morphogen gradients, and systemic cues, such as nutrient status. The conserved Hippo (Hpo) pathway, which promotes both cell cycle exit and apoptosis, is a major determinant of size control.The pathway core is a kinase cassette, comprising the kinases Hpo and Warts (Wts) and the scaffold proteins Salvador (Sav) and Mats, which inactivates the pro-growth transcriptional co-activator Yorkie (Yki). We performed a split TEV-based genome-wide RNAi screen for modulators of Hpo signalling. We characterise the Drosophila salt-inducible kinases (Sik2 and Sik3) as negative regulators of Hpo signalling. Activated Sik kinases increase Yki target expression and promote tissue overgrowth through phosphorylation of Sav at Ser413. Since Sik kinases have been implicated in nutrient sensing, this suggests a link between the Hippo pathway and systemic growth control.
Hpo signalling; proliferation; Sik2; Sik3; split TEV; RNAi screening
Organisms are constantly challenged by stresses and privations and require adaptive responses for their survival. The transcription factor DAF-16/FOXO is central nexus in these responses, but despite its importance little is known about how it regulates its target genes. Proteomic identification of DAF-16/FOXO binding partners in Caenorhabditis elegans and their subsequent functional evaluation by RNA interference (RNAi) revealed several candidate DAF-16/FOXO cofactors, most notably the chromatin remodeller SWI/SNF. DAF-16/FOXO and SWI/SNF form a complex and globally colocalize at DAF-16/FOXO target promoters. We show that specifically for gene-activation, DAF-16/FOXO depends on SWI/SNF, facilitating SWI/SNF recruitment to target promoters, in order to activate transcription by presumed remodelling of local chromatin. For the animal, this translates into an essential role of SWI/SNF for DAF-16/FOXO-mediated processes, i.e. dauer formation, stress resistance, and the promotion of longevity. Thus we give insight into the mechanisms of DAF-16/FOXO-mediated transcriptional regulation and establish a critical link between ATP-dependent chromatin remodelling and lifespan regulation.
daf-16; FOXO; SWI/SNF; longevity; stress response; dauer formation; chromatin remodelling; proteomics
Eps8, a bi-functional actin cytoskeleton remodeler, is a positive regulator of cell proliferation and motility. Here, we describe an unrecognized mechanism regulating Eps8 that is required for proper mitotic progression: While Eps8 is stable in G1 and S phase, its half-life drops sharply in G2. This requires G2-specific proteasomal degradation mediated by the Ubiquitin E3 ligase SCFFbxw5. Consistent with a short window of degradation, Eps8 disappears from the cell cortex early in mitosis, but reappears at the midzone of dividing cells. Failure to reduce Eps8 levels in G2 prolongs its localization at the cell cortex and markedly delays cell rounding and prometaphase duration. However, during late stages of mitosis and cytokinesis, Eps8 capping activity is required to prevent membrane blebbing and cell shape deformations. Our findings identify SCFFbxw5-driven fluctuation of Eps8 levels as an important mechanism that contributes to cell-shape changes during entry into – and exit from - mitosis.
The cyclical protrusion and retraction of the leading edge is a hallmark of many migrating cells involved in processes such as development, inflammation, and tumorigenesis. The molecular identity of signaling mechanisms that control these cycles has remained unknown. Here, we used live cell imaging of biosensors to monitor spontaneous morphodynamic and signaling activities, and employed correlative image analysis to examine the role of cAMP-activated Protein Kinase A (PKA) in protrusion regulation. PKA activity at the leading edge is closely synchronized with rapid protrusion and with the activity of RhoA. Ensuing PKA phosphorylation of RhoA and the resulting increased interaction between RhoA and RhoGDI establishes a negative feedback that controls the cycling of RhoA activity at the leading edge. Thus, cooperation between PKA, RhoA, and a RhoGDI forms a pacemaker that governs the morphodynamic behavior of migrating cells.
Cell migration; cAMP activated Protein Kinase A; Rho GTPase; cell protrusion; RhoGDI
The dynamin-related GTPase Dnm1 controls mitochondrial morphology in yeast. Here we show that dnm1 mutations convert the mitochondrial compartment into a planar ‘net’ of interconnected tubules. We propose that this net morphology results from a defect in mitochondrial fission. Immunogold labelling localizes Dnm1 to the cytoplasmic face of constricted mitochondrial tubules that appear to be dividing and to the ends of mitochondrial tubules that appear to have recently completed division. The activity of Dnm1 is epistatic to that of Fzo1, a GTPase in the outer mitochondrial membrane that regulates mitochondrial fusion. dnm1 mutations prevent mitochondrial fragmentation in fzo1 mutant strains. These findings indicate that Dnm1 regulates mitochondrial fission, assembling on the cytoplasmic face of mitochondrial tubules at sites at which division will occur.
We have identified Rab10 as an ER specific Rab GTPase that regulates ER structure and dynamics. We show that Rab10 localizes to the ER and to dynamic ER-associated structures that track along microtubules and mark the position of new ER tubule growth. Rab10 depletion or expression of a Rab10 GDP-locked mutant alters ER morphology, resulting in decreased ER tubules. We demonstrate that this defect is due to a reduced ability of dynamic ER tubules to grow out and successfully fuse with adjacent ER. Consistent with this function, Rab10 partitions to dynamic ER-associated domains found at the leading edge of almost half of all ER tubule dynamics. Interestingly, this Rab10 domain is highly enriched with at least two ER enzymes that regulate phospholipid synthesis, PI Synthase and CEPT1. Both the formation and function of this Rab10/PIS/CEPT1 dynamic domain is inhibited by expression of a GDP-locked Rab10 mutant. Together, these data demonstrate that Rab10 regulates ER dynamics and further suggests that these dynamics could be coupled to phospholipid synthesis.
Understanding the molecular mechanisms that promote successful tissue regeneration is critical for continued advancements in regenerative medicine. Vertebrate amphibian tadpoles of the species Xenopus laevis and Xenopus tropicalis have remarkable abilities to regenerate their tails following amputation 1, 2, via the coordinated activity of numerous growth factor signaling pathways, including the Wnt, Fgf, BMP, notch, and TGFβ pathways 3-6. Little is known, however, about the events that act upstream of these signalling pathways following injury. Here, we show that Xenopus tadpole tail amputation induces a sustained production of reactive oxygen species (ROS) during tail regeneration. Lowering ROS levels, via pharmacological or genetic approaches, reduces cell proliferation and impairs tail regeneration. Genetic rescue experiments restored both ROS production and the initiation of the regenerative response. Sustained increased ROS levels are required for Wnt/β-catenin signaling and the activation of one of its major downstream targets, fgf20
7, which, in turn, is essential for proper tail regeneration. These findings demonstrate that injury-induced ROS production is an important regulator of tissue regeneration.