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1.  Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation 
The Journal of Cell Biology  2013;202(2):331-349.
SNX18 promotes autophagosome formation by remodeling membranes and providing membrane to forming autophagosomes.
The membrane remodeling events required for autophagosome biogenesis are still poorly understood. Because PX domain proteins mediate membrane remodeling and trafficking, we conducted an imaging-based siRNA screen for autophagosome formation targeting human PX proteins. The PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation, and its Drosophila melanogaster homologue SH3PX1 was found to be required for efficient autophagosome formation in the larval fat body. We show that SNX18 is required for recruitment of Atg16L1-positive recycling endosomes to a perinuclear area and for delivery of Atg16L1- and LC3-positive membranes to autophagosome precursors. We identify a direct interaction of SNX18 with LC3 and show that the pro-autophagic activity of SNX18 depends on its membrane binding and tubulation capacity. We also show that the function of SNX18 in membrane tubulation and autophagy is negatively regulated by phosphorylation of S233. We conclude that SNX18 promotes autophagosome formation by virtue of its ability to remodel membranes and provide membrane to forming autophagosomes.
doi:10.1083/jcb.201205129
PMCID: PMC3718966  PMID: 23878278
2.  Base excision repair AP endonucleases and mismatch repair act together to induce checkpoint-mediated autophagy 
Nature Communications  2013;4:2578.
Cellular responses to DNA damage involve distinct DNA repair pathways, such as mismatch repair (MMR) and base excision repair (BER). Using Caenorhabditis elegans as a model system, we present genetic and molecular evidence of a mechanistic link between processing of DNA damage and activation of autophagy. Here we show that the BER AP endonucleases APN-1 and EXO-3 function in the same pathway as MMR, to elicit DNA-directed toxicity in response to 5-fluorouracil, a mainstay of systemic adjuvant treatment of solid cancers. Immunohistochemical analyses suggest that EXO-3 generates the DNA nicks required for MMR activation. Processing of DNA damage via this pathway, in which both BER and MMR enzymes are required, leads to induction of autophagy in C. elegans and human cells. Hence, our data show that MMR- and AP endonuclease-dependent processing of 5-fluorouracil-induced DNA damage leads to checkpoint activation and induction of autophagy, whose hyperactivation contributes to cell death.
The chemotherapeutic drug 5-fluorouracil causes cell toxicity by inducing DNA lesions. Here, SenGupta et al. use C. elegans to show that components of the base excision repair and the mismatch repair pathways function together in the response to 5-fluorouracil, resulting in activation of the DNA damage checkpoint and induction of autophagy.
doi:10.1038/ncomms3674
PMCID: PMC3826653  PMID: 24154628
3.  Receptor protein complexes are in control of autophagy 
Autophagy  2012;8(11):1701-1705.
In autophagic processes a variety of cargos is delivered to the degradative compartment of cells. Recent progress in autophagy research has provided support for the notion that when autophagic processes are operating in selective mode, a receptor protein complex will process the cargo. Here we present a concept of receptor protein complexes as comprising a functional tetrad of components: a ligand, a receptor, a scaffold and an Atg8 family protein. Our current understanding of each of the four components and their interaction in the context of cargo selection are considered in turn.
doi:10.4161/auto.21332
PMCID: PMC3494607  PMID: 22874568
autophagic cargo; ligand; receptor; scaffold protein; Atg8 family protein; phagophore
4.  A comprehensive glossary of autophagy-related molecules and processes (2nd edition) 
Autophagy  2011;7(11):1273-1294.
The study of autophagy is rapidly expanding, and our knowledge of the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. The vocabulary associated with autophagy has grown concomitantly. In fact, it is difficult for readers—even those who work in the field—to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors and chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, and the roles of accessory components and structures that are associated with autophagy.
doi:10.4161/auto.7.11.17661
PMCID: PMC3359482  PMID: 21997368
autophagy; lysosome; mitophagy; pexophagy; stress; vacuole
5.  Selective Types of Autophagy 
doi:10.1155/2012/156272
PMCID: PMC3432363  PMID: 22956958
6.  The elimination of accumulated and aggregated proteins: A role for aggrephagy in neurodegeneration 
Neurobiology of disease  2010;43(1):17-28.
The presence of ubiquitinated protein inclusions is a hallmark of most adult onset neurodegenerative disorders. Although the toxicity of these structures remains controversial, their prolonged presence in neurons is indicative of some failure in fundamental cellular processes. It therefore may be possible that driving the elimination of inclusions can help re-establish normal cellular function. There is growing evidence that macroautophagy has two roles; first, as a non-selective degradative response to cellular stress such as starvation, and the other as a highly selective quality control mechanism whose basal levels are important to maintain cellular health. One particular form of macroautophagy, aggrephagy, may have particular relevance in neurodegeneration, as it is responsible for the selective elimination of accumulated and aggregated ubiquitinated proteins. In this review, we will discuss the molecular mechanisms and role of protein aggregation in neurodegeneration, as well as the molecular mechanism of aggrephagy and how it may impact disease.
doi:10.1016/j.nbd.2010.08.015
PMCID: PMC2998573  PMID: 20732422
autophagy; protein aggregates; neurodegeneration; ubiquitination; p62; ALFY; aggresome; neurons
7.  p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects 
Autophagy  2011;7(6):572-583.
Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.
doi:10.4161/auto.7.6.14943
PMCID: PMC3127048  PMID: 21325881
p62; Ref(2)P; insoluble ubiquitinated proteins; aggregates; neural degeneration; Alzheimer disease; aging; macroautophagy
8.  Organelle biogenesis and autophagy 
doi:10.1091/mbc.E11-12-0979
PMCID: PMC3302756
9.  Autophagy: More Than a Nonselective Pathway 
Autophagy is a catabolic pathway conserved among eukaryotes that allows cells to rapidly eliminate large unwanted structures such as aberrant protein aggregates, superfluous or damaged organelles, and invading pathogens. The hallmark of this transport pathway is the sequestration of the cargoes that have to be degraded in the lysosomes by double-membrane vesicles called autophagosomes. The key actors mediating the biogenesis of these carriers are the autophagy-related genes (ATGs). For a long time, it was assumed that autophagy is a bulk process. Recent studies, however, have highlighted the capacity of this pathway to exclusively eliminate specific structures and thus better fulfil the catabolic necessities of the cell. We are just starting to unveil the regulation and mechanism of these selective types of autophagy, but what it is already clearly emerging is that structures targeted to destruction are accurately enwrapped by autophagosomes through the action of specific receptors and adaptors. In this paper, we will briefly discuss the impact that the selective types of autophagy have had on our understanding of autophagy.
doi:10.1155/2012/219625
PMCID: PMC3362037  PMID: 22666256
10.  Alfy-dependent elimination of aggregated proteins by macroautophagy 
Autophagy  2011;7(3):346-350.
Degradation of different cargo by macroautophagy is emerging as a highly selective process which relies upon specific autophagy receptors and adapter molecules that link the cargo with the autophagic molecular machinery. We have recently reported that the large phsophatidylinositol-3-phosphate (PtdIns(3)P)-binding protein Alfy (Autophagy-linked FYVE protein) is required for selective degradation of aggregated proteins. Although depletion of Alfy inhibits Atg5-dependent aggregate degradation, overexpression of Alfy results in Atg5-dependent aggregate clearance and neuroprotection. Alfy-mediated degradation requires the ability of Alfy to directly interact with Atg5. This ability to interact with the core autophagic machinery may cause Alfy to diminish the responsiveness to nonselective autophagic degradation as measured by long-lived protein degradation. Thus, increasing Alfy-mediated protein degradation may be beneficial in some organs, but may be detrimental in others.
doi:10.4161/auto.7.3.14234
PMCID: PMC3060415  PMID: 21150266
autophagy; protein aggregates; neurodegeneration; Alfy; aggregation; degradation
11.  The selective macroautophagic degradation of aggregated proteins requires the phosphatidylinositol 3-phosphate binding protein Alfy 
Molecular cell  2010;38(2):265-279.
There is growing evidence that macroautophagic cargo is not limited to bulk cytosol in response to starvation, and can occur selectively for substrates including aggregated proteins. It remains unclear, however, if starvation-induced and selective macroautophagy share identical adapter molecules to capture their cargo. Here we report that Alfy, a phosphatidylinositol 3-phosphate binding protein, is central to the selective elimination of aggregated proteins. We report that the loss of Alfy inhibits the clearance of inclusions, with little to no effect on the starvation response. Alfy is recruited to intracellular inclusions and scaffolds a complex between p62(SQSTM1)-positive proteins and the autophagic effectors Atg5, Atg12, Atg16L and LC3. Alfy overexpression leads to elimination of aggregates in an Atg5-dependent manner, and likewise, to protection in a neuronal and Drosophila model of polyglutamine toxicity. We propose that Alfy plays a key role in selective macroautophagy, by bridging cargo to the molecular machinery that builds autophagosomes.
doi:10.1016/j.molcel.2010.04.007
PMCID: PMC2867245  PMID: 20417604
12.  Coordination of membrane events during autophagy by multiple class III PI3-kinase complexes 
The Journal of Cell Biology  2009;186(6):773-782.
Autophagy or “self-eating” is a highly conserved pathway that enables cells to degrade pieces of themselves in autolysosomes to enable their survival in times of stress, including nutrient deprivation. The formation of these degradative compartments requires cytosolic proteins, some of which are autophagy specific, as well as intracellular organelles, such as the ER and Golgi, and the endosome–lysosome system. Here we discuss the cross talk between autophagy and intracellular compartments, highlighting recent exciting data about the role and regulation of the Vps34 class III phosphatidylinositol (PI) 3-kinase in autophagy.
doi:10.1083/jcb.200907014
PMCID: PMC2753151  PMID: 19797076
13.  Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes 
Klionsky, Daniel J. | Abeliovich, Hagai | Agostinis, Patrizia | Agrawal, Devendra K. | Aliev, Gjumrakch | Askew, David S. | Baba, Misuzu | Baehrecke, Eric H. | Bahr, Ben A. | Ballabio, Andrea | Bamber, Bruce A. | Bassham, Diane C. | Bergamini, Ettore | Bi, Xiaoning | Biard-Piechaczyk, Martine | Blum, Janice S. | Bredesen, Dale E. | Brodsky, Jeffrey L. | Brumell, John H. | Brunk, Ulf T. | Bursch, Wilfried | Camougrand, Nadine | Cebollero, Eduardo | Cecconi, Francesco | Chen, Yingyu | Chin, Lih-Shen | Choi, Augustine | Chu, Charleen T. | Chung, Jongkyeong | Clarke, Peter G.H. | Clark, Robert S.B. | Clarke, Steven G. | Clavé, Corinne | Cleveland, John L. | Codogno, Patrice | Colombo, María I. | Coto-Montes, Ana | Cregg, James M. | Cuervo, Ana Maria | Debnath, Jayanta | Demarchi, Francesca | Dennis, Patrick B. | Dennis, Phillip A. | Deretic, Vojo | Devenish, Rodney J. | Di Sano, Federica | Dice, J. Fred | DiFiglia, Marian | Dinesh-Kumar, Savithramma | Distelhorst, Clark W. | Djavaheri-Mergny, Mojgan | Dorsey, Frank C. | Dröge, Wulf | Dron, Michel | Dunn, William A. | Duszenko, Michael | Eissa, N. Tony | Elazar, Zvulun | Esclatine, Audrey | Eskelinen, Eeva-Liisa | Fésüs, László | Finley, Kim D. | Fuentes, José M. | Fueyo, Juan | Fujisaki, Kozo | Galliot, Brigitte | Gao, Fen-Biao | Gewirtz, David A. | Gibson, Spencer B. | Gohla, Antje | Goldberg, Alfred L. | Gonzalez, Ramon | González-Estévez, Cristina | Gorski, Sharon | Gottlieb, Roberta A. | Häussinger, Dieter | He, You-Wen | Heidenreich, Kim | Hill, Joseph A. | Høyer-Hansen, Maria | Hu, Xun | Huang, Wei-Pang | Iwasaki, Akiko | Jäättelä, Marja | Jackson, William T. | Jiang, Xuejun | Jin, Shengkan | Johansen, Terje | Jung, Jae U. | Kadowaki, Motoni | Kang, Chanhee | Kelekar, Ameeta | Kessel, David H. | Kiel, Jan A.K.W. | Kim, Hong Pyo | Kimchi, Adi | Kinsella, Timothy J. | Kiselyov, Kirill | Kitamoto, Katsuhiko | Knecht, Erwin | Komatsu, Masaaki | Kominami, Eiki | Kondo, Seiji | Kovács, Attila L. | Kroemer, Guido | Kuan, Chia-Yi | Kumar, Rakesh | Kundu, Mondira | Landry, Jacques | Laporte, Marianne | Le, Weidong | Lei, Huan-Yao | Lenardo, Michael J. | Levine, Beth | Lieberman, Andrew | Lim, Kah-Leong | Lin, Fu-Cheng | Liou, Willisa | Liu, Leroy F. | Lopez-Berestein, Gabriel | López-Otín, Carlos | Lu, Bo | Macleod, Kay F. | Malorni, Walter | Martinet, Wim | Matsuoka, Ken | Mautner, Josef | Meijer, Alfred J. | Meléndez, Alicia | Michels, Paul | Miotto, Giovanni | Mistiaen, Wilhelm P. | Mizushima, Noboru | Mograbi, Baharia | Monastyrska, Iryna | Moore, Michael N. | Moreira, Paula I. | Moriyasu, Yuji | Motyl, Tomasz | Münz, Christian | Murphy, Leon O. | Naqvi, Naweed I. | Neufeld, Thomas P. | Nishino, Ichizo | Nixon, Ralph A. | Noda, Takeshi | Nürnberg, Bernd | Ogawa, Michinaga | Oleinick, Nancy L. | Olsen, Laura J. | Ozpolat, Bulent | Paglin, Shoshana | Palmer, Glen E. | Papassideri, Issidora | Parkes, Miles | Perlmutter, David H. | Perry, George | Piacentini, Mauro | Pinkas-Kramarski, Ronit | Prescott, Mark | Proikas-Cezanne, Tassula | Raben, Nina | Rami, Abdelhaq | Reggiori, Fulvio | Rohrer, Bärbel | Rubinsztein, David C. | Ryan, Kevin M. | Sadoshima, Junichi | Sakagami, Hiroshi | Sakai, Yasuyoshi | Sandri, Marco | Sasakawa, Chihiro | Sass, Miklós | Schneider, Claudio | Seglen, Per O. | Seleverstov, Oleksandr | Settleman, Jeffrey | Shacka, John J. | Shapiro, Irving M. | Sibirny, Andrei | Silva-Zacarin, Elaine C.M. | Simon, Hans-Uwe | Simone, Cristiano | Simonsen, Anne | Smith, Mark A. | Spanel-Borowski, Katharina | Srinivas, Vickram | Steeves, Meredith | Stenmark, Harald | Stromhaug, Per E. | Subauste, Carlos S. | Sugimoto, Seiichiro | Sulzer, David | Suzuki, Toshihiko | Swanson, Michele S. | Tabas, Ira | Takeshita, Fumihiko | Talbot, Nicholas J. | Tallóczy, Zsolt | Tanaka, Keiji | Tanaka, Kozo | Tanida, Isei | Taylor, Graham S. | Taylor, J. Paul | Terman, Alexei | Tettamanti, Gianluca | Thompson, Craig B. | Thumm, Michael | Tolkovsky, Aviva M. | Tooze, Sharon A. | Truant, Ray | Tumanovska, Lesya V. | Uchiyama, Yasuo | Ueno, Takashi | Uzcátegui, Néstor L. | van der Klei, Ida | Vaquero, Eva C. | Vellai, Tibor | Vogel, Michael W. | Wang, Hong-Gang | Webster, Paul | Wiley, John W. | Xi, Zhijun | Xiao, Gutian | Yahalom, Joachim | Yang, Jin-Ming | Yap, George | Yin, Xiao-Ming | Yoshimori, Tamotsu | Yu, Li | Yue, Zhenyu | Yuzaki, Michisuke | Zabirnyk, Olga | Zheng, Xiaoxiang | Zhu, Xiongwei | Deter, Russell L.
Autophagy  2007;4(2):151-175.
Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
PMCID: PMC2654259  PMID: 18188003
autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
14.  Self-eating from an ER-associated cup 
The Journal of Cell Biology  2008;182(4):621-622.
Since the first morphological description of autophagosomes in the early 1960s, two critical questions have been a matter of intense investigation and debate: what is the origin of the autophagosomal membrane and how is it formed? A study by Axe et al. (E.L. Axe, S.A. Walker, M. Manifava, P. Chandra, H.L. Roderick, A. Habermann, G. Griffiths, and N.T. Ktistakis. 2008. J. Cell Biol. 182:685–701) provides evidence that cup-shaped protrusions from the endoplasmic reticulum, named omegasomes, serve as platforms for autophagosome biogenesis in mammalian cells.
doi:10.1083/jcb.200807061
PMCID: PMC2518700  PMID: 18725534
15.  Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain 
The Journal of Cell Biology  2008;180(6):1065-1071.
p62 has been proposed to mark ubiquitinated protein bodies for autophagic degradation. We report that the Drosophila melanogaster p62 orthologue, Ref(2)P, is a regulator of protein aggregation in the adult brain. We demonstrate that Ref(2)P localizes to age-induced protein aggregates as well as to aggregates caused by reduced autophagic or proteasomal activity. A similar localization to protein aggregates is also observed in D. melanogaster models of human neurodegenerative diseases. Although atg8a autophagy mutant flies show accumulation of ubiquitin- and Ref(2)P-positive protein aggregates, this is abrogated in atg8a/ref(2)P double mutants. Both the multimerization and ubiquitin binding domains of Ref(2)P are required for aggregate formation in vivo. Our findings reveal a major role for Ref(2)P in the formation of ubiquitin-positive protein aggregates both under physiological conditions and when normal protein turnover is inhibited.
doi:10.1083/jcb.200711108
PMCID: PMC2290837  PMID: 18347073
16.  Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease 
The Journal of Cell Biology  2007;179(3):485-500.
The endosomal sorting complexes required for transport (ESCRTs) are required to sort integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB). Mutations in the ESCRT-III subunit CHMP2B were recently associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. We show here that autophagic degradation is inhibited in cells depleted of ESCRT subunits and in cells expressing CHMP2B mutants, leading to accumulation of protein aggregates containing ubiquitinated proteins, p62 and Alfy. Moreover, we find that functional MVBs are required for clearance of TDP-43 (identified as the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits), and of expanded polyglutamine aggregates associated with Huntington's disease. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations.
doi:10.1083/jcb.200702115
PMCID: PMC2064794  PMID: 17984323
17.  Phosphoinositides and phagocytosis 
The Journal of Cell Biology  2001;155(1):15-18.
Phosphoinositide 3 kinases (PI3Ks)* are known as regulators of phagocytosis. Recent results demonstrate that class I and III PI3Ks act consecutively in phagosome formation and maturation, and that their respective products, phosphatidylinositol 3,4,5-trisphosphate (PI[3,4,5]P3) and phosphatidylinositol 3-phosphate (PI[3]P), accumulate transiently at different stages. Phagosomes containing Mycobacterium tuberculosis do not acquire the PI(3)P-binding protein EEA1, which is required for phagosome maturation. This suggests a possible mechanism of how this microorganism evades degradation in phagolysosomes.
doi:10.1083/jcb.200109001
PMCID: PMC2150801  PMID: 11581282

Results 1-17 (17)