Local synthesis of dynein cofactors matches retrograde transport to acutely changing demands - PubMed (original) (raw)

Local synthesis of dynein cofactors matches retrograde transport to acutely changing demands

Joseph M Villarin et al. Nat Commun. 2016.

Abstract

Cytoplasmic dynein mediates retrograde transport in axons, but it is unknown how its transport characteristics are regulated to meet acutely changing demands. We find that stimulus-induced retrograde transport of different cargos requires the local synthesis of different dynein cofactors. Nerve growth factor (NGF)-induced transport of large vesicles requires local synthesis of Lis1, while smaller signalling endosomes require both Lis1 and p150Glued. Lis1 synthesis is also triggered by NGF withdrawal and required for the transport of a death signal. Association of Lis1 transcripts with the microtubule plus-end tracking protein APC is required for their translation in response to NGF stimulation but not for their axonal recruitment and translation upon NGF withdrawal. These studies reveal a critical role for local synthesis of dynein cofactors for the transport of specific cargos and identify association with RNA-binding proteins as a mechanism to establish functionally distinct pools of a single transcript species in axons.

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Figures

Figure 1. Local protein synthesis mediates NGF-regulated changes in axonal transport.

(a) Representation of a microfluidic chamber used to isolate axons. DRG neurons are seeded in the cell body compartment (green), and the axons extend through two microgroove barriers (blue) into the axonal compartments (orange). All axon-specific treatments were applied to both axonal compartments, and analyses were performed in the distal most compartment. (b–d) DRG neurons were cultured in microfluidic chambers for 3 DIV, at which point the NGF concentration in the axonal chamber was changed to 5 ng ml−1 for 24 h. On DIV 4, axons were pretreated with protein synthesis inhibitors (anisomycin and emetine) or vehicle (dimethylsulphoxide, DMSO) for 2 h before application of medium containing the inhibitors or DMSO and either 5 ng ml−1 NGF (b), no NGF (c), or 100 ng ml−1 NGF (d) and LysoTracker Green for 15 min. Live-imaging time-lapse series of axonal fields were acquired, with images being taken every 13 s for 4 min. Kymographs of representative 100-μm-long axonal segments are shown. Scale bar, 10 μm. LysoTracker-positive particles with diameters ≥1 μm were scored as anterograde, retrograde, bidirectional or stationary. Percentage point differences to baseline condition are plotted. Data represent the means±s.e.m. of nine fields per conditions (_n_=3 biological replicates). *_P_≥0.05; **_P_≥0.01; ***_P_≥0.001. One-way ANOVA with Bonferroni's multiple comparison test. (e) DRG neurons were cultured as in b. After 10 min of different NGF treatments, axonal levels of 4EBP1 and p-4EBP1 were determined by immunofluorescence. Scale bar, 5 μm. Data represent the means±s.e.m. of 15 fields per conditions (_n_=3 biological replicates). *_P_≥0.05; ***_P_≥0.001. Two-way ANOVA with Dunnett's multiple comparison test. (f,g) DRG neurons were cultured and axons were treated with NGF and inhibitors as in b. Puromycin was added to all compartments of the chambers during the NGF treatment period. *_P_≥0.01; ***_P_≥0.001. Two-way ANOVA with Bonferroni's multiple comparison test. Scale bars, 10 μm. NS, not significant.

Figure 2. NGF signalling differentially regulates Lis1 and p150_Glued_ levels in axons.

DRG neurons were cultured and treated as in Fig. 1. (a) Transcripts coding for dynein regulators have been found in transcriptomes derived from embryonic rat DRG axons using microarray, embryonic mouse DRG using RNAseq and embryonic rat hippocampal axons using RNAseq. Transcripts found in all three studies are highlighted in red, and Lis1 and p150_Glued_ are outlined in blue. (b) Pafah1b1 and Dctn1 levels were measured by quantitative FISH in axons kept for 12 h at the baseline NGF level (5 ng ml−1). Background fluorescence was determined using a Gfp probe and subtracted. Means±s.e.m. of 15 optical fields per condition (_n_=3 biological replicates). *_P_≥0.05; **_P_≥0.01. Kruskal–Wallis test with Dunn's multiple comparison test. (c) Axons were pretreated with protein synthesis inhibitors (anisomycin and emetine) or vehicle, followed by exposure to different concentrations of NGF (0, 5 or 100 ng ml−1) for 10 min. Axonal Lis1 levels were measured by quantitative immunofluorescence. Means±s.e.m. of 15–20 optical fields per conditions (_n_=3–4 biological replicates). *P_≥0.05. Two-way ANOVA with Dunnett's multiple comparison test. (d) Neurons were cultured and treated as in b. Axonal p150_Glued levels were measured by quantitative immunofluorescence. Means±s.e.m. of 15 optical fields per conditions (_n_=3 biological replicates). *_P_≥0.05. Two-way ANOVA with Dunnett's multiple comparison test. Scale bars, 5 μm. NS, not significant.

Figure 3. NGF induces local synthesis of Lis1 and p150_Glued_.

DRG neurons were cultured in microfluidic chambers. On DIV 3, the NGF concentration in the axonal chamber was changed to 5 ng ml−1, and axons were selectively transfected with a non-targeting control siRNA or siRNAs targeting Pafah1b1 or Dctn1. (a,b) Twenty-four hours after transfection, axons were treated with 0, 5 or 100 ng ml−1 NGF for 10 min, and Lis1 (a) and p150_Glued_ (b) levels in the cell bodies were determined by immunofluorescence. Means±s.e.m. of 15 optical fields per conditions (n_=3 biological replicates). No significant changes. Two-way ANOVA with Dunnett's multiple comparison test. Scale bars, 20 μm. (c,d) Neurons were cultured and treated as before, and axonal Lis1 (c) and p150_Glued (d) levels were determined by immunofluorescence. Scale bars, 5 μm. Means±s.e.m. of 20–75 optical fields per conditions (_n_=4–15 biological replicates). *_P_≥0.05; **_P_≥0.01; ***_P_≥0.001. Two-way ANOVA with Dunnett's multiple comparison test. See also Supplementary Fig. 1. NS, not significant.

Figure 4. NGF-induced changes in axonal trafficking require local synthesis of Lis1 or p150_Glued_.

DRG neurons were cultured in microfluidic chambers. On DIV 3, the NGF concentration in the axonal chamber was changed to 5 ng ml−1, and axons were selectively transfected with a non-targeting control siRNA or siRNAs targeting Pafah1b1 or Dctn1. (a–c) After 24 h, fresh medium was added to the axonal chamber containing 5 ng ml−1 NGF, no NGF or 100 ng ml−1 NGF together with LysoTracker Green for 15 min. Live-imaging time-lapse series of axonal fields were acquired, with images being taken every 13 s for 4 min. Kymographs of representative 100-μm-long axonal segments are shown. Scale bar, 10 μm. LysoTracker-positive particles with diameters ≥1 μm were scored as anterograde, retrograde, bidirectional or stationary. Means±s.e.m. of 12–18 optical fields per conditions (_n_=3–6 biological replicates). **_P_≥0.01; ***_P_≥0.001. One-way ANOVA with Bonferroni's multiple comparisons test. (d) On DIV 4, axons were treated with 100 ng ml−1 QD-NGF for 15 min and live imaged as above. QD-labelled particles <1-μm diameter were scored as anterograde, retrograde, bidirectional or stationary. Means±s.e.m. of nine optical fields per conditions (_n_=3 biological replicates). **_P_≥0.01; ***_P_≥0.001. Kruskal–Wallis test with Dunn's multiple comparison test. NS, not significant.

Figure 5. Pro-apoptotic signalling from NGF-deprived axons requires axonally synthesized Lis1 and active GSK3β.

(a) DRG neurons were cultured and transfected with siRNAs as in Fig. 4. On DIV 4, the medium in the somatic compartment was changed to NGF-free medium containing NGF-neutralizing antibody, and axonal compartments were changed to 100 ng ml−1 NGF or NGF-free medium with NGF-neutralizing antibody plus vehicle for 24 h. Cell death was assessed by TUNEL assay. Means±s.e.m. of 15–25 optical fields per conditions (_n_=3–5 biological replicates). ***_P_≥0.001. Two-way ANOVA with Dunnett's multiple comparison test. (b) Neurons were cultured and treated as in a. Survival was assessed by calcein AM staining. Means±s.e.m. of 15 optical fields per conditions (_n_=3 biological replicates). ***_P_≥0.001. Two-way ANOVA with Dunnett's multiple comparison test. (c) DRG neurons were cultured as in Fig. 4. On DIV 4, the medium in the somatic compartment was changed to NGF-free medium containing NGF-neutralizing antibody, and the medium in the axonal chamber was changed to 100 ng ml−1 NGF or NGF-free medium with NGF-neutralizing antibody plus the mixed lineage kinase inhibitor, CEP-1347, the p38 MAP kinase inhibitor, SB239063, or the GSK3β inhibitors, LiCl or SB216763, or vehicle for 24 h. Cell death was assessed by TUNEL assay. Means±s.e.m. of 15–25 optical fields per conditions (_n_=3–5 biological replicates). ***_P_≥0.001. Two-way ANOVA with Dunnett's multiple comparison test. Scale bars, 20 μm. NS, not significant.

Figure 6. NGF signalling regulates axonal transcript levels of dynein regulators.

(a–d) DRG neurons were cultured in microfluidic chambers for 3 DIV, at which time the NGF concentration in the axonal chamber was changed to 5 ng ml−1, and axons were selectively transfected with a non-targeting control siRNA or siRNAs targeting Pafah1b1 (a,c) or Dctn1 (b,d). Twenty-four hours after the transfection, the NGF concentration in the axonal chamber was adjusted to 0, 5 or 100 ng ml−1 NGF for 12 h, and cell body Pafah1b1 (a) or Dctn1 (b) or axonal Pafah1b1 (c) or Dctn1 (d) mRNA levels were determined by FISH. Means±s.e.m. of 15–25 optical fields per condition (_n_=3–5 biological replicates). *_P_≥0.05; **_P_≥0.01; ***_P_≥0.001. Two-way ANOVA. Scale bars, 20 μm (a,b); 5 μm (c,d). (e) Neurons were cultured and axons treated with NGF in microfluidic chambers as before. Axonal RNAs were collected after the 12 h NGF treatment, and Pafah1b1 and Dctn1 levels were determined by quantitative real-time RT–PCR. Relative quantification with Gapdh as reference was done using the 2−ΔΔCT method. The means of the 5 ng ml−1 NGF conditions for Pafah1b1 and Dctn1 were defined as 1.0. Means±s.e.m. of 3–5 biological replicates. *_P_≥0.05. Kruskal–Wallis test with Dunn's multiple comparison test. NS, not significant.

Figure 7. Association with APC separates axonally localized Lis1 transcripts into two functionally distinct pools.

(a) Partial sequence of the 3′-UTR of rat Pafah1b1 starting at the stop codon (*). The binding regions of the CUGU and control LNAs are indicated in maroon and grey, respectively. The CUGU element is underlined. (b) Dissociated DRG were transfected with control and CUGU LNA, and 24 h later, APC RNA immunoprecipitation was performed. Pafah1b1 was quantified by RT–PCR. 2−ΔΔCT values are reported relative to Tubb3 (positive control, binds APC but is not targeted by the LNAs). Gfp was included as a control (no reads detected). Means±s.e.m. (_n_=2 biological replicates with two technical replicates each). *_P_≥0.05. _t_-test. (c) DRG neurons were cultured in microfluidic chambers. On DIV 3, the NGF concentration in the axonal chamber was changed to 5 ng ml−1, and cell bodies were selectively transfected with the control or CUGU LNAs. Twenty-four hours after transfection, axons were treated with 0, 5 or 100 ng ml−1 NGF for 12 h, and axonal Pafah1b1 mRNA levels were determined by FISH. Background fluorescence was determined using a Gfp probe and subtracted. Means±s.e.m. of 15 optical fields per condition (_n_=3 biological replicates). *_P_≥0.05. Two-way ANOVA with Fisher's least significant difference test. Scale bar, 5 μm. (d) DRG neurons were cultured and transfected as in a. Twenty-four hours after transfection, axons were treated with 0, 5 or 100 ng ml−1 NGF for 10 min, and axonal Lis1 protein levels were measured by quantitative immunofluorescence. Means±s.e.m. of 20–30 optical fields per conditions (_n_=4–6 biological replicates). *_P_≥0.05. Two-way ANOVA with Fisher's LSD test. (e–g) DRG neurons were cultured and transfected as in a. Twenty-four hours after transfection, transport of LysoTracker-positive particles was observed in axons at baseline NGF (e), without NGF (f) or stimulated with NGF (g). Live-imaging time-lapse series of axonal fields were acquired, with images being taken every 13 s for 4 min. LysoTracker-positive particles with diameters ≥1 μm were scored as anterograde, retrograde, bidirectional or stationary. Means±s.e.m. of nine optical fields per conditions (_n_=3 biological replicates). **_P_≥0.01. One-way ANOVA with Bonferroni's multiple comparisons test. NS, not significant.

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Local synthesis of dynein cofactors matches retrograde transport to acutely changing demands - PubMed (original) (raw)