Transactive response DNA-binding protein 43 is enriched at the centrosome in human cells

Abstract

The centrosome, as the main microtubule organizing centre, plays key roles in cell polarity, genome stability and ciliogenesis. The recent identification of ribosomes, RNA-binding proteins and transcripts at the centrosome suggests local protein synthesis. In this context, we hypothesized that TDP-43, a highly conserved RNA binding protein involved in the pathophysiology of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, could be enriched at this organelle. Using dedicated high magnification sub-diffraction microscopy on human cells, we discovered a novel localization of TDP-43 at the centrosome during all phases of the cell cycle. These results were confirmed on purified centrosomes by western blot and immunofluorescence microscopy. In addition, the co-localization of TDP-43 and pericentrin suggested a pericentriolar enrichment of the protein, leading us to hypothesize that TDP-43 might interact with local mRNAs and proteins. Supporting this hypothesis, we found four conserved centrosomal mRNAs and 16 centrosomal proteins identified as direct TDP-43 interactors. More strikingly, all the 16 proteins are implicated in the pathophysiology of TDP-43 proteinopathies, suggesting that TDP-43 dysfunction in this organelle contributes to neurodegeneration. This first description of TDP-43 centrosomal enrichment paves the way for a more comprehensive understanding of TDP-43 physiology and pathology.

Keywords: ALS; FTLD; TDP-43; centrosome; pericentriolar matrix.

Figure 1

Centrosomal enrichment of TDP-43 in human cells. (A and B) Total internal reflection fluorescence (TIRF) microscopy images of fixed healthy human skin fibroblasts (A), and pseudo-neuronal HeLa, U-87 MG and SH-SY5Y cells (B) immune-stained with antibodies against TDP-43 (10782-2-AP) and pericentrin, showing a co-localization of the two signals when merged. (C) Similar results were obtained in human skin fibroblasts after a 2-h treatment with nocodazole (5 mg/ml) inducing microtubule depolymerization. (D) Western blot performed on purified centrosome fractions from human T-lymphoblastoid KE-37 cells using primary antibodies against TDP-43 (10782-2-AP), γ-Tubulin and Lamin B1, showing an enrichment of TDP-43 at the centrosome. I = insoluble fraction; S = soluble fraction; CTR = centrosome fraction. (E) Immunofluorescence microscopy performed on purified centrosomes (arrowheads) fixed and immune-stained for TDP-43 (60019-2-Ig) and γ-Tubulin showing a co-localization of the two signals.

Figure 2

Cell cycle independent pericentriolar enrichment of TDP-43 in physiological and pathological conditions. (A) Merged images of TIRF microscopy performed on human skin fibroblasts fixed and immune-stained for TDP-43, pericentrin and GT335, showing the presence of TDP-43 at the centrosome in all phases of the cell cycle, including mitosis. (B) Surface area of the TDP-43 centrosomal fraction (μm²) measured at each phase of the cell cycle (ns = not significant). (C) Schematic representation of the different parts of the centrosome, with corresponding protein markers. (D) High magnification TIRF microscopy images of the centrosome of human skin fibroblasts immune-stained with antibodies against TDP-43, centrin, CEP-164 and pericentrin. The co-localization profiles (fluorescence intensity of each channel) shown on the right and the Pearson coefficients of co-localization (E) indicate a pericentriolar enrichment of TDP-43 (P < 0.001). (F and G) Super resolution stochastic optical reconstruction microscopy (STORM) images of human skin fibroblasts immune-stained with antibodies against TDP-43 and centrin (F) or pericentrin (G). (H) TIRF microscopy images of TDP-43 and centrosomal TDP-43 partners associated to familial ALS/FTLD in fixed healthy human skin fibroblasts (magnification at the centrosome) disclosing a co-localization. (I) TIRF microscopy images of fixed human skin fibroblasts derived from sporadic and familial ALS (fALS) patients immune-stained with antibodies against TDP-43 and pericentrin, showing a co-localization of the two signals.