Figure 4.
Characterization of cytosolic Spc105 oligomers. (A) Representative time-lapse confocal imaging of cells with comparable expression of the mCherry-Cry2 tag alone or tagged Spc105 truncations subjected to the same activation protocol. The average cytosolic mCherry fluorescence intensities are indicated for each circular region. (B) Box and whisker plots of the average cytosolic mCherry fluorescence prior to activation of all the cells that formed clusters in the activation/imaging protocol. (C) Representative confocal time-lapse of 266–384-mCherry-Cry2 clusters (yellow and blue circles) fusing (green circle). (D) Representative confocal time-lapse of optogenetic clustering in a 266–384-mCherry-Cry2-expressing cell being rapidly reversed upon the addition of 3.5% 1,6-HD. (E) Schematic of D. melanogaster Spc105-B highlighting the N-terminal MT-(orange) and PP1-binding (magenta) motifs and the seven regions across Spc105 with a catGRANULE LLPS propensity score above 0 (blue) including the catGRANULE plot of the first 400 aa of Spc105-B with region 266–384 highlighted in the inset showing T-COFFEE alignments of the overlapping KI motifs (Hs: Homo sapiens; Dm: D. melanogaster). (F) Representative confocal Z-plane of a cell coexpressing BubR1-EGFP (green) and oligomerized clusters of the KI motif-containing region from human KNL1 (196–276) fused to FUS-mCherry-Cry2 (magenta). The boxed region is highlighted in the insets. The average fold enrichment for the cell (from five clusters) is indicated in the image. (G) Quantification of the fold enrichment of the signal of BubR1-EGFP relative to the local background signal (266–384-mCherry-Cry2; n = 70 clusters from 14 cells [same data as plotted in Fig. 2 K], HsKI-FUS-mCherry-Cry2; n = 65 clusters from 13 cells). Whiskers indicate min-max, box is 25th–75th percentile, and bar is median in B. Bar plots report mean ± SD in G. Different letters above each column in B indicate significant differences (P < 0.05) determined by a randomization method (PlotsOfDifferences) for all pairwise combinations. The reported P value in G was generated by a two-tailed t test: n.s. is not significant (P > 0.05). Scale bars, 10 μm (A, D, and F); 1 μm (C and insets). Displayed times are min:s.

Characterization of cytosolic Spc105 oligomers. (A) Representative time-lapse confocal imaging of cells with comparable expression of the mCherry-Cry2 tag alone or tagged Spc105 truncations subjected to the same activation protocol. The average cytosolic mCherry fluorescence intensities are indicated for each circular region. (B) Box and whisker plots of the average cytosolic mCherry fluorescence prior to activation of all the cells that formed clusters in the activation/imaging protocol. (C) Representative confocal time-lapse of 266–384-mCherry-Cry2 clusters (yellow and blue circles) fusing (green circle). (D) Representative confocal time-lapse of optogenetic clustering in a 266–384-mCherry-Cry2-expressing cell being rapidly reversed upon the addition of 3.5% 1,6-HD. (E) Schematic of D. melanogaster Spc105-B highlighting the N-terminal MT-(orange) and PP1-binding (magenta) motifs and the seven regions across Spc105 with a catGRANULE LLPS propensity score above 0 (blue) including the catGRANULE plot of the first 400 aa of Spc105-B with region 266–384 highlighted in the inset showing T-COFFEE alignments of the overlapping KI motifs (Hs: Homo sapiens; Dm: D. melanogaster). (F) Representative confocal Z-plane of a cell coexpressing BubR1-EGFP (green) and oligomerized clusters of the KI motif-containing region from human KNL1 (196–276) fused to FUS-mCherry-Cry2 (magenta). The boxed region is highlighted in the insets. The average fold enrichment for the cell (from five clusters) is indicated in the image. (G) Quantification of the fold enrichment of the signal of BubR1-EGFP relative to the local background signal (266–384-mCherry-Cry2; n = 70 clusters from 14 cells [same data as plotted in Fig. 2 K], HsKI-FUS-mCherry-Cry2; n = 65 clusters from 13 cells). Whiskers indicate min-max, box is 25th–75th percentile, and bar is median in B. Bar plots report mean ± SD in G. Different letters above each column in B indicate significant differences (P < 0.05) determined by a randomization method (PlotsOfDifferences) for all pairwise combinations. The reported P value in G was generated by a two-tailed t test: n.s. is not significant (P > 0.05). Scale bars, 10 μm (A, D, and F); 1 μm (C and insets). Displayed times are min:s.

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