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LAMP-2 is required for incorporating syntaxin-17 into autophagosomes and for their fusion
with lysosomes
Virginie Hubert1, Andrea Peschel1, Brigitte Langer1, Marion Gröger2, Andrew Rees1, and
Renate Kain1
1Clinical Institute of Pathology, Medical University of Vienna, Austria
2Core Facilities, Medical University of Vienna, Austria
Authors for correspondence:
Name: Virginie Hubert
Address: Clinical Institute of Pathology, Medical University of Vienna, Währinger Gürtel 18-
deficiency severely reduces STX17 expression on autophagosomes and this provides a
potential mechanism for their failure to fuse with lysosomes.
LAMP-2 deficiency prevents SNAP-29 localisation to autophagosomes
SNAP-29 complexes with STX17 on the surface of autophagosomes and enhances fusion by
binding VAMP8 on lysosomes. 30, 52, 59 SNAP-29 positive puncta were rare in wild-type MEF
under resting conditions and did not co-localize with LC3 (Fig 6A, Fig 6B) (Table S11).
After induction of autophagy, their number increased markedly and co-localization with LC3
was obvious (Fig. 6C) whereas co-localization with LAMP-1 was rare (Fig. 6D). By contrast,
SNAP-29 positive puncta were significantly more common in LAMP-2 deficient MEF under
basal conditions but were unaffected by rapamycin treatment (Fig. 6A, Fig. 6B). SNAP-29
did not co-localize with LC3 (Fig. 6E) but occasional co-localization with LAMP-1 was
observed and slightly increased after induction of autophagy (Fig. 6F) as confirmed by
quantification of co-localization. By measuring co-localization we also confirmed that the
overlapping of SNAP-29 with LC3 was decreased non-significantly in the absence of LAMP-
2 (Mander’s overlapping coefficient: 0,0770,023 and 0.0550,028 in wild-type treated with
FCS and rapamycin-0.0320.017 and 0.0340.038 in LAMP-2 deficient cells pre- and post-
rapamycin treatment). Thus, autophagosomes in LAMP-2 deficient MEF fail to recruit
SNAP-29 as well as STX17.
VPS33A does not co-localize with LC3 in LAMP-2 deficient MEF
The HOPS complex also binds to STX17 on autophagosomes and tethers them to Rab7 on
lysosomes 33, 39 prior to fusion. 58 Accordingly, we investigated the influence of LAMP-2
deficiency on Rab7 and the HOPS complex subunit Vacuolar protein sorting-associated
protein 33A (VPS33A). The three cell lines contained similar numbers of Rab7 positive
puncta (Fig. S8A, Fig. S8B) that also expressed LAMP-1 (Fig. S8C, Fig. S8D); Rab7 did not
co-localize with EGFP-LC3 (Fig. S8E, Fig. S8F). As previously reported, Rab7 positive
vacuoles were restricted to the perinuclear regions of wild-type and LAMP-2 deficient MEF 14, 30 but were more diffusely distributed in the LAMP-1/2 doubly deficient cells (Fig. S8A).
Thus, Rab7 retains its normal expression in lysosomes/late endosomes in LAMP-2 singly
deficient MEF.
Under resting conditions, VPS33A partially co-localized with LAMP-1 (Fig. 7A) and had a
similar perinuclear distribution in wild-type and LAMP-2 deficient MEF (Fig. 7B). In wild-
type MEF, treatment with rapamycin induced a partial redistribution of VPS33A from
LAMP-1 expressing vacuoles to LC3 positive autophagosomes (Fig. 7C) but this did not
occur in LAMP-2 deficient MEF (Fig. 7D) in which co-localization of VPS33A and LC3 was
reduced, albeit not significantly (Mander’s overlapping coefficient: 0.1150.075 and
0.1050.080 in wild-type treated with FCS and rapamycin-0.0700.063 and 0.0820.042 in
LAMP-2 deficient cells pre- and post-rapamycin treatment). By contrast, treatment with
rapamycin increased co-localization between VPS33A and LAMP-1 in the LAMP-2 deficient
cells (Fig. 7E) This provides further confirmation of the functional consequences of the lack
of STX17 on autophagosomes.
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Sup. Figure 1: Model for autophagosome-lysosome fusion. Tethering of the autophagosome with the lysosomes is mediated by the interaction of the Rab7 effector RILP which respectively binds the HOPS complex. The autophagosomal SNARE syntaxin 17 binds HOPS and mediates fusion with the lysosome through its interaction with SNAP-29 and lysosomal VAMP8. Further stabilization of the syntaxin 17-SNAP-29 complex is achieved by Atg14.
Biology Open (2016): doi:10.1242/bio.018648: Supplementary information
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Sup. Figure 2: Characterization of cell lines. (A) Immortalization of the three cell lines by insertion of the SV40 large T antigen was confirmed by immunohistochemistry. Scale bar = 100µm. (B) LAMP-2 is expressed in wild type MEF while LAMP-1 is expressed in wild type and LAMP-2 deficient cells but not in doubly deficient fibroblast. Scale bar = 20µm. (C) Western blot analysis of total cell extracts confirm the absence of LAMP-2 expression in single and double knock out while LAMP-1 was not expressed in LAMP-1/2 -/- fibroblasts (D). A Pearson’s correlation coefficient confirms the high degree o f colocalization of EGFP with mRFP and thus the efficiency of the construct. Datas are expressed as mean±SD of at least 10 cells.
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Sup. Figure 3: The autophagic flux is impaired in LAMP-2 single and LAMP-1/2 doublydeficient cells after autophagy induction. Autophagy induction in wild type MEF is reflected by an increased number of autophagosomes and autolysosomes while in LAMP-2 single deficient and LAMP-1/2 double deficient cells the number of autolysosomes remains unchanged after treatment with rapamycin (A) or HBSS (B). The number of LC3 dots was quantified from at least 25 cells in each condition and is representative of 3 independent experiments. Datas are expressed as mean±SD. *, P<0.05; **, P<0.01***; P<0.001; ****, P<0.0001 (Mann Whitney test).
Biology Open (2016): doi:10.1242/bio.018648: Supplementary information
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Sup. Figure 4: Complementation of LAMP-2 and LAMP-1/2 deficient cells with LAMP-2A restores CMA. Wild type (A), LAMP-2 deficient (B) and LAMP-1/2 deficient (C) cells were transfected with the photoswitchable reporter pKFERQ-PS-CFP2 able to monitor CMA (B). While CMA was efficiently induced in wild type MEF, LAMP-2 single and LAMP-1/2 doubly deficient cell were unable to conduct it. Scale bar = 5µm. Complementation of the deficient cell lines with LAMP-2A restores CMA (D). ). Scale bar = 10µm.
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Sup. Figure 5: Autophagic flux is not restored by LAMP-2B. Autophagic flux remains altered inLAMP-2 deficient MEF reconstituted with LAMP-2B (A) both after treatment with rapamycin (B) or HBSS (C). Scale bar = 10µm. Datas are expressed as mean±SD of 3 independent experiments. *, P<0.05; **, P<0.01***; P<0.001; ****, P<0.0001 (Mann Whitney test).
Biology Open (2016): doi:10.1242/bio.018648: Supplementary information
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Sup. Figure 6: VAMP8 expression and localization is not altered in the absence of LAMP-2. The number of VAMP8 positive puncta is similar in the three cell lines before and after autophagy induction with rapamycin or HBSS for 4h00 (A). VAMP8 co-localized with LAMP-1 (B-C) but not with EGFP-LC3 (D-E) indicating its lysosomal localization both in LAMP-2 sufficient (B and D) or deficient cells (C and E). Scale bar = 10µm. Immunoelectron microscopy confirms thelocalization of VAMP8 in the lysosomes in the three cell lines after treatment with HBSS (F) (Scale bar = 500nm) as well as its co-localization with LAMP-1 particles (G) (Scale bar = 200nm). Datas are expressed as mean±SD from at least 15 cells in each conditions and is representative of 3 independent experiments.
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Sup. Figure 7: Intact lysosome like structures are observed in the autophagosomes of LAMP-2 single and LAMP-1/2 doubly deficient MEF. A VAMP8/LAMP-1 ratio was established by counting positive particles per vesicles and was similar in both cell lines, independently of the cell treatment (A). Under rapamycin treatment, undigested VAMP8 (B) lysosome like structures were observed in the autophagosomes of LAMP-2 and LAMP-1/2 deficient fibroblast while fusion of the autophagosome with the lysosomes was observed in wild-type. The ratio STX17 particles/LAMP-1 particles was increased in LAMP-2 deficient cells under autophagy induction with rapamycin (C). The presence of STX17 positive lysosome like structures (D) in LAMP-2 single deficient and LAMP-1/2 double deficient cells was observed and associated with an increased ratio of STX17 particles/LAMP-1 particles in these vesicles (E). This ratio was similar in both cell lines when quantification was performed uniquely in normal vesicles (F). Scale bar = 500nm. P<0.01*** (Mann Whitney test).
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Sup. Figure 8: The number of Rab7 positive vesicles is similar in the three cell lines despite an altered distribution in LAMP-1/2 deficient cell switching form perinuclear to diffuse staining (A-B). Scale bar = 20µm. The number of Rab7 positive vesicles was quantified from at least 15 cells in each condition and is representative of 3 independent experiments. In wild-type (C) and LAMP-2-/- cell (D) Rab7 co-localized with LAMP-1 but not with EGFP-LC3 (E-F). Scale bar = 10µm.
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Sup. Figure 9: Blockage of autophagosome-lysosome fusion in LAMP-2 deficient cells. In these cells, STX17 does not translocated to the autophagosome and therefore cannot be bound by the HOPS complex. The absence of binding between HOPS and STX17 prevents the tethering and the fusion of the autophagosome with the lysosome.
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Parameter quantified Cell Treatment Value expressed as mean± SD
CMA Wild type HBSS 8.429±3.625
CMA LAMP-2 -/- HBSS 3.050±1.504
CMA LAMP-2 -/- transfected with LAMP-2A HBSS 9.909±4.323
Sup. Table 2: Confirmation of the ability of LAMP-2A to restore CMA. Cells were co-transfected with the CMA reporter pKFERQ-PS-CFP2 and LAMP-2 followed by quantitation of CMA.
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Parameter quantified Cell Treatment Value expressed as mean± SD
Macroautophagy Autophagosome
Wild type Control media 14.524±12.135
Macroautophagy Autolysosome
Wild type Control media 12.810±10.083
Macroautophagy Autophagosome
Wild type Rapamycin 13.6±6.597
Macroautophagy Autolysosome
Wild type Rapamycin 22.4±10.515
Macroautophagy Autophagosome
LAMP-2 -/- Control media 6.4±2.746
Macroautophagy Autolysosome
LAMP-2 -/- Control media 6.333±3.374
Macroautophagy Autophagosome
LAMP-2 -/- Rapamycin 11.875±5.045
Macroautophagy Autolysosome
LAMP-2 -/- Rapamycin 5.25±3.941
Macroautophagy Autophagosome
LAMP-2 -/- transfected with LAMP-2A
Control media 6.765±4.452
Macroautophagy Autolysosome
LAMP-2 -/- transfected with LAMP-2A
Control media 8±5.831
Macroautophagy Autophagosome
LAMP-2 -/- transfected with LAMP-2A
Rapamycin 11.316±6.183
Macroautophagy Autolysosome
LAMP-2 -/- transfected with LAMP-2A
Rapamycin 12.053±4.365
Macroautophagy Autophagosome
LAMP-2 -/- transfected with LAMP-2B
Control media 5.867±3.159
Macroautophagy Autolysosome
LAMP-2 -/- transfected with LAMP-2B
Control media 4.733±2.604
Macroautophagy Autophagosome
LAMP-2 -/- transfected with LAMP-2B
Rapamycin 11.353±5.373
Macroautophagy Autolysosome
LAMP-2 -/- transfected with LAMP-2B
Rapamycin 5.059±4.723
Sup. Table 3: Quantitative evaluation of macroautophagy using the tfLC3 construct in wild-type or LAMP-2 deficient cells reconstituted or not with LAMP-2A or LAMP-2B, following treatment with rapamycin.
Biology Open (2016): doi:10.1242/bio.018648: Supplementary information
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Parameter quantified Cell Treatment Value expressed as mean± SD
Macroautophagy Autophagosome
Wild type Control media 14.524±12.135
Macroautophagy Autolysosome
Wild type Control media 12.810±10.083
Macroautophagy Autophagosome
Wild type HBSS 14.923±10.423
Macroautophagy Autolysosome
Wild type HBSS 22.962±12.571
Macroautophagy Autophagosome
LAMP-2 -/- Control media 6.4±2.746
Macroautophagy Autolysosome
LAMP-2 -/- Control media 6.333±3.374
Macroautophagy Autophagosome
LAMP-2 -/- HBSS 15.188±6.102
Macroautophagy Autolysosome
LAMP-2 -/- HBSS 7.313±3.807
Macroautophagy Autophagosome
LAMP-2 -/- transfected with LAMP-2A
Control media 6.765±4.452
Macroautophagy Autolysosome
LAMP-2 -/- transfected with LAMP-2A
Control media 8±5.831
Macroautophagy Autophagosome
LAMP-2 -/- transfected with LAMP-2A
HBSS 10.375±5.303
Macroautophagy Autolysosome
LAMP-2 -/- transfected with LAMP-2A
HBSS 15.938±4.959
Macroautophagy Autophagosome
LAMP-2 -/- transfected with LAMP-2B
Control media 5.867±3.159
Macroautophagy Autolysosome
LAMP-2 -/- transfected with LAMP-2B
Control media 4.733±2.604
Macroautophagy Autophagosome
LAMP-2 -/- transfected with LAMP-2B
HBSS 8.4±4.867
Macroautophagy Autolysosome
LAMP-2 -/- transfected with LAMP-2B
HBSS 8.467±6.010
Sup. Table 4: Quantitative evaluation of macroautophagy using the tfLC3 construct in wild-type or LAMP-2 deficient cells reconstituted or not with LAMP-2A or LAMP-2B, following treatment with HBSS.
Biology Open (2016): doi:10.1242/bio.018648: Supplementary information
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Parameter quantified Cell Treatment Value expressed as mean± SD
STX17+ vesicles Wild type Control media 10.267±6.713
STX17+ vesicles Wild type Rapamycin 19.200±5.846
STX17+ vesicles LAMP-2 -/- Control media 7±6.245
STX17+ vesicles LAMP-2 -/- Rapamycin 6.692±4.111
STX17+ vesicles LAMP-2 -/- transfected with LAMP-2A Control media 9.765±3.784
STX17+ vesicles LAMP-2 -/- transfected with LAMP-2A Rapamycin 13.235±8.467
Sup. Table 8: Quantitative evaluation of the number of STX17 positive vesicles in wild-type, LAMP-2 deficient and LAMP-2 deficient cells reconstituted with LAMP-2A
Biology Open (2016): doi:10.1242/bio.018648: Supplementary information
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