Lysosomal targeting of autophagosomes by the TECPR domain of TECPR2

Abstract

TECPR2 (tectonin beta-propeller repeat containing 2) is a large, multi-domain protein comprised of an amino-terminal WD domain, a middle unstructured region and a carboxy-terminal TEPCR domain comprises of six TECPR repeats followed by a functional LIR motif. Human TECPR2 mutations are linked to spastic paraplegia type 49 (SPG49), a hereditary neurodegenerative disorder. Here we show that basal macroautophagic/autophagic flux is impaired in SPG49 patient fibroblasts in the form of accumulated autophagosomes. Ectopic expression of either full length TECPR2 or the TECPR domain rescued autophagy in patient fibroblasts in a LIR-dependent manner. Moreover, this domain is recruited to the cytosolic leaflet of autophagosomal and lysosomal membranes in a LIR- and VAMP8-dependent manner, respectively. These findings provide evidence for a new role of the TECPR domain in particular, and TECPR2 in general, in lysosomal targeting of autophagosomes via association with Atg8-family proteins on autophagosomes and VAMP8 on lysosomes.Abbreviations: HOPS: homotypic fusion and vacuole protein sorting; LIR: LC3-interacting region; SPG49: spastic paraplegia type 49; STX17: syntaxin 17; TECPR2: tectonin beta-propeller repeat containing 2; VAMP8: vesicle associated membrane protein 8.

Keywords: Autophagy; SPG49; TECPR2; lysosome; neurodegeneration.

Figure 1.

Basal accumulation of autophagosomes in SPG49 patient fibroblasts. (A) Total protein extracts from primary fibroblast cells derived from SPG49 patients (and healthy control) were analyzed by western blot for TECPR2, LC3B and SQSTM1 using corresponding antibodies. The accumulation of autophagosomes under basal conditions is indicated by higher ACTA1/actin-normalized levels of LC3B-II and SQSTM1, which were calculated and presented (lower panel) with the SEM of three independent experiments, *p < 0.05, determined by one-way ANOVA with post-hoc Dunnett’s Multiple Comparison Test. (B) Visual assessment of autophagy markers in primary fibroblasts cells derived from SPG49 patients (and healthy control) upon treatment with 0.1 µM bafilomycin A₁ (Baf. A₁ where indicated) for 4 h. Cells were fixed with methanol, immunostained for LC3B and SQSTM1 and analyzed by confocal microscopy. Scale bar: 20 μm. The accumulation of LC3B and SQSTM1 was calculated as relative intensity (lower panel) and presented with the SEM of three independent experiments, *p < 0.05, determined by one-way ANOVA with post-hoc Dunnett’s Multiple Comparison Test. (C) Primary fibroblast cells derived from SPG49 patients (and healthy control) were grown to confluency in complete medium and treated (where indicated) for the last 4 h with 0.1 µM bafilomycin A₁. Total protein extracts were analyzed by western blotting for TECPR2, LC3B and SQSTM1. Levels of LC3B-II and SQSTM1 with the SEM of three independent experiments, *p < 0.05, determined by one-way ANOVA with post-hoc Dunnett’s Multiple Comparison Test. (D) Primary fibroblast BJ cells were transfected with nontargeting (control) siRNA (siNT) or TECPR2 siRNA using DharmaFECT1 transfection reagent for 72 h and treated (where indicated) for the last 4 h with 0.1 µM bafilomycin A₁. Cells were fixed with methanol, immunostained for LC3B and SQSTM1 and analyzed by confocal microscopy. Scale bar: 20 μm. The accumulation of LC3B and SQSTM1 was calculated as relative intensity (lower panel) and presented with the SEM of three independent experiments, *p < 0.05, determined by one-way ANOVA (analysis of variance) with post-hoc Dunnett’s Multiple Comparison Test

Figure 2.

Starvation rescues autophagosomes accumulation in fibroblasts of SPG49 patients. (A) Visual assessment of autophagy markers, LC3B and SQSTM1, in primary fibroblasts cells derived from SPG49 patients. Cells were maintained in complete medium (“DMEM”) under basal conditions or switched to starvation medium (“EBSS”), or treated with 0.1 µM rapamycin and in addition were treated (where indicate) with 0.1 µM bafilomycin A₁ for 4 h. After 4 h, cells were fixed with methanol, immunostained for LC3B and SQSTM1 and analyzed by confocal microscopy. Scale bar: 20 μm. (B) Primary fibroblasts from SPG49 patients were switched to starvation medium (EBSS) for 4 h with 0.1 µM bafilomycin A₁ (where indicated). Total protein extracts were analyzed by western blotting for TECPR2, LC3B and SQSTM1, and ACTA1/actin-normalized levels of LC3B-II and SQSTM1 were calculated and presented (right panel) with the SEM of three independent experiments, *p < 0.05, determined by one-way ANOVA with post-hoc Dunnett’s Multiple Comparison Test. (C) SPG49 Ex.8 mutant fibroblasts (and fibroblasts from healthy control) were grown to confluence and treated as indicated with 0.1 µM rapamycin and 0.1 µM bafilomycin A₁ (where indicated) for 4 h. Total protein extracts were analyzed by western blotting for TECPR2, LC3B and SQSTM1. Levels of LC3B-II and SQSTM1 were calculated and presented (right panel) with SEM of three independent experiments, *p < 0.05, determined by one-way ANOVA with post-hoc Dunnett’s Multiple Comparison Test

Figure 3.

Replenishment of TECPR2 alleviates basal accumulation of autophagosomes in SPG49 patient fibroblasts in a LIR-dependent manner. (A) Primary fibroblasts of SPG49 Ex.8 mutant patient (and healthy control) were transfected for expression of TECPR2-Flag, TECPR2[ΔLIR]-Flag or empty Flag construct control using JetPrime transfection reagent for 48 h. Cells were then fixed with methanol, immunostained for LC3B and Flag and analyzed by confocal microscopy. Scale bar: 20 μm. The level of LC3B was calculated as relative intensity and presented with the SEM of three independent experiments, *p < 0.05, determined by one-way ANOVA with post-hoc Dunnett’s Multiple Comparison Test. (B) Endogenous TECPR2 was immunoprecipitated from HeLa cells with anti-TECPR2 antibody by protein G beads and co-co-precipitation of LC3B was probed by anti-LC3B antibody. (C) Lysates derived from parental HeLa and LC3B^endoHA cells were immunoprecipitated with anti-HA followed by immunoblot analysis with indicated antibodies

Figure 4.

TECPR2 promotes basal lysosomal consumption of autophagosomes. (A) Primary fibroblasts from SPG49 patients (and healthy control) were treated (where indicated) with 0.1 µM bafilomycin A₁ for 4 h. Cells were fixed with methanol, immunostained with LC3B and LAMP1 and analyzed by confocal microscopy. Scale bar: 20 μm and 2 μm for zoomed images. The colocalization events were calculated manually from at least three independent fields and presented with the SEM, ***p < 0.001, determined by one-way ANOVA with post-hoc Dunnett’s Multiple Comparison Test. (B) Primary fibroblasts from SPG49 Ex. 8 or Ex. 8 + 16 mutant patients (and healthy control) were transfected for mRFP-GFP-LC3B expression using JetPrime transfection reagent for 48 h. Cells were fixed with methanol and analyzed by confocal microscopy. Scale bar of original images: 20 μm and 2 μm for zoomed images. Large magnifications of stained cells are presented. The number of autophagosomes (yellow) and autolysosomes (red) was calculated and presented with the SEM, *p < 0.05, determined by one-way ANOVA with post-hoc Dunnett’s Multiple Comparison Test. (C) Homogenates of HeLa cells were floated over a sucrose gradient as described in Materials and Methods, proteins of collected fraction were immunoblotted for TECPR2, LC3B and VAMP8 and quantified by ImageJ software. (D) Endogenous VAMP8 from HeLa cells was immunoprecipitated with anti-VAMP8 antibody and co-precipitation of TECPR2 was probed by anti-TECPR2 antibody

Figure 5.

The TECPR domain of TECPR2 associates with autophagosomes and lysosomes. (A) Schematic presentation of TECPR2 domains fused to mCherry. (B) HeLa cells were transfected for mCherry-TECPR2 expression using JetPrime reagent for 24 h. Cells were fixed with methanol, immunostained for LC3B and LAMP1 and analyzed by confocal microscopy. Scale bar: 20 μm and 2 μm for zoomed images. (C) Proteins extracted from cells transfected as (B) were floated on a sucrose gradient as described in Materials and Methods, collected fraction proteins were immunoblotted for mCherry, LC3B and VAMP8. (D) HeLa cells were transfected with siRNA targeting VAMP8 and/or STX17, or a non-targeting (siNT) control, using DharmaFECT1 transfection reagent. After 24 h cells were transfected for mCherry-TECPR or mCherry-TECPR[ΔLIR] expression using JetPrime reagent for additional 24 h. Cells were fixed with methanol, immunostained for LC3B and LAMP1 and examined by confocal microscopy. Scale bar: 2 μm. Large magnifications of stained cells are presented. Colocalization of structures positive for mCherry-TECPR2 or mCherry-TECPR2[ΔLIR] with LC3B, LAMP1 or both was quantified manually

Figure 6.

The TECPR domain of TECPR2 is sufficient for alleviating basal accumulation of autophagosomes in SPG49 patient fibroblasts. (A) Schematic presentation of TECPR2 variants fused to Flag tag. (B) Primary fibroblasts of SPG49 Ex.8 mutant were transfected for expression of Flag tag-fused TECPR2 variants (or empty Flag cassette control) using JetPrime transfection reagent for 48 h. Cells were fixed with methanol, immunostained for Flag and LC3B and analyzed by confocal microscopy. Scale bar: 20 μm. The accumulation of LC3B was calculated as relative intensity and presented with the SEM of three independent experiments, *p < 0.05, determined by one-way ANOVA with post-hoc Dunnett’s Multiple Comparison Test. (C) Model – the TECPR domain of TECPR2 mediates autophagosome-lysosome fusion