RESUMO
Compartmentalization of cellular material in droplet-like structures is a hallmark of liquid-liquid phase separation1,2, but the mechanisms of droplet removal are poorly understood. Evidence suggests that droplets can be degraded by autophagy3,4, a highly conserved degradation system in which membrane sheets bend to isolate portions of the cytoplasm within double-membrane autophagosomes5-7. Here we examine how autophagosomes sequester droplets that contain the protein p62 (also known as SQSTM1) in living cells, and demonstrate that double-membrane, autophagosome-like vesicles form at the surface of protein-free droplets in vitro through partial wetting. A minimal physical model shows that droplet surface tension supports the formation of membrane sheets. The model also predicts that bending sheets either divide droplets for piecemeal sequestration or sequester entire droplets. We find that autophagosomal sequestration is robust to variations in the droplet-sheet adhesion strength. However, the two sides of partially wetted sheets are exposed to different environments, which can determine the bending direction of autophagosomal sheets. Our discovery of this interplay between the material properties of droplets and membrane sheets enables us to elucidate the mechanisms that underpin droplet autophagy, or 'fluidophagy'. Furthermore, we uncover a switching mechanism that allows droplets to act as liquid assembly platforms for cytosol-degrading autophagosomes8 or as specific autophagy substrates9-11. We propose that droplet-mediated autophagy represents a previously undescribed class of processes that are driven by elastocapillarity, highlighting the importance of wetting in cytosolic organization.
Assuntos
Autofagossomos/metabolismo , Autofagia , Compartimento Celular , Citosol/metabolismo , Molhabilidade , Adesividade , Autofagossomos/química , Linhagem Celular , Citosol/química , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Proteína Sequestossoma-1/metabolismo , Tensão SuperficialRESUMO
Autophagosome formation requires multiple autophagy-related (ATG) factors. However, we find that a subset of autophagy substrates remains robustly targeted to the lysosome in the absence of several core ATGs, including the LC3 lipidation machinery. To address this unexpected result, we performed genome-wide CRISPR screens identifying genes required for NBR1 flux in ATG7KO cells. We find that ATG7-independent autophagy still requires canonical ATG factors including FIP200. However, in the absence of LC3 lipidation, additional factors are required including TAX1BP1 and TBK1. TAX1BP1's ability to cluster FIP200 around NBR1 cargo and induce local autophagosome formation enforces cargo specificity and replaces the requirement for lipidated LC3. In support of this model, we define a ubiquitin-independent mode of TAX1BP1 recruitment to NBR1 puncta, highlighting that TAX1BP1 recruitment and clustering, rather than ubiquitin binding per se, is critical for function. Collectively, our data provide a mechanistic basis for reports of selective autophagy in cells lacking the lipidation machinery, wherein receptor-mediated clustering of upstream autophagy factors drives continued autophagosome formation.
Assuntos
Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Autofagia/fisiologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Autofagossomos/metabolismo , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Morte Celular , Análise por Conglomerados , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células K562 , Lisossomos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina/metabolismoRESUMO
As malignant tumours develop, they interact intimately with their microenvironment and can activate autophagy, a catabolic process which provides nutrients during starvation. How tumours regulate autophagy in vivo and whether autophagy affects tumour growth is controversial. Here we demonstrate, using a well characterized Drosophila melanogaster malignant tumour model, that non-cell-autonomous autophagy is induced both in the tumour microenvironment and systemically in distant tissues. Tumour growth can be pharmacologically restrained using autophagy inhibitors, and early-stage tumour growth and invasion are genetically dependent on autophagy within the local tumour microenvironment. Induction of autophagy is mediated by Drosophila tumour necrosis factor and interleukin-6-like signalling from metabolically stressed tumour cells, whereas tumour growth depends on active amino acid transport. We show that dormant growth-impaired tumours from autophagy-deficient animals reactivate tumorous growth when transplanted into autophagy-proficient hosts. We conclude that transformed cells engage surrounding normal cells as active and essential microenvironmental contributors to early tumour growth through nutrient-generating autophagy.
Assuntos
Autofagia , Drosophila melanogaster/citologia , Modelos Biológicos , Neoplasias/patologia , Microambiente Tumoral , Aminoácidos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Transporte Biológico , Proliferação de Células , Modelos Animais de Doenças , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Feminino , Interleucina-6/metabolismo , Proteínas de Membrana , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
As part of the lysosomal degradation pathway, the endosomal sorting complexes required for transport (ESCRT-0 to -III/VPS4) sequester receptors at the endosome and simultaneously deform the membrane to generate intraluminal vesicles (ILVs). Whereas ESCRT-III/VPS4 have an established function in ILV formation, the role of upstream ESCRTs (0 to II) in membrane shape remodeling is not understood. Combining experimental measurements and electron microscopy analysis of ESCRT-III-depleted cells with a mathematical model, we show that upstream ESCRT-induced alteration of the Gaussian bending rigidity and their crowding in concert with the transmembrane cargo on the membrane induce membrane deformation and facilitate ILV formation: Upstream ESCRT-driven budding does not require ATP consumption as only a small energy barrier needs to be overcome. Our model predicts that ESCRTs do not become part of the ILV, but localize with a high density at the membrane neck, where the steep decline in the Gaussian curvature likely triggers ESCRT-III/VPS4 assembly to enable neck constriction and scission.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/fisiologia , Modelos Biológicos , Endossomos/ultraestrutura , Células HeLa , HumanosRESUMO
The power of forward genetics in yeast is the foundation on which the field of autophagy research firmly stands. Complementary work on autophagy in higher eukaryotes has revealed both the deep conservation of this process, as well as novel mechanisms by which autophagy is regulated in the context of development, immunity, and neuronal homeostasis. The recent emergence of new clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based technologies has begun facilitating efforts to define novel autophagy factors and pathways by forward genetic screening in mammalian cells. Here, we set out to develop an expanded toolkit of autophagy reporters amenable to CRISPR/Cas9 screening. Genome-wide screening of our reporters in mammalian cells recovered virtually all known autophagy-related (ATG) factors as well as previously uncharacterized factors, including vacuolar protein sorting 37 homolog A (VPS37A), transmembrane protein 251 (TMEM251), amyotrophic lateral sclerosis 2 (ALS2), and TMEM41B. To validate this data set, we used quantitative microscopy and biochemical analyses to show that 1 novel hit, TMEM41B, is required for phagophore maturation. TMEM41B is an integral endoplasmic reticulum (ER) membrane protein distantly related to the established autophagy factor vacuole membrane protein 1 (VMP1), and our data show that these two factors play related, albeit not fully overlapping, roles in autophagosome biogenesis. In sum, our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.
Assuntos
Autofagia/genética , Autofagia/fisiologia , Proteínas de Membrana/genética , Sistemas CRISPR-Cas , Retículo Endoplasmático/metabolismo , Humanos , Células K562 , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Transporte ProteicoRESUMO
At the onset of metazoan cell division the nuclear envelope breaks down to enable capture of chromosomes by the microtubule-containing spindle apparatus. During anaphase, when chromosomes have separated, the nuclear envelope is reassembled around the forming daughter nuclei. How the nuclear envelope is sealed, and how this is coordinated with spindle disassembly, is largely unknown. Here we show that endosomal sorting complex required for transport (ESCRT)-III, previously found to promote membrane constriction and sealing during receptor sorting, virus budding, cytokinesis and plasma membrane repair, is transiently recruited to the reassembling nuclear envelope during late anaphase. ESCRT-III and its regulatory AAA (ATPase associated with diverse cellular activities) ATPase VPS4 are specifically recruited by the ESCRT-III-like protein CHMP7 to sites where the reforming nuclear envelope engulfs spindle microtubules. Subsequent association of another ESCRT-III-like protein, IST1, directly recruits the AAA ATPase spastin to sever microtubules. Disrupting spastin function impairs spindle disassembly and results in extended localization of ESCRT-III at the nuclear envelope. Interference with ESCRT-III functions in anaphase is accompanied by delayed microtubule disassembly, compromised nuclear integrity and the appearance of DNA damage foci in subsequent interphase. We propose that ESCRT-III, VPS4 and spastin cooperate to coordinate nuclear envelope sealing and spindle disassembly at nuclear envelope-microtubule intersection sites during mitotic exit to ensure nuclear integrity and genome safeguarding, with a striking mechanistic parallel to cytokinetic abscission.
Assuntos
Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fusão de Membrana , Membrana Nuclear/metabolismo , Fuso Acromático/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Anáfase , Pontos de Checagem do Ciclo Celular , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , Humanos , Microtúbulos/metabolismo , Espastina , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The main organelles of the secretory and endocytic pathways--the endoplasmic reticulum (ER) and endosomes, respectively--are connected through contact sites whose numbers increase as endosomes mature. One function of such sites is to enable dephosphorylation of the cytosolic tails of endosomal signalling receptors by an ER-associated phosphatase, whereas others serve to negatively control the association of endosomes with the minus-end-directed microtubule motor dynein or mediate endosome fission. Cholesterol transfer and Ca(2+) exchange have been proposed as additional functions of such sites. However, the compositions, activities and regulations of ER-endosome contact sites remain incompletely understood. Here we show in human and rat cell lines that protrudin, an ER protein that promotes protrusion and neurite outgrowth, forms contact sites with late endosomes (LEs) via coincident detection of the small GTPase RAB7 and phosphatidylinositol 3-phosphate (PtdIns(3)P). These contact sites mediate transfer of the microtubule motor kinesin 1 from protrudin to the motor adaptor FYCO1 on LEs. Repeated LE-ER contacts promote microtubule-dependent translocation of LEs to the cell periphery and subsequent synaptotagmin-VII-dependent fusion with the plasma membrane. Such fusion induces outgrowth of protrusions and neurites, which requires the abilities of protrudin and FYCO1 to interact with LEs and kinesin 1. Thus, protrudin-containing ER-LE contact sites are platforms for kinesin-1 loading onto LEs, and kinesin-1-mediated translocation of LEs to the plasma membrane, fuelled by repeated ER contacts, promotes protrusion and neurite outgrowth.
Assuntos
Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Neuritos/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos , Microtúbulos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Ratos , Sinaptotagminas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Several proteins have been identified as amyloid forming in humans, and independent of protein origin, the fibrils are morphologically similar. Therefore, there is a potential for structures with amyloid seeding ability to induce both homologous and heterologous fibril growth; thus, molecular interaction can constitute a link between different amyloid forms. Intravenous injection with preformed fibrils from islet amyloid polypeptide (IAPP), proIAPP, or amyloid-beta (Aß) into human IAPP transgenic mice triggered IAPP amyloid formation in pancreas in 5 of 7 mice in each group, demonstrating that IAPP amyloid could be enhanced through homologous and heterologous seeding with higher efficiency for the former mechanism. Proximity ligation assay was used for colocalization studies of IAPP and Aß in islet amyloid in type 2 diabetic patients and Aß deposits in brains of patients with Alzheimer disease. Aß reactivity was not detected in islet amyloid although islet ß cells express AßPP and convertases necessary for Aß production. By contrast, IAPP and proIAPP were detected in cerebral and vascular Aß deposits, and presence of proximity ligation signal at both locations showed that the peptides were <40 nm apart. It is not clear whether IAPP present in brain originates from pancreas or is locally produced. Heterologous seeding between IAPP and Aß shown here may represent a molecular link between type 2 diabetes and Alzheimer disease.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Amiloidose/metabolismo , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Amiloidose/patologia , Animais , Encéfalo/patologia , Diabetes Mellitus Tipo 2/patologia , Feminino , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-IdadeRESUMO
Activated transmembrane receptors continue to signal following endocytosis and are only silenced upon ESCRT-mediated internalization of the receptors into intralumenal vesicles (ILVs) of the endosomes. Accordingly, endosomes with dysfunctional receptor internalization into ILVs can cause sustained receptor signaling which has been implicated in cancer progression. Here, we describe a surveillance mechanism that allows cells to detect and clear physically intact endosomes with aberrant receptor accumulation and elevated signaling. Proximity biotinylation and proteomics analyses of ESCRT-0 defective endosomes revealed a strong enrichment of the ubiquitin-binding macroautophagy/autophagy receptors SQSTM1 and NBR1, a phenotype that was confirmed in cell culture and fly tissue. Live cell microscopy demonstrated that loss of the ESCRT-0 subunit HGS/HRS or the ESCRT-I subunit VPS37 led to high levels of ubiquitinated and phosphorylated receptors on endosomes. This was accompanied by dynamic recruitment of NBR1 and SQSTM1 as well as proteins involved in autophagy initiation and autophagosome biogenesis. Light microscopy and electron tomography revealed that endosomes with intact limiting membrane, but aberrant receptor downregulation were engulfed by phagophores. Inhibition of autophagy caused increased intra- and intercellular signaling and directed cell migration. We conclude that dysfunctional endosomes are surveyed and cleared by an autophagic process, simaphagy, which serves as a failsafe mechanism in signal termination.Abbreviations: AKT: AKT serine/threonine kinase; APEX2: apurinic/apyrimidinic endodoexyribonuclease 2; ctrl: control; EEA1: early endosome antigen 1; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HGS/HRS: hepatocyte growth factor-regulated tyrosine kinase substrate; IF: immunofluorescence; ILV: intralumenal vesicle; KO: knockout; LIR: LC3-interacting region; LLOMe: L-leucyl-L-leucine methyl ester (hydrochloride); MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; NBR1: NBR1 autophagy cargo receptor; PAG10: Protein A-conjugated 10-nm gold; RB1CC1/FIP200: RB1 inducible coiled-coil 1; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TUB: Tubulin; UBA: ubiquitin-associated; ULK1: unc-51 like autophagy activating kinase 1; VCL: Vinculin; VPS37: VPS37 subunit of ESCRT-I; WB: western blot; WT: wild-type.
RESUMO
During autophagy, cytosolic cargo is sequestered into double-membrane vesicles called autophagosomes. The contributions of specific lipids, such as cholesterol, to the membranes that form the autophagosome, remain to be fully characterized. Here, we demonstrate that short term cholesterol depletion leads to a rapid induction of autophagy and a corresponding increase in autophagy initiation events. We further show that the ER-localized cholesterol transport protein GRAMD1C functions as a negative regulator of starvation-induced autophagy and that both its cholesterol transport VASt domain and membrane binding GRAM domain are required for GRAMD1C-mediated suppression of autophagy initiation. Similar to its yeast orthologue, GRAMD1C associates with mitochondria through its GRAM domain. Cells lacking GRAMD1C or its VASt domain show increased mitochondrial cholesterol levels and mitochondrial oxidative phosphorylation, suggesting that GRAMD1C may facilitate cholesterol transfer at ER-mitochondria contact sites. Finally, we demonstrate that expression of GRAMD family proteins is linked to clear cell renal carcinoma survival, highlighting the pathophysiological relevance of cholesterol transport proteins.
Assuntos
Autofagia , Proteínas de Transporte , Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Colesterol/metabolismo , Metabolismo Energético , Transporte ProteicoRESUMO
Phase-separated droplets with liquid-like properties can be degraded by macroautophagy/autophagy, but the mechanism underlying this degradation is poorly understood. We have recently derived a physical model to investigate the interaction between autophagic membranes and such droplets, uncovering that intrinsic wetting interactions underlie droplet-membrane contacts. We found that the competition between droplet surface tension and the increasing tendency of growing membrane sheets to bend determines whether a droplet is completely engulfed or isolated in a piecemeal fashion, a process we term fluidophagy. Intriguingly, we found that another critical parameter of droplet-membrane interactions, the spontaneous curvature of the membrane, determines whether the droplet is degraded by autophagy or - counterintuitively - serves as a platform from which autophagic membranes expand into the cytosol. We also discovered that the interaction of membrane-associated LC3 with the LC3-interacting region (LIR) found in the autophagic cargo receptor protein SQSTM1/p62 and many other autophagy-related proteins influences the preferred bending directionality of forming autophagosomes in living cells. Our study provides a physical account of how droplet-membrane wetting underpins the structure and fate of forming autophagosomes.
Assuntos
Autofagossomos , Autofagia , Citosol , Macroautofagia , Proteínas Associadas aos MicrotúbulosRESUMO
The mechanisms involved in programmed or damage-induced removal of mitochondria by mitophagy remains elusive. Here, we have screened for regulators of PRKN-independent mitophagy using an siRNA library targeting 197 proteins containing lipid interacting domains. We identify Cyclin G-associated kinase (GAK) and Protein Kinase C Delta (PRKCD) as regulators of PRKN-independent mitophagy, with both being dispensable for PRKN-dependent mitophagy and starvation-induced autophagy. We demonstrate that the kinase activity of both GAK and PRKCD are required for efficient mitophagy in vitro, that PRKCD is present on mitochondria, and that PRKCD facilitates recruitment of ULK1/ATG13 to early autophagic structures. Importantly, we demonstrate in vivo relevance for both kinases in the regulation of basal mitophagy. Knockdown of GAK homologue (gakh-1) in C. elegans or knockout of PRKCD homologues in zebrafish led to significant inhibition of basal mitophagy, highlighting the evolutionary relevance of these kinases in mitophagy regulation.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitofagia , Proteína Quinase C-delta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Caenorhabditis elegans , Linhagem Celular Tumoral , Deferiprona/farmacologia , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Peixe-ZebraRESUMO
p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 domain filament formation in vitro remain to be determined and the role of p62 filaments inside the cell is currently unclear. We here determine four high-resolution cryo-EM structures of different human and Arabidopsis PB1 domain assemblies and observed a filamentous ultrastructure of p62/SQSTM1 bodies using correlative cellular EM. We show that oligomerization or polymerization, driven by a double arginine finger in the PB1 domain, is a general requirement for lysosomal targeting of p62. Furthermore, the filamentous assembly state of p62 is required for autophagosomal processing of the p62-specific cargo KEAP1. Our results show that using such mechanisms, p62 filaments can be critical for cargo uptake in autophagy and are an integral part of phase-separated p62 bodies.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Transporte/química , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/química , Autofagia/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Células HeLa , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lisossomos/metabolismo , Polimerização , Conformação Proteica , Domínios Proteicos , Proteína Sequestossoma-1/genéticaRESUMO
The ESCRT-III membrane fission machinery maintains the integrity of the nuclear envelope. Although primary nuclei resealing takes minutes, micronuclear envelope ruptures seem to be irreversible. Instead, micronuclear ruptures result in catastrophic membrane collapse and are associated with chromosome fragmentation and chromothripsis, complex chromosome rearrangements thought to be a major driving force in cancer development. Here we use a combination of live microscopy and electron tomography, as well as computer simulations, to uncover the mechanism underlying micronuclear collapse. We show that, due to their small size, micronuclei inherently lack the capacity of primary nuclei to restrict the accumulation of CHMP7-LEMD2, a compartmentalization sensor that detects loss of nuclear integrity. This causes unrestrained ESCRT-III accumulation, which drives extensive membrane deformation, DNA damage and chromosome fragmentation. Thus, the nuclear-integrity surveillance machinery is a double-edged sword, as its sensitivity ensures rapid repair at primary nuclei while causing unrestrained activity at ruptured micronuclei, with catastrophic consequences for genome stability.
Assuntos
Núcleo Celular/patologia , Cromatina/metabolismo , Aberrações Cromossômicas , Dano ao DNA , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Instabilidade Genômica , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HeLa , HumanosRESUMO
Hedgehog (Hh) signaling is a key regulatory pathway during development and also has a functional role in mature neurons. Here, we show that Hh signaling regulates the odor response in adult Drosophila olfactory sensory neurons (OSNs). We demonstrate that this is achieved by regulating odorant receptor (OR) transport to and within the primary cilium in OSN neurons. Regulation relies on ciliary localization of the Hh signal transducer Smoothened (Smo). We further demonstrate that the Hh- and Smo-dependent regulation of the kinesin-like protein Cos2 acts in parallel to the intraflagellar transport system (IFT) to localize ORs within the cilium compartment. These findings expand our knowledge of Hh signaling to encompass chemosensory modulation and receptor trafficking.
Assuntos
Cílios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Odorantes/metabolismo , Animais , Comportamento Animal , Cálcio/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Cinesinas/metabolismo , Mutagênese , Odorantes , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Odorantes/genética , Transdução de Sinais , Receptor SmoothenedRESUMO
A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1- and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.
Assuntos
Autofagia/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Ácidos Fosfatídicos/metabolismo , Animais , Animais Geneticamente Modificados , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular , Cortactina/metabolismo , Células HEK293 , Células HeLa , Humanos , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Proteínas do Tecido Nervoso/química , Fosfolipase D/metabolismo , Domínios Proteicos , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
UNLABELLED: Tankyrase (TNKS) enzymes, due to their poly(ADP-ribose) polymerase activity, have emerged as potential targets in experimental cancer therapy. However, the functional consequences of TNKS inhibition remain incompletely resolved because of the binding promiscuity of TNKS. One of the hallmarks of small-molecule TNKS inhibitors (TNKSi) is the stabilization of AXIN, which plays a pivotal role in the WNT/ß-catenin signaling pathway. The present study focused on the known ability of TNKSi to induce cytoplasmic puncta (degradasomes) consisting of components of the signal-limiting WNT/ß-catenin destruction complex. Using the colorectal cancer cell line SW480 stably transfected with GFP-TNKS1, it was demonstrated that a TNKS-specific inhibitor (G007-LK) induces highly dynamic and mobile degradasomes that contain phosphorylated ß-catenin, ubiquitin, and ß-TrCP. Likewise, G007-LK was found to induce similar degradasomes in other colorectal cancer cell lines expressing wild-type or truncated versions of the degradasome component APC. Super-resolution and electron microscopy revealed that the induced degradasomes in SW480 cells are membrane-free structures that consist of a filamentous assembly of high electron densities and discrete subdomains of various destruction complex components. Fluorescence recovery after photobleaching experiments further demonstrated that ß-catenin-mCherry was rapidly turned over in the G007-LK-induced degradasomes, whereas GFP-TNKS1 remained stable. In conclusion, TNKS inhibition attenuates WNT/ß-catenin signaling by promoting dynamic assemblies of functional active destruction complexes into a TNKS-containing scaffold even in the presence of an APC truncation. IMPLICATIONS: This study demonstrates that ß-catenin is rapidly turned over in highly dynamic assemblies of WNT destruction complexes (degradasomes) upon tankyrase inhibition and provides a direct mechanistic link between degradasome formation and reduced WNT signaling in colorectal cancer cells.
Assuntos
Complexo de Sinalização da Axina/metabolismo , Sulfonas/farmacologia , Tanquirases/antagonistas & inibidores , Tanquirases/metabolismo , Triazóis/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , HumanosRESUMO
Autophagy represents an intracellular degradation process which is involved in both regular cell homeostasis and disease settings. In recent years, the molecular machinery governing this process has been elucidated. The ULK1 kinase complex consisting of the serine/threonine protein kinase ULK1 and the adapter proteins ATG13, RB1CC1, and ATG101, is centrally involved in the regulation of autophagy initiation. This complex is in turn regulated by the activity of different nutrient- or energy-sensing kinases, including MTOR, AMPK, and AKT. However, next to phosphorylation processes it has been suggested that ubiquitination of ULK1 positively influences ULK1 function. Here we report that the inhibition of deubiquitinases by the compound WP1130 leads to increased ULK1 ubiquitination, the transfer of ULK1 to aggresomes, and the inhibition of ULK1 activity. Additionally, WP1130 can block the autophagic flux. Thus, treatment with WP1130 might represent an efficient tool to inhibit the autophagy-initiating ULK1 complex and autophagy.
Assuntos
Autofagia/efeitos dos fármacos , Cianoacrilatos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Agregados Proteicos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Ubiquitinação/efeitos dos fármacosRESUMO
Cilia mediate Hedgehog (Hh) signaling in vertebrates and Hh deregulation results in several clinical manifestations, such as obesity, cognitive disabilities, developmental malformations, and various cancers. Drosophila cells are nonciliated during development, which has led to the assumption that cilia-mediated Hh signaling is restricted to vertebrates. Here, we identify and characterize a cilia-mediated Hh pathway in Drosophila olfactory sensory neurons. We demonstrate that several fundamental key aspects of the vertebrate cilia pathway, such as ciliary localization of Smoothened and the requirement of the intraflagellar transport system, are present in Drosophila. We show that Cos2 and Fused are required for the ciliary transport of Smoothened and that cilia mediate the expression of the Hh pathway target genes. Taken together, our data demonstrate that Hh signaling in Drosophila can be mediated by two pathways and that the ciliary Hh pathway is conserved from Drosophila to vertebrates.
Assuntos
Cílios/metabolismo , Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Cílios/patologia , Proteínas de Drosophila/análise , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Humanos , Cinesinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/metabolismo , Receptor SmoothenedRESUMO
Amyloid formation is cytotoxic and can activate the caspase cascade. Here, we monitor caspase-3-like activity as reduction of fluorescence resonance energy transfer (FRET) using the contstruct pFRET2-DEVD containing enhanced cyan fluorescent protin (EYFP) linked by the caspase-3 specific cleavage site residues DEVD. Beta-TC-6 cells were transfected, and the fluoorescence was measured at 440 nm excitation and 535 nm (EYFP) and 480 nm (ECFP) emission wavelength. Cells were incubated with recombinant pro lset Amyloid Polypeptide (rec prolAPP) or the processing metabolites of prolAPP; the N-terminal flanking peptide withIAPP (recN+IAPP); IAPP with the C-terminal flanking peptied (recIAPP+C) and lslet Amyloid Polypeptide (recIAPP) . Peptides were added in solubilized from (50 microM) or as performed amyloid-like fibrils, or as a combination of these. FRET was measured and incubation with a mixture of solubilized peptide and performed fibrils resulted in loss of FRET and apoptosis was determined to occure in cells incubated with recproIAPP (49%), recN+IAPP (46%), recIAPP (72%) and recIAPP+C (59%). These results show that proIAPP and the processing intermediates reside the same cell toxic capacity as IAPP, and they can all have a central role in the reduction of beta-cell number in type 2 diabetes.