TipC and the chorea-acanthocytosis protein VPS13A regulate autophagy in Dictyostelium and human HeLa cells

Abstract

Deficient autophagy causes a distinct phenotype in Dictyostelium discoideum, characterized by the formation of multitips at the mound stage. This led us to analyze autophagy in a number of multitipped mutants described previously (tipA(-), tipB(-), tipC(-), and tipD(-)). We found a clear autophagic dysfunction in tipC(-) and tipD(-) while the others showed no defects. tipD codes for a homolog of Atg16, which confirms the role of this protein in Dictyostelium autophagy and validates our approach. The tipC-encoded protein is highly similar to human VPS13A (also known as chorein), whose mutations cause the chorea-acanthocytosis syndrome. No member of the VPS13 protein family has been previously related to autophagy despite the presence of a region of similarity to Atg2 at the C terminus. This region also contains the conserved domain of unknown function DUF1162. Of interest, the expression of the TipC C-terminal coding sequence containing these 2 motifs largely complemented the mutant phenotype. Dictyostelium cells lacking TipC displayed a reduced number of autophagosomes visualized with the markers GFP-Atg18 and GFP-Atg8 and an impaired autophagic degradation as determined by a proteolytic cleavage assay. Downregulation of human VPS13A in HeLa cells by RNA interference confirmed the participation of the human protein in autophagy. VPS13A-depleted cells showed accumulation of autophagic markers and impaired autophagic flux.

Keywords: ATG, autophagy related; AX4, axenic strain 4; DUF, domain of unknown function; Dictyostelium; GFP, green fluorescent protein; HeLa cells; LC3, microtubule-associated protein 1 light chain 3; PtdIns3K, phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol 3-phosphate; TipC; VPS13; VPS13, vacuolar protein sorting 13 homolog (S. cerevisiae); VPS13A; WIPI, WD repeat domain, phosphoinoside interacting; autophagy; chorea-acanthocytosis; chorein.

Figure 1.

Autophagic flux is decreased in tipC⁻ and tipD⁻ cells. Proteolytic cleavage assay was performed in strains transfected with the marker GFP-Tkt-1. Protein extracts were analyzed by western blot using anti-GFP antibody. (A) The accumulation of cleaved GFP fragment (black arrows) in the presence of 100 mM NH₄Cl is reduced in tipC⁻ and tipD⁻ mutants compared to the wild-type AX4 strain. (B) tipA⁻ and tipB⁻ levels of cleaved GFP are similar to those of AX4. The complete GFP-Tkt-1fusion protein is marked by white arrows and the stars indicate nonspecific immunoreactive bands.

Figure 2 (See previous page).

The conserved C terminus of TipC largely complements the tipC⁻ phenotype. (A) Scheme of the 3848-amino acid TipC protein and the conserved domains (colored boxes). The line under the scheme depicts the fragment expressed in the complementation experiments. Alignment of the N-terminal (B) and C-terminal (C) regions of D. discoideum TipC (EAL73163.1) and the human VPS13A (NP_150648.2) proteins using the ClustalW algorithm and shaded using the Boxshade tool at the SDSC Biology WorkBench server (http://workbench.sdsc.edu/). Identical residues are shaded black, and similar residues are shaded gray. Colored boxes frame the conserved domains. (D) Western blot using anti-GFP antibody confirmed the presence of a unique band of 150 kDa in the transformed strain with the C-terminal region of TipC (amino acids 2725 to 3848) fused to GFP. Lanes 1 and 3 correspond to protein extracts of AX4 and tipC⁻ that do not express the fused proteins and were used as controls of antibody specificity. (E) The C-terminal TipC polypeptide fused to GFP is localized in the cytoplasm according to in vivo confocal microscopy visualization of transformed cells. (F) The complemented strain rescues fruiting body development. Scale bar: 10 μm. (G) Calcofluor staining shows details of stalk differentiation and spore shape of the different strains. For tipC⁻ mainly cell debris could be visualized. Scale bar: 10 μm. Spore efficiency of each strain (sp. eff.) is indicated. (H) Phagocytosis rate of the wild-type, the mutant, and the complemented strain was measured through the internalized fluorescent signal of cells that were previously incubated with fluorescent beads during the indicated times.

Figure 3.

The pattern of GFP-Atg8 is altered in tipC⁻ and tipD⁻ cells. (A) In vivo confocal analysis of cells expressing GFP-Atg8 in growing and starvation conditions. Puncta formation is inhibited in starved tipC⁻ cells compared with AX4. In contrast, large aggregates of the autophagosome marker are evident in tipD⁻ cells in both growing conditions. (B) Immunofluorescence of ubiquitin in the cells expressing GFP-Atg8 demonstrates the presence of ubiquitin-positive protein aggregates in both mutants. The ubiquitin structures are smaller in tipC⁻ cells and do not always colocalize with GFP-Atg8, while the autophagosome marker is clearly contained in the large tipD⁻ ubiquitinated aggregates. Scale bar: 10 μm.

Figure 4.

GFP-Atg18 puncta signaling is decreased in tipC⁻ cells and accumulates in tipD⁻ cells. (A) Cells expressing GFP-Atg18 were analyzed in vivo by confocal microscopy. Puncta formation is induced under starvation conditions in AX4 cells. However, most of tipC⁻ cells do not display any puncta. Conversely, many puncta are observed tipD⁻ cells regardless of the nutritional conditions. Scale bar: 10 μm. (B) Quantification of GFP-Atg18 puncta of at least 100 cells in blinded images from 3 independent experiments. Results are shown as mean values with standard deviations of the percentage of cells showing the indicated number of puncta. Asterisks indicate the level of significance of the Student t test (* < 0.05; ** < 0.01).

Figure 5.

VPS13A downregulation causes GFP-LC3 and GFP-WIPI1 accumulation. (A) Western blot of control and VPS13A siRNA-transfected cells. A band of the VPS13A-predicted molecular weight decreases in the VPS13A-silenced cells. The asterisks indicate nonspecific immunoreactive bands. (B and C) HeLa cells stably expressing GFP-LC3 were transfected with control or VPS13A siRNAs and the pattern observed by confocal microscopy. VPS13A-depleted cells show accumulated puncta and less translocation from the nucleus to the cytoplasm during starvation. (D and E) HeLa cells were transfected with GFP-WIPI1 and the pattern observed by confocal microscopy. The number of puncta is higher in VPS13A siRNA-treated cells and does not significantly increase following starvation. The graphs show the mean and the standard deviation of puncta quantification of more than 40 cells from 3 independent experiments. Asterisks indicate the level of significance of the Student t test (* < 0.05; ** < 0.01). Scale bar: 10 μm.

Figure 6.

A reduced autophagic flux is observed in VPS13A siRNA-treated cells. (A) GFP-LC3, free GFP, and endogenous LC3-I/-II were analyzed by western blot from control or VPS13A siRNA-treated cells under growth or starvation conditions with or without 5 μM chloroquine (CQ). (B–E) Densitometric quantification showing the mean and the standard deviations of at least 3 independent experiments. The data was normalized against GAPDH. Asterisks indicate the level of significance of the Student t test (* < 0.05; ** < 0.01; *** < 0.001).