1. Cell Biology
  2. Neuroscience

Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration

  1. Andrew T Moehlman
  2. Amanda K Casey
  3. Kelly Servage
  4. Kim Orth  Is a corresponding author
  5. Helmut Krämer  Is a corresponding author
  1. UT Southwestern Medical Center, United States
  2. Howard Hughes Medical Institute, United States
https://doi.org/10.7554/eLife.38752
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Cite this article
  1. Andrew T Moehlman
  2. Amanda K Casey
  3. Kelly Servage
  4. Kim Orth
  5. Helmut Krämer
(2018)
Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration
eLife 7:e38752.
https://doi.org/10.7554/eLife.38752
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5 figures, 1 table and 1 additional file

Figures

Figure 1 with 3 supplements
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BiP is a target of Fic AMPylation and deAMPylation in vivo.

(A) BiP AMPylation during times of low ER stress reserves a portion of the chaperone to allow for a rapid, deAMPylation-driven, response to high ER stress (Casey et al., 2017; Preissler et al., 2017a). (B) Bar graphs show the percentage of null mutant BiPG0102/y males rescued by the indicated genomic BiPWT, BiPT366A or BiPT518A genomic transgene (Tg) relative to sibling controls. N = 3 biological replicas. At least 50 flies scored for each replica. Bar graphs show means ± Standard Deviation (SD). (C) Bar graphs show the percentage of viable flies of the indicated wild-type or fic30C genotypes expressing the overactive FicE247G under the ubiquitous Da-Gal4 driver relative to sibling controls. Among the indicated genomic BiP transgenes, only BiPT366A provides partial rescue of lethality in the BiP+/+ background and near complete rescue in a BiPG0102 null background. N = 3 biological replicas. At least 100 total flies scored for each replica. Bar graphs show means ± SD.

https://doi.org/10.7554/eLife.38752.002
Figure 1—source data 1

Relates to Figure 1B and C.

Quantification of fly survival.

https://doi.org/10.7554/eLife.38752.006
Figure 1—figure supplement 1
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Expression of genomic BiP transgenes.

(A) Western blots for FLAG-tagged BiP transgenes and total BiP in whole head lysates in BiP wild type or homozygous mutant background as indicated. Actin (JLA-20) served as a loading control. (B) Kar2T538A mutants have temperature-sensitive growth defects. Yeast strains kar2Δ + pKar2, kar2Δ + pkar2T386A, and kar2Δ + pkar2T538A were grown at 25°C and five-fold serially diluted onto plates of rich media incubated at the indicated temperatures.

https://doi.org/10.7554/eLife.38752.003
Figure 1—figure supplement 2
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AMPylation-resistant BiPT366A phenocopies fic.

(A) ERGs of fic30C flies show reduced ON transients (arrows). Flies homozygous for a lethal BiPG0102 allele rescued by BiPWT transgene have normal vision but flies rescued with the mutant BiPT366A transgene display reduced ON transients. (B) Quantification of ERG traces. Bar graphs show means ± SD. ****p<0.0001; ***p<0.001; *p<0.05; n = 24 flies per genotype and condition.

https://doi.org/10.7554/eLife.38752.004
Figure 1—figure supplement 2—source data 1

Relates to Figure 1—figure supplement 2B.

Quantification of ERG components (white-).

https://doi.org/10.7554/eLife.38752.007
Figure 1—figure supplement 3
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Red eye pigment suppresses ERG phenotypes of fic30C and BiPT366A mutants.

(A) ERGs of OreR and red-eyed fic30C flies as well as BiPWT and BiPT366A animals. (B) Quantification of ERG data. Bar graphs show means ± SD. **p<0.01, n = 18 flies per genotype and condition.

https://doi.org/10.7554/eLife.38752.005
Figure 1—figure supplement 3—source data 2

Relates to Figure 1—figure supplement 3B.

Quantification of ERG components (white+).

https://doi.org/10.7554/eLife.38752.008
Figure 2 with 1 supplement
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Genetic interactions between Fic and UPR genes.

Representative SEM images of heterozygote mutant fic30C/+ eyes expressing the indicated UAS-RNAi transgenes with (A–F) or without (A’–F’) UAS-FicE247G under longGMR-Gal4 control. See Figure 2—figure supplement 1 for quantification. Scale bar: 100 µM.

https://doi.org/10.7554/eLife.38752.009
Figure 2—figure supplement 1
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Genetic interactions between Fic and UPR genes.

UASScer-driven RNAi transgenes (either TRIP or VDRC lines) were used to silence candidate UPR and ER stress-related genes in a fic30C/+heterozygous background, with either LongGMR-Gal4, UAS-FicE247G or LongGMR-Gal4 only. Eye roughness was scored for individual flies and averaged for each cross. Table reports number of flies scored in each group (0 = no roughness, 2 = mildly rough (control flies), 4 = severely rough, 1 and 3 are intermediate phenotypes) and the weighted average of the eye roughness. Significance differences are highlighted in yellow, and p-values were determined using Fisher’s Exact Test for categorical data, comparing the effects of each gene knockdown with the control group (top line, fic30C/+; LongGMR-Gal4, UAS-FicE247G). Interactions were considered significant for any individual test if p<0.003 as determined using Bonferroni’s multiple comparison adjustment.

https://doi.org/10.7554/eLife.38752.010
Figure 3 with 3 supplements
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Fic-mediated AMPylation of BiP is required for photoreceptor maintenance.

(A) A representative ERG trace in response to a 1 s light pulse displaying the sustained negative potential (SNP), representing the depolarization within photoreceptor neurons, and the ON and OFF transients, reflecting post-synaptic activity of lamina neurons. (B) Representation of the different light treatments of flies before ERG recordings: 3 days of 12 hr light (500 lux) and 12 hr dark (LD), 3 days of continuous light (LL) or 3 days of continuous light followed by 3 days of LD (Rec). 1 s light pulses were performed at 4 s intervals. (C) Representative traces from w1118, fic30C, BiPWT and BiPT366A flies. Under LL, fic30C and BiPT366A mutants lose ON and OFF transients (red arrows) and have reduced SNPs. The changes are reversed after 3 days of recovery (Rec). (D) Quantification of key components of ERGs shown in panel C. Bar graphs show means ± SD. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; n = 24 flies for each genotype/condition, pooled from three independent biological replicas.

https://doi.org/10.7554/eLife.38752.011
Figure 3—source data 1

Relates to Figure 3D.

Quantification of ERG components with LD, LL, and Recovery.

https://doi.org/10.7554/eLife.38752.015
Figure 3—figure supplement 1
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Light-induced defects in light-startle activity in fic30C mutants.

(A, B, and C) Actogram of w1118 or fic30C flies reared in LD for three days (A), LL for three days (B), or recovery condition (three days in LL then three days in LD) (C). Light pulse is indicated by upper bars. Data is averaged from three biological replicas, each containing 16 flies per genotype. Data were collected in two-minute bins. Error bars represent Standard Error. (D) Quantification of change in beam breaks per 2 min bin for the 10 min intervals before and after the onset of the light pulse in each experiment. Bar graphs show means ± SD. ***p<0.01, n = 3 experimental repeats with 16 flies per genotype and condition. Dead flies and those with a change in activity greater than three deviations from the median were excluded.

https://doi.org/10.7554/eLife.38752.012
Figure 3—figure supplement 1—source data 1

Relates to Figure 3—figure supplement 1A, B, C and D.

Quantification of light-induced startle behavior- average activity and delta calculations.

https://doi.org/10.7554/eLife.38752.016
Figure 3—figure supplement 2
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fic30C mutants are sensitive to constant light, regardless of total intensity.

(A) Representative ERGs of flies following 3 days of LL or LD with either 500 or 5000 lux light, showing fic30C null animals lose ON/OFF transients and have reduced SNPs with constant light, regardless of intensity, but under LD conditions, even at 5000 lux, have healthy ERG responses. (B) Quantification of ERG data. Bar graphs show means ± SD. ****p<0.0001; ***p<0.001; *p<0.05; n = 10 flies per genotype and condition.

https://doi.org/10.7554/eLife.38752.013
Figure 3—figure supplement 2—source data 2

Relates to Figure 3—figure supplement 2B.

Quantification of ERG components with 500 and 5000 lux.

https://doi.org/10.7554/eLife.38752.017
Figure 3—figure supplement 3
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Fic30C mutants recover ERG properties in 72 hours after 10 days of LL.

(A) Representative ERGs of flies following 10 days of LL, 10 days LD (500 lux), and 3 days Recovery following 10 days. (B) Quantification of ERG data. Bar graphs show means ± SD. ****p<0.0001; ***p<0.001; *p<0.05; n = 8 flies per genotype and condition.

https://doi.org/10.7554/eLife.38752.014
Figure 3—figure supplement 3—source data 3

Relates to Figure 3—figure supplement 3B.

Quantification of ERG components with 10 days of LL.

https://doi.org/10.7554/eLife.38752.018
Figure 4
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AMPylation of BiP is required for maintaining structural plasticity in the retina.

(A–C) Representative TEM images of retina thin sections from the indicated genotypes with either standard LD (A), the stress-inducing LL (B) or recovery treatment (C). Scale bars: 1 µM. Yellow boxes indicate rhabdomeres shown in high magnification images. High magnification scale bars: 0.5 µM. (D) Percentages of flies with intact deep pseudopupil following LD, LL and Rec. N = 3 independent biological replicas with approximately 50 flies scored per genotype per replica. Bar graphs show means ± SD.

https://doi.org/10.7554/eLife.38752.019
Figure 4—source data 1

Relates to Figure 4D.

Scoring of flies for deep pseudopupil defects.

https://doi.org/10.7554/eLife.38752.020
Figure 5
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ER homeostasis is disturbed in fic mutants during prolonged light stimulation.

(A) Representative images of BiP immunohistochemistry in sections of w1118 and fic30C flies following 3 days LD, LL or Recovery treatments. (B) Quantification of BiP fluorescence intensity, normalized to wild-type LD controls, in the lamina neuropil and retina from two independent experiments. (C) Representative images of a Xbp1-GFP splicing reporter in either a Fic wild-type or the null fic30C background following LD, 1 day LL, 2 day LL, 3 day LL, and Recovery conditions. (D) Quantification of GFP fluorescence intensity, normalized to wild-type LD controls, in the lamina neuropil and retina from two independent experiments. (E) Representative images of an Atf4-dsRed reporter in either a wild-type or fic30C background following LD, 1 day LL, 2 day LL, 3 day LL, and Recovery conditions. (F) Quantification of Atf4-dsRed intensity, normalized to wild-type LD controls, in the lamina neuropil and retina from two independent experiments. For all experiments, n = 8 flies per genotype/condition, with exceptions of outliers falling three standard deviations outside the mean. Bar graphs show means ± SD. For all experiments, significance is indicated for treatment compared to the LD condition for the corresponding genotype. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05. All scale bars: 50 µM.

https://doi.org/10.7554/eLife.38752.021
Figure 5—source data 1

Relates to Figures 5B, D and F.

Quantification of integrated intensity of BiP (5B), Xbp1-GFP (5D), and Atf4-DsRed (5F).

https://doi.org/10.7554/eLife.38752.022

Tables

Key resources table
Reagent type (species)
or resource		DesignationSource or referenceIdentifiersAdditional information
Gene
(Drosophila
melanogaster)		ficNAFLYB:FBgn0263278
Gene
(D. melanogaster)		Hsc3-70NAFLYB:FBgn0001218
Genetic reagent
(D. melanogaster)		Da-Gal4Bloomington Drosophila
Stock Center		BDSC:55851;
FLYB:FBst0055851;
RRID: BDSC_55851
Genetic reagent
(D. melanogaster)		LongGMR-Gal4Bloomington Drosophila
Stock Center		BDSC:8121;
FLYB:FBst0008121;
RRID:BDSC_8121
Genetic reagent
(D. melanogaster)		W[1118]Bloomington Drosophila
Stock Center		BDSC:3605;
FLYB:FBst0003605;
RRID:BDSC_3605
Genetic reagent
(D. melanogaster)		BiP[G0102]/FM7cBloomington Drosophila
Stock Center		BDSC:11815;
FLYB:FBal0098203;
RRID:BDSC_11815
Genetic reagent
(D. melanogaster)		UAS[Scer]-Xbp1-
GFP.hg		Bloomington Drosophila
Stock Center		BDSC:60731;
FLYB:FBst0060731;
RRID:BDSC_60731		Sone et al. (2013)
Genetic reagent
(D. melanogaster)		dsRed.crc(ATF4).
5'UTR.tub		DOI: 10.1371/journal.pone.
0126795; PMID:25978358		FLYB: FBal0304834Gift from Don Ryoo, NYU.
Tubulin promoter and ATF4
5'UTR drive DsRed expression
(Flybase FBal0304834)
Genetic reagent
(D. melanogaster)		genomic 3xFLAG-
BiP[WT]		This paperpAttb_gen3xFLAG-BiP[WT]
inserted in AttP landing site
at 89E11
Genetic reagent
(D. melanogaster)		genomic 3xFLAG-
BiP[T366A]		This paperpAttb_gen3xFLAG-BiP[T366A]
inserted in AttP landing site
at 89E11
Genetic reagent
(D. melanogaster)		genomic 3xFLAG-
BiP[T518A]		This paperpAttb_gen3xFLAG-BiP[T518A]
inserted in AttP landing site
at 89E11
Genetic reagent
(D. melanogaster)		GMR-dsRNA[white]This paperpAttb_GMR-dsRNA[white]
inserted in AttP landing site
at 43A1
Genetic reagent
(D. melanogaster)		fic[30C]DOI: 10.1074/jbc.M117.
799296; PMID:29089387		Casey et al. (2017)
Genetic reagent
(D. melanogaster)		UAS[Scer]-V5-
Fic[E247G]		DOI: 10.1074/jbc.M117.
799296; PMID:29089387		Casey et al. (2017)
Genetic reagent
(D. melanogaster)		UPR and ER protein RNAi lines
screened are contained in
Supplemental Table 1
Genetic reagent
(Saccharomyces
cerevisiae)		KAR2::KANGE Healthcare Life
Sciences		SGD:S000003571gene replacement generated using
PCR-based gene deletion strategy
yielding start- to stop-codon
deletion
Strain, strain
background
(Saccharomyces
cerevisiae)		BY4741 MATa
his3Δ1 leu2Δ0
met15Δ0 ura3Δ0		DOI: 10.1002/(SICI)1097-
0061(19980130)14:2 < 115::
AID-YEA204 > 3.0.CO;2–2;
PMID: 9483801		GenBank: JRIS00000000.1
Antibodyanti-Hsc70-3 (BiP)
(Guinea Pig polyclonal)		DOI: 10.1038/sj.emboj.
7601477; PMID:17170705		FLYB: FBgn0001218;
RRID: AB_2569409		Gift from Don Ryoo, NYU
(1:2000 IHC, 1:8000 WB)
Antibodyanti-RFP
(Rabbit polyclonal)		RocklandRockland:600-401-379;
RRID:AB_2209751		(1:1000 IHC)
Antibodyanti-GFP
(Chicken polyclonal)		ThermoFisher
Scientific		ThermoFisher
Scientific:A10262;
RRID: AB_2534023		(1:1000 IHC)
Antibodyanti-Flag
(mouse monoclonal)		SigmaSigma:F-3165;
RRID:AB_259529		(1:2000 WB)
Antibodyanti-Actin
(mouse monoclonal)		Developmental Studies
Hybridoma Bank		DSHB:JLA20;
RRID: AB_528068		1:2000 (WB)
AntibodyAlexa 488- or 568-
secondaries		Molecular Probes(1:1000 IHC)
AntibodyLICOR 800 or 700-
secondaries		LICOR Biosciences(1:20,000 WB)
Recombinant
DNA reagent		pAttb_gen3xFLAG-
BiP[WT]		This paperPCR in multiple steps from genomic
DNA {sequence location = X:
11,801,696..11,807,117 [-]}.
Cloned into modified pAttb vector
Recombinant
DNA reagent		pAttb_gen3xFLAG-
BiP[T366A]		This paperProgenitors: pAttb_gen3xFLAG-
BiP[WT]. Mutated sequence
synthesized with Geneblock (IDT)
Recombinant
DNA reagent		pAttb_gen3xFLAG-
BiP[T518A]		This paperProgenitors: pAttb_gen3xFLAG-
BiP[WT]. Mutated sequence
synthesized with Geneblock (IDT)
Recombinant
DNA reagent		pAttb_GMR-
dsRNA[white]		This paperProgenitors: pUASt_dsRNA[white]
(gift from Dean Smith, UT
Southwestern, PMID: 11804566).
GMR sequence: Geneblock (IDT)
Recombinant
DNA reagent		pKAR2:LEU2This paperCloned from amplification
of endogenous KAR2 with primers
(see below)
Recombinant
DNA reagent		pKAR2[T386A]:
LEU2		This paperProgenitor: pKAR2:LEU2. Site
directed mutagenesis used to
make mutation
Recombinant
DNA reagent		pKAR2[T538A]:
LEU2		This paperProgenitor: pKAR2:LEU2. Site
directed mutagenesis used to
make mutation
Sequence-based
reagent		5’-GCATCCGCGGATACT
CTCGTACCCTGCCGC-3’		This paperCloning for pKAR2:LEU2
Sequence-based
reagent		5’-ATGCGAGCTCCGTAT
ATACTCAGTATAATC-3’		This paperCloning for pKAR2:LEU2
Sequence-based
reagent		5’-GGTTGGTGGTTCTG
CTAGAATTCCAAAGGT
CCAACAATTGTTAGAA
TCATACTTTGATGG-3’		This paperMutagenesis primer for
pKAR2[T386A]:LEU2
Sequence-based
reagent		5’-ACCTTTGGAATTCT
AGCAGAACCACCAAC
CAAAACGATATCATCA
ACATCCTTCTTTTCC-3’.		This paperMutagenesis primer for
pKAR2[T386A]:LEU2
Sequence-based
reagent		5’-AGATAAGGGAGCTGG
TAAATCCGAATCTATCAC
CATCACTAACG-3’		This paperMutagenesis primer for
pKAR2[T538A]:LEU2
Sequence-based
reagent		5’-GGATTTACCAGCTCC
CTTATCTGTGGCAGACA
CCTTCAGAATACC-3’.		This paperMutagenesis primer for
pKAR2[T538A]:LEU2
Chemical compound,
drug		VECTASHIELD Antifade
Mounting Medium
with DAPI		Vector LaboratoriesVector Laboratories:
H-1200
Software, algorithmAdobe PhotoshopAdobeRRID:SCR_014199
Software, algorithmImageJNIHRRID:SCR_003070

Additional files

Transparent reporting form
https://doi.org/10.7554/eLife.38752.023

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  1. Andrew T Moehlman
  2. Amanda K Casey
  3. Kelly Servage
  4. Kim Orth
  5. Helmut Krämer
(2018)
Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration
eLife 7:e38752.
https://doi.org/10.7554/eLife.38752