VCP-dependent muscle degeneration is linked to defects in a dynamic tubular lysosomal network in vivo
- University of California, San Francisco, United States
Figures
Figure 1
Lysosomes adopt an extended dynamic tubular array in Drosophila sarcoplasms.
(A) Muscles of third instar larvae from segment A2. (B) Representative live image of Spin-RFP expressed in muscles at 63× magnification. Muscle 4 (B) is shown. DNA was stained with Hoescht. (C) Representative live image of Spin-RFP expressed in muscles using the muscle-specific MHC-Gal4 driver. DNA was stained with Hoescht. (D) Representative image of Spin-GFP localization in a muscle that was fixed with 4% PFA prior to imaging. (E) Time-lapse images of the Spin-RFP network. Time 0 is represented in magenta, and the 5 min time-point is represented in green. The 2 time points were merged to show new and lost tubule formations over the course of 5 min. Arrows indicate examples of de novo tubule formations and the asterisk indicates a retracted tubule. (F) Representative time-lapse sequence of a Spin-GFP tubule fission event. (G) Representative time-lapse sequence of a Spin-GFP tubule fusion event. In the last frame, a de novo tubule can be seen extruding from the middle of a pre-existing tubule. (H–J) Spin-RFP localization in muscles treated with DMSO (H), Nocodazole (I) or LatA (J). (K) Spin-RFP localization in muscles expressing Clathrin heavy chain (Chc) RNAi.
Figure 2
Spin-RFP tubules do not co-localize with mitochondria, ER, golgi or early endosomes.
(A) Co-imaging of Spin-GFP and Lysotracker Red staining. (B) Lysotracker staining of wild type muscles. (C–H) Co-imaging of Spin-RFP with ER tracker (C), Mito tracker (D), YFP-Rab5 (E), Rab11-GFP (F), ManII-GFP (G), GalT-YFP (H).
Figure 3
VCP inhibition disrupts the lysosome tubule lattice and human VCP rescues this defect.
(A) Representative live image of Spin-RFP expressed in muscle using the muscle-specific BG57-Gal4 driver. (B) Live image of Spin-RFP in muscles expressing VCP-RNAi using the muscle-specific BG57-Gal4 driver. (C, D) Live images of Spin-RFP expressed in muscles that were treated with DMSO (C) or the VCP-specific inhibitor DBeQ (D) for 4 hr. (E) Live image of Spin-GFP expressed in muscles using the muscle-specific BG57-Gal4 driver. (F) Live image of Spin-GFP in muscles expressing VCP-RNAi using the muscle-specific BG57-Gal4 driver. (G) Live image of Spin-GFP in muscles that co-express VCP-RNAi and human VCP (hVCP) using the muscle-specific BG57-Gal4 driver. (H–J) Lysotracker staining in wild type (H) muscles or muscles expressing parkin-RNAi (I) or tbph-RNAi (J). (K, L) Spin-RFP localization in muscles treated with DMSO (K) or tunicamycin (TM) (L). (M) Western blot analysis of total Hsc70/BiP protein levels. Tubulin serves as a loading control.
Figure 4
Autophagosomes co-localize with the tubular lysosomal network.
(A) Representative live image of Spin-GFP and mCherry-Atg8a co-expressed in muscles using the muscle-specific BG57-Gal4 driver. (B) Representative time-lapse sequence of Spin-GFP and mCherry-Atg8a in muscle. The arrow follows a mCherry-Atg8a positive puncta trafficking along a Spin-GFP tubule. (C) Spin-GFP and mCherry-Atg8a no longer co-localize in muscles expressing VCP-RNAi. White box indicates region shown at higher magnification and separate channels at right. D. Live image of GFP-mCherry-Atg8a in muscles treated with DMSO for 3 hr. Note the lack of GFP signal. (E) Live image of GFP-mCherry-Atg8a in muscles treated with the V-ATPase specific inhibitor Concanamycin A (ConA) for 3 hr. Note the presence of GFP-positive tubules. (F) Live image of GFP-mCherry-Atg8a in muscles treated with the VCP-specific inhibitor DBeQ for 3 hr. Note the presence of GFP-positive vesicles. (G) Spin-RFP localization in WT muscles or muscles expressing Atg7-RNAi using the muscle specific BG57-Gal4 driver.
Figure 5 with 1 supplement
VCP co-localizes with the tubular auto-lysosomes.
(A) Representative live image of VCP-Venus and Spin-RFP expressed in muscles using the muscle-specific BG57-Gal4 driver. White box indicates region shown at higher magnification and separate channels at right. (B) Representative live image of VCP-Venus and mCherry-Atg8a expressed in muscles using the muscle-specific BG57-Gal4 driver. Inset as in A. (C, D) VCP-Venus and mCherry-Atg8a localization in muscles treated with the VCP inhibitor DBeQ (C) or the proteasome inhibitor MG132 (D) for 3 hr. (E) Western blot analysis of total VCP protein levels from muscles in the treatments indicated. Tubulin serves as a loading control. (F) Representative time-lapse sequence of VCP-Venus and mCherry-Atg8a after MG132 was washed out. The arrow indicates a tubule extending from a mCherry-Atg8a positive vesicle.
Figure 5—figure supplement 1
VCP-Venus localization in Drosophila muscle.
https://doi.org/10.7554/eLife.07366.013
Figure 6 with 2 supplements
Disruption of the tubular auto-lysosomal network correlates with increased poly-Ubiquitin aggregates, impaired mitochondria and increased lipofuscin granules.
(A, B) Wild type (A) and VCP-RNAi (B) expressing muscles were fixed and stained with a poly-Ubiquitin antibody. Nuclei with localized poly-Ubiquitin staining are apparent in A. Nuclei are indicated (dashed circle) in B. (C, D) Wild type animals were treated with DMSO (C) or the VCP-specific inhibitor DBeQ (D), fixed and stained with a poly-Ubiquitin antibody. (E) Quantitation of the number of poly-Ubiquitin aggregates per 50 µm2 from wild type muscles treated with DMSO for 4 hr or DBeQ for various times (n = 9, *p < 0.05, **p < 0.01). (F) Localization of Spin-GFP and poly-Ubiquitin in muscles expressing VCP-RNAi. (G, H) Mitotracker-C2TMRos staining in control (G) and VCP-RNAi (H) muscles. (I–K) Autofluoresence at 488 nm and lysotracker staining in wild type (I), muscles expressing VCP-RNAi (J), and wild type muscles treated with the VCP-specific inhibitor DBeQ for 4 hr (K).
Figure 6—figure supplement 1
Loss of tubular lysosomes correlates with impaired muscle function.
(A) Wild type and VCP-RNAi gross muscle morphology. (B) BG57-Gal4 control animals and animals expressing VCP-RNAi in muscles were assessed for their crawling ability on a petri dish. (C) The total distance traveled in 1 min was measured for each animal and averaged (n = 7, **p < 0.01). (C) Representative traces for wildtype muscles and muscles expressing VCP-RNAi. (D) Quantitation of EPSPs, mEPSPs and resistance input (n = 10, *p < 0.05, **p < 0.01).
Figure 6—figure supplement 2
Lysosomal acidity and Cathepsin processing are maintained in VCP-RNAi expressing muscles.
(A) Spin-GFP co-imaging with lysotracker in muscles expressing VCP-RNAi. (B) Western blot analysis of Cathepsin L processing in the genotypes indicated. Tubulin serves as a loading control.
Figure 7
Pathogenic VCP alleles disrupt the tubular auto-lysosomal network.
(A) Schematic diagram of VCP protein. Top: Human pathogenic VCP mutations are labeled on the cartoon. Bottom: sequence alignment of human VCP and Drosophila VCP/Ter94 pathogenic mutant regions. (B–F) Autofluoresence at 488 nm and lysotracker staining in wild type muscles expressing VCP-WT (B), VCP-RNAi (C) VCP-R152H (D), VCP-R188Q (E) or VCPA-229E (F) transgenes. (G) Quantitation of the number of auto-fluorescent puncta per 50 µm2 in the genotypes indicated (n = 9, *p < 0.05, **p < 0.01).
Videos
Video 1
Spin-RFP tubular network in Drosophila muscle.
Spin-RFP was expressed in muscles and imaged live. Average Z-stacks were assembled to produce a 3D volume projection and various angles of the projections are shown.
Video 2
Spin-RFP tubule dynamics in Drosophila muscle.
Representative time-lapse video of Spin-RFP expressed in muscles. Frames were taken at 10 s intervals.
Video 3
Spin-RFP tubule dynamics in an intact larva.
A whole un-dissected larva was immobilized in a mircrofluidics chamber and Spin-RFP was imaged in the body-wall muscle through the transparent cuticle. Frames were taken at 10 s intervals.
Video 4
Spin-GFP dynamics in muscles expressing VCP-RNAi.
Representative time-lapse video of Spin-GFP in muscles expressing VCP-RNAi. Frames were taken at 10 s intervals.
Video 5
Spin-GFP and mCherry-Atg8a dynamics in Drosophila muscle.
Representative time-lapse video of Spin-GFP and mCherry-Atg8 co-expressed in muscles. Frames were taken at 10 s intervals.
Video 6
VCP-Venus and mCherry-Atg8a dynamics after MG132 wash out.
Muscles co-expressing VCP-Venus and mCherry-Atg8 were treated with the proteasome inhibitor MG132 for 3 hr. MG132 was washed out and time-lapse images were taken every 10 s.
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VCP-dependent muscle degeneration is linked to defects in a dynamic tubular lysosomal network in vivo
eLife 4:e07366.
https://doi.org/10.7554/eLife.07366
