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Tyramine induces dynamic RNP granule remodeling and translation activation in the Drosophila brain

  1. Nadia Formicola
  2. Marjorie Heim
  3. Jérémy Dufourt
  4. Anne-Sophie Lancelot
  5. Akira Nakamura
  6. Mounia Lagha
  7. Florence Besse  Is a corresponding author
  1. Université Côte d’Azur, CNRS, Inserm, Institut de Biologie Valrose, France
  2. Institut de Génétique Moléculaire de Montpellier, University of Montpellier, France
  3. Department of Germline Development, Institute of Molecular Embryology and Genetics, and Graduate School of Pharmaceutical Sciences, Kumamoto University, Japan
Research Article
Cite this article as: eLife 2021;10:e65742 doi: 10.7554/eLife.65742
6 figures, 9 videos, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
Neuronal ribonucleoprotein (RNP) granules undergo remodeling in response to Tyramine.

(A, B) Cell bodies of adult Mushroom Body (MB) γ neurons stained with anti-Imp (A, B, green in A’, B’) and anti-Me31B (A’, B’, red in A’, B’) antibodies. Brain explants were incubated for 30 min in saline (A–A’, control) or in saline supplemented with 10 mM Tyramine (B–B”). Note that most of the cell body volume is occupied by the nucleus, and thus that the cytoplasm is visible as a ring on confocal sections. Scale bar in A, B: 5 µm. (C) Schematic representation of an adult Drosophila head, with MBs highlighted. The morphology of a single MB γ neuron is represented in red. The region imaged to analyze RNP granule behavior is boxed (turquoise dotted lines). (D, E) Normalized numbers of Imp- (D) or Me31B- (E) containing granules (per image field). Individual data points were color-coded based on the experimental replicate they belong to. Three (D) to four (E) replicates were performed and the mean value of each replicate is indicated as a symbol (triangle). At least 20 (D) or 12 (E) data points were collected for each replicate. ***, p<0.001 (t-test on individual data points). n.s. stands for not significant. (F) Distribution of Me31B partition coefficients. Partition coefficients were estimated by dividing the intensity of Me31B signal in individual RNP granules to the intensity of the cytoplasmic diffuse pool (see Materials and methods) and calculated for each granule detected in the imaged fields. The individual data points displayed on the graph were extracted from a single replicate. Three replicates were performed and the mean value of each is indicated as a symbol (triangle). Number of RNP granules: 622 granules distributed across 18 fields (control), 777 granules distributed across 18 fields (+ Tyramine). ***, p<0.001 (t-test on individual data points). Note that the p-value obtained when comparing the distributions of replicate means is 0.7 (Mann–Whitney test). For the list of values used to generate the graphs shown in D–F see Figure 1—source data 1.

Figure 1—figure supplement 1
Quantification and detection of ribonucleoprotein (RNP) granule number in response to neurotransmitters and reversibility.

(A) Normalized numbers of Imp-containing granules (per image field) after treatment with 10 mM Acetylcholine (Ach); 10 mM Dopamine (Dopa); 10 mM Octopamine (Oct); or 10 mM Tyramine (Tyr). Individual data points were color-coded based on the experimental replicate they belong to. Three replicates were performed for each condition and the mean value of each replicate is indicated as a symbol (triangle). At least 10 data points were collected for each replicate. **, p<0.01; ***, p<0.001 (Kruskal–Wallis test on individual data points with Dunn’s post-test). (B) Automatic detection of RNP granules using the SPaDe algorithm. Both panels display cell bodies of adult Mushroom Body γ neurons expressing a GFP-Imp fusion from the endogenous locus (G080-GFP-Imp protein-trap line). While the lower sample was treated with 10 mM Tyramine, the upper sample was not. For each panel, the left image represents the field cropped from confocal images (single sections) and used for automated detection. The right image displays the masks of the granules detected with SPaDe (highlighted in purple). (C) Normalized numbers of Imp-containing granules (per image field; red) and Me31B partition coefficient (green). Brain explants were incubated for 30 min in saline (control) or in saline supplemented with 10 mM Tyramine (+ Tyr). For the recovery condition (+ Tyr + recovery), brains were transferred back to regular saline after Tyramine treatment and incubated for 60 min. Two and three replicate experiments were performed to monitor Me31B and Imp condensation respectively. Mean values were calculated for each replicate and their average is shown for each condition. Error bars represent S.E.M. (D) Normalized numbers of Imp-containing granules (per image field). Kir2.1 was expressed under the control of the tub-Gal80ts;;OK107-Gal4 driver. Individual data points in D were color-coded based on the experimental replicate they belong to. Four replicates were performed and the mean value of each replicate is indicated as a symbol (triangle). At least 13 data points were collected for each replicate. **, p<0.01; ***, p<0.001 (Kruskal–Wallis test on individual data points with Dunn’s post-test). n.s. stands for not significant. For the list of values used to generate the graphs shown in A, C, D see Figure 1—figure supplement 1—source data 1.

Figure 1—figure supplement 2
Quantification of Me31B and Imp protein levels after Tyramine treatment.

(A) Western blot performed on lysates from adult G080-GFP-Imp brains treated (+ Tyr) or not (control) with 10 mM Tyramine. Anti-GFP antibodies were used to detect GFP-Imp fusions (MW ≈ 90 kDa), and anti-Me31B antibodies to detect Me31B (MW ≈ 50 kDa). Tubulin was used as a loading control. (B) Me31B-GFP and GFP-Imp overall expression levels quantified from movies at t = 0 and t = 32 min. Tyramine was added at t = 2 min. Data were normalized to t = 0. Box plots are represented using the min to max convention, where the middle line defines the median and the whiskers go down to the smallest value and up to the largest. Numbers of imaged brains for Me31B-GFP: 7 (control) and 11 (+ Tyramine) and for GFP-Imp: 7 (control) and 7 (+ Tyramine). n.s. stands for not significant. For the list of values used to generate the graph shown in B see Figure 1—figure supplement 2—source data 1. For original western blot images see Figure 1—figure supplement 2—source data 2.

Figure 2 with 1 supplement
Dynamics of Tyramine-induced ribonucleoprotein (RNP) granule remodeling.

(A) Image sequences extracted from movies following Me31B-GFP-positive granules over time. Both fusion (upper panel) and fission (lower panel) events are shown. GFP intensities are represented using the ‘Fire’ LUT of ImageJ. Scale bar: 2 µm. (B) Me31B-GFP mean partition coefficients in function of time in brain explants treated (red) or not (black) with 10 mM Tyramine. Each data point represents the mean of the average partition coefficients measured for all granules present in fields imaged at a given time point. Tyramine was added at t = 2 min (orange arrow). (C, D) Image sequences extracted from movies recording the cell bodies of adult Mushroom Body (MB) γ neurons endogenously expressing Me31B-GFP proteins. Brain explants were either maintained in saline (C), or supplemented with 10 mM Tyramine (D) at t = 2 min. Images were originally acquired every 30 s. Intensities are displayed using the ‘Fire’ LUT of ImageJ. Scale bar: 5 μm. Numbers of movies: 7 (ctrl) and 11 (+ Tyr). Note that in these experiments MB γ neurons could not be unambiguously distinguished from other MB neuronal subpopulations. No difference could however be observed in the behavior of Me31B-GFP-positive granules within MB neurons. For the list of values used to generate the graphs shown in B see Figure 2—source data 1.

Figure 2—figure supplement 1
Mean numbers of GFP-Imp-positive granules in function of time in brain explants.

Each data point represents the mean number of granules detected in fields imaged at a given time point. Tyramine was added at t = 2 min (orange arrow). Error bars correspond to S.E.M. Numbers of movies: 8 (ctrl) and 15 (+ Tyr). For the list of values used to generate the graph see Figure 2—figure supplement 1—source data 1.

Tyramine induces TyrR-dependent responses in MB neurons.

(A–C) Cell bodies of control (A, B) or TyrRGal4 mutant (C) adult Mushroom Body γ neurons stained with anti-Imp (A–C) and anti-Me31B (A’–C’) antibodies. Brains were incubated in saline (A, A’, control) or in saline supplemented with 10 mM Tyramine (B–C’) Scale bar: 5 μm. (D) Normalized numbers of Imp-containing granules (per image field). Individual data points were color-coded based on the experimental replicate they belong to. Three replicates were performed for each condition and the mean value of each replicate is indicated as a symbol (triangle). At least 10 data points were collected for each replicate. ***, p<0.001 (Kruskal–Wallis test on individual data points with Dunn’s post-test). n.s. stands for not significant. (E) Distribution of Me31B partition coefficients. Partition coefficients were estimated by dividing the intensity of Me31B signal in individual ribonucleoprotein (RNP) granules to the intensity of the cytoplasmic diffuse pool and calculated for each granule detected in the imaged fields. The individual data points displayed on the graph were extracted from a single replicate. Three replicates were performed and the mean value of each is indicated as a symbol (triangle). At least 10 fields were analyzed per condition. Number of RNP granules: 875 granules distributed across 18 fields (control), 558 granules distributed across 14 fields (+ Tyramine), and 475 granules distributed across 10 fields (+ Tyramine in TyrRGal4 mutants). ***, p<0.001 (one-way ANOVA on individual data points with Dunnett’s post-tests). n.s. stands for not significant. (F) Average fluorescence intensity (F) of GCamp3.0 signal in MB calyx upon exposure of control (green) or TyrRGal4 mutants (orange) brain explants to 10 mM Tyramine. The MB247-homer::GCamp3.0 reporter was used to monitor Ca2+ levels. Data are plotted as F(t)-F(t = 0)/F(t = 0) (ΔF/F(0); see Materials and methods). Tyramine was added at t = 2 min (red arrow). Error bars correspond to S.E.M. Number of brains analyzed: 18 (control) and 13 (TyrRGal4 mutants). (G) Dose-dependent long-term increase in Ca2+ levels upon exposure to Tyramine. Intensities of GCamp3.0 signal are plotted as F(t = 30 min) – F(t = 0). Numbers of brains analyzed: 5 (-), 5 (1 nM), 6 (300 nM), 6 (30 μM), and 8 (10 mM). For the list of values used to generate the graphs shown in D–G see Figure 3—source data 1.

Figure 4 with 1 supplement
CamkII interacts with Imp and is required for Tyramine-induced Imp decondensation.

(A) CamkII co-immunoprecipitates with Imp. FLAG-CamkII constructs were co-transfected with either GFP-Imp or GFP (negative control) in S2R+ cells. GFP proteins were immunoprecipitated and the bound fractions (right) used for western blot. Input fractions (left) were used as a control of expression. Anti-FLAG and anti-GFP antibodies were used to detect respectively CamkII (MW ≈ 55–60 kDa) and Imp (MW ≈ 90 kDa) fusion proteins. Cell lysates were treated (+) or not (−) with RNase prior to immunoprecipitation. (B) Normalized numbers of Imp-containing granules in brain explants treated (+ Tyramine) or not (ctrl) with 10 mM Tyramine. + ala refers to the condition where the CamkII inhibitory peptide ala was expressed specifically in MB neurons, using the tub-Gal80ts;;OK107-Gal4 driver. Individual data points were color-coded based on the experimental replicate they belong to. Three replicates were performed for each condition and the mean value of each replicate is indicated as a symbol (triangle). At least 12 data points were collected for each replicate. (C) Distribution of Me31B partition coefficients. Me31B partition coefficients were estimated by dividing the intensity of Me31B signal in individual ribonucleoprotein (RNP) granules to the intensity of the cytoplasmic diffuse pool and calculated for all the granules detected in imaged fields. The individual data points displayed on the graph were extracted from a single replicate. Three replicates were performed and the mean value of each is indicated as a symbol (triangle). Number of RNP granules: 622 granules distributed across 18 fields (control), 777 granules distributed across 18 fields (+ Tyramine) and 663 granules distributed across 16 fields (+ Tyramine +ala). *, p<0.05; ***, p<0.001 (one-way ANOVA on individual data points with Dunnett’s post-tests). n.s. stands for not significant. For the list of values used to generate the graphs shown in B, C see Figure 4—source data 1. For original western blot images see Figure 4—figure supplement 1—source data 1.

Figure 4—figure supplement 1
CamkII interacts with Imp.

(A) Schematic representation of the procedure used to identify Imp interactors in MB γ neurons. Lysates of adult 201Y-Gal4, UAS-GFP-Imp heads were used for the IP-MS experiments and control IPs (201Y-Gal4, UAS-GFP) were performed in parallel. (B) Pie chart representing the distribution of Imp protein interactors in function of their biological GO category. 51 proteins were reproducibly identified by mass spectrometry (MS) analysis of the bound fraction recovered upon immunoprecipitation (IP) of GFP-tagged Imp proteins expressed in Mushroom Body (MB) neurons. (C–C’’’) Cell bodies of adult MB γ neurons expressing endogenously produced GFP-CamkII proteins (C’, blue in C’’’) and stained with α-Imp (C, green in C’’’) and α-phospho-CamkII (C’’, red in C’’’) antibodies. Scale bar: 5 μm. (D) A variant of Imp with phosphomutant RXXS/T CamkII consensus sites still responds to Tyramine. Normalized numbers of Imp-containing granules (per image field) after treatment with 10 mM Tyramine (+ Tyr). Individual data points were color-coded based on the experimental replicate they belong to. Three replicates were performed and the mean value of each replicate is indicated as a symbol (triangle). At least 12 data points were collected for each replicate. ***, p<0.001 (t-test). For a table listing identified Imp interactors see Supplementary file 1. For the list of values used to generate the graph in D see Figure 4—source data 2.

Figure 5 with 1 supplement
The translation of granule-associated mRNAs is increased upon Tyramine treatment.

(A) Normalized GFP signal intensities produced by reporters in which the 3’UTR of different transcripts was fused to GFP. (B) Normalized GFP signal intensities produced by the GFP-profilin 3’UTR reporter. In A and B, reporters were expressed for 3 days under the control of tub-Gal80ts;;OK107-Gal4. Brain explants were treated (+Tyr) or not (ctrl) with 10 mM Tyramine for 30 min. In B, anisomycin (aniso) was added in addition to Tyramine to block translation. For the profilin 3’UTR reporter in A, three outlier data were omitted from the graph (although they were considered to calculate the mean of the corresponding replicate and to perform statistical tests). (C) Normalized GFP signal intensities produced by the GFP-profilin 3’UTR reporter. GFP-profilin 3’UTR was expressed solely (ctrl), or together with ala (+ ala), and brain explants were treated (+) or not (−) with 10 mM Tyramine for 30 min. The ala inhibitory peptide was expressed conditionally in adult MB neurons using tub-Gal80ts;;OK107-Gal4. (D) Proportion of gfp-profilin 3’UTR RNA molecules contained in Me31B-mTomato-positive granules. Co-localization was measured using the JACoP plugin of ImageJ (see Materials and methods and Figure 4—figure supplement 1C) and values normalized to controls. Individual data points in A–D were color-coded based on the experimental replicate they belong to. Three replicates were performed for each condition and the mean value of each replicate is indicated as a symbol (triangle). At least 10 data points were collected for each replicate. ***, p<0.001 (t-tests on individual data points for A, D and Kruskal–Wallis test on individual data points with Dunn’s post-tests for B, C). n.s. stands for not significant. For the list of values used to generate the graphs shown in A–D see Figure 5—source data 1.

Figure 5—figure supplement 1
Release of endogenous profilin mRNA from granules after 10 min Tyramine treatment.

(A) Proportion of endogenous profilin smFISH spots contained in GFP-Imp-positive granules in brains treated (+ Tyr) or not (control) with 10 mM Tyramine for 10 min. Co-localization was measured using the JACoP plugin of ImageJ (see Materials and methods) and values normalized to controls. (B) Total number of endogenous profilin smFISH spots in brains treated (+ Tyr) or not (control) with 10 mM Tyramine for 10 min. Numbers in A, B were normalized to the control condition. Individual data points in A and B were color-coded based on the experimental replicate they belong to. Three replicates were performed for each condition and the mean value of each replicate is indicated as a symbol (triangle). At least 11 data points were collected for each replicate. **, p<0.01 (unpaired t-test). n.s. stands for not significant. (C–E) Colocalization of gfp- profilin 3’UTR spots with Me31B+ granules. Cell bodies of adult MB neurons expressing Me31B-mTomato and the gfp-profilin 3’UTR reporter. Me31B-mTomato signal is shown in C (gray) and E (red) and gfp-profilin 3’UTR signal is shown in D (gray) and E (green). The masks of objects detected with SPaDe are shown in C’ and D’. These masks are used to perform object-based colocalization analysis using the JACoP plugin of ImageJ, in which RNA spots are defined by their center of mass (green dots) and granules are detected as particles (red masks). Co-localization is indicated as a yellow mark. Scale bar: 5 μm. For the list of values used to generate the graphs in A and B see Figure 5—figure supplement 1—source data 1.

Figure 6 with 1 supplement
Dynamics of profilin translation upon Tyramine treatment.

(A) Image sequences extracted from movies recording the distribution of SunTag-tagged Profilin peptides in brain explants. ScFv-GFP-NLS was expressed in MB γ neurons with (upper and lower panels) or without (middle panel) SunTag-profilin mRNAs. Images were recorded every 30 s and Tyramine was added at t = 0. The translation inhibitor puromycin (puro) was added prior to Tyramine (lower panel). Complete genotype: UAS-SunTag-profilin/+; UASp-ScFv-GFP-NLS/VT44966-Gal4. Scale bar: 3 μm. (B) Distributions of the number of Tyramine-induced ScFv-GFP-positive cytoplasmic foci observed per cell in the presence (+) or absence (−) of SunTag-profilin transcripts. Puromycin (puro) significantly inhibited the formation of ScFv-GFP-positive foci. ***, p<0.001 (Kruskal–Wallis test with Dunn’s post-tests on individual data points). At least 45 cells and five movies were analyzed per condition. (C) Normalized number of cells with ScFv-GFP cytoplasmic foci (red) observed for each time point in brain explants treated with 10 mM Tyramine. Numbers of cells are normalized so that one represents for each movie the maximal number of cells with ScFv-GFP foci (n = 5 movies). Fluorescence intensity of GCamp3.0 is shown for comparison (green; see Figure 3F). Tyramine was added at t = 2 min (orange arrow). Error bars correspond to S.E.M. For the list of values used to generate the graphs shown in B, C see Figure 6—source data 1.

Figure 6—figure supplement 1
Model of Tyramine signaling and its impact on MB neuronal ribonucleoprotein (RNP) granules.

Based on our data, we propose that Tyramine signals through TyrR-expressing neurons to generate calcium transients in Mushroom Body neurons. Calcium transients in turn activate CamkII, thus promoting the partial disassembly of Imp+ Me31+ neuronal RNP granules and the translation of granule-associated mRNAs such as profilin.

Videos

Video 1
Fusion between two Me31B-GFP-containing granules.

Real-time imaging of Me31B-GFP-containing granules in the cell body of an intact adult brain explant. Signal intensities are displayed using the ‘Fire’ LUT of ImageJ. Images were acquired every 30 s. Scale bar: 0.5 μm.

Video 2
Fission of a Me31B-GFP-containing granule.

Real-time imaging of Me31B-GFP-containing granules in the cell body of an intact adult brain explant. Signal intensities are displayed using the ‘Fire’ LUT of ImageJ. Images were acquired every 30 s. Scale bar: 0.5 μm.

Video 3
Behavior of Me31B-GFP-containing granules in brain explants treated with control saline.

Real-time imaging of MB γ cell bodies expressing endogenously expressed Me31B-GFP. Signal intensities are displayed using the ‘Fire’ LUT of ImageJ. Images were acquired every 30 s for 30 min. Control HL3 buffer was added at t = 2 min. Scale bar: 3 μm.

Video 4
Dynamic response of Me31B-GFP- containing granules to Tyramine.

Real-time imaging of MB γ cell bodies expressing endogenously expressed Me31B-GFP. Signal intensities are displayed using the ‘Fire’ LUT of ImageJ. Images were acquired every 30 s for 30 min, from intact adult brain explants. Tyramine was added at t = 2 min to reach a 10 mM final concentration. Scale bar: 3 μm.

Video 5
Tyramine induces a rise in the intracellular Ca2+ concentration of control MB neurons.

Real-time imaging of MB neurons (calyx region) expressing a Homer::GCamp3.0 construct under the control of the MB247 promoter. Signal intensities are displayed using the ‘Fire’ LUT of ImageJ. Images were acquired every 30 s for 15 min, from intact adult brain explants. Tyramine was added at t = 2 min. Scale bar: 10 μm.

Video 6
Tyramine-induced calcium response is inhibited in TyrR-/- mutant context.

Real-time imaging of a TyrRGal4 brain in which MB neurons expressed a Homer::GCamp3.0 construct under the control of the MB247 promoter. MB calyx region is shown. Signal intensities are displayed using the ‘Fire’ LUT of ImageJ. Images were acquired every 30 s for 15 min, from intact adult brain explants. Tyramine was added at t = 2 min. Scale bar: 10 μm.

Video 7
Tyramine induces the assembly of SunTag-profilin foci.

Real-time imaging of MB γ cell bodies co-expressing SunTag-profilin RNAs and ScFv-GFP-NLS fusions under the control of VT44966-Gal4. Note that VT44966-Gal4 is expressed at high level only in a subset of MB γ neurons. Some cells initially contained a big cluster of ScFv-GFP fusions; these cells were excluded from the analysis. Signal intensities are displayed using the ‘Fire’ LUT of ImageJ. Images were acquired every 30 s for 30 min, from intact adult brain explants. Tyramine was added at t = 2 min to reach a 10 mM final concentration. Scale bar: 3 μm.

Video 8
ScFv-GFP-positive foci are not observed in the absence of SunTag-profilin transcripts.

Real-time imaging of MB γ cell bodies expressing ScFv-GFP-NLS fusions under the control of VT44966-Gal4. Note that VT44966-Gal4 is expressed at high level only in a subset of MB γ neurons. Signal intensities are displayed using the ‘Fire’ LUT of ImageJ. Images were acquired every 30 s for 12 min, from intact adult brain explants. Tyramine was added at t = 2 min to reach a 10 mM final concentration. Scale bar: 3 μm.

Video 9
Puromycin disrupts the assembly of SunTag-profilin foci.

Real-time imaging of MB γ cell bodies co-expressing SunTag-profilin RNAs and ScFv-GFP-NLS fusions under the control of VT44966-Gal4. Note that VT44966-Gal4 is expressed at high level only in a subset of MB γ neurons. Signal intensities are displayed using the ‘Fire’ LUT of ImageJ. Images were acquired every 30 s for 12 min, from intact adult brain explants. Brain explants were incubated with 250 μM Puromycin for 15 min before addition of Tyramine. Tyramine was added 2 min after the movie starts, to reach a 10 mM final concentration. Scale bar: 3 μm.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (D. melanogaster)
w1118BDSCRRID:BDSC_3605
Genetic reagent (D. melanogaster)
Tubulin-Gal80ts;; OK107-Gal4
Besse team, IBV, Nice, FRThis stock was obtained via genetic crosses so as to establish a stock carrying both the P{tubP GAL80ts} and the OK107 Gal4 driver.
Genetic reagent (D. melanogaster)
Canton-S
PMID:23083740Dr K. Keleman (HHMI/ Janelia Farm, USA)
Genetic reagent (D. melanogaster)
MB247:homer:GCamp3.0
PMID:24065891
Genetic reagent (D. melanogaster)
TyrRGal4
PMID:27498566
Genetic reagent (D. melanogaster)
G080-GFP-Imp
PMID:24656828Dr L. Cooley (Yale University, USA)
Genetic reagent (D. melanogaster)
VT44966-Gal4PMID:29322941RRID:FlyBase_FBst0488404Dr K. Keleman (HHMI/ Janelia Farm, USA)
Genetic reagent (D. melanogaster)
YFP-CamkIIPMID:25294944RRID:DGGR_115127
Genetic reagent (D. melanogaster)
UAS-EGFP-kir2,1PMID:11343651RRID:BDSC_6595
Genetic reagent (D. melanogaster)
UAS-CamkII-alaPMID:8384859RRID:BDSC_29666
Genetic reagent (D. melanogaster)
UASp-EGFP-SV40-3'UTR-this study
(Besse team, IBV, Nice, FR)
Expresses EGFP coding sequence upstream of SV40 3’UTR under UAS control, insertion on chromosome III. For further information, see “Generation of Drosophila lines”.
Genetic reagent (D. melanogaster)
UASp-EGFP-profilin-3'UTRthis study
(Besse team, IBV, Nice, FR)
Expresses EGFP coding sequence upstream of profilin 3’UTR under UAS control, insertions on chromosome II or III. For further information, see “Generation of Drosophila lines”.
Genetic reagent (D. melanogaster)
UASp-EGFP-cofilin-3'UTRthis study
(Besse team, IBV, Nice, FR)
Expresses EGFP-coding sequence upstream of cofilin 3’UTR under UAS control, insertion on chromosome III. For further information, see “Generation of Drosophila lines”.
Genetic reagent (D. melanogaster)
UASp-EGFP-camk2-3'UTRthis study
(Besse team, IBV, Nice, FR)
Expresses EGFP-coding sequence upstrream of camk2 3’UTR under UAS control, insertion on chromosome II. For further information, see “Generation of Drosophila lines”.
Genetic reagent (D. melanogaster)
Me31B-GFPthis study
(Besse team, IBV, Nice, FR ; Nakamura team, Kumamoto University, Kumamoto, Japan)
Knock-in line generated using the CRISPR-Cas9 technology. The GFP tag is C-terminal. For further information, see “Generation of Drosophila lines”.
Genetic reagent (D. melanogaster)
Me31B-mTomatothis study
(Besse team, IBV, Nice, FR ; Nakamura team, Kumamoto University, Kumamoto, Japan)
Knock-in line generated using the CRISPR-Cas9 technology. The mTomato tag is C-terminal. For further information, see “Generation of Drosophila lines”.
Genetic reagent (D. melanogaster)
UAS-SunTag-profilinthis study
(Besse team, IBV, Nice, FR ; Lagha team, IGMM, Montpellier, FR)
Expresses a SunTagged Profilin (isoform RB) under UAS-control, insertion on chromosome II. For further information, see “Generation of Drosophila lines”.
Genetic reagent (D. melanogaster)
UASp-scFv-GFP-NLSthis study
(Besse team, IBV, Nice, FR ; Lagha team, IGMM, Montpellier, FR)
Expresssd a scFvGFP under UAS-control, insertion on chromosome III. For further information, see “Generation of Drosophila lines”.
Genetic reagent (D. melanogaster)G080-GFP-Imp- RXXA mutantThis study
(Besse team, IBV, Nice, FR)
Mutant line generated using the CRISPR-Cas9 gene-editing technology. The four potential CamkII consensus sites RXXS/T are mutated into RXXA. For further information, see “Generation of Drosophila lines”.
AntibodyAnti-Imp (Rabbit polyclonal)PMID:24656828IF (1:1000)
AntibodyAnti-Imp (Rat polyclonal)PMID:24656828IF (1:1000)
Antibodyanti-Me31B (Rabbit polyclonal)PMID:28388438Dr C. Lim (School of Life Sciences, Korea) IF (1:3000) and WB (1:5000)
Antibodyanti-Me31B (Mouse monoclonal)PMID:11546740RRID:AB_2568986IF (1:3000)
Antibodyanti-pCamkII (rabbit polyclonal)Santa Cruz BiotechnologyCat# sc-12886-R RRID:AB_2067915IF (1:1000)
Antibodyanti-GFP (Chicken polyclonal)AbcamCat# ab13970 RRID:AB_300798IF (1:1000)
Antibodyanti-GFP (Rabbit polyclonal)Torrey Pines BiolabsCat# TP401 071519 RRID:AB_10013661WB (1:2500)
Antibodyanti-FLAG (Mouse monoclonal)Sigma-AldrichCat# F1804, RRID:AB_262044WB (1:2500)
Antibodyanti-Tubulin (Mouse monoclonal)Sigma-AldrichCat# T9026, RRID:AB_477593WB (1:5000)

Additional files

Supplementary file 1

Imp protein interactors identified through IP-MS.

Number of peptides found for each protein in the GFP-Imp- or GFP- bound fractions are indicated for replicates 1 and 2.

https://cdn.elifesciences.org/articles/65742/elife-65742-supp1-v2.xlsx
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https://cdn.elifesciences.org/articles/65742/elife-65742-transrepform-v2.pdf

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