Mapping the self-association domains of ataxin-1: identification of novel non overlapping motifs

Abstract

The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) is caused by aggregation and misfolding of the ataxin-1 protein. While the pathology correlates with mutations that lead to expansion of a polyglutamine tract in the protein, other regions contribute to the aggregation process as also non-expanded ataxin-1 is intrinsically aggregation-prone and forms nuclear foci in cell. Here, we have used a combined approach based on FRET analysis, confocal microscopy and in vitro techniques to map aggregation-prone regions other than polyglutamine and to establish the importance of dimerization in self-association/foci formation. Identification of aggregation-prone regions other than polyglutamine could greatly help the development of SCA1 treatment more specific than that based on targeting the low complexity polyglutamine region.

Keywords: Confocal microscopy; FRET; Foci; Misfolding diseases; Spinocerebellar ataxia type 1.

Figure 1. Summary of the ataxin-1 constructs used in the present study.

Full-length ataxin-1 protein is represented by a black line. The positions of the polyQ tract, the AXH domain and NLS are explicitly indicated.

Figure 2. Identifying the region responsible for foci formation in ataxin-1.

Various deletion constructs of ataxin-1 were expressed as YFP fusion proteins in COS cells. (A) Full-length ataxin-1 30Q-YFP. Overlay with DAPI is shown in B. Expression analysis shows a diffused expression pattern in cells expressing ataxin-1 N terminal regions without (C) or with the polyQ tract (D). In contrast, cells expressing the C-terminal regions either without (E) or with (F) the polyQ tract readily formed foci.

Figure 3. AXH domain is not involved in foci formation.

A YFP construct starting at the ataxin-1 AXH domain and ending at the last ataxin-1 amino acid (AXH2End) was expressed as a YFP fusion in COS cells (A and B). These cells showed a diffused YFP fluorescence which was nuclear, as evidenced by DAPI overlay (B). YFP tagged AXH domain also failed to form foci (C and D). Upon N-terminal extension of the AXH2End construct to include further residues starting from amino acids TLND, nuclear foci formation was observed (Figs. 4E and 4F). YFP fluorescence in left panels is overlaid with DAPI in right panels.

Figure 4. Foci forming analysis of the CT2AXH region.

Residues TLND to AXH from ataxin-1 in fusion with YFP forms small foci that are extranuclear (A and B). One cell from the image is enlarged in C for clarity. N terminal extension of this region where the construct started after the polyQ tract (CT2AXH) formed larger foci which also were extranuclear (D and E). Addition of an NLS to this construct resulted in nuclear foci formation (F and G). Deletion of CT2AXH from ataxin-1 resulted in diffused expression of the protein (H and I). A, D, F and H show YFP fluorescence. YFP fluorescence is overlaid with DAPI in B, C, E, G and I.

Figure 5. Analytical size exclusion chromatography.

Analytical gel-filtration chromatograms of AXH (continuous line) and TLND2AXH (dotted line) constructs. The position of molecular weight markers is indicated for comparison. Calculated molecular weights are 13.9 kDa for AXH and 16 kDa for TLND2AXH.

Figure 6. ‘Rainbow’ pseudocolour look-up table (LUT)-encoded pre- and post-bleach images of CFP and YFP fusion proteins.

Magnified crops of both CFP and YFP signals in the bleach region (black circles) are depicted for pre- and post-bleach for each FRET pair (C, F, I, L, O and R). All scale bars are 5 µm. The FRET pairs are, (A–C) CFP-C-Atx1 vs YFP-C-Atx1; (A) CFP fluorescence, (B) YFP fluorescence; (D–F) CFP-C-Atx1 vs YFP-N-Atx1; (D) CFP fluorescence, (E) YFP fluorescence; (G–I) CFP-567-Atx1 vs YFP-wt-Atx1; (G) CFP fluorescence, (H) YFP fluorescence; (J–L) CFP-CT2AXH vs YFP-CT2AXH; (J) CFP fluorescence, (K) YFP fluorescence; (M–O) CFP-AXH vs YFP-AXH; (M) CFP fluorescence, (N) YFP fluorescence; (P–R) CFP-TLND2AXH vs YFP-TLND2AXH; (P) CFP fluorescence, (Q) YFP fluorescence.

Figure 7. Box and whisker plots depicting population distribution of percentage corrected FRET and showing maximum, minimum, upper and lower quartiles, and sample median.

The individual FRET pairs are shown in the X axis. These are: (1) CFP-C–Atx1 vs YFP-C-Atx1; (2) CFP-C-Atx1 vs YFP-N-Atx1; (3) CFP-567-Atx1 vs YFP-wt-Atx1; (4) CFP-CT2AXH vs YFP-CT2AXH; (5) CFP-AXH vs YFP-AXH; (6) CFP-TLND2AXH vs YFP-TLND2AXH. Means ± standard errors, rounded to one decimal place, are shown above each boxplot. Statistical significance bars are shown and represent results of unpaired t-tests of mean difference = 0 and represent number of individual bleach events pooled from at least 4 individual cells.