Figure 1.
Expression patterns of ataxin-1 fusion proteins.
Figure 1A. Distribution of ataxin-1 and ataxin-1-GFP. Upper panel; untagged ataxin-1 stained with 1C2 with a DAPI merge; lower panel, GFP-ataxin-1 with DAPI merge. Figure 1B. DsRed1-E5-ataxin-1 emits both red (r) and green (g) fluorescence. The merged red green and red/DAPI fluorescence micrographs are also shown. An orthogonal slice through a reticular IB seeded by DsRed1-E5-ataxin is shown lower right. Scale bars: 8 microns. Figure 1C. IBs of DsRed1-E5-ataxin express protein of comparable age. Synchronous peaks and troughs of red/green fluorescence emitted by IBs of DsRed1-E5-ataxin in shared nuclei. IBs are ranked according to maturity; with high g/r ratios indicating immature protein. Figure 1D. Synchronous seeding of DsRed1-E5-ataxin IBs. HeLa nuclei were ranked according to mean g/r ratio of their IB populations (24hr after transfection). 169 IBs in 16 nuclei were included in this data set.
Figure 2.
IB properties and lethality of ataxin-1 fusion proteins.
Figure 2A. Distribution of DsRed1-E5-PML and dual transfected ataxin-1 fusions. LH panel; puncta of DsRed1-E5-PML reveal heterogeneous red/green fluorescence (arrowed) in a HeLa transfectant. Ne denotes nuclear envelope. Scale bar: 8 microns. RH panel; cytoplasmic enrichment of aged DsRed1-E5-ataxin in dual transfectants. Green and red indicates the wavelengths scanned in each micrograph, with DAPI co-stain. Scale bar: 15 microns. Figure 2B. Red/green fluorescence in single and co-transfectants of ataxin-1 fusion proteins. LH panel; GFP-ataxin-1; Middle panel; DsRed1-E5-ataxin-1; RH panel; dual transfectants. Figure 2C. Enriched red fluorescence in co-transfectants occurs in the region indicated. Figure 2D. Dye uptake shows increased lethality of DsRed1-E5-ataxin-1. Increased DAPI uptake is seen in DsRed1-E5-ataxin-1 versus GFP-ataxin-1 and Dual transfectants. Figure 2E. Cell proliferation assay shows reduced viability in DsRed1-E5-ataxin-1 transfectants. MTT based assay of DsRed1-E5 (Ds), GFP (G) and dual (x2) transfectants, comparing ataxin-1 expressing both normal [Q30] and expanded polyQ repeats [Q82]. Figure 2F. PML-NDs are sequestered by IBs of DsRed1-E5-ataxin-1. Histogram of red/green fluorescence across a DsRed1-E5-ataxin-1 transfectant, stained with N19. Spikes of red fluorescence (darker line) denote PML-NDs tethered to IBs in a single nucleus. Figure 2G. PML/DsRed1-E5-ataxin-1 sequestration. Scale bar: 8 microns. Inset; ataxin-1/PML sequestration captured by immunofluorescent staining of endogenous PML (red) with GFP-ataxin-1. Scale bar: 15 microns. Figure 2H. Mobility of DsRed1-E5-ataxin-1 IBs revealed by time-lapsed confocal microscopy. 9-section Z series were collected every 5 minutes for an hour. The four frames shown indicate fusion events over twenty minutes in a single optical section. Multiple fusion events occur within the boxed region. The arrowed IB also tracks towards this region. The disordered fluorescence at the extremities of the nucleus corresponds to cytoplasmic material.
Table 1.
Green/Red Fluorescence ratios in IBs of DsRed1-E5-ataxin-1 IBs.
Table 2.
Non-linear relationship between g/r ratio and IB diameter.