Title / Authors / Details Open Access Download

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).
Klionsky DJ, Ktistakis NT et al

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

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Autophagy, 1, 1, , 08 Feb 2021


Sorting nexin 5 mediates virus-induced autophagy and immunity.
Dong X, Yang Y, Zou Z, Zhao Y, Ci B, Zhong L, Bhave M, Wang L, Kuo YC, Zang X, Zhong R, Aguilera ER, Richardson RB, Simonetti B, Schoggins JW, Pfeiffer JK, Yu L, Zhang X, Xie Y, Schmid SL, Xiao G, Gleeson PA, Ktistakis NT, Cullen PJ, Xavier RJ, Levine B

Autophagy, a process of degradation that occurs via the lysosomal pathway, has an essential role in multiple aspects of immunity, including immune system development, regulation of innate and adaptive immune and inflammatory responses, selective degradation of intracellular microorganisms, and host protection against infectious diseases. Autophagy is known to be induced by stimuli such as nutrient deprivation and suppression of mTOR, but little is known about how autophagosomal biogenesis is initiated in mammalian cells in response to viral infection. Here, using genome-wide short interfering RNA screens, we find that the endosomal protein sorting nexin 5 (SNX5) is essential for virus-induced, but not for basal, stress- or endosome-induced, autophagy. We show that SNX5 deletion increases cellular susceptibility to viral infection in vitro, and that Snx5 knockout in mice enhances lethality after infection with several human viruses. Mechanistically, SNX5 interacts with beclin 1 and ATG14-containing class III phosphatidylinositol-3-kinase (PI3KC3) complex 1 (PI3KC3-C1), increases the lipid kinase activity of purified PI3KC3-C1, and is required for endosomal generation of phosphatidylinositol-3-phosphate (PtdIns(3)P) and recruitment of the PtdIns(3)P-binding protein WIPI2 to virion-containing endosomes. These findings identify a context- and organelle-specific mechanism-SNX5-dependent PI3KC3-C1 activation at endosomes-for initiation of autophagy during viral infection.

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Nature, 1, 1, , 16 Dec 2020


CDK1, the Other 'Master Regulator' of Autophagy.
Odle RI, Florey O, Ktistakis NT, Cook SJ

Autophagy and cap-dependent mRNA translation are tightly regulated by the mechanistic target of rapamycin complex 1 (mTORC1) signalling complex in response to nutrient availability. However, the regulation of these processes, and mTORC1 itself, is different during mitosis, and this has remained an area of significant controversy; for example, studies have argued that autophagy is either repressed or highly active during mitosis. Recent studies have shown that autophagy initiation is repressed, and cap-dependent mRNA translation is maintained during mitosis despite mTORC1 activity being repressed. This is achieved in large part by a switch from mTORC1- to cyclin-dependent kinase 1 (CDK1)-mediated regulation. Here, we review the history and recent advances and seek to present a unifying model to inform the future study of autophagy and mTORC1 during mitosis.

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Trends in cell biology, 1, 1, , 30 Nov 2020


Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform.
Zachari M, Gudmundsson SR, Li Z, Manifava M, Cugliandolo F, Shah R, Smith M, Stronge J, Karanasios E, Piunti C, Kishi-Itakura C, Vihinen H, Jokitalo E, Guan JL, Buss F, Smith AM, Walker SA, Eskelinen EL, Ktistakis NT


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Developmental cell, 55, 2, , 26 Oct 2020


Mitochondrial Oxidative Damage Underlies Regulatory T Cell Defects in Autoimmunity.
Alissafi T, Kalafati L, Lazari M, Filia A, Kloukina I, Manifava M, Lim JH, Alexaki VI, Ktistakis NT, Doskas T, Garinis GA, Chavakis T, Boumpas DT, Verginis P

Regulatory T cells (Tregs) are vital for the maintenance of immune homeostasis, while their dysfunction constitutes a cardinal feature of autoimmunity. Under steady-state conditions, mitochondrial metabolism is critical for Treg function; however, the metabolic adaptations of Tregs during autoimmunity are ill-defined. Herein, we report that elevated mitochondrial oxidative stress and a robust DNA damage response (DDR) associated with cell death occur in Tregs in individuals with autoimmunity. In an experimental autoimmune encephalitis (EAE) mouse model of autoimmunity, we found a Treg dysfunction recapitulating the features of autoimmune Tregs with a prominent mtROS signature. Scavenging of mtROS in Tregs of EAE mice reversed the DDR and prevented Treg death, while attenuating the Th1 and Th17 autoimmune responses. These findings highlight an unrecognized role of mitochondrial oxidative stress in defining Treg fate during autoimmunity, which may facilitate the design of novel immunotherapies for diseases with disturbed immune tolerance.

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Cell metabolism, 1, 1, , 22 Jul 2020


ATG13 dynamics in nonselective autophagy and mitophagy: insights from live imaging studies and mathematical modeling.
Dalle Pezze P, Karanasios E, Kandia V, Manifava M, Walker SA, Gambardella Le Novère N, Ktistakis NT

During macroautophagy/autophagy, the ULK complex nucleates autophagic precursors, which give rise to autophagosomes. We analyzed, by live imaging and mathematical modeling, the translocation of ATG13 (part of the ULK complex) to the autophagic puncta in starvation-induced autophagy and ivermectin-induced mitophagy. In nonselective autophagy, the intensity and duration of ATG13 translocation approximated a normal distribution, whereas wortmannin reduced this effect and shifted to a log-normal distribution. During mitophagy, multiple translocations of ATG13 with increasing time between peaks were observed. We hypothesized that these multiple translocations arise because the engulfment of mitochondrial fragments required successive nucleation of phagophores on the same target, and a mathematical model based on this idea reproduced the oscillatory behavior. Significantly, model and experimental data were also in agreement that the number of ATG13 translocations is directly proportional to the diameter of the targeted mitochondrial fragments. Thus, our data provide novel insights into the early dynamics of selective and nonselective autophagy. ATG: autophagy related 13; CFP: cyan fluorescent protein; dsRED: red fluorescent protein; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; IVM: ivermectin; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: PtdIns-3-phosphate; ULK: unc-51 like autophagy activating kinase.

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Autophagy, 1, 1, , 22 Apr 2020

DOI: 10.1080/15548627.2020.1749401

Mammalian Mitophagosome Formation: A Focus on the Early Signals and Steps.
Zachari M, Ktistakis NT

Mitophagy, a conserved intracellular process by which mitochondria are eliminated via the autophagic machinery, is a quality control mechanism which facilitates maintenance of a functional mitochondrial network and cell homeostasis, making it a key process in development and longevity. Mitophagy has been linked to multiple human disorders, especially neurodegenerative diseases where the long-lived neurons are relying on clearance of old/damaged mitochondria to survive. During the past decade, the availability of novel tools to study mitophagy both and has significantly advanced our understanding of the molecular mechanisms governing this fundamental process in normal physiology and in various disease models. We here give an overview of the known mitophagy pathways and how they are induced, with a particular emphasis on the early events governing mitophagosome formation.

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Frontiers in cell and developmental biology, 8, 1, , 2020

DOI: 10.3389/fcell.2020.00171

Open Access

Editorial: Autophagy and Ageing: Ideas, Methods, Molecules.
Proikas-Cezanne T, Ktistakis NT

Ever since it was first discussed in evolutionary terms by Haldane (1941), Medawar (1952), and Williams (1957), [reviewed in Partridge and Gems (2006)] aging has become a focus of much current research interest, especially following discoveries pointing to molecular, genetic, and biochemical hallmarks that control its progression and severity (López-Otín et al., 2013). Amongst the many cellular processes that provide physiological connections to the aging process, autophagy is perhaps one of the most compelling (Rubinsztein et al., 2011; Nakamura and Yoshimori, 2018).

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Frontiers in cell and developmental biology, 8, 1, , 2020

DOI: 10.3389/fcell.2020.00141

Open Access

Ultrastructural insights into pathogen clearance by autophagy.
Kishi-Itakura C, Ktistakis NT, Buss F

Autophagy defends cells against proliferation of bacteria such as Salmonella in the cytosol. After escape from a damaged Salmonella-containing vacuole (SCV) exposing luminal glycans that bind to Galectin-8, the host cell ubiquitination machinery deposits a dense layer of ubiquitin around the cytosolic bacteria. The nature and spatial distribution of this ubiquitin coat in relation to other autophagy-related membranes are unknown. Using Transmission Electron Microscopy we determined the exact localisation of ubiquitin, the ruptured SCV membrane and phagophores around cytosolic Salmonella. Ubiquitin was not predominantly present on the Salmonella surface, but enriched on the fragmented SCV. Cytosolic bacteria without SCVs were less efficiently targeted by phagophores. Single bacteria were contained in single phagophores but multiple bacteria could be within large autophagic vacuoles reaching 30 μm in circumference. These large phagophores followed the contour of the engulfed bacteria, they were frequently in close association with endoplasmic reticulum membranes and, within them, remnants of the SCV were seen associated with each engulfed particle. Our data suggest that the Salmonella SCV has a major role in the formation of autophagic phagophores and highlight evolutionary conserved parallel mechanisms between xenophagy and mitophagy with the fragmented SCV and the damaged outer mitochondrial membrane serving similar functions. This article is protected by copyright. All rights reserved.

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Traffic (Copenhagen, Denmark), 1, 1, , 21 Feb 2020

DOI: 10.1111/tra.12723

Temporal inhibition of autophagy reveals segmental reversal of ageing with increased cancer risk.
Cassidy LD, Young ARJ, Young CNJ, Soilleux EJ, Fielder E, Weigand BM, Lagnado A, Brais R, Ktistakis NT, Wiggins KA, Pyrillou K, Clarke MCH, Jurk D, Passos JF, Narita M

Autophagy is an important cellular degradation pathway with a central role in metabolism as well as basic quality control, two processes inextricably linked to ageing. A decrease in autophagy is associated with increasing age, yet it is unknown if this is causal in the ageing process, and whether autophagy restoration can counteract these ageing effects. Here we demonstrate that systemic autophagy inhibition induces the premature acquisition of age-associated phenotypes and pathologies in mammals. Remarkably, autophagy restoration provides a near complete recovery of morbidity and a significant extension of lifespan; however, at the molecular level this rescue appears incomplete. Importantly autophagy-restored mice still succumb earlier due to an increase in spontaneous tumour formation. Thus, our data suggest that chronic autophagy inhibition confers an irreversible increase in cancer risk and uncovers a biphasic role of autophagy in cancer development being both tumour suppressive and oncogenic, sequentially.

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Nature communications, 11, 1, , 16 Jan 2020

DOI: 10.1038/s41467-019-14187-x

Open Access

An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis.
Odle RI, Walker SA, Oxley D, Kidger AM, Balmanno K, Gilley R, Okkenhaug H, Florey O, Ktistakis NT, Cook SJ

Since nuclear envelope breakdown occurs during mitosis in metazoan cells, it has been proposed that macroautophagy must be inhibited to maintain genome integrity. However, repression of macroautophagy during mitosis remains controversial and mechanistic detail limited to the suggestion that CDK1 phosphorylates VPS34. Here, we show that initiation of macroautophagy, measured by the translocation of the ULK complex to autophagic puncta, is repressed during mitosis, even when mTORC1 is inhibited. Indeed, mTORC1 is inactive during mitosis, reflecting its failure to localize to lysosomes due to CDK1-dependent RAPTOR phosphorylation. While mTORC1 normally represses autophagy via phosphorylation of ULK1, ATG13, ATG14, and TFEB, we show that the mitotic phosphorylation of these autophagy regulators, including at known repressive sites, is dependent on CDK1 but independent of mTOR. Thus, CDK1 substitutes for inhibited mTORC1 as the master regulator of macroautophagy during mitosis, uncoupling autophagy regulation from nutrient status to ensure repression of macroautophagy during mitosis.

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Molecular cell, S1097-2765, 19, , 06 Nov 2019

DOI: 10.1016/j.molcel.2019.10.016

Open Access

Autophagosome biogenesis machinery.
Walker SA, Ktistakis NT

We review current knowledge of the process of autophagosome formation with special emphasis on the very early steps: turning on the autophagy pathway, assembling the autophagy machinery, and building the autophagosome. The pathway is remarkably well co-ordinated spatially and temporally, and it shows broad conservation across species and cell types, including neurons. In addition, although much current knowledge derives mostly from settings of non-selective autophagy, recent work also indicates that selective autophagy, and more specifically mitophagy, shows similar dynamics. Having an understanding of this remarkable process may help the design of novel therapeutics for neurodegeneration and other pathologies.

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Journal of molecular biology, , 1089-8638, , 2019


Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling.
Fraser J, Simpson J, Fontana R, Kishi-Itakura C, Ktistakis NT, Gammoh N

Despite recently uncovered connections between autophagy and the endocytic pathway, the role of autophagy in regulating endosomal function remains incompletely understood. Here, we find that the ablation of autophagy-essential players disrupts EGF-induced endocytic trafficking of EGFR. Cells lacking ATG7 or ATG16L1 exhibit increased levels of phosphatidylinositol-3-phosphate (PI(3)P), a key determinant of early endosome maturation. Increased PI(3)P levels are associated with an accumulation of EEA1-positive endosomes where EGFR trafficking is stalled. Aberrant early endosomes are recognised by the autophagy machinery in a TBK1- and Gal8-dependent manner and are delivered to LAMP2-positive lysosomes. Preventing this homeostatic regulation of early endosomes by autophagy reduces EGFR recycling to the plasma membrane and compromises downstream signalling and cell survival. Our findings uncover a novel role for the autophagy machinery in maintaining early endosome function and growth factor sensing.

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EMBO reports, , 1469-3178, , 2019


Open Access

Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform.
Zachari M, Gudmundsson SR, Li Z, Manifava M, Shah R, Smith M, Stronge J, Karanasios E, Piunti C, Kishi-Itakura C, Vihinen H, Jokitalo E, Guan JL, Buss F, Smith AM, Walker SA, Eskelinen EL, Ktistakis NT

The dynamics and coordination between autophagy machinery and selective receptors during mitophagy are unknown. Also unknown is whether mitophagy depends on pre-existing membranes or is triggered on the surface of damaged mitochondria. Using a ubiquitin-dependent mitophagy inducer, the lactone ivermectin, we have combined genetic and imaging experiments to address these questions. Ubiquitination of mitochondrial fragments is required the earliest, followed by auto-phosphorylation of TBK1. Next, early essential autophagy proteins FIP200 and ATG13 act at different steps, whereas ULK1 and ULK2 are dispensable. Receptors act temporally and mechanistically upstream of ATG13 but downstream of FIP200. The VPS34 complex functions at the omegasome step. ATG13 and optineurin target mitochondria in a discontinuous oscillatory way, suggesting multiple initiation events. Targeted ubiquitinated mitochondria are cradled by endoplasmic reticulum (ER) strands even without functional autophagy machinery and mitophagy adaptors. We propose that damaged mitochondria are ubiquitinated and dynamically encased in ER strands, providing platforms for formation of the mitophagosomes.

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Developmental cell, , 1878-1551, , 2019


Open Access

Autophagy, Inflammation, and Metabolism (AIM) Center in its second year.
Deretic V, Prossnitz E, Burge M, Campen MJ, Cannon J, Liu KJ, Liu M, Hall P, Sklar LA, Allers L, Mariscal L, Garcia SA, Weaver J, Baehrecke EH, Behrends C, Cecconi F, Codogno P, Chen GC, Elazar Z, Eskelinen EL, Fourie B, Gozuacik D, Hong W, Jo EK, Johansen T, Juhász G, Kimchi A, Ktistakis N, Kroemer G, Mizushima N, Münz C, Reggiori F, Rubinsztein D, Ryan K, Schroder K, Shen HM, Simonsen A, Tooze SA, Vaccaro M, Yoshimori T, Yu L, Zhang H, Klionsky DJ

The NIH-funded center for autophagy research named Autophagy, Inflammation, and Metabolism (AIM) Center of Biomedical Research Excellence, located at the University of New Mexico Health Science Center is now completing its second year as a working center with a mission to promote autophagy research locally, nationally, and internationally. The center has thus far supported a cadre of 6 junior faculty (mentored PIs; mPIs) at a near-R01 level of funding. Two mPIs have graduated by obtaining their independent R01 funding and 3 of the remaining 4 have won significant funding from NIH in the form of R21 and R56 awards. The first year and a half of setting up the center has been punctuated by completion of renovations and acquisition and upgrades for equipment supporting autophagy, inflammation and metabolism studies. The scientific cores usage, and the growth of new studies is promoted through pilot grants and several types of enablement initiatives. The intent to cultivate AIM as a scholarly hub for autophagy and related studies is manifested in its Vibrant Campus Initiative, and the Tuesday AIM Seminar series, as well as by hosting a major scientific event, the 2019 AIM symposium, with nearly one third of the faculty from the International Council of Affiliate Members being present and leading sessions, giving talks, and conducting workshop activities. These and other events are often videostreamed for a worldwide scientific audience, and information about events at AIM and elsewhere are disseminated on Twitter and can be followed on the AIM web site. AIM intends to invigorate research on overlapping areas between autophagy, inflammation and metabolism with a number of new initiatives to promote metabolomic research. With the turnover of mPIs as they obtain their independent funding, new junior faculty are recruited and appointed as mPIs. All these activities are in keeping with AIM's intention to enable the next generation of autophagy researchers and help anchor, disseminate, and convey the depth and excitement of the autophagy field.

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Autophagy, 15, 1554-8635, , 2019


Who plays the ferryman: ATG2 channels lipids into the forming autophagosome.
Ktistakis NT

Expansion of the autophagosomal membrane requires a mechanism to supply lipids while excluding most membrane proteins. In this issue, Valverde et al. (2019. identify ATG2, a member of the autophagy-related protein family, as a lipid transfer protein and provide important novel insights on how autophagosomes grow.

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The Journal of cell biology, , 1540-8140, , 2019


ER platforms mediating autophagosome generation.
Ktistakis NT

The origin of the autophagosomal membrane started to be debated by scientists working in the field within one year of the modern definition of autophagy in 1963. There is now converging evidence from older and newer studies that the endoplasmic reticulum is involved in formation of autophagosomes. Thus, it is possible to trace from early morphological work - done without the benefit of molecular descriptions - to recent studies - dissecting how specific proteins nucleate autophagosome biogenesis - a long series of experimental findings that are beginning to answer the 55-year old question with some confidence. The view that has emerged is that specialised regions of the endoplasmic reticulum, in dynamic cross talk with most intracellular organelles via membrane contact sites, provide a platform for autophagosome biogenesis.

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Biochimica et biophysica acta. Molecular and cell biology of lipids, , 1879-2618, , 2019


Phosphorylation of Syntaxin 17 by TBK1 Controls Autophagy Initiation.
Kumar S, Gu Y, Abudu YP, Bruun JA, Jain A, Farzam F, Mudd M, Anonsen JH, Rusten TE, Kasof G, Ktistakis N, Lidke KA, Johansen T, Deretic V

Syntaxin 17 (Stx17) has been implicated in autophagosome-lysosome fusion. Here, we report that Stx17 functions in assembly of protein complexes during autophagy initiation. Stx17 is phosphorylated by TBK1 whereby phospho-Stx17 controls the formation of the ATG13FIP200 mammalian pre-autophagosomal structure (mPAS) in response to induction of autophagy. TBK1 phosphorylates Stx17 at S202. During autophagy induction, Stx17 transfers from the Golgi, where its steady-state pools localize, to the ATG13FIP200 mPAS. Stx17 was in complexes with ATG13 and FIP200, whereas its non-phosphorylatable mutant Stx17 was not. Stx17 or TBK1 knockouts blocked ATG13 and FIP200 puncta formation. Stx17 or TBK1 knockouts reduced the formation of ATG13 protein complexes with FIP200 and ULK1. Endogenous Stx17 colocalized with LC3B following induction of autophagy. Stx17 knockout diminished LC3 response and reduced sequestration of the prototypical bulk autophagy cargo lactate dehydrogenase. We conclude that Stx17 is a TBK1 substrate and that together they orchestrate assembly of mPAS.

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Developmental cell, , 1878-1551, , 2019


Alpha-synuclein fibrils recruit TBK1 and OPTN to lysosomal damage sites and induce autophagy in microglial cells.
Bussi C, Peralta Ramos JM, Arroyo DS, Gallea JI, Ronchi P, Kolovou A, Wang JM, Florey O, Celej MS, Schwab Y, Ktistakis NT, Iribarren P

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most work was done in neurons and not in microglial cells. Here we report that exogenous fibrillar but not monomeric alpha-synuclein (AS) induces autophagy in microglial cells. We extensively studied the dynamics of this response by both live-cell imaging and correlative light-electron microscopy (CLEM) and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and Optineurin (OPTN) to ubiquitinated lysosomes. In addition, we observed that LC3 recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.

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Journal of cell science, , 1477-9137, , 2018


Open Access

Autophagy, Inflammation, and Metabolism (AIM) Center of Biomedical Research Excellence: supporting the next generation of autophagy researchers and fostering international collaborations.
Deretic V, Prossnitz E, Burge M, Campen MJ, Cannon J, Liu KJ, Sklar LA, Allers L, Garcia SA, Baehrecke EH, Behrends C, Cecconi F, Codogno P, Chen GC, Elazar Z, Eskelinen EL, Fourie B, Gozuacik D, Hong W, Hotamisligi G, Jäättelä M, Jo EK, Johansen T, Juhász G, Kimchi A, Ktistakis N, Kroemer G, MIzushima N, Münz C, Reggiori F, Rubinsztein D, Ryan K, Schroder K, Simonsen A, Tooze S, Vaccaro M, Yoshimori T, Yu L, Zhang H, Klionsky DJ

Recently, NIH has funded a center for autophagy research named the Autophagy, Inflammation, and Metabolism (AIM) Center of Biomedical Research Excellence, located at the University of New Mexico Health Science Center (UNM HSC), with aspirations to promote autophagy research locally, nationally, and internationally. The center has 3 major missions: (i) to support junior faculty in their endeavors to develop investigations in this area and obtain independent funding; (ii) to develop and provide technological platforms to advance autophagy research with emphasis on cellular approaches for high quality reproducible research; and (iii) to foster international collaborations through the formation of an International Council of Affiliate Members and through hosting national and international workshops and symposia. Scientifically, the AIM center is focused on autophagy and its intersections with other processes, with emphasis on both fundamental discoveries and applied translational research.

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Autophagy, , 1554-8635, , 2018


In praise of M. Anselmier who first used the term "autophagie" in 1859.
Ktistakis NT

Autophagy, , 1554-8635, , 2017


Molecular definitions of autophagy and related processes.
Galluzzi L, Baehrecke EH, Ballabio A, Boya P, Bravo-San Pedro JM, Cecconi F, Choi AM, Chu CT, Codogno P, Colombo MI, Cuervo AM, Debnath J, Deretic V, Dikic I, Eskelinen EL, Fimia GM, Fulda S, Gewirtz DA, Green DR, Hansen M, Harper JW, Jäättelä M, Johansen T, Juhasz G, Kimmelman AC, Kraft C, Ktistakis NT, Kumar S, Levine B, Lopez-Otin C, Madeo F, Martens S, Martinez J, Melendez A, Mizushima N, Münz C, Murphy LO, Penninger JM, Piacentini M, Reggiori F, Rubinsztein DC, Ryan KM, Santambrogio L, Scorrano L, Simon AK, Simon HU, Simonsen A, Tavernarakis N, Tooze SA, Yoshimori T, Yuan J, Yue Z, Zhong Q, Kroemer G

Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.

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The EMBO journal, , 1460-2075, , 2017


Phospholipase D activity couples plasma membrane endocytosis with retromer dependent recycling.
Thakur R, Panda A, Coessens E, Raj N, Yadav S, Balakrishnan S, Zhang Q, Georgiev P, Basak B, Pasricha R, Wakelam MJ, Ktistakis NT, Padinjat R

During illumination, the light sensitive plasma membrane (rhabdomere) of Drosophila photoreceptors undergoes turnover with consequent changes in size and composition. However the mechanism by which illumination is coupled to rhabdomere turnover remains unclear. We find that photoreceptors contain a light-dependent phospholipase D (PLD) activity. During illumination, loss of PLD resulted in an enhanced reduction in rhabdomere size, accumulation of Rab7 positive, rhodopsin1-containing vesicles (RLVs) in the cell body and reduced rhodopsin protein. These phenotypes were associated with reduced levels of phosphatidic acid, the product of PLD activity and were rescued by reconstitution with catalytically active PLD. In wild type photoreceptors, during illumination, enhanced PLD activity was sufficient to clear RLVs from the cell body by a process dependent on Arf1-GTP levels and retromer complex function. Thus, during illumination, PLD activity couples endocytosis of RLVs with their recycling to the plasma membrane thus maintaining plasma membrane size and composition.

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eLife, 5, 2050-084X, , 2016


Open Access

Assembly of early machinery for autophagy induction: novel insights from high resolution microscopy.
Ktistakis NT, Walker SA, Karanasios E

Oncotarget, , 1949-2553, , 2016


Open Access

Dynamics of mTORC1 activation in response to amino acids.
Manifava M, Smith M, Rotondo S, Walker S, Niewczas I, Zoncu R, Clark J, Ktistakis NT

Amino acids are essential activators of mTORC1 via a complex containing RAG GTPases, RAGULATOR and the vacuolar ATPase. Sensing of amino acids causes translocation of mTORC1 to lysosomes, an obligate step for activation. To examine the spatial and temporal dynamics of this translocation, we used live imaging of the mTORC1 component RAPTOR and a cell permeant fluorescent analogue of di-leucine methyl ester. Translocation to lysosomes is a transient event, occurring within 2 min of aa addition and peaking within 5 min. It is temporally coupled with fluorescent leucine appearance in lysosomes and is sustained in comparison to aa stimulation. Sestrin2 and the vacuolar ATPase are negative and positive regulators of mTORC1 activity in our experimental system. Of note, phosphorylation of canonical mTORC1 targets is delayed compared to lysosomal translocation suggesting a dynamic and transient passage of mTORC1 from the lysosomal surface before targetting its substrates elsewhere.

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eLife, 5, 2050-084X, , 2016


Open Access

Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex.
Ohashi Y, Soler N, García Ortegón M, Zhang L, Kirsten ML, Perisic O, Masson GR, Burke JE, Jakobi AJ, Apostolakis AA, Johnson CM, Ohashi M, Ktistakis NT, Sachse C, Williams RL

The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.

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Autophagy, , 1554-8635, , 2016


Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles.
Karanasios E, Walker SA, Okkenhaug H, Manifava M, Hummel E, Zimmermann H, Ahmed Q, Domart MC, Collinson L, Ktistakis NT

Autophagosome formation requires sequential translocation of autophagy-specific proteins to membranes enriched in PI3P and connected to the ER. Preceding this, the earliest autophagy-specific structure forming de novo is a small punctum of the ULK1 complex. The provenance of this structure and its mode of formation are unknown. We show that the ULK1 structure emerges from regions, where ATG9 vesicles align with the ER and its formation requires ER exit and coatomer function. Super-resolution microscopy reveals that the ULK1 compartment consists of regularly assembled punctate elements that cluster in progressively larger spherical structures and associates uniquely with the early autophagy machinery. Correlative electron microscopy after live imaging shows tubulovesicular membranes present at the locus of this structure. We propose that the nucleation of autophagosomes occurs in regions, where the ULK1 complex coalesces with ER and the ATG9 compartment.

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Nature communications, 7, 2041-1723, , 2016


Open Access

Digesting the Expanding Mechanisms of Autophagy.
Ktistakis NT, Tooze SA

Autophagy is a catabolic 'self-eating' pathway that is emerging as a crucial integration point in cell physiology. With its own set of genes, the autophagy pathway communicates with virtually all signalling networks and organelles. Recent advances have been made in understanding the origin of the autophagosomal membrane, novel regulators, and the mechanisms by which specific intracellular membranes become autophagy substrates. New studies on noncanonical autophagy, mediated by subsets of autophagy proteins, and the role of autophagy proteins in non-autophagy pathways are also emerging in many different biological contexts. Our understanding of canonical autophagy, including membrane origin and autophagy proteins, needs to be considered together with emerging noncanonical pathways.

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Trends in cell biology, , 1879-3088, , 2016


Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
Klionsky DJ, Ktistakis NT, O'Donnell VB, et al


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Autophagy, 12, 1554-8635, , 2016


Open Access

The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix.
Barneda D, Planas-Iglesias J, Gaspar ML, Mohammadyani D, Prasannan S, Dormann D, Han GS, Jesch SA, Carman GM, Kagan V, Parker MG, Ktistakis NT, Dixon AM, Klein-Seetharaman J, Henry S, Christian M

Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD-LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat.

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eLife, 4, 2050-084X, , 2015


Open Access

Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes.
Rostislavleva K, Soler N, Ohashi Y, Zhang L, Pardon E, Burke JE, Masson GR, Johnson C, Steyaert J, Ktistakis NT, Williams RL

Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis, and autophagy. We present the 4.4 angstrom crystal structure of the 385-kilodalton endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1), and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm, where the Vps15 kinase domain engages the Vps34 activation loop to regulate its activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, suggesting a path for complex assembly. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal conformational changes accompanying membrane binding and identify a Vps30 loop that is critical for the ability of complex II to phosphorylate giant liposomes on which complex I is inactive.

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Science (New York, N.Y.), 350, 1095-9203, , 2015


ERES: sites for autophagosome biogenesis and maturation?
Sanchez-Wandelmer J, Ktistakis NT, Reggiori F

Autophagosomes are the hallmark of autophagy, but despite their central role in this degradative pathway that involves vesicle transport to lysosomes or vacuoles, the mechanism underlying their biogenesis still remains largely unknown. Our current concepts about autophagosome biogenesis are based on models suggesting that a small autonomous cisterna grows into an autophagosome through expansion at its extremities. Recent findings have revealed that endoplasmic reticulum (ER) exit sites (ERES), specialized ER regions where proteins are sorted into the secretory system, are key players in the formation of autophagosomes. Owing to the morphological connection of nascent autophagosomes with the ER, this has raised several questions that challenge our current perception of autophagosome biogenesis, such as are ERES the compartments where autophagosome formation takes place? What is the functional relevance of this connection? Are these compartments providing essential molecules for the generation of autophagosomes and/or are they structural platforms where these vesicles emerge? In this Hypothesis, we discuss recent data that have implicated the ERES in autophagosome biogenesis and we propose two models to describe the possible role of this compartment at different steps in the process of autophagosome biogenesis. This article is part of a Focus on Autophagosome biogenesis. For further reading, please see related articles: 'Membrane dynamics in autophagosome biogenesis' by Sven R. Carlsson and Anne Simonsen (J. Cell Sci. 128, 193-205) and 'WIPI proteins: essential PtdIns3P effectors at the nascent autophagosome' by Tassula Proikas-Cezanne et al. (J. Cell Sci. 128, 207-217).

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Journal of cell science, 128, 1477-9137, , 2015


Open Access

Live-cell imaging for the assessment of the dynamics of autophagosome formation: Focus on early steps.
Karanasios E, Ktistakis NT

Autophagy is a cytosolic degradative pathway, which through a series of complicated membrane rearrangements leads to the formation of a unique double membrane vesicle, the autophagosome. The use of fluorescent proteins has allowed visualizing the autophagosome formation in live cells and in real time, almost 40years after electron microscopy studies observed these structures for the first time. In the last decade, live-cell imaging has been extensively used to study the dynamics of autophagosome formation in cultured mammalian cells. Hereby we will discuss how the live-cell imaging studies have tried to settle the debate about the origin of the autophagosome membrane and how they have described the way different autophagy proteins coordinate in space and time in order to drive autophagosome formation.

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Methods (San Diego, Calif.), , 1095-9130, , 2014


Dynamics of autophagosome formation: a pulse and a sequence of waves.
Ktistakis NT, Karanasios E, Manifava M

Autophagosomes form in eukaryotic cells in response to starvation or to other stress conditions brought about by the unwanted presence in the cytosol of pathogens, damaged organelles or aggregated protein assemblies. The uniqueness of autophagosomes is that they form de novo and that they are the only double-membraned vesicles known in cells, having arisen from flat membrane sheets which have expanded and self-closed. The various steps describing their formation as well as most of the protein and lipid components involved have been identified. Furthermore, the hierarchical relationships among the components are well documented, and the mechanistic rationale for some of these hierarchies has been revealed. In the present review, we try to provide a current view of the process of autophagosome formation in mammalian cells, emphasizing along the way gaps in our knowledge that need additional work.

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Biochemical Society transactions, 42, 1470-8752, , 2014


Imaging autophagy.
Karanasios E, Stapleton E, Manifava M, Ktistakis NT

Autophagy is a membrane-trafficking pathway activated to deliver cytosolic material for degradation to lysosomes through a novel membrane compartment, the autophagosome. Fluorescence microscopy is the most common method used to visualize proteins inside cells, and it is widely used in the autophagy field. To distinguish it from the cellular background, the protein of interest (POI) is either fused with a genetically encoded fluorescent protein or stained with an antibody that is conjugated to an inorganic fluorescent compound. Genetic tagging of the POI allows its visualization in live cells, while immunostaining of the POI requires the fixation of cells and the permeabilization of cell membranes. Here we describe detailed protocols on how to visualize autophagy dynamics using fluorescence microscopy in live and fixed cells. We discuss the critical parameters of each technique, their advantages, and why the robustness is increased when they are used in tandem.

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Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.], 69, 1934-9300, , 2014


The immune system GTPase GIMAP6 interacts with the Atg8 homologue GABARAPL2 and is recruited to autophagosomes.
Pascall JC, Rotondo S, Mukadam AS, Oxley D, Webster J, Walker SA, Piron J, Carter C, Ktistakis NT, Butcher GW

The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux.

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PloS one, 8, 1932-6203, , 2013


Open Access

Omegasomes: PI3P platforms that manufacture autophagosomes.
Roberts R, Ktistakis NT

Autophagy is a conserved survival pathway, which cells and tissues will activate during times of stress. It is characterized by the formation of double-membrane vesicles called autophagosomes inside the cytoplasm. The molecular mechanisms and the signalling components involved require specific control to ensure correct activation. The present chapter describes the formation of autophagosomes from within omegasomes, newly identified membrane compartments enriched in PI3P (phosphatidylinositol 3-phosphate) that serve as platforms for the formation of at least some autophagosomes. We discuss the signalling events required to nucleate the formation of omegasomes as well as the protein complexes involved.

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Essays in biochemistry, 55, 1744-1358, , 2013


Dynamic association of the ULK1 complex with omegasomes during autophagy induction.
Karanasios E, Stapleton E, Manifava M, Kaizuka T, Mizushima N, Walker SA, Ktistakis NT

Induction of autophagy requires the ULK1 protein kinase complex and the Vps34 lipid kinase complex. PtdIns3P synthesised by Vps34 accumulates in omegasomes, membrane extensions of the ER within which some autophagosomes form. The ULK1 complex is thought to target autophagosomes independently of PtdIns3P, and its functional relationship to omegasomes is unclear. Here we show that the ULK1 complex colocalises with omegasomes in a PtdIns3P-dependent way. Live-cell imaging of Atg13 (a ULK1 complex component), omegasomes and LC3 establishes and annotates for the first time a complete sequence of steps leading to autophagosome formation, as follows. Upon starvation, the ULK1 complex forms puncta associated with the ER and sporadically with mitochondria. If PtdIns3P is available, these puncta become omegasomes. Subsequently, the ULK1 complex exits omegasomes and autophagosomes bud off. If PtdIns3P is unavailable, ULK1 puncta are greatly reduced in number and duration. Atg13 contains a region with affinity for acidic phospholipids, required for translocation to punctate structures and autophagy progression.

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Journal of cell science, 126, 1477-9137, , 2013


Open Access

Differential isolation and identification of PI(3)P and PI(3,5)P2 binding proteins from Arabidopsis thaliana using an agarose-phosphatidylinositol-phosphate affinity chromatography.
D Oxley, N Ktistakis, T Farmaki

A phosphatidylinositol-phosphate affinity chromatographic approach combined with mass spectrometry was used in order to identify novel PI(3)P and PI(3,5)P2 binding proteins from Arabidopsis thaliana suspension cell extracts. Most of the phosphatidylinositol-phosphate interacting candidates identified from this differential screening are characterized by lysine/arginine rich patches. Direct phosphoinositide binding was identified for important membrane trafficking regulators as well as protein quality control proteins such as the ATG18p orthologue involved in autophagosome formation and the lipid Sec14p like transfer protein. A pentatricopeptide repeat (PPR) containing protein was shown to directly bind to PI(3,5)P2 but not to PI(3)P. PIP chromatography performed using extracts obtained from high salt (0.4M and 1M NaCl) pretreated suspensions showed that the association of an S5-1 40S ribosomal protein with both PI(3)P and PI(3,5)P2 was abolished under salt stress whereas salinity stress induced an increase in the phosphoinositide association of the DUF538 domain containing protein SVB, associated with trichome size. Additional interacting candidates were co-purified with the phosphoinositide bound proteins. Binding of the COP9 signalosome, the heat shock proteins, and the identified 26S proteasomal subunits, is suggested as an indirect effect of their interaction with other proteins directly bound to the PI(3)P and the PI(3,5)P2 phosphoinositides.

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Journal of proteomics, , , , 2013

DOI: 10.1016/j.jprot.2013.08.020

Live cell imaging of early autophagy events: omegasomes and beyond.
E Karanasios, E Stapleton, SA Walker, M Manifava, NT Ktistakis

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer. Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution. Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

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Journal of visualized experiments : JoVE, , 77, , 2013

DOI: 10.3791/50484

Characteristics and requirements of basal autophagy in HEK 293 cells.
Musiwaro P, Smith M, Manifava M, Walker SA, Ktistakis NT

Basal autophagy-here defined as macroautophagic activity during cellular growth in normal medium containing amino acids and serum-appears to be highly active in many cell types and in animal tissues. Here we characterized this pathway in mammalian HEK 293 cells. First, we examined, side by side, three compounds that are widely used to reveal basal autophagy by blocking maturation of autophagosomes: bafilomycin A 1 (BafA1), chloroquine and vinblastine. Only BafA1 appeared to be without complicating side effects. Chloroquine partially inhibited mechanistic target of rapamycin (MTOR) activity, which would induce autophagy induction as well as block autophagosome maturation. Vinblastine caused the distribution of early omegasome components into punctate phagophore assembly sites, and therefore it would also induce autophagy, complicating interpretation. Basal autophagy was significantly sensitive to inhibition by wortmannin, and therefore required formation of phosphatidylinositol 3-phosphate (PtdIns3P), but it was twice as resistant to wortmannin as starvation-induced autophagy. We also determined that basal autophagy was significantly suppressed by MTOR activation brought about by overexpression of RHEB or activated RAGs. Finally we investigated the spatial relationship of nascent autophagosomes to the endoplasmic reticulum (ER) or to mitochondria by live imaging experiments under conditions that reveal basal autophagy (with BafA1 treatment), or upon MTOR inactivation (which would result in autophagy induction). Side-by-side comparison showed that under both basal and induced autophagy, 100% of autophagosomes first appeared in close proximity to ER strands. In parallel measurements, 40% were in close proximity to mitochondria under both conditions. We concluded that in HEK 293 cells, basal autophagy is mechanistically similar to that induced by MTOR inactivation in all aspects examined.

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Autophagy, 9, 1554-8635, , 2013


PIPing on lysosome tubes.
NT Ktistakis, SA Tooze

The EMBO journal, 32, 3, , 2013

DOI: 10.1038/emboj.2012.355

Open Access

The Arabidopsis thaliana immunophilin ROF1 directly interacts with PI(3)P and PI(3,5)P2 and affects germination under osmotic stress.
D Karali, D Oxley, J Runions, N Ktistakis, T Farmaki

A direct interaction of the Arabidopsis thaliana immunophilin ROF1 with phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate was identified using a phosphatidylinositol-phosphate affinity chromatography of cell suspension extracts, combined with a mass spectrometry (nano LC ESI-MS/MS) analysis. The first FK506 binding domain was shown sufficient to bind to both phosphatidylinositol-phosphate stereoisomers. GFP-tagged ROF1 under the control of a 35S promoter was localised in the cytoplasm and the cell periphery of Nicotiana tabacum leaf explants. Immunofluorescence microscopy of Arabidopsis thaliana root tips verified its cytoplasmic localization and membrane association and showed ROF1 localization in the elongation zone which was expanded to the meristematic zone in plants grown on high salt media. Endogenous ROF1 was shown to accumulate in response to high salt treatment in Arabidopsis thaliana young leaves as well as in seedlings germinated on high salt media (0.15 and 0.2 M NaCl) at both an mRNA and protein level. Plants over-expressing ROF1, (WSROF1OE), exhibited enhanced germination under salinity stress which was significantly reduced in the rof1(-) knock out mutants and abolished in the double mutants of ROF1 and of its interacting homologue ROF2 (WSrof1(-)/2(-)). Our results show that ROF1 plays an important role in the osmotic/salt stress responses of germinating Arabidopsis thaliana seedlings and suggest its involvement in salinity stress responses through a phosphatidylinositol-phosphate related protein quality control pathway.

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PloS one, 7, 11, , 2012

DOI: 10.1371/journal.pone.0048241

Open Access

How phosphoinositide 3-phosphate controls growth downstream of amino acids and autophagy downstream of amino acid withdrawal.
NT Ktistakis, M Manifava, P Schoenfelder, S Rotondo

The simple phosphoinositide PtdIns3P has been shown to control cell growth downstream of amino acid signalling and autophagy downstream of amino acid withdrawal. These opposing effects depend in part on the existence of distinct complexes of Vps34 (vacuolar protein sorting 34), the kinase responsible for the majority of PtdIns3P synthesis in cells: one complex is activated after amino acid withdrawal to induce autophagy and another regulates mTORC1 (mammalian target of rapamycin complex 1) activation when amino acids are present. However, lipid-dependent signalling almost always exhibits a spatial dimension, related to the site of formation of the lipid signal. In the case of PtdIns3P-regulated autophagy induction, recent data suggest that PtdIns3P accumulates in a membrane compartment dynamically connected to the endoplasmic reticulum that constitutes a platform for the formation of some autophagosomes. For PtdIns3P-regulated mTORC1 activity, a spatial context is not yet known: several possibilities can be envisaged based on the known effects of PtdIns3P on the endocytic system and on recent data suggesting that activation of mTORC1 depends on its localization on lysosomes.

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Biochemical Society transactions, 40, 1, , 2012

DOI: 10.1042/BST20110684

Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate.
EM Cottam, HJ Maier, M Manifava, LC Vaux, P Chandra-Schoenfelder, W Gerner, P Britton, NT Ktistakis, T Wileman

Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can provide an innate defence against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, Infectious Bronchitis Virus (IBV) activates autophagy. A screen of individual IBV non-structural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses Mouse Hepatitis Virus, and Severe Acute Respiratory Syndrome Virus, and the equivalent nsp5-7 of the arterivirus Porcine Reproductive and Respiratory Syndrome Virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II-positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double FYVE-domain containing protein (DFCP) indicating localised concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signalling, activation of sirtuin1 or induction of ER stress.

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Autophagy, 7, 11, , 2011

DOI: 10.4161/auto.7.11.16642

Open Access

The autoimmunity-related GIMAP5 GTPase is a lysosome-associated protein.
VW Wong, AE Saunders, A Hutchings, JC Pascall, C Carter, NA Bright, SA Walker, NT Ktistakis, GW Butcher

A mutation in the rat GIMAP5 gene predisposes for autoimmunity, most famously in the BB rat model of autoimmune type 1 diabetes mellitus. This mutation is associated with severe peripheral T lymphopenia, as is mutation of the same gene in mice, but the mechanism by which GIMAP5 normally protects T cells from death is unknown. GIMAP5 is a putative small GTPase, a class of proteins which often fulfil their functions in the vicinity of cellular membranes. The objective of this study was to determine the normal intracellular location of GIMAP5 in lymphoid cells. Combining studies in rat, mouse and human systems, novel monoclonal antibodies (mAbs) were used to examine the localization of GIMAP5 and the closely-related protein, GIMAP1, in lymphoid cells by means of confocal microscopy and sub-cellular fractionation combined with immunoblotting. Additionally, human Jurkat T cells that inducibly express epitope-tagged GIMAP5 were established and used in electron microscopy (EM). Endogenous GIMAP5 was found to be located in a membraneous compartment/s which was also detected by established markers of lysosomes. GIMAP1, by contrast, was found to be located in the Golgi apparatus. EM studies of the inducible Jurkat T cells also found GIMAP5 in lysosomes and, in addition, in multivesicular bodies. This study establishes that the endogenous location of GIMAP5 is in lysosomes and related compartments and provides a clearer context for hypotheses about its mechanism of action.

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Self/nonself, 1, 3, , 2010

DOI: 10.4161/self.1.3.12819

Open Access

Lipid signaling and homeostasis: PA- is better than PA-H, but what about those PIPs?
NT Ktistakis

Although cellular membranes are composed of hundreds of distinct lipid species, the lipid composition is maintained within a narrow range. The regulatory circuit responsible for this homeostasis in yeast depends on a membrane-bound transcriptional repressor that translocates to the nucleus in response to the abundance of its lipid ligand on the membrane. Feedback control in this system is provided because the lipid ligand is also an end product of the activity of the transcription factor. This basic design is also evident in higher eukaryotes such as Drosophila and mammals, but with important differences in the lipid being sensed, the composition of the sensors, and the fine-tuning of the response. New work indicates that regulation of intracellular pH levels in yeast by glucose availability may fine-tune the binding of the repressor to its lipid ligand, providing a mechanism that connects phospholipid metabolism to nutrient sensing. The importance of pH effects in this pathway raises the possibility that additional lipid-signaling pathways may be regulated by the protonation state of the lipid or its effector.

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Science signaling, 3, 151, , 2010

DOI: 10.1126/scisignal.3151pe46

Antibacterial autophagy occurs at PI(3)P-enriched domains of the endoplasmic reticulum and requires Rab1 GTPase.
J Huang, CL Birmingham, S Shahnazari, J Shiu, YT Zheng, AC Smith, KG Campellone, WD Heo, S Gruenheid, T Meyer, MD Welch, NT Ktistakis, PK Kim, DJ Klionsky, JH Brumell

Autophagy mediates the degradation of cytoplasmic components in eukaryotic cells and plays a key role in immunity. The mechanism of autophagosome formation is not clear. Here we examined two potential membrane sources for antibacterial autophagy: the ER and mitochondria. DFCP1, a marker of specialized ER domains known as 'omegasomes,' associated with Salmonella-containing autophagosomes via its PtdIns(3)P and ER-binding domains, while a mitochondrial marker (cytochrome b5-GFP) did not. Rab1 also localized to autophagosomes, and its activity was required for autophagosome formation, clearance of protein aggregates and peroxisomes, and autophagy of Salmonella. Overexpression of Rab1 enhanced antibacterial autophagy. The role of Rab1 in antibacterial autophagy was independent of its role in ER-to-Golgi transport. Our data suggest that antibacterial autophagy occurs at omegasomes and reveal that the Rab1 GTPase plays a crucial role in mammalian autophagy.

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Autophagy, 7, 1, , 2011


Open Access

What is the advantage of a transient precursor in autophagosome biogenesis?
NT Ktistakis, S Andrews, J Long

We have recently proposed that some autophagosomes are formed within omegasomes, membrane sites connected to the endoplasmic reticulum and enriched in phosphatidylinositol 3-phosphate. In order to understand if there is any biological advantage to having such a precursor in autophagosome biogenesis, we generated a simple computer program that simulates omegasome and autophagosome formation under a variety of conditions. We concluded from running this simulation that having a transient precursor permits a bigger dynamic range of the autophagic response and allows a more efficient approach to steady state after autophagy stimulation.

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Autophagy, 7, 1, , 2011

DOI: 10.1083/jcb.200803137

Open Access

Autophagosome formation in mammalian cells.
C Burman, NT Ktistakis

Autophagy is a fundamental intracellular trafficking pathway conserved from yeast to mammals. It is generally thought to play a pro-survival role, and it can be up regulated in response to both external and intracellular factors, including amino acid starvation, growth factor withdrawal, low cellular energy levels, endoplasmic reticulum (ER) stress, hypoxia, oxidative stress, pathogen infection, and organelle damage. During autophagy initiation a portion of the cytosol is surrounded by a flat membrane sheet known as the isolation membrane or phagophore. The isolation membrane then elongates and seals itself to form an autophagosome. The autophagosome fuses with normal endocytic traffic to mature into a late autophagosome, before fusing with lysosomes. The molecular machinery that enables formation of an autophagosome in response to the various autophagy stimuli is almost completely identified in yeast and-thanks to the observed conservation-is also being rapidly elucidated in higher eukaryotes including mammals. What are less clear and currently under intense investigation are the mechanism by which these various autophagy components co-ordinate in order to generate autophagosomes. In this review, we will discuss briefly the fundamental importance of autophagy in various pathophysiological states and we will then review in detail the various players in early autophagy. Our main thesis will be that a conserved group of heteromeric protein complexes and a relatively simple signalling lipid are responsible for the formation of autophagosomes in mammalian cells.

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Seminars in immunopathology, 32, 4, , 2010

DOI: 10.1007/s00281-010-0222-z

Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L.
K Matsunaga, E Morita, T Saitoh, S Akira, NT Ktistakis, T Izumi, T Noda, T Yoshimori

Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.

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The Journal of cell biology, 190, 4, , 2010

DOI: 10.1083/jcb.200911141

Open Access

Regulation of autophagy by phosphatidylinositol 3-phosphate.
C Burman, NT Ktistakis

The simple phosphoinositide phosphatidylinositol 3-phosphate (PI(3)P) has been known to have important functions in endocytic and phagocytic traffic, and to be required for the autophagic pathway. In all of these settings, PI(3)P appears to create platforms that serve to recruit specific effectors for membrane trafficking events. In autophagy, PI(3)P may form the platform for autophagosome biogenesis.

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FEBS letters, 584, 7, , 2010

DOI: 10.1016/j.febslet.2010.01.011

Open Access

Modulation of local PtdIns3P levels by the PI phosphatase MTMR3 regulates constitutive autophagy.
N Taguchi-Atarashi, M Hamasaki, K Matsunaga, H Omori, NT Ktistakis, T Yoshimori, T Noda

Autophagy is a catabolic process that delivers cytoplasmic material to the lysosome for degradation. The mechanisms regulating autophagosome formation and size remain unclear. Here, we show that autophagosome formation was triggered by the overexpression of a dominant-negative inactive mutant of Myotubularin-related phosphatase 3 (MTMR3). Mutant MTMR3 partially localized to autophagosomes, and PtdIns3P and two autophagy-related PtdIns3P-binding proteins, GFP-DFCP1 and GFP-WIPI-1alpha (WIPI49/Atg18), accumulated at sites of autophagosome formation. Knock-down of MTMR3 increased autophagosome formation, and overexpression of wild-type MTMR3 led to significantly smaller nascent autophagosomes and a net reduction in autophagic activity. These results indicate that autophagy initiation depends on the balance between PI 3-kinase and PI 3-phosphatase activity. Local levels of PtdIns3P at the site of autophagosome formation determine autophagy initiation and the size of the autophagosome membrane structure.

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Traffic (Copenhagen, Denmark), 11, 4, , 2010

DOI: 10.1111/j.1600-0854.2010.01034.x

Synthesis and biological evaluation of phosphatidylinositol phosphate affinity probes.
SJ Conway, J Gardiner, SJ Grove, MK Johns, ZY Lim, GF Painter, DE Robinson, C Schieber, JW Thuring, LS Wong, MX Yin, AW Burgess, B Catimel, PT Hawkins, NT Ktistakis, LR Stephens, AB Holmes

The synthesis of the complete family of phosphatidylinositol phosphate analogues (PIPs) from five key core intermediates A-E is described. These core compounds were obtained from myo-inositol orthoformate 1 via regioselective DIBAL-H and trimethylaluminium-mediated cleavages and a resolution-protection process using camphor acetals 10. Coupling of cores A-E with phosphoramidites 34 and 38, derived from the requisite protected lipid side chains, afforded the fully-protected PIPs. Removal of the remaining protecting groups was achieved via hydrogenolysis using palladium black or palladium hydroxide on carbon in the presence of sodium bicarbonate to afford the complete family of dipalmitoyl- and amino-PIP analogues 42, 45, 50, 51, 58, 59, 67, 68, 76, 77, 82, 83, 92, 93, 99 and 100. Investigations using affinity probes incorporating these compounds have identified novel proteins involved in the PI3K intracellular signalling network and have allowed a comprehensive proteomic analysis of phosphoinositide interacting proteins.

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Organic & biomolecular chemistry, 8, 1, , 2010

DOI: 10.1039/b913399b

Galpha(q)-mediated plasma membrane translocation of sphingosine kinase-1 and cross-activation of S1P receptors.
M ter Braak, K Danneberg, K Lichte, K Liphardt, NT Ktistakis, SM Pitson, T Hla, KH Jakobs, D Meyer zu Heringdorf

Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of Gq-coupled receptors induced a profound, rapid (half-life 3-5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein alpha-subunits specifically of the Gq family. Classical Gq signalling pathways, or phosphorylation at Ser225, phospholipase D and Ca2+/calmodulin were not involved in M3 receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of Gq-coupled receptors, linking them via cross-activation of S1P receptors to G(i) and G12/13 signalling pathways.

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Biochimica et biophysica acta, 1791, 5, , 2009


Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels.
P Raghu, E Coessens, M Manifava, P Georgiev, T Pettitt, E Wood, I Garcia-Murillas, H Okkenhaug, D Trivedi, Q Zhang, A Razzaq, O Zaid, M Wakelam, CJ O'Kane, N Ktistakis

Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. In this study, we show that before a critical time period during rhabdomere biogenesis in Drosophila melanogaster photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. We propose that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis.

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The Journal of cell biology, 185, 1, , 2009

DOI: 10.1083/jcb.200807027

Open Access

Emerging findings from studies of phospholipase D in model organisms (and a short update on phosphatidic acid effectors).
P Raghu, M Manifava, J Coadwell, NT Ktistakis

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate phosphatidic acid and choline. Historically, much PLD work has been conducted in mammalian settings although genes encoding enzymes of this family have been identified in all eukaryotic organisms. Recently, important insights on PLD function are emerging from work in yeast, but much less is known about PLD in other organisms. In this review we will summarize what is known about phospholipase D in several model organisms, including C. elegans, D. discoideum, D. rerio and D. melanogaster. In the cases where knockouts are available (C. elegans, Dictyostelium and Drosophila) the PLD gene(s) appear not to be essential for viability, but several studies are beginning to identify pathways where this activity has a role. Given that the proteins in model organisms are very similar to their mammalian counterparts, we expect that future studies in model organisms will complement and extend ongoing work in mammalian settings. At the end of this review we will also provide a short update on phosphatidic acid targets, a topic last reviewed in 2006.

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Biochimica et biophysica acta, 1791, 9, , 2009

DOI: 10.1016/j.bbalip.2009.03.013

The sphingosine 1-phosphate receptor 5 and sphingosine kinases 1 and 2 are localised in centrosomes: possible role in regulating cell division.
L Gillies, SC Lee, JS Long, N Ktistakis, NJ Pyne, S Pyne

We show here that the endogenous sphingosine 1-phosphate 5 receptor (S1P(5), a G protein coupled receptor (GPCR) whose natural ligand is sphingosine 1-phosphate (S1P)) and sphingosine kinases 1 and 2 (SK1 and SK2), which catalyse formation of S1P, are co-localised in the centrosome of mammalian cells, where they may participate in regulating mitosis. The centrosome is a site for active GTP-GDP cycling involving the G-protein, G(i) and tubulin, which are required for spindle pole organization and force generation during cell division. Therefore, the presence of S1P(5) (which normally functions as a plasma membrane guanine nucleotide exchange factor, GEF) and sphingosine kinases in the centrosome might suggest that S1P(5) may function as a ligand activated GEF in regulating G-protein-dependent spindle formation and mitosis. The addition of S1P to cells inhibits trafficking of S1P(5) to the centrosome, suggesting a dynamic shuttling endocytic mechanism controlled by ligand occupancy of cell surface receptor. We therefore propose that the centrosomal S1P(5) receptor might function as an intracellular target of S1P linked to regulation of mitosis.

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Cellular signalling, 21, 5, , 2009

DOI: 10.1016/j.cellsig.2009.01.023

PA binding of phosphatidylinositol 4-phosphate 5-kinase.
C Stace, M Manifava, C Delon, J Coadwell, S Cockcroft, NT Ktistakis

Advances in enzyme regulation, 48, , , 2008

DOI: 10.1016/j.advenzreg.2007.11.008

Phosphatidic acid- and phosphatidylserine-binding proteins.
CL Stace, NT Ktistakis

Phosphatidic acid and phosphatidylserine are negatively charged abundant phospholipids with well-recognized structural roles in cellular membranes. They are also signaling lipids since their regulated formation (or appearance) can constitute an important signal for downstream responses. The list of potential effectors for these lipids is expanding rapidly and includes proteins involved in virtually all aspects of cellular regulation. Because it is not always clear whether these effectors recognize the specific phospholipids or a general negatively-charged membrane environment, questions about specificity must be addressed on a case by case basis. In this review we present an up to date list of potential phosphatidic acid- and phosphatidylserine-binding proteins.

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Biochimica et biophysica acta, 1761, 8, , 2006

DOI: 10.1016/j.bbalip.2006.03.006

Sphingosine kinase 1 is an intracellular effector of phosphatidic acid.
C Delon, M Manifava, E Wood, D Thompson, S Krugmann, S Pyne, NT Ktistakis

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to generate sphingosine 1-phosphate (S1P). Because both substrate and product of the enzyme are potentially important signaling molecules, the regulation of SK1 is of considerable interest. We report that SK1, which is ordinarily a cytosolic enzyme, translocates in vivo and in vitro to membrane compartments enriched in phosphatidic acid (PA), the lipid product of phospholipase D. This translocation depends on direct interaction of SK1 with PA, because recombinant purified enzyme shows strong affinity for pure PA coupled to Affi-Gel. The SK1-PA interaction maps to the C terminus of SK1 and is independent of catalytic activity or of the diacylglycerol kinase-like domain of the enzyme. Thus SK1 constitutes a novel, physiologically relevant PA effector.

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The Journal of biological chemistry, 279, 43, , 2004

DOI: 10.1074/jbc.M405771200

Open Access