EMBO/soda-nel huggingface dataset:
Interplay of LRRK2 with chaperone-mediated autophagy (2013)
figf5

(a) Immunoblots of rat liver lysosomes incubated with WT or G/S LRRK2 alone or with 1 mM GTP and/or GAPDH. Duplicate samples are shown. Bottom: quantification of lysosome-bound LRRK2 expressed as a multiple of the amount bound without additions (n = 6 independent experiments). (b) Effect of kinase inhibitor on WT, G/S, or the kinase-dead mutant D/A LRRK2 incubated with rat liver lysosomes in presence or absence of GAPDH (5 μg). Right: quantification expressed as a multiple of the association without additions (n = 3–5 independent experiments). (c,d) Transient transfection of HEK293 cells with the myc- or GFP-tagged LRRK2 constructs indicated at top left in c. Full, full length; M, KFERQ-like motifs (M1–M8). (c) Binding to GST-hsc70. Left, representative immunoblots; input lanes, one-tenth of input material. NT, non-transfected cells. Immunoblot for hsc70 is shown as loading control. Top right: quantification of LRRK2 bound to hsc70. Where indicated, samples were also incubated with RNase A to compete the KFERQ-mediated binding of hsc70 (n = 3 or 4 independent experiments). (d) Coimmunoprecipitation (IP) of hsc70 with anti-myc or anti-GFP in the same cells. Inputs (Inp), one-quarter of starting volume. (e) Binding or LRRK2 to rat liver lysosomes treated with the indicated concentrations of trypsin. LAMP-1 is shown to confirm the integrity of the lysosomes. (f) Co-IP of LAMP-2A with anti-LRRK2 in lysosomes incubated with WT or G/S LRRK2. Inp, one-quarter of starting sample; IgG, immunoglobulin G. Values: LAMP-2A recovered in the IP (IP L2A) corrected for the amount of LRRK2 pulled down and expressed as a multiple of WT value. (g,h) Duplicate LAMP-2A immunoblots of rat liver lysosomes incubated alone (−) or with WT or G/S LRRK2 (g) or with lysosomes from control (Ctr) or from WT-LRRK2 or G/S-LRRK2 transgenic mice (h) and subjected to blue native electrophoresis. Arrow, 700-kDa multimeric complex. All values are mean + s.e.m.; differences compared to none or untreated (*) or to WT or full-length protein (§) were significant at P 0.05; unpaired t-test in a and ANOVA and Bonferroni test in b,c. Full-length blots and gels in Supplementary Figure 12.
Interplay of LRRK2 with chaperone-mediated autophagy (2013)
figf6

(a) Immunofluorescence for LAMP-2A and LAMP-1 of sections from the dorsal motor nuclei of the vagus nerves of brains from two different unaffected control (Ctr) individuals and two individuals with Parkinson's disease with the G/S LRRK2 mutation. Left: images from representative fields. Right: number of fluorescent puncta per cell. Black dots, values in individual cells; red lines, mean. (b) Immunohistochemistry for LAMP-2A in the same samples. (c) Immunofluorescence for LAMP-2A (red) and LRRK2 (green) in the same samples. Merged images from the two fluorophore channels are shown (for colocalization quantification, see Supplementary Fig. 7b). Scale bars, 10 μm. Values are mean + s.e.m.; *P 0.05, ANOVA and Bonferroni test.
Interplay of LRRK2 with chaperone-mediated autophagy (2013)
figf7

(a) Top: LRRK2 immunoblot of rat liver lysosomes incubated with WT, G/S, D/A (kinase-dead), I/T or R/C mutant LRRK2, alone or in the presence of protease inhibitors (PI). Input lanes, one-quarter of input material. Bottom: quantification of binding and uptake (calculated as in Fig. 2b; n = 4 or 5 independent experiments). (b) Left: immunoblots of lysosomes incubated as in a, alone or in the presence of increasing concentrations of RNase A (left) or GAPDH (right). Right: quantification of LRRK2 bound, as a multiple of the binding when incubated with lysosomes alone (n = 3 independent experiments). (c) Top: coimmunoprecipitation (co-IP) of LAMP-2A (L2A) with anti-LRRK2 from lysosomes incubated with LRRK2 proteins as in a. Co-immunoprecipitated fractions were immunoblotted for LAMP-2A and LRRK2. Input: immunoblot for LAMP-2A before the affinity purification. Bottom: amount of LAMP-2A coimmunoprecipitated, expressed as a multiple of the value with WT LRRK2 (n = 3 or 4 independent experiments). (d) Co-IP of LAMP-2A with anti-GFP in tet-on HEK293 cells expressing GFP-LRRK2 proteins and pulsed for 24 h with doxycycline. Inputs (Inp), one-quarter of material added. (e) The same cells as in d and control cells transduced with KFERQ-PA-mCherry1. Left: representative images. Nuclei are highlighted with DAPI in blue. Right: Quantification after photoactivation and 16 h of serum deprivation (n > 150 cells). All values are mean + s.e.m. Differences with WT (*) or with the indicated mutants (§) were significant at P 0.05; unpaired t-test in a–c and ANOVA and Bonferroni test in e. Scale bars, 10 μm. Full-length blots and gels in Supplementary Figure 12.
Interplay of LRRK2 with chaperone-mediated autophagy (2013)
figf8

(a) α-syn immunoblot of rat liver lysosomes incubated with WT or mutant (A53T) α-syn alone (−) or with WT LRRK2 or G/S LRRK2. Inp, input; Mon, monomers. Right: quantification of α-syn oligomers, expressed as a multiple of amounts in samples without LRRK2 (n = 4 or 5 independent experiments). (b–d) Degradation of radiolabeled proteins (b), binding of GST–α-syn (c) and levels of endogenous α-syn (d) in intact brain lysosomes (Lysos) from non-transgenic (Ctr) and G/S LRRK2 transgenic mice. Bottom panel in c shows lower exposure to visualize monomers of α-syn. Homog, homogenates; olig, oligomers; I, input (n = 3 independent experiments). (e) Ventral midbrain dopaminergic neuronal cultures from control (Ctr) or WT-LRRK2 or G/S-LRRK2 transgenic mice untreated or treated with RNAi for LAMP-2A (L2A(−)). (f) Colocalization between α-syn and total LAMP-2 from cells in e (n > 50 tyrosine hydroxylase (TH)-positive neurons). Total LAMP-2 is not reduced in L2A knockdown cells because LAMP-2B is upregulated. (g) iPSC lines from controls and from patients with G/S LRRK2-mutation Parkinson's disease, differentiated for 30 d and immunostained for LAMP-2A, α-syn and TH. Merged channels (left) and magnified green and red channels from boxed region (right). Arrows, colocalization LAMP-2A and α-syn. (h) Percentage of LAMP-2A+ puncta colocalizing with α-syn in TH+ cells (n > 50) for two control and two LRRK2 iPSC lines treated as in g. (i,j) iPSC lines as in g, differentiated for 30 or 75 d and untreated or treated with RNAi for LAMP-2A. (i) Immunofluorescence for LAMP-2A, α-syn and GFP in LAMP-2A-RNAi iPSC lines differentiated for 30 d. Bottom: higher magnifications of boxed areas. (j) Percentage of TH+ cells showing cytoplasmic accumulation of α-syn. n ≥ 2 experiments using two control and two LRRK2 iPSC lines (n > 70 and n > 150 TH+ cells in Ctr and LRRK2, respectively). Scale bars, 10 μm. All values are mean + s.e.m. Differences from WT or control (*) or between control or knockdown cells (§) are significant at P 0.05; unpaired t-test in a,h and ANOVA and Bonferroni test in f,j. Full-length blots and gels in Supplementary Figure 12.
Gatekeeper role of brain antigen-presenting CD11c+ cells in neuroinflammation (2015)
Figure 1

Figure 1

Adoptive transfer EAE of encephalitogenic CD4+ T cells into CD11c-GFP+-depleted and control mice.

(A) Mean clinical scores (±SEM) of C57BL/6→C57BL/6 controls treated with DTX and CD11c-DTR/GFP→C57BL/6 treated with DTX or PBS during the whole observation period starting from day -1. All animals received in vitro-generated 2d2.tdRFP Th17 intravenously on day 0. DC depletion in CD11c-DTR/GFP→C57BL/6 reduces the encephalitogenicity of adoptively transferred 2d2.tdRFP Th17. Mann-Whitney-U-test was performed on mean clinical scores (n=11PBS/13DTX); * p < 0.05, ** p < 0.01; see also Appendix Table S1. Pooled data from two independent experiments.

(B) Mononuclear cells were isolated on day 30 of animals shown in (A) from the CNS of PBS- and DTX-treated CD11c-DTR/GFP→C57BL/6 that had been transferred with 2d2.tdRFP Th17. Flow cytometry was performed on surface stained cell samples. Cells were pre-gated on lymphocyte cells (FSC SSC gate) and PI-negative; pooled data of three animals; representative of two independent experiments.

(C) Mononuclear cells were isolated from the CNS of PBS- and DTX-treated 2d2.tdRFP Th17-transferred CD11c-DTR/GFP→C57BL/6 mice 2–3 d after onset of the disease. Upper panels: cells were stimulated with plate-bound anti-CD3/anti-CD28, stained for CD4 and cytokines IFN-, IL-17 (pre-gated in addition to FSC/SSC on CD45+CD4+). Lower panels: expression of transcription factor FoxP3; pooled data from three animals, representative for two independent experiments.

(D), (E) Quantification of cytokine expression data as shown in (C) for pooled data from 2 independent experiments; Mann-Whitney-U-Test * p < 0.05.

(F) 2d2 Th17 were transferred into lymphopenic Rag2-/-c-/-. Mononuclear cells were isolated from the spleen and brain at the peak of the disease and analyzed for IL-17 and GM-CSF production after stimulation with anti-CD3/anti-CD28.

(G) GM-CSF production of IL-17 producers versus IL-17 non-producers of CNS-isolated 2d2 Th17 cells (see also (F)). Each dot/square represents a single animal; Kruskal-Wallis-test; * p < 0.05, ** p < 0.01, **** p < 0.0001.
Gatekeeper role of brain antigen-presenting CD11c+ cells in neuroinflammation (2015)
Figure 3

Figure 3

Preferential interaction of CNS CD11c-GFP+ cells with IL-17hi 2d2 Th17 cells at the onset of the disease.

TPLSM of EAE lesions in the brainstem of adoptive transfer EAE at the onset of the disease. EAE was induced by transfer of in vitro differentiated 2d2 Th17 cells (tdRFP-red, IL-17-EGFP-green) into CD11c-DTR/GFP mice (CD11c-GFP - green); imaging area 300 × 300 µm.

(A) Intravascular IL-17hi 2d2 Th17 cell (arrow, double positive -> green: IL-17EGFP and red: 2d2.tdRFP) rolling cell towards a CD11c-GFP+ cell (arrowhead). White dotted lines mark a venous vessel.

(B) Time-lapse TPLSM (maximal intensity projections) of the boxed area in (A) shows that the perivascular elongated CD11c-GFP+ cell (asterisk) enters into contact with IL-17-expressing (GFP – green and 2d2.tdRFP – red double-positive; arrow and arrow head) Th17 cells (scale bar: 20 µm).

(C) XYZ-resolved TPLSM depiction reveals that the elongated perivascular CD11c-GFP+ cell makes contact with the intravascular 2d2 Th17 cells via a filopodium-like dendrite (xy-plane, 300 × 300 µm, z-depth 70 µm).

(D) Strong interaction of a perivascular IL-17hi 2d2 Th17 with a perivascular CD11c-GFP+ cell (insert upper left contact).

(E) Time lapse of the insert in (D): Stopping motility of the round-shaped IL-17hi 2d2 Th17 cell is followed by entry into the CNS parenchyma.

(F) Magnified image of interaction of the upper left and (G) lower right perivascular T cell–DC contacts as shown in (D) and (E).

(H) Time-lapse imaging of parenchymal CD11c-GFP+ cells (arrowhead), targeted migration of an IL-17hi 2d2 Th17 cell (arrow, double positive -> green: IL-17EGFP and red: 2d2.tdRFP) towards the CD11c-GFP+ cell (arrowhead). Scale bar is 20 µm.

(I) Quantification of the motility pattern of IL-17hi 2d2.tdRFP vs. all 2d2.tdRFP in CD11c-DTR/GFP brainstem lesions before and after (2–5 h) intraperitoneal DTX injection. Evaluation of mean track velocity of the single cell tracking (each dot represents a track); pooled data from at least two independent experiments (>3 imaging areas). For the statistical analysis, the Mann-Whitney-U-test was performed (*** p < 0.0001, n.s. = not significant).

(K) Percentage of stopping cells (red bar; instantaneous velocity <2 µm/min) of the data set as shown in (I).
Gatekeeper role of brain antigen-presenting CD11c+ cells in neuroinflammation (2015)
Figure 4

Figure 4

Characterization of CD11c+ subpopulations in the CNS in the course of EAE.

Active EAE was induced by subcutaneous immunization with CFA/MOG35-55. Cells were isolated from the CNS of EAE animals at different stages of the disease (pre-onset [day 8–9], peak [day 14–17], chronic [day 22–27]; data from at least two independent experiments). Representative samples are shown, which were analyzed by flow cytometry for the expression of the markers CD11c, CD11b and Ly6C/G (pre-gated on lympho-monocytic cells by FSC/SSC; IAb+ expression was confirmed for CD11c+ cells in separate stainings).

(A) In the CNS, there was a strong increase of CD11c+ proportions over the different phases of the disease. The composition of CD11c+ subpopulations changed over time, starting with a preponderance of CD11c+Ly6C/G- conventional DCs before onset of EAE. In the peak and chronic phase there was a continuous increase in the proportion of monocyte-derived CD11c+ cells.

(B) For the CNS, the quantification of the absolute numbers shows a strong increase in CD11c+ cells during the course of EAE.

(C) In the CNS, the proportions of the subpopulations show an increasing proportion of monocyte-derived CD11c+ cells.
Gatekeeper role of brain antigen-presenting CD11c+ cells in neuroinflammation (2015)
Figure 5

Figure 5

Chemokine expression profiling of CD11c+ cells in the spleen and CNS reveal a key role of CNS CD11c+ cells for inflammatory chemokines.

(A) CD11c+ cells, CD4+ cells and CX3CR1+CD45intIAb+ (microglia) cells were isolated from peak EAE animals from the CNS. Samples from 6–11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleen CD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). CNS CD11c+ cells show gene expression of the inflammatory chemokines Ccl2, Ccl5, Cxcl9, Cxcl10 but not Ccl20 (mean ± SEM).

(B) CD11c+ cells, CD4+ cells and CD11c-CD11b+IAb+ (monocytes/macrophages) were isolated from EAE affected mice as described in (A). Relevantly regulated chemokines in other cell populations than CNS CD11c+ cells are shown (mean ± SEM).

(C) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the CNS samples as described above and (D) the spleen samples.

Statistical significance was determined using Kruskal-Wallis test. P-values < 0.05 were regarded as statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Gatekeeper role of brain antigen-presenting CD11c+ cells in neuroinflammation (2015)
Figure 6

Figure 6

Cluster formation of DC within the CNS parenchyma at the peak of the disease.

(A) CD11c-GFP+ DC cluster formation near venous vessel with (B) protrusions attracting 2d2.tdRFP Th17 cells. (C) Low-velocity motility of 2d2.tdRFP T cells in DC cluster compared to outside of the cluster. SHG in upper panel on the left (arrow). In addition, intercellular matrix fibers (arrow) are visualized that are only found in inflamed CNS parenchyma and are due to second harmonic generation (SHG) at 1110 nm wavelength excitation and detection at 535 ± 50nm. Tracking of 2d2.tdRFP Th17 in right panel (color-coded tracks, low velocity: red, high velocity: yellow). (D) Quantitative analysis of T cell tracking: higher mean velocity of 2d2.tdRFP Th17 cells in the onset of the disease with differential distribution at the peak of the disease. Cluster associated tracks have significantly lower mean velocities than Th17 cells outside the CD11c-GFP clusters. (E) Analogously for track straightness; Kruskal-Wallis Test and Dunn’s Multiple Comparison Test (*** p < 0.0001) were performed; pooled data from at least three independent experiments per group.
Regulators of autophagosome formation in Drosophila muscles. (2015)
Fig 1

An assay for monitoring autophagy levels in primary cultured muscles.

(A-D) Primary cultures from D. melanogaster embryos expressing GFP-Atg8a under the control of Dmef2-Gal4, stained with Phalloidin (actin) in red. Inset are magnifications of areas denoted by dotted line. (A) In untreated control cultures, GFP-Atg8a localizes throughout the muscle cytoplasm and nuclei. (B) Addition of rapamycin (Rap) for 12 hrs results in the formation of small GFP-Atg8a labeled punctae. (C) Addition of chloroquine (CQ) for 12 hrs results in the accumulation of large GFP-Atg8a labeled vesicles that localize around the nucleus and between the myofibers. (D) Addition of both CQ and Rap for 12hrs, triggers the formation of greater numbers of enlarged vesicles in the muscles than either drug alone. (E) Knockdown of the core autophagy gene, Atg1, prevents the formation of GFP-Atg8a vesicles in CQ+Rap treated cultures. (F) Quantification of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). Error bars indicate SEM with **p<.01; ***p<.001.
Regulators of autophagosome formation in Drosophila muscles. (2015)
Fig 3

Sustained proteasome dysfunction inhibits autophagosome formation.

(A) Primary cultured muscles with control lacZ RNAi, treated for 12 hrs with Rap+CQ. Note the formation of large GFP-Atg8a vesicles throughout the muscles. GFP-Atg8a vesicles are much reduced in primary cultured muscles with Rpn1 (PSMD2) RNAi (B) or Rpt1 (PSMC2) RNAi (C), treated for 12 hrs with Rap+CQ. (D) Quantification for A-C of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). SEM is indicated with **p<.01. (E) Control white RNAi muscles starved and treated with CQ accumulate GFP-Atg8a punctae, but very few colocalize with ubiquitin (Ubi). (F) Knockdown of Rpn1 in starved + CQ muscle strongly inhibits the formation of GFP-Atg8a punctae. The remainder colocalize with ubiquitin. (G) Quantification of the mean GFP-Atg8a punctae area and Mander’s overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in E and F. Error bars indicate SEM for n = 8 ventral longitudinal muscles from individual animals, with p-values for Rpn1 RNAi relative to white RNAi control (***p<.001). (H, I) Autophagosome number is reduced, but autophagic flux is not significantly altered in starved Rpn1 vs. white RNAi muscles expressing GFP-mCherry-Atg8. (J) Flux measurement is shown on the right as the ratio of mCherry punctae to GFP-mCherry (n = 6). SEM is indicated. (K) Anti-Atg8a Western blot of third instar muscle lysate from Dmef2-Gal4; UAS-RNAi animals. In fed white RNAi control, endogenous Atg8a appears as a single band (Atg8a-I). 6 hrs starvation induces lipid modification of Atg8a and the appearance of a second band (Atg8a-II). Knockdown of Atg1 or Rpn1 inhibits Atg8a modification, indicating reduced autophagy levels. In all panels green = GFP-Atg8a. Red = Phalloidin (A-C), anti-ubiquitin (E, F), or mCherry (G, H).
Regulators of autophagosome formation in Drosophila muscles. (2015)
Fig 4

Short-term, but not long-term, proteasome disruption triggers autophagic clearance of aggregates

(A-D) Inhibition of the proteasome by overexpression of a temperature sensitive proteasome mutant (DTS). Dmef2-Gal4, UAS-GFP-Atg8a control and UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a and animals were shifted to the restrictive temperature then starved and CQ treated 6 hrs prior to dissection. 18 hrs post-shift (PS), both control (A) and DTS muscles (B) accumulate GFP-Atg8a punctae, but the latter also accumulate ubiquitin (Ubi) aggregates. 48 hrs PS, control muscles (C) maintain strong autophagy induction, but DTS mutant muscles (D) have reduced GFP-Atg8a punctae accumulation, similar to the Rpn1 knockdown phenotype. (E) Quantification of the mean GFP-Atg8a punctae and Mander’s overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in A-D (SEM is indicated for n = 8 ventral longitudinal muscles from individual animals with p-values of DTS relative to control). Dark green = Dmef2-Gal4, UAS-GFP-Atg8a; Light green = UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a. (F) Western blot analysis of endogenous Atg8a from third instar muscle lysate. 6 hrs starvation induces lipidated Atg8a-II in Dmef2-Gal4 control at both 18 hrs and 48 hrs PS, but not in DTS at 48 hrs PS.
Regulators of autophagosome formation in Drosophila muscles. (2015)
Fig 5

Tyramine (Tyr) treatment reduces autophagy induced by proteasome inhibition.

(A-C) Time course of effect of temperature shift on aggregates, autophagosomes, and larval locomotion in UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a animals starved 6 hrs prior to analysis. (A) The number of ubiquitin aggregates peaks at 18–24 hrs PS, and decreases significantly from 18 to 48 hrs PS (n = 10 and p-values shown relative to 18 hrs PS). (B) The number of GFP-Atg8a punctae peaks at 12–24 hrs PS and decreases significantly from 18 to 48 hrs PS (n = 10 and p-values shown relative to 18 hrs PS). (C) Larval locomotion, measured as the time to crawl 3cm, gradually and significantly worsens from 18 to 48 hrs PS (n = 6 and p-values shown relative to 0 hrs PS. (D-E) tyramine (Tyr) treatment reduces GFP-Atg8a punctae in larval muscle. UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a animals were placed on 100mg/ml Tyr (D) or control food (E), shifted to restrictive temperature for 18hrs, and starved 6 hrs prior to dissection. (F-H) Effect of Tyr treatment on aggregates, autophagosomes, and larval locomotion in UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a and Dmef2-Gal4, UAS-GFP-Atg8a animals, shifted to restrictive temperature for 18hrs, and starved 6 hrs prior to dissection. Aggregate (F) and GFP-Atg8a punctae (G) numbers in muscles are significantly reduced with 100 mg/ml Tyr treatment in DTS but not control animals 18 hrs post-shift (PS). (H) 100 mg/ml Tyr treatment significantly worsens larval locomotion in both control and DTS animals 18 hrs PS. n = 12 and p-values shown relative to non-Tyr treated animals. For all measurements, SEM is shown with *p<.05; **p<.01; ***p<.001. (I) Western blot analysis of endogenous Atg8a from third instar muscle lysate 18 hrs PS. 6 hrs starvation induces lipidated Atg8a-II in Dmef2-G4 control with or without 100 mg/ml Tyr. After 6hrs starvation, Atg8a-II levels are reduced in DTS + 100 mg/ml Tyr relative to untreated DTS.
Regulators of autophagosome formation in Drosophila muscles. (2015)
Fig 6

Rab GTPase-activating proteins and a Rab effector protein are required for autophagy <italic>in vivo</italic>.

(A-C) Protein interactors of ANKFY1 (D.m. gene = CG41099), RABGAP1 and RABGAP1L (D.m gene = CG7112), RAB3GAP1 (D.m. gene = CG31935), and RAB3GAP2 (D.m. gene = Rab3-GAP), identified in mammalian mass spec. Note that each of these proteins interacts with an Atg8a ortholog. D. melanogaster orthologs of other protein interactors of ANKYF1 (D.m. genes Nipsnap and CG7053) were also identified as hits in the primary muscle screen. Based on our RNAi experiments red spheres are assigned to genes/proteins that promote autophagosome formation and/or expansion; blue spheres are assigned to genes/proteins that suppress autophagosome formation and/or expansion. (D-H)Dmef2-Gal4 drives expression of UAS-GFP-Atg8a in animals starved on low nutrient food for 6 hrs + 2.5 mg/ml CQ for 6 hrs. RNAi knockdown of CG41099
(E), CG7112
(F), CG31935
(G), and Rab3-GAP
(H), blocks the formation of GFP-Atg8a labeled autophagosomes compared to knockdown of white (D). (I-M)
Dmef2-Gal4 drives expression of UAS-myc-Atg8a in animals starved on low nutrient food for 6 hrs + 2.5 mg/ml CQ for 6 hrs. The effect of the RAB3GAPs is not via Rab3 or Rab5 function, as a Rab3 null mutation (J), expression of a constitutive active form of Rab3 (Q80L) (K) or Rab5 (Q88L) (L) has no effect on muscle autophagy compared to control (I). Expression of a constitutive active form of Rab39 (Q69L) (M) reduces myc-Atg8a labeled vesicles. (N) Quantification of autophagy phenotypes from panels D-M. SEM is indicated, with n = 8 ventral longitudinal muscles from individual animals and **p<.01; ***p<.001. (O) Atg8a and Rab3-GAP colocalize to autophagosomes. Dmef2-Gal4 drives expression of UAS-myc-Atg8a and UAS-CFP-Rab3-GAP in animals starved and treated with CQ for 6 hrs. In all panels green = GFP-Atg8a and red = myc-Atg8a, and blue = CFP-Rab3Gap2. (P) Anti-Atg8a Western blot of third instar muscle lysate from Dmef2-Gal4 driving the RNAi or overexpression constructs shown in D-N. Total levels of endogenous Atg8a are variable, but only knockdown of CG41099, CG7112, CG31935, Rab3-GAP, or overexpression of Rab39 (Q69L) block induction of lipidated Atg8a-II following 6 hrs starvation.