Short and long sleeping mutants reveal links between sleep and macroautophagy
- Chronobiology and Sleep Institute, Perelman Medical School of University of Pennsylvania, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1 with 1 supplement
Sleep phenotype of argus mutants.
All sleep metrics were measured under a 12 hr:12 hr light:dark cycle in female iso31 (gray), aus/+ (pink) and aus/aus (red) flies. (A) Mean activity (top panel) and sleep (bottom panel) over time …
-
Figure 1—source data 1
Sleep Phenotype of Argus Mutants.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Circadian rhythms are intact in the aus mutant.
(A) Sample actograms from iso31 control and aus mutant flies. (B) Activity data from flies assayed during constant darkness were assessed for circadian rhythmicity. Average circadian period length …
-
Figure 1—figure supplement 1—source data 1
Circadian rhythms are intact in the aus mutant.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig2-data2-v1.xlsx
Figure 2 with 2 supplements
Mapping the argus sleep phenotype to a single gene: cg16791.
(A) The genomic location of argus is indicated as a star within a 5 Mb region on the right arm of the third chromosome, following genetic mapping with visible mutations and SNP markers. (B) …
-
Figure 2—source data 1
Mapping the argus sleep phenotype to a single gene: cg16791.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
A mutation in Nrx1 does not underlie the aus reduced sleep phenotype.
(A) Total sleep in nrx/nrx mutants is comparable to iso31 control and greater than aus/aus mutants. n = 12–16; individual brain values overlaid with population median±interquartiles; Tukey test. (B) …
-
Figure 2—figure supplement 1—source data 1
A mutation in Nrx1 does not underlie the aus reduced sleep phenotype.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig4-data4-v1.xlsx
Figure 2—figure supplement 2
Knockdown of aus in adult neurons via RNAi reduces sleep.
(A–B) Pan-neuronal knockdown of cg16791 with elav-Gal4> dcr,cg16791-RNAi#1 reduced total sleep length in both female (A) and male (B) flies. n = 10–13 (females) and 10–12 (males); individual fly …
-
Figure 2—figure supplement 2—source data 1
Knockdown of aus in adult neurons via RNAi reduces sleep.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig5-data5-v1.xlsx
Figure 3 with 1 supplement
CG16791 underlies the argus sleep phenotype.
(A) Transheterozygotes of male aus and cg16791 insertional mutant (P1) have reduced total sleep compared to aus/+ and cg16791-P1/+ controls. n = 6–13; individual flies overlaid with …
-
Figure 3—source data 1
CG16791 underlies the argus sleep phenotype.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
CRISPR-targeting of argus to generate a null mutant; supplemental Crispr-KO and full-length rescue data.
(A) CRISPR-targeting of argus exon1 to replace it with a selectable marker, Dsred. (B) Southern blot analysis of CG16791 (KO) using part of the Dsred gene as DIGI-probe. A single ~3 kb DIGI-positive …
-
Figure 3—figure supplement 1—source data 1
CRISPR-targeting of argus to generate a null mutant; supplemental Crispr-KO and full-length rescue data.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig7-data7-v1.xlsx
Figure 4
Argus functions in dimmed positive neurons to regulate sleep.
(A) The aus promoter region was subcloned, and a ~ 2000 bp sequence was inserted upstream of Gal4 and used to drive GFP (left). aus2kGal4 driving uas-cg16791 partially rescues the short sleep …
-
Figure 4—source data 1
Argus Functions in Dimmed Positive Neurons to Regulate Sleep.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig4-data1-v1.xlsx
Figure 5
The argus mutant displays accumulation of autophagosomes.
Female iso31 control and aus/aus brains with elav-Gal4> UAS-GFP-mCherry-Atg8a driving pan-neuronal autophagy sensor were live imaged from ZT0-2. mCherry fluoresces in all Atg8a(+) puncta, while GFP …
-
Figure 5—source data 1
The argus mutant displays accumulation of autophagosomes.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig5-data1-v1.xlsx
Figure 6 with 2 supplements
Blocking autophagosome production rescues the short sleep phenotype of the argus mutant.
(A) Total, day, and night sleep were measured under 12 hr:12 hr light:dark in iso31 control, bchs/+, and bchs/bchs female flies. n = 31–32; individual flies overlaid with median±interquartiles; …
-
Figure 6—source data 1
Blocking autophagosome production rescues the short sleep phenotype of the argus mutant.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Effects of aus and bchs on sleep consolidation, latency, and activity index.
(A–E) All sleep metrics were measured under a 12 hr:12 hr light:dark cycle in iso31 (gray), bchs/+ (pink) or bchs/bchs (red) flies. (A) Male total sleep amount during the whole 24-hr cycle (left), …
-
Figure 6—figure supplement 1—source data 1
Effects of aus and bchs on Sleep Consolidation, Latency, and Activity Index.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig11-data11-v1.xlsx
Figure 6—figure supplement 2
Rescue of aus mutants by atg5/atg7 RNAi.
(A–B) Total sleep amount with pan-neuronal atg5 (A) or atg7 (B) knockdown in female flies. elav+ Dicer2/+, UAS-atg RNAi/+ or elav+ Dicer2> UAS atg RNAi. n = 16–25 (atg5) or n = 16 (atg7); individual …
-
Figure 6—figure supplement 2—source data 1
Rescue of aus mutants by atg5/atg7 RNAi.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig12-data12-v1.xlsx
Figure 7 with 2 supplements
Blocking neuronal or whole-fly autophagosome formation increases sleep.
(A) Difference in first-pass population median sleep on RU+ food for a range of female nsybGS> dcr;autophagy-RNAi crosses compared with nsybGS> dcr control (x-axis) and RNAi control (y-axis). Red, …
-
Figure 7—source data 1
Blocking Neuronal or Whole-Fly Autophagosome Formation Increases Sleep.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Validated hits from autophagy RNAi screens.
(A–E) Total sleep amount in female UAS-dcr/+;nsybGS/+, UAS-RNAi/+, or nsybGS> dcr,RNAi flies on RU+ food. (F–P) Total sleep amount in female UAS-dcr/+;actinGS/+, UAS-RNAi/+, or actinGS> dcr,RNAi …
-
Figure 7—figure supplement 1—source data 1
Validated hits from autophagy RNAi screens.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig14-data14-v1.xlsx
Figure 7—figure supplement 2
atg1 and atg8b RNAi additional sleep metrics, activity index, and validation of knockdown.
Additional metrics comparing GS+ UAS dcr control, UAS-RNAi control, and GS> dcr,RNAi females back-crossed to iso31, on both RU+ and RU- food. All data shown as individual flies overlaid with …
-
Figure 7—figure supplement 2—source data 1
atg1 and atg8b RNAi additional sleep metrics, activity index, and validation of knockdown.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig15-data15-v1.xlsx
Figure 8 with 1 supplement
Sleep regulates autophagosome production.
elav-Gal4> UAS-GFP-mCherry-Atg8a flies expressing pan-neuronal autophagy sensor were live imaged as follows. All quantification shows individual brain values overlaid with population …
-
Figure 8—source data 1
Sleep regulates autophagosome production.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig8-data1-v1.xlsx
Figure 8—figure supplement 1
Validation of the Ilastik algorithm for scoring autophagy and the gaboxadol effect on sleep.
(A–D) Female elav-Gal4> UAS-GFP-mCherry-Atg8a brains driving pan-neuronal autophagy sensor were live imaged from ZT2-4, after ~2 hr of pre-incubation in either vehicle or 2 µM rapamycin supplemented …
-
Figure 8—figure supplement 1—source data 1
Validation of the Ilastik algorithm for scoring autophagy and the gaboxadol effect on sleep.
- https://cdn.elifesciences.org/articles/64140/elife-64140-fig17-data17-v1.xlsx
Figure 9
Model for sleep-autophagy interaction.
This schematic details our model for how sleep and macroautophagy interact, based on our results. (A) Sleep decreases autophagosome number under normal conditions, in a manner that is sensitive to …
Additional files
-
Supplementary file 1
Lines and Primers.
Tab 1: A figure-by-figure breakdown of alleles, sources, and backgrounds for each fly line used in most figures of the manuscript. Tab2: A list of all primer sequences used in producing and validating the novel fly lines described in the manuscript.
- https://cdn.elifesciences.org/articles/64140/elife-64140-supp1-v1.xlsx
-
Supplementary file 2
Bioinformatic analysis of the CG16791/ Aus protein product.
An unbiased ProDom analysis of the full-length CG16791, Isoform A protein sequence identified a number of candidate transmembrane domains. Validation with TMPred produced a similar 5-transmembrane best-fit topological prediction for all naturally occurring isoforms of CG16791, as well as our UAS-aus construct protein product. Deep-Loc-1.0 predicted the cell membrane as the most likely initial insertion site for all of these same CG16791 sequences, with the endoplasmic reticulum and Golgi apparatus as possible alternative insertion sites.
- https://cdn.elifesciences.org/articles/64140/elife-64140-supp2-v1.xlsx
-
Supplementary file 3
Autophagy RNAi Screen, First-Pass Sleep for All Crosses.
Tab 1: A list of all RNAi’s used in the screens, including unambiguous stock center IDs. Tab 2: First-pass medians, interquartiles, and n’s for total sleep in females on RU+ food for each nsybGS> dcr,RNAi cross with appropriate controls. Crosses that passed primary criterion are indicated, and annotated with whether they passed subsequent criteria or not and, if not, why. Tab 3: First-pass medians, interquartiles, and n’s for total sleep in females on RU+ food for each actinGS> dcr,RNAi cross with appropriate controls. Crosses that passed primary criterion are indicated, and annotated with whether they passed subsequent criteria or not and, if not, why.
- https://cdn.elifesciences.org/articles/64140/elife-64140-supp3-v1.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/64140/elife-64140-transrepform1-v1.docx
