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How and why to study autophagy in Drosophila: it's more than just a garbage chute.

Nagy P, Varga Á, Kovács AL, Takáts S, Juhász G - Methods (2014)

Bottom Line: This way, autophagy contributes to the homeodynamic turnover of proteins, lipids, nucleic acids, glycogen, and even whole organelles.Autophagic activity is increased by adverse conditions such as nutrient limitation, growth factor withdrawal and oxidative stress, and it generally protects cells and organisms to promote their survival.Here we discuss the different microscopy-based, biochemical and genetic methods currently available to study autophagy in various tissues of the popular model Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Pázmány s. 1/C. 6.520, Budapest H-1117, Hungary.

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Related in: MedlinePlus

Ultrastructural analysis of autophagy. (A) Transmission electron microscopy image of fat body from a starved larva. Autophagosomes (ap) bordered by a double membrane (with the often seen characteristic cleft between them) contain undegraded cargo, which appears largely similar to the surrounding cytoplasm. In contrast, the content of the single-membrane bound autolysosome (al) is typically dark and condensed due to ongoing breakdown, although remnants of cytoplasmic material can often be recognized. (B) Autophagic structures are practically never seen in ultrastructural images of wild type adult brains. (C) Double-membrane autophagosomes accumulate in large numbers in neurons of Syntaxin 17 mutant adult flies. (D) Apoptotic bodies (apopt) induced by clonal overexpression of Hid are engulfed by neighboring eye imaginal disc cells, and contain cytoplasmic remnants of the fragmented dying cell, but these are not of autophagic origin. Their recognition is facilitated by the presence of the condensed nucleus in some of the engulfed cell fragments, and apoptotic bodies may be found outside of healthy cells as well, as illustrated by the one situated between two neighboring cells in the bottom right corner of this panel. Note that apoptotic cells usually appear darker than healthy cells in ultrastructural images even before being engulfed, due to ongoing protein degradation by caspases and acidification of the cytoplasm. (E) Large protein aggregates form in neurons of Atg mutant adult flies. The aggregate (arrowhead) can be easily recognized by its homogenous appearance, as they mostly exclude cytoplasmic structures, for example vesicles and ribosomes. This image shows a single protein aggregate with a rarely seen phagophore (p) attached to its surface in an Atg2 mutant brain. (F) Protein aggregates (arrowheads) accumulating in Atg mutant neurons contain ubiquitinated proteins and their selective autophagic receptor p62 (also known as Ref(2)P in flies). This image shows immunogold labeling (black dots) of p62 in Atg8a mutant neurons. Abbreviations: ap, autophagosome; apopt, apoptotic cell fragment; al, autolysosome; p, phagophore; m, mitochondrion. Bars equal 1 μm in all panels.
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f0010: Ultrastructural analysis of autophagy. (A) Transmission electron microscopy image of fat body from a starved larva. Autophagosomes (ap) bordered by a double membrane (with the often seen characteristic cleft between them) contain undegraded cargo, which appears largely similar to the surrounding cytoplasm. In contrast, the content of the single-membrane bound autolysosome (al) is typically dark and condensed due to ongoing breakdown, although remnants of cytoplasmic material can often be recognized. (B) Autophagic structures are practically never seen in ultrastructural images of wild type adult brains. (C) Double-membrane autophagosomes accumulate in large numbers in neurons of Syntaxin 17 mutant adult flies. (D) Apoptotic bodies (apopt) induced by clonal overexpression of Hid are engulfed by neighboring eye imaginal disc cells, and contain cytoplasmic remnants of the fragmented dying cell, but these are not of autophagic origin. Their recognition is facilitated by the presence of the condensed nucleus in some of the engulfed cell fragments, and apoptotic bodies may be found outside of healthy cells as well, as illustrated by the one situated between two neighboring cells in the bottom right corner of this panel. Note that apoptotic cells usually appear darker than healthy cells in ultrastructural images even before being engulfed, due to ongoing protein degradation by caspases and acidification of the cytoplasm. (E) Large protein aggregates form in neurons of Atg mutant adult flies. The aggregate (arrowhead) can be easily recognized by its homogenous appearance, as they mostly exclude cytoplasmic structures, for example vesicles and ribosomes. This image shows a single protein aggregate with a rarely seen phagophore (p) attached to its surface in an Atg2 mutant brain. (F) Protein aggregates (arrowheads) accumulating in Atg mutant neurons contain ubiquitinated proteins and their selective autophagic receptor p62 (also known as Ref(2)P in flies). This image shows immunogold labeling (black dots) of p62 in Atg8a mutant neurons. Abbreviations: ap, autophagosome; apopt, apoptotic cell fragment; al, autolysosome; p, phagophore; m, mitochondrion. Bars equal 1 μm in all panels.

Mentions: Autophagic structures have certain unique features that make it possible to reliably recognize them. Depending on the sample preparation technique and type of fixation used, the closely apposed membranes of both phagophore cisterns and autophagosomes may open up, and thus a characteristic cleft appears between the two membrane sheets (as seen in Fig. 2A,C and E; note that such vesicles are very rarely observed in neurons of wild type adult flies, shown in Fig. 2B). This phenomenon is often seen in samples prepared by chemical fixation of cells and tissues by a glutaraldehyde-containing isoosmotic solution, which is followed by dehydration of fixed samples and embedding into a resin [6,10,13,21–23]. Intracellular structures likely undergo a variable extent of shrinkage during fixation and embedding according to local conditions in the surrounding cytoplasm, which is why this cleft may be seen only in a subset of autophagic structures. The appearance of this cleft is unlikely to be specific for different cell types or organisms, as it has been documented elsewhere as well [9,24]. The membranes of these early autophagic structures are of the thin type, based on which autophagosomes can be distinguished from interdigitations observed between neighboring cells in some tissues, as the membrane of these are of the thick type (plasma membrane). Autophagosomes contain undigested material, the morphology of which appears very similar to the surrounding cytoplasm (Fig. 2A and C). In contrast, the content of autolysosomes is mostly heterogeneous, as it shows morphological signs of ongoing degradation ranging from recognizable to finally unidentifiable cytoplasmic material like mitochondria, RER, ribosomes and so on. The heterogeneous morphology can be the result of multiple subsequent fusion events resulting in a multifocal appearance. Both primary lysosomes and actively digesting autolysosomes and endolysosomes contain acid phosphatase, which can be detected in the electron microscope using a classical, very simple enzymatic reaction [25,26]. In addition, immunogold labeling for lysosomal proteins using antibodies (or tagged reporters and anti-tag antibodies) to cathepsin proteases or lysosomal membrane proteins can also be used to identify lysosomes in ultrastructural studies [26].


How and why to study autophagy in Drosophila: it's more than just a garbage chute.

Nagy P, Varga Á, Kovács AL, Takáts S, Juhász G - Methods (2014)

Ultrastructural analysis of autophagy. (A) Transmission electron microscopy image of fat body from a starved larva. Autophagosomes (ap) bordered by a double membrane (with the often seen characteristic cleft between them) contain undegraded cargo, which appears largely similar to the surrounding cytoplasm. In contrast, the content of the single-membrane bound autolysosome (al) is typically dark and condensed due to ongoing breakdown, although remnants of cytoplasmic material can often be recognized. (B) Autophagic structures are practically never seen in ultrastructural images of wild type adult brains. (C) Double-membrane autophagosomes accumulate in large numbers in neurons of Syntaxin 17 mutant adult flies. (D) Apoptotic bodies (apopt) induced by clonal overexpression of Hid are engulfed by neighboring eye imaginal disc cells, and contain cytoplasmic remnants of the fragmented dying cell, but these are not of autophagic origin. Their recognition is facilitated by the presence of the condensed nucleus in some of the engulfed cell fragments, and apoptotic bodies may be found outside of healthy cells as well, as illustrated by the one situated between two neighboring cells in the bottom right corner of this panel. Note that apoptotic cells usually appear darker than healthy cells in ultrastructural images even before being engulfed, due to ongoing protein degradation by caspases and acidification of the cytoplasm. (E) Large protein aggregates form in neurons of Atg mutant adult flies. The aggregate (arrowhead) can be easily recognized by its homogenous appearance, as they mostly exclude cytoplasmic structures, for example vesicles and ribosomes. This image shows a single protein aggregate with a rarely seen phagophore (p) attached to its surface in an Atg2 mutant brain. (F) Protein aggregates (arrowheads) accumulating in Atg mutant neurons contain ubiquitinated proteins and their selective autophagic receptor p62 (also known as Ref(2)P in flies). This image shows immunogold labeling (black dots) of p62 in Atg8a mutant neurons. Abbreviations: ap, autophagosome; apopt, apoptotic cell fragment; al, autolysosome; p, phagophore; m, mitochondrion. Bars equal 1 μm in all panels.
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Related In: Results  -  Collection

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Show All Figures
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f0010: Ultrastructural analysis of autophagy. (A) Transmission electron microscopy image of fat body from a starved larva. Autophagosomes (ap) bordered by a double membrane (with the often seen characteristic cleft between them) contain undegraded cargo, which appears largely similar to the surrounding cytoplasm. In contrast, the content of the single-membrane bound autolysosome (al) is typically dark and condensed due to ongoing breakdown, although remnants of cytoplasmic material can often be recognized. (B) Autophagic structures are practically never seen in ultrastructural images of wild type adult brains. (C) Double-membrane autophagosomes accumulate in large numbers in neurons of Syntaxin 17 mutant adult flies. (D) Apoptotic bodies (apopt) induced by clonal overexpression of Hid are engulfed by neighboring eye imaginal disc cells, and contain cytoplasmic remnants of the fragmented dying cell, but these are not of autophagic origin. Their recognition is facilitated by the presence of the condensed nucleus in some of the engulfed cell fragments, and apoptotic bodies may be found outside of healthy cells as well, as illustrated by the one situated between two neighboring cells in the bottom right corner of this panel. Note that apoptotic cells usually appear darker than healthy cells in ultrastructural images even before being engulfed, due to ongoing protein degradation by caspases and acidification of the cytoplasm. (E) Large protein aggregates form in neurons of Atg mutant adult flies. The aggregate (arrowhead) can be easily recognized by its homogenous appearance, as they mostly exclude cytoplasmic structures, for example vesicles and ribosomes. This image shows a single protein aggregate with a rarely seen phagophore (p) attached to its surface in an Atg2 mutant brain. (F) Protein aggregates (arrowheads) accumulating in Atg mutant neurons contain ubiquitinated proteins and their selective autophagic receptor p62 (also known as Ref(2)P in flies). This image shows immunogold labeling (black dots) of p62 in Atg8a mutant neurons. Abbreviations: ap, autophagosome; apopt, apoptotic cell fragment; al, autolysosome; p, phagophore; m, mitochondrion. Bars equal 1 μm in all panels.
Mentions: Autophagic structures have certain unique features that make it possible to reliably recognize them. Depending on the sample preparation technique and type of fixation used, the closely apposed membranes of both phagophore cisterns and autophagosomes may open up, and thus a characteristic cleft appears between the two membrane sheets (as seen in Fig. 2A,C and E; note that such vesicles are very rarely observed in neurons of wild type adult flies, shown in Fig. 2B). This phenomenon is often seen in samples prepared by chemical fixation of cells and tissues by a glutaraldehyde-containing isoosmotic solution, which is followed by dehydration of fixed samples and embedding into a resin [6,10,13,21–23]. Intracellular structures likely undergo a variable extent of shrinkage during fixation and embedding according to local conditions in the surrounding cytoplasm, which is why this cleft may be seen only in a subset of autophagic structures. The appearance of this cleft is unlikely to be specific for different cell types or organisms, as it has been documented elsewhere as well [9,24]. The membranes of these early autophagic structures are of the thin type, based on which autophagosomes can be distinguished from interdigitations observed between neighboring cells in some tissues, as the membrane of these are of the thick type (plasma membrane). Autophagosomes contain undigested material, the morphology of which appears very similar to the surrounding cytoplasm (Fig. 2A and C). In contrast, the content of autolysosomes is mostly heterogeneous, as it shows morphological signs of ongoing degradation ranging from recognizable to finally unidentifiable cytoplasmic material like mitochondria, RER, ribosomes and so on. The heterogeneous morphology can be the result of multiple subsequent fusion events resulting in a multifocal appearance. Both primary lysosomes and actively digesting autolysosomes and endolysosomes contain acid phosphatase, which can be detected in the electron microscope using a classical, very simple enzymatic reaction [25,26]. In addition, immunogold labeling for lysosomal proteins using antibodies (or tagged reporters and anti-tag antibodies) to cathepsin proteases or lysosomal membrane proteins can also be used to identify lysosomes in ultrastructural studies [26].

Bottom Line: This way, autophagy contributes to the homeodynamic turnover of proteins, lipids, nucleic acids, glycogen, and even whole organelles.Autophagic activity is increased by adverse conditions such as nutrient limitation, growth factor withdrawal and oxidative stress, and it generally protects cells and organisms to promote their survival.Here we discuss the different microscopy-based, biochemical and genetic methods currently available to study autophagy in various tissues of the popular model Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Pázmány s. 1/C. 6.520, Budapest H-1117, Hungary.

Show MeSH
Related in: MedlinePlus