Centaurin-α₂ interacts with β-tubulin and stabilizes microtubules

Abstract

Centaurin-α₂ is a GTPase-activating protein for ARF (ARFGAP) showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols. Taking into account that Centaurin-α₂ can localize in cytoplasm and that its cytoplasmatic function is not well defined, we searched for further interactors by yeast two-hybrid assay to investigate its biological function. We identified a further Centaurin-α₂ interacting protein, β-Tubulin, by yeast two-hybrid assay. The interaction, involving the C-terminal region of β-Tubulin, has been confirmed by coimmunoprecipitation experiments. After Centaurin-α₂ overexpression in HeLa cells and extraction of soluble (αβ dimers) and insoluble (microtubules) fractions of Tubulin, we observed that Centaurin-α₂ mainly interacts with the polymerized Tubulin fraction, besides colocalizing with microtubules (MTs) in cytoplasm accordingly. Even following the depolimerizing Tubulin treatments Centaurin-α₂ remains mainly associated to nocodazole- and cold-resistant MTs. We found an increase of MT stability in transfected HeLa cells, evaluating as marker of stability the level of MT acetylation. In vitro assays using purified Centaurin-α₂ and tubulin confirmed that Centaurin-α₂ promotes tubulin assembly and increases microtubule stability. The biological effect of Centaurin-α₂ overexpression, assessed through the detection of an increased number of mitotic HeLa cells with bipolar spindles and with the correct number of centrosomes in both dividing and not dividing cells, is consistent with the Centaurin-α₂ role on MT stabilization. Centaurin-α₂ interacts with β-Tubulin and it mainly associates to MTs, resistant to destabilizing agents, in vitro and in cell. We propose Centaurin-α₂ as a new microtubule-associated protein (MAP) increasing MT stability.

Figure 1. Centaurin-α₂ interacts with β-Tubulin.

A) Yeast two-hybrid assay on L40 yeast cotransformed with Tubulin β chain and different baits (pSTT91-Centaurin-α₂, pBTM116-CoRest, pBTM116-laminin or pBTM116-bars. B) Immunoprecipitation of β-Tubulin Centaurin-α₂. Immunoblot of Centaurin-α₂ (HA, upper panel) and of β-Tubulin (β-Tub, lower panel) were performed on total extracts from HeLa cells transfected with pCGN-Centaurin-α₂ (Input), and on extracts immunoprecipitated with anti-HA antibody (IP:HA) and Preimmune serum.

Figure 2. Centaurin-α₂ binds to stable MTs in HeLa cells.

Immunoblots (A) and densitometric analyses (B) of Triton-soluble (S, white bars) and insoluble (I, black bars) fractions of pCGN-Centaurin-α₂ transfected HeLa cells, in control conditions (CONT) or treated with nocodazole (NOC) and cold (COLD). ***p<0.005 vs CONT. C) Cytoskeletal fractions of Hela cells transfected with pCGN-Centaurin-α₂ or with pCGN vector were immonustained with anti-HA (green) and anti-α-Tubulin (red) antibodies. For pCGN-Centaurin-α₂ merged image, the inset (white rectangle) show the discrete distribution of HA spots along microtubules. Scale bar: 20 µm. D) Analyses of the intensity of fluorescence of green signal (HA) and red signal (MTs) in pCGN (white bars) or pCGN-Centaurin-α₂ (black bars) expressing HeLa cells. ***p<0.005 vs pCGN. E) Immunoblots of the total and free (S) or polymerized (I) α-Tubulin (α-Tub) and densitometric analyses of whole cell extracts (F) or Triton-soluble (S, white bars) and insoluble (I, black bars) fractions (G) of pCGN (pCGN) or pCGN-Centaurin-α₂ (pCenta) transfected HeLa cells. **p<0.01 vs pCGN.

Figure 3. Centaurin-α₂ increases MT formation and stability.

A) Comassie Blue stained SDS-PAGE gel of supernatant (S) and pellet (P) fractions of 40 µM Tubulin (Tub), 5 µM Centaurin-α₂ (Centa) or 40 µM Tubulin plus 5 µM Centaurin-α₂ (Tub+Centa) after 90 minutes of polymerization at 37°C. B) Anti-His staining (Green) on in vitro MTs assembled in the absence (Tub) or in the presence of 5 µM Centaurin-α₂ (Tub + Centa), w/o (37°C) or with (37°C→4°C) 30 minutes of incubation on ice. MTs were visualized by DIC microscopy. Scale bar: 2 µm. Comassie Blue stained SDS-PAGE gel (C) and densitometric analyses of tubulin content (D) of supernatant (S, white bars) and pellet (P, black bars) fractions of MTs polymerized at 18 µM tubulin (Cc) or at 40 µM and then destabilized 30 minutes on ice (37°C→4°C), in the absence (Tub) or in the presence of 5 µM Centaurin-α₂ (Tub + Centa). **p<0.02, ***p<0.005 vs Tub. E) densitometric analyses of Centaurin-α₂ associate to supernatant (S, white bars) and pellet (P, black bars) fractions of 5 µM Centaurin-α₂ (Centa) or 40 µM Tubulin plus 5 µM Centaurin-α₂ (Tub+Centa) after 90 minutes of polymerization at 37°C (37°C) or after 30 minutes of incubation on ice (37°C→4°C).

Figure 4. Centaurin-α₂ increases MT acetylation.

Immunoblot (A) and densitometric analyses (B) of acetylated-α-Tubulin (Ac-Tub) and tyrosinated-α-Tubulin (Tyr-Tub) in whole-cells extracts derived from HeLa cells transfected with pCGN (pCGN, white bars) or pCGN-Centaurin-α₂ (pCenta, black bars). *p<0.05 vs pCGN. Immunoblots (C) and densitometric analyses (D) of acetylated-α-Tubulin (Ac-Tub) associated to Triton-soluble (S, white bars) and insoluble (I, black bars) of pCGN (pCGN) or pCGN-Centaurin-α₂ (pCenta) expressing HeLa cells. **p<0.02 vs pCGN.

Figure 5. Centaurin-α₂ promotes the correct assembly of mitotic apparatus.

A) Representative microphotographs of the spindle morphologies observed in pCGN or pCGN-Centaurin-α₂ expressing HeLa cells. B) Quantification of the percentage of cells displaying bipolar (white bars) or aberrant (black bars) spindles, after pCGN (pCGN) or pCGN-Centaurin-α₂ (pCenta) transfection. C) Quantification of the percentage of cells displaying bipolar (white bars) or aberrant (black bars) spindles, after ON-TARGET plus SMART pool siRNA Centa2 or ON-TARGET plus Non-targeting Pool transfection, *p<0.05, **p<0.02, ***p<0.005 vs pCGN. Scale bar = 20 µm.

Figure 6. Centaurin-α₂ decreases the HeLa cells with abnormal centrosome number.

Representative microphotographs (A) and quantification (B) of centrosomes numbers associate to the pCGN (pCGN, white bars) or pCGN-Centaurin-α₂ (pCenta, black bars) expressing HeLa cells, in not dividing and dividing cells. *p<0.05, **p<0.02 vs pCGN. Scale bar = 20 µm.