Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM)

Duke, Elizabeth; Razi, Minoo; Weston, Anne; Guttmann, Peter; Werner, Stephan; Henzler, Katja; Schneider, Gerd; Tooze, Sharon A.; Collinson, Lucy M.

doi:10.1016/j.ultramic.2013.10.006

Cited by 113 publications

(104 citation statements)

References 54 publications

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“…Electron tomography examinations of the omegasome ultrastructure have revealed that the extremities of the expanding phagophore are indeed physically connected with the ER, supporting this idea (Hayashi-Nishino et al, 2009;Ylä-Anttila et al, 2009). In addition, two more recent studies that combined correlative light and electron microscopy have shown that omegasomes arise from hotspots in the ER and connect to it through thin tubular membranes (Duke et al, 2014;Uemura et al, 2014). The association between these two organelles could be essential for the efficient supply of the enormous quantities of lipids that are required to form autophagosomes.…”

Section: Model II -Eres As a Scaffold For Autophagosome Biogenesismentioning

confidence: 99%

ERES: sites for autophagosome biogenesis and maturation?

Sánchez

Ktistakis

Reggiori

2015

Journal of Cell Science

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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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Section: Model II -Eres As a Scaffold For Autophagosome Biogenesismentioning

confidence: 99%

ERES: sites for autophagosome biogenesis and maturation?

Sánchez

Ktistakis

Reggiori

2015

Journal of Cell Science

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“…Most work performed on SXT microscopes addresses samples a few microns thick [4,[12][13][14][15][16][17][18]. Depending on the ratio between the sample thickness and the DOF, standard reconstruction algorithms introduce different artifacts which can be better estimated once the experimental DOF is known [19].…”

Section: Introductionmentioning

confidence: 99%

Characterization of transfer function, resolution and depth of field of a soft X-ray microscope applied to tomography enhancement by Wiener deconvolution

Otón

Pereiro

Pérez-Berná

et al. 2016

Biomed. Opt. Express

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Full field soft X-ray microscopy is becoming a powerful imaging technique to analyze whole cells preserved under cryo conditions. Images obtained in these X-ray microscopes can be combined by tomographic reconstruction to quantitatively estimate the three-dimensional (3D) distribution of absorption coefficients inside the cell. The impulse response of an imaging system is one of the factors that limits the quality of the X-ray microscope reconstructions. The main goal of this work is to experimentally measure the 3D impulse response and to assess the optical resolution and depth of field of the Mistral microscope at ALBA synchrotron (Barcelona, Spain). To this end we measure the microscope apparent transfer function (ATF) and we use it to design a deblurring Wiener filter, obtaining an increase in the image quality when applied to experimental datasets collected at ALBA. References and links1. G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. B. Heymann, W. G. Muller, J. G.McNally, and W. G. Müller, "Three-dimensional cellular ultrastructure resolved by X-ray microscopy," Nat. Methods 7, 985-987 (2010). 2. J. Kirz, C. Jacobsen, and M. Howells, "Soft X-ray microscopes and their biological applications," Q. Rev. Biophys. 28, 33-130 (1995). Collinson, "Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM)," Ultramicroscopy 143, 77-87 (2014). 14. C. Hagen, S. Werner, and S. Carregal-Romero, "Multimodal nanoparticles as alignment and correlation markers in fluorescence/soft X-ray cryo-microscopy/tomography of nucleoplasmic reticulum and apoptosis in mammalian cells," Ultramicroscopy

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“…In contrast, mammalian ATG9 is not stably incorporated into the isolation membrane or autophagosomes but is instead transiently associated with the omegasome, a phosphatidylinositol 3-phosphate (PI3P)-enriched endoplasmic reticulum (ER) subdomain (5). Cryomicroscopy studies have shown a close association between ATG9 vesicles and the omegasome structure (7), together with the presence of ATG9 on tubulovesicular membranes surrounding autophagosomes (5). A recent finding by livecell imaging indicates that autophagosome formation occurs where ATG9 vesicles coalesce with the ER (8).…”

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confidence: 99%

ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis

Zhuang

Chung

Cui

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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162

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Autophagy is a conserved pathway for bulk degradation of cytoplasmic material by a double-membrane structure named the autophagosome. The initiation of autophagosome formation requires the recruitment of autophagy-related protein 9 (ATG9) vesicles to the preautophagosomal structure. However, the functional relationship between ATG9 vesicles and the phagophore is controversial in different systems, and the molecular function of ATG9 remains unknown in plants. Here, we demonstrate that ATG9 is essential for endoplasmic reticulum (ER)-derived autophagosome formation in plants. Through a combination of genetic, in vivo imaging and electron tomography approaches, we show that Arabidopsis ATG9 deficiency leads to a drastic accumulation of autophagosome-related tubular structures in direct membrane continuity with the ER upon autophagic induction. Dynamic analyses demonstrate a transient membrane association between ATG9 vesicles and the autophagosomal membrane during autophagy. Furthermore, trafficking of ATG18a is compromised in atg9 mutants during autophagy by forming extended tubules in a phosphatidylinositol 3-phosphatedependent manner. Taken together, this study provides evidence for a pivotal role of ATG9 in regulating autophagosome progression from the ER membrane in Arabidopsis.O ne long-lasting question regarding autophagosome biogenesis is its membrane origin (1). The initiation site for autophagosomes is termed the preautophagosomal structure or phagophore assembly site (PAS). However, the source of the phagophore membrane remains controversial in different systems, and exactly how the phagophore is initiated from its membrane origin is still unclear. The core autophagy-related (ATG) machinery regulates phagophore assembly in a spatiotemporally coordinated manner whereas some of the ATG components will disassociate from the completed autophagosome and some are turned over together with the autophagosome (1-3).As the sole transmembrane protein, autophagy-related protein 9 (ATG9) has long been suggested to provide a lipid/membrane source for autophagosome formation because ATG9-deficient mutants in yeast or mammal fail to form autophagosomes (4, 5). Although ATG9 is conserved in all eukaryotes (6), it seems that ATG9 might perform its function divergently in different systems. In yeast, ATG9 participates in an early step by shuttling from a non-PAS site to the PAS site and supports an assembly model for yeast autophagosome biogenesis (4). In contrast, mammalian ATG9 is not stably incorporated into the isolation membrane or autophagosomes but is instead transiently associated with the omegasome, a phosphatidylinositol 3-phosphate (PI3P)-enriched endoplasmic reticulum (ER) subdomain (5). Cryomicroscopy studies have shown a close association between ATG9 vesicles and the omegasome structure (7), together with the presence of ATG9 on tubulovesicular membranes surrounding autophagosomes (5). A recent finding by livecell imaging indicates that autophagosome formation occurs where ATG9 vesicles coalesce with the ER ...

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Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM)

Cited by 113 publications

References 54 publications

ERES: sites for autophagosome biogenesis and maturation?

ERES: sites for autophagosome biogenesis and maturation?

Characterization of transfer function, resolution and depth of field of a soft X-ray microscope applied to tomography enhancement by Wiener deconvolution

ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis

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