SPG20 protein spartin is recruited to midbodies by ESCRT-III protein Ist1 and participates in cytokinesis

Abstract

Hereditary spastic paraplegias (HSPs, SPG1-46) are inherited neurological disorders characterized by lower extremity spastic weakness. Loss-of-function SPG20 gene mutations cause an autosomal recessive HSP known as Troyer syndrome. The SPG20 protein spartin localizes to lipid droplets and endosomes, and it interacts with tail interacting protein 47 (TIP47) as well as the ubiquitin E3 ligases atrophin-1-interacting protein (AIP)4 and AIP5. Spartin harbors a domain contained within microtubule-interacting and trafficking molecules (MIT) at its N-terminus, and most proteins with MIT domains interact with specific ESCRT-III proteins. Using yeast two-hybrid and in vitro surface plasmon resonance assays, we demonstrate that the spartin MIT domain binds with micromolar affinity to the endosomal sorting complex required for transport (ESCRT)-III protein increased sodium tolerance (Ist)1 but not to ESCRT-III proteins charged multivesicular body proteins 1-7. Spartin colocalizes with Ist1 at the midbody, and depletion of Ist1 in cells by small interfering RNA significantly decreases the number of cells where spartin is present at midbodies. Depletion of spartin does not affect Ist1 localization to midbodies but markedly impairs cytokinesis. A structure-based amino acid substitution in the spartin MIT domain (F24D) blocks the spartin-Ist1 interaction. Spartin F24D does not localize to the midbody and acts in a dominant-negative manner to impair cytokinesis. These data suggest that Ist1 interaction is important for spartin recruitment to the midbody and that spartin participates in cytokinesis.

Figure 1.

Spastin binds to MIM1 of the ESCRT-III protein Ist1. (A) Top, yeast two-hybrid interactions between the spastin MIT domain bait (residues 110-195) and the indicated ESCRT-III prey constructs were assayed using the HIS3 reporter (sequential 10-fold yeast dilutions are shown). Bottom, amino acid sequence alignment of the C-terminal MIM domains of CHMP1B and Ist1. Hydrophobic residues are in green, positively charged residues are in blue, and negatively charged residues are in red. Conserved residues important for MIM–MIT domain interactions in several other studies are boxed. (B) Spastin MIT bait was tested for yeast two-hybrid interactions as described in A with the indicated Ist1 prey constructs. Boundary amino acid residues are indicated. BD, binding domain. (C) Wild-type spastin and mutant spastin F124D MIT baits were tested for yeast two-hybrid interactions with the Ist1 prey. Schematic diagrams of the Ist1 and spastin (M87 isoform) protein structures are at the top, with amino acid numbers shown. (D) SPR analysis of wild-type or mutant spastin F124D MIT domain (analyte) binding to immobilized Ist1-CTR or CHMP1B-CTR, as shown.

Figure 2.

Spartin MIT domain binds selectively to Ist1. (A) Yeast two-hybrid interactions between the spartin MIT domain bait and the indicated ESCRT-III prey constructs were assayed using the HIS3 reporter (sequential 10-fold yeast dilutions are shown). (B) Spartin MIT bait was tested for yeast two-hybrid interactions as described in A with the indicated Ist1 prey constructs. Boundary amino acid residues are indicated. Schematic diagrams for Ist1 and spartin are shown at the top, with amino acid residues numbered. BD, binding domain. (C) Structural model of spartin MIT domain with Ist1 MIM. Spartin is colored in cyan and Ist1 in orange. Residue Phe24 (F24) in the spartin MIT domain is shown in green. (D) Crystal structure of spastin MIT in complex with CHMP1B, from Yang et al. (2008). Spastin is colored in cyan and CHMP1B in orange. Residue Phe124 (F124) in the spastin MIT domain is colored in green. (E) Structural alignment model for the spartin (cyan) and spastin (orange) MIT domains. Spartin residue F24 is colored in green and spastin residue F124 is in yellow. (F) Wild-type and mutant spartin F24D MIT baits were tested for yeast two-hybrid interactions with Ist1. BD, binding domain. (G) SPR analysis of wild-type or mutant spartin F24D MIT domain (analyte) binding to immobilized Ist1-CTR or CHMP1B-CTR.

Figure 3.

Ist1 localizes to centrosome and midbody during mitosis. (A–C) HA-Ist1 (green) accumulates at the microtubule-organizing center/centrosome during prophase, metaphase (arrowheads), and early anaphase (A), and at the midbody during late cytokinesis (A–C). Corresponding images show the localizations of β-tubulin (red; B) and CEP55 (red; C), and merged images are at the right. A DIC image centered on the midbody is shown in an inset in B. (D) HeLa cells were costained for endogenous Ist1 (green) and β-tubulin (red), with the merged image at the right. Boxed areas are enlarged in the panels below. Bars, 10 μm.

Figure 4.

Spartin localizes to midbodies during cytokinesis. (A) HA-spartin (green) accumulates at the midbody during late cytokinesis. Corresponding images show the localization of β-tubulin (red), and merged images are at the right. The boxed area is enlarged in the panels below. (B) HA-spartin (green) localizes to the midbody, as shown with costaining for CEP55 (red). The merged image is at the right. Boxed area is enlarged in the panels below. (C) HeLa cells were stained for endogenous spartin (green) and β-tubulin (red), with the merged image at the right. Boxed area is enlarged in the bottom panels. Bars, 10 μm.

Figure 5.

Ist1 depletion decreases spartin recruitment to midbodies and impairs cell division. (A) HeLa cells were transfected with control siRNA or else Ist1 siRNA and immunostained for Ist1 (green) or β-tubulin (red). Boxed areas are enlarged in the panels below. Merged images are shown at the right. (B) Ist siRNA cells were imunostained for spartin (green) and β-tubulin (red). There is no spartin at the midbody, as shown in the boxed area enlarged in the panels below. (C) Cell extracts from HeLa cells treated with control or Ist1-specific siRNA were immunoblotted for Ist1. PLCγ levels were monitored to control for protein loading (left). Percentages of control and Ist1 siRNA cells with spartin (middle) or Ist1 (right) present at midbodies are shown (means ± SD of at least three trials, with 100 cells/experiment). (D and E) Multinucleated cells were evident upon Ist1 siRNA treatment of HeLa cells (D). Nuclei were identified by costaining with DAPI. (E) Quantification of the percentage of multinucleated cells after control versus Ist1 siRNA treatment (means ± SD of at least three trials, with 100 cells per experiment). *p < 0.001. Bars, 10 μm.

Figure 6.

Spartin is not required for Ist1 localization to midbodies. (A) HeLa cells were transfected with control siRNA or else spartin-specific siRNA and immunostained for spartin (green) or β-tubulin (red). Boxed areas are enlarged in the panels below. Merged images are shown at the right. (B) Cells treated with spartin siRNA were immunostained for Ist1 (green) and β-tubulin (red). Ist1 is present at the midbody, as shown in the boxed area enlarged in the panels below. A DIC image centered on the midbody is shown in the inset. (C) Cell extracts from HeLa cells treated with control or spartin-specific siRNA were immunoblotted for spartin. GAPDH levels were monitored to control for protein loading (left). Quantification of the percentage of control and Ist1 siRNA cells with spartin (middle) or Ist1 (right) present at midbodies (means ± SD of at least three trials, with 100 cells per experiment). (D–G) Quantifications of cells prepared as described in C are shown for percentages of mitotic cells (D), cells in interphase (E), cells interconnected with a visible midbody (F), and multinucleated cells (G). Means ± SD of at least three trials are shown, with 100 cells per experiment. **p < 0.001. Bars, 10 μm.

Figure 7.

Spartin depletion inhibits the abscission phase of cytokinesis. (A) HeLa cells transfected with control or spartin-specific siRNA were imaged live at the indicated times (in minutes). Bars, 10 μm. (B) Quantification of the time required to complete cytokinesis (12 cells/condition). *p < 0.001. (C) Plot of cleavage furrow diameter over time after anaphase onset (10 cells/condition, means ± SD).

Figure 8.

Spartin F24D does not localize to midbodies and causes dominant-negative impairment in cell division. (A) HA-spartin F24D (green) does not accumulate at the midbody during cytokinesis in HeLa cells. Corresponding images show the localization of β-tubulin (red), and merged images are at the right. The boxed area is enlarged in the panels below. (B) Multinucleated cells, with nuclei identified using DAPI staining (blue), were frequently seen upon HA-spartin F24D expression (green). β-Tubulin staining is shown in red. (C) Quantification of the percentage of multinucleated cells upon expression of empty vector, HA-spartin, HA-spartin F24D, and HA-Ist1 (means ± SD of at least three trials, with 100 cells/experiment). **p < 0.001 for wild type (WT) versus F24D. Bars, 10 μm.