Spastin-interacting protein NA14/SSNA1 functions in cytokinesis and axon development

Abstract

Hereditary spastic paraplegias (HSPs) are a genetically diverse group of inherited neurological disorders (SPG1-72) with the cardinal feature of prominent lower-extremity spasticity due to a length-dependent axonopathy of corticospinal motor neurons. The most frequent form of autosomal dominant HSP results from mutations of the SPG4 gene product spastin. This is an ATPase associated with diverse cellular activities (AAA) protein that binds to and severs microtubules. While spastin participates in crucial cellular processes such as cytokinesis, endosomal tubulation, and axon development, its role in HSP pathogenesis remains unclear. Spastin interacts in cells with the NA14 protein, a major target for auto-antibodies in Sjögren's syndrome (nuclear autoantigen 1; SSNA1). Our analysis of endogenous spastin and NA14 proteins in HeLa cells and rat cortical neurons in primary culture revealed a clear distribution of both proteins to centrosomes, with NA14 localizing specifically to centrioles. Stable NA14 knockdown in cell lines dramatically affected cell division, in particular cytokinesis. Furthermore, overexpression of NA14 in neurons significantly increased axon outgrowth and branching, while also enhancing neuronal differentiation. We postulate that NA14 may act as an adaptor protein regulating spastin localization to centrosomes, temporally and spatially regulating the microtubule-severing activity of spastin that is particularly critical during the cell cycle and neuronal development.

Figure 1. Endogenous NA14 and spastin proteins localize to the centrosome.

(A) Endogenous NA14 and spastin (red) co-localize at the centrosome in HeLa cells, which were co-stained for the centrosomal markers pericentrin or γ-tubulin (green). Merged images are at the right, and boxed areas are enlarged in the insets. Images were acquired using confocal immunofluorescence microscopy, and relative fluorescence intensities for the indicated linear regions in the merged images, measured using Zeiss LSM710 software, are graphed. Note the high degree of line-scan overlap (right). AU, arbitrary units. (B) HeLa cells co-immunostained for NA14 (two different antibodies), spastin and γ-tubulin are shown. Merged images with DAPI nuclear staining are at the right. Boxed areas are enlarged in the upper right-hand corner insets. Scale bar: 10 µm.

Figure 2. NA14 localizes to the centrosome and interacts with the M87 isoform of spastin.

(A) Top, Schematic diagrams showing the domain organizations of spastin isoforms generated through use of 2 different translation start codons (exon 4 splice cassettes are also indicated). Bottom, HeLa cells were transfected with Myc-tagged spastin M1 or spastin M87 and immunoblotted (IB) with Myc-tag antibody. Migrations of molecular weight standards (in kDa) are indicated at the left. (B) Myc-spastin isoforms M1 and M87 (green) were co-stained with pericentrin antibodies (red) and visualized by confocal microscopy. Scale bars: 10 µm. (C) Myc-spastin isoforms M1 and M87 (red) were co-stained with pericentrin antibodies (green) and visualized by confocal microscopy. Relative fluorescence intensities for the indicated linear regions in merged images (boxed areas enlarged in insets at the bottom left) were measured using Zeiss LSM710 software and graphed (right). Scale bar: 10 µm. (D) Relative numbers of HeLa cells accumulating the indicated endogenous or recombinant spastin proteins in the centrosome were quantified (means ±SEM; n = 3, with 100 cells per trial). (E) HeLa cells were transfected with Myc–spastin M1 or M87 as indicated, immunoprecipitated (IP) with NA14 antibodies or non-immune IgG, and immunoblotted (IB) with Myc-epitope antibodies. The input represents 5% of the starting material. Migrations of molecular weight standards (in kDa) are indicated at the left, and the spastin isoform to the right.

Figure 3. Wild-type NA14 but not NA14 (33–119) localizes to the centrosome.

(A) Endogenous NA14 and HA-NA14 from stable cell lines (green) were co-stained with pericentrin antibodies (red) and visualized using confocal microscopy. Hatched boxes are enlarged in the lower right-hand corner insets. (B) HA-NA14 (33–119) stably expressed in HEK293T cells (red) was co-stained with pericentrin antibodies (green). Relative fluorescence intensities for the indicated linear regions in merged images (boxed area enlarged in lower left-hand corner inset) were measured using Zeiss LSM710 software and graphed. AU, arbitrary units. Scale bars: 10 µm. (C) Relative numbers of HeLa cells accumulating the indicated endogenous or recombinant NA14 proteins in the centrosome were quantified (means ±SEM; n = 3, with 100 cells per trial).

Figure 4. NA14 and spastin localize to the centrosome and midbody during cytokinesis.

(A) Merged images of endogenous NA14 (green) accumulated at the centrosome during interphase and at the midbodies during late cytokinesis, along with β-tubulin (red). (B) Endogenous spastin (green) localizes to the centrosome and the midbodies, as shown by co-staining for β-tubulin (red). Merged images are at the right. (C) HeLa cell lines stably expressing control shRNA (shCTL) or shRNAs against NA14 (sh3 shown) were immunostained for endogenous β-tubulin (green) and γ-tubulin (red), with merged images at the right. Bar: 10 µm. (D) Cell extracts from cell lines stably expressing the indicated shRNAs were immunoblotted for NA14. PLCγ1 (149 kDa) and β-tubulin levels were monitored to control for protein loading. # denotes a cross-reacting band. (E and F) Mitotic and multinucleated cells were quantified in control and NA14 shRNA stable cell lines (means ±SEM; n = 3, with 100 cells per experiment). Nuclei were identified by co-staining with DAPI. (G) Quantification of cell death in control and NA14 shRNA stable cells lines by measuring lactate dehydrogenase release from cells (means ±SEM; n = 3, with 100 cells per experiment). *p<0.05; **p<0.01.

Figure 5. NA14 accumulates at the base of axons in cultured cortical neurons at 6DIV.

(A) Mixed cortical neurons at 1, 3 and 6DIV were immunostained for endogenous NA14 (green) and γ-tubulin (red); NA14 localizes to the centrosome at 6DIV. Scale bars: 40 µm. Relative fluorescence intensities for the indicated linear regions in merged images were measured using Zeiss LSM710 software and graphed (at the right). AU, arbitrary units. (B) Neurons at 1, 3 and 6DIV were immunostained for endogenous NA14 (red), neurofilaments (gray) and pericentrin (green). The merged images are indicated. Scale bars: 40 µm.

Figure 6. Morphological analysis of control and NA14-overexpressing cortical neurons.

(A) Representative neurons at developmental stages I, II, and III were co-stained for tau-1 (red) and MAP-2 (green). Merged images are at the right. Scale bar: 40 µm. (B) Pie graphs showing the percentages of cortical neurons in stages I, II, and III in each experimental group (n>100). More neurons remained in stages I and II in untransfected and NA14 (33–119) expressing cultures than in HA-NA14 expressing neurons. (C) β-tubulin staining (black) reveals processes of transfected and control cultured neurons at 3 and 6DIV. Scale bar: 40 µm. (D and E) Quantifications of primary axon length as well as number of primary axon branches in cortical neurons in primary culture (means ±SEM; n = 3, with 30–60 neurons per trial). (F) Numbers of dendrites per cell are shown graphically (means ±SD; n = 3, with 30–60 neurons per trial). *p<0.05, ***p<0.001.