Electron tomography reveals the endoplasmic reticulum as a membrane source for autophagosome formation

Hayashi-Nishino, Mitsuko; Fujita, Naonobu; Noda, Tetsuji; Yamaguchi, Akihito; Yoshimori, Tamotsu; Yamamoto, Akira

doi:10.4161/auto.6.2.11134

Cited by 72 publications

(49 citation statements)

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“…Interactions between omegasomes, isolation membranes and the ER are well documented in mammalian cells 8,24,25,43 . Notably, the continuity of the isolation membrane with the ER led to a model implying direct differentiation of the phagophore from this compartment, and the systematic inclusion of ER fragments in the autophagosome, captured by folding and invagination of the isolation membrane, sandwiched between cradle-shaped ER sheets 25 .…”

Section: Discussionmentioning

confidence: 99%

ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants

Bars

Marion

Borgne³

et al. 2014

Nat Commun

125

103

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Autophagosomes are the organelles responsible for macroautophagy and arise, in yeast and animals, from the sealing of a cup-shaped double-membrane precursor, the phagophore. How the phagophore is generated and grows into a sealed autophagosome is still not clear in detail, and unknown in plants. This is due, in part, to the scarcity of structurally informative, real-time imaging data of the required protein machinery at the phagophore formation site. Here we find that in intact living Arabidopsis tissue, autophagy-related protein ATG5, which is essential for autophagosome formation, is present at the phagophore site from early, sub-resolution stages and later defines a torus-shaped structure on a flat cisternal early phagophore. Movement and expansion of this structure are accompanied by the underlying endoplasmic reticulum, suggesting tight connections between the two compartments. Detailed real-time and 3D imaging of the growing phagophore are leveraged to propose a model for autophagosome formation in plants.

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Section: Discussionmentioning

confidence: 99%

ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants

Bars

Marion

Borgne³

et al. 2014

Nat Commun

125

103

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“…Membranes derived from the Golgi, endoplasmic reticulum, mitochondria, and plasma membrane have been implicated as the source membranes. [20][21][22][23][24][25] It is possible that vesicles from multiple membrane sources may fuse heterotypically to generate APs. This early fusion steps may involve lipidhydrolyzing enzymes, possibly PLD.…”

Section: Discussionmentioning

confidence: 99%

Phospholipase D1 regulates autophagic flux and clearance of α-synuclein aggregates

et al. 2014

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Many neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, are characterized by abnormal accumulations of aggregated proteins. Brains in these diseases also show accumulation of autophagic vesicles in the neuronal cytoplasm, suggesting impairment of the autophagic process. As autophagy involves de novo membrane production and vesicle fusion, extensive changes in lipid molecules are necessary. However, the involvement of signaling lipid-modifying enzymes in autophagy and their roles in neurodegenerative diseases are not clear. Using specific inhibitor, we show that loss of phospholipase D1 (PLD1) activity resulted in an accumulation of microtubule-associated protein light chain 3 (LC3), p62, and polyubiquitinated proteins, signs representing malfunction in autophagic flux. Fluorescence and electron microscopic analyses demonstrated impaired fusion of autophagosomes with lysosomes, resulting in accumulation of autophagosomes. Within the cells with impaired autophagic flux, a-synuclein aggregates accumulated in autophagosomes. Knockdown of PLD1 expression using small interfering RNA also resulted in impaired autophagic flux and accumulation of a-synuclein aggregates in autophagosomes. Neuronal toxicity caused by a-synuclein accumulation was rescued by overexpression of PLD1; however, expression of activity-deficient mutant, PLD1-KRM, showed reduced rescue effects. Finally, we demonstrated that both PLD activity and expression levels were reduced in brain tissues of dementia with Lewy bodies (DLB) patients, whereas the amounts of a-synuclein and p62 were increased in the same tissue samples. Collectively, these results suggest that insufficient PLD activity, and therefore, the changes in phospholipid compositions within membranes, might be an important contributor to impaired autophagic process and protein accumulation in Lewy body diseases.

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“…100 Combinatorial assays using tandem mRFP-GFP-LC3 (see Tandem mRFP/ mCherry-GFP fluorescence microscopy) along with static TEM images should help in the analysis of flux and the visualization of cargo structures. 101 Another technique that has proven quite useful for analyzing the complex membrane structures that participate in autophagy is three-dimensional electron tomography, 102,103 and cryoelectron microscopy (Fig. 4).…”

Section: Introductionmentioning

confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are 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 monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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Electron tomography reveals the endoplasmic reticulum as a membrane source for autophagosome formation

Cited by 72 publications

References 0 publications

ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants

ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants

Phospholipase D1 regulates autophagic flux and clearance of α-synuclein aggregates

Guidelines for the use and interpretation of assays for monitoring autophagy

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