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. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: Nat Cell Biol. 2014 Aug 31;16(9):852–863. doi: 10.1038/ncb3030

Figure 8.

Figure 8

Morphological analysis of asters induced by augmin complexes and schematic for augmin organization and function. Augmin complexes and Ran(Q69L)(15 μM) were added to extracts, fixed and processed. (a, b) Two examples of microtubule asters induced by GFP-tagged tetramer-II (a) and GFP-tagged octamer[hDgt6Δ (433–955)] (b) at 60 nM. Defined angular coordinates are indicated (blue and yellow lines). (c, d) Representative images after polar transformation of asters induced by tetramer-II (c) and octamer[hDgt6Δ (433–955)] (d). The microtubule fluorescence intensity along the radial direction (x-axis, where radius=0 corresponds to the center of the aster), is shown for all angles (y-axis, from 0 to 360 degrees). Labelled radial profiles (blue and yellow lines) correspond to those shown in (a) and (b). (e) Normalized angular intensity values at radius= 8 μm in asters induced by teramer-II and octamer[hDgt6Δ (433–955)]. (f) Coefficient of variation (CV, equal to the standard deviation divided by the mean of the intensity) of tetramer-II and octamer[hDgt6Δ (433–955)] induced asters at 4 and 8μm radii. CV was calculated for half-circle (180 degree) regions containing the highest detected microtubule signal. (Tetramer-II, N=27 asters; octamer, N=37 asters; S.D. was determined from data pooled from 3 independent experiments.) (g) Normalized intensity averaged across all angles plotted as a function of radial distance from the aster center. (h) Normalized angular intensity values at radius=8μm in asters induced by Ran-alone, holo-complex (15 nM) and octamer[hDgt6Δ (433–955)] (15 nM). (i) Coefficient of variation calculated from angular intensity values (Ran(Q69L) alone, N=28 asters; holo-complex, N=31 asters, octamer[hDgt6Δ (433–955)], N=35 asters; S.D. was determined from data pooled across from 3 or more independent experiments.) (j) Normalized intensity averaged across all angles plotted as a function of radial distance from the aster center. (k) Schematic for augmin’s subunit organization and function. Direct interaction between subunits is indicated as a red bar. Hice1 and hDgt6 (1–432) form hetero-dimers. Distinct tetrameric complexes have a common Hice1·hDgt6Δ (433–955) core. The six proteins in these two tetramers form a stable hexamer. Together with hDgt5 and hDgt3, these proteins form an octameric complex. Table provides a summary of the results for microtubule interaction in vitro and microtubule aster formation of augmin complexes.