Spastic paraplegia proteins spastizin and spatacsin mediate autophagic lysosome reformation
Jaerak Chang, Seongju Lee, Craig Blackstone
Jaerak Chang, Seongju Lee, Craig Blackstone
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Research Article Neuroscience

Spastic paraplegia proteins spastizin and spatacsin mediate autophagic lysosome reformation

Abstract

Autophagy allows cells to adapt to changes in their environment by coordinating the degradation and recycling of cellular components and organelles to maintain homeostasis. Lysosomes are organelles critical for terminating autophagy via their fusion with mature autophagosomes to generate autolysosomes that degrade autophagic materials; therefore, maintenance of the lysosomal population is essential for autophagy-dependent cellular clearance. Here, we have demonstrated that the two most common autosomal recessive hereditary spastic paraplegia gene products, the SPG15 protein spastizin and the SPG11 protein spatacsin, are pivotal for autophagic lysosome reformation (ALR), a pathway that generates new lysosomes. Lysosomal targeting of spastizin required an intact FYVE domain, which binds phosphatidylinositol 3-phosphate. Loss of spastizin or spatacsin resulted in depletion of free lysosomes, which are competent to fuse with autophagosomes, and an accumulation of autolysosomes, reflecting a failure in ALR. Moreover, spastizin and spatacsin were essential components for the initiation of lysosomal tubulation. Together, these results link dysfunction of the autophagy/lysosomal biogenesis machinery to neurodegeneration.

Authors

Jaerak Chang, Seongju Lee, Craig Blackstone

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Figure 7

Autolysosomes in spastizin- or spatacsin-depleted cells are functional.

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Autolysosomes in spastizin- or spatacsin-depleted cells are functional. ...
(A) HeLa cells were transfected with siCTL, siSPG15, or siSPG11 siRNAs. LysoSensor Green DND-189 (1 mM) was added for 1 hour to stain acidic lysosomes. To neutralize acidic lysosomes, bafilomycin A1 (Baf A1, 40 μM) was added in serum-free media for 16 hours before imaging. (B) HeLa cells stably expressing mRFP-GFP-LC3 were transfected with siCTL, siSPG15, or siSPG11 siRNAs. To elevate the lysosomal pH, chloroquine (CQ, 50 μM) was added to siCTL-transfected, serum-deprived cells for 4 hours. Cells were visualized by confocal microscopy, and images from red and green channels were merged. (C) Cells with >20 yellow puncta from B were quantified (n = 3; >200 cells per experiment). (D) Lysates were prepared from control (II-1), SPG15 (II-3), or SPG11 (SPG11-1 and SPG11-2) fibroblasts. HeLa cell lysates were obtained from cells transfected with siCTL, siSPG15, siSPG11, or AP5B1 (siAP5B1-1) siRNAs. Lysates were immunoblotted as shown. Molecular weight standards (in kDa) are shown to the left. Scale bar: 10 μm. Mean ± SD are shown. One-way ANOVA followed by Tukey’s multiple comparison test, ***P < 0.001.