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. 2016 Jan 7;5:e10147. doi: 10.7554/eLife.10147

Figure 2. Evolution of a novel spindle-orientation function in ancestral GKPID.

(A) Anc-gkdup (circles) is an active nucleotide kinase in a coupled enzyme assay for the reaction shown; Anc-GK1PID (boxes) is inactive. Activity of the human gk enzyme (triangles) is shown for reference. Error bars show SEM for three replicates. (B) The more recent ancestral protein Anc-GK1PID (boxes) binds a 20 amino-acid peptide (see methods) from the Pins protein in a fluorescent anisotropy assay, but Anc-gkdup (cirlcles) does not. Pins binding by the GKPID of the Drosophila melanogaster Dlg protein (triangles) is shown for reference. Error bars show SEM for three replicates. (C–F) Evolution of spindle orientation function as assayed in cultured S2 cells that do not express endogenous Dlg protein. Cells were transfected with a GK construct (C, –control: empty transfection vector; D, + control: GKPID from extant Drosophila Dlg) and scored for alignment of the mitotic spindle (red, tubulin, visualized immunocytochemically) relative to the Pins cortical crescent (green, a GFP-tagged Pins-Ecd fusion). In the example images for each experiment, two cells are shown, the bottom one of which is dividing. The angle of the mitotic spindle relative to a line bisecting the Pins crescent (from 0°, precisely aligned, to 90°) was recorded in many dividing cells; the radial histogram (right) shows the distribution of observed angles among all cells scored with a given genotype. Cells transfected with Anc-gkdup (E) do not display robust spindle orientation, but those transfected with Anc-GK1PID do (F). SEM: Standard error of the mean.

DOI: http://dx.doi.org/10.7554/eLife.10147.009

Figure 2.

Figure 2—figure supplement 1. Properties of ancestral protein AncGK2-PID.

Figure 2—figure supplement 1.

(A) Distribution over sites of posterior probabilities of ML amino acid states in AncGK2PID . Mean posterior probability = 0.87. (B) Anc-GK2PID(squares) binds the Pins peptide ligand with high affinity in a fluorescence anisotropy assay. Binding by the Drosophila melanogaster Dlg GKPID is shown for comparison (circles). Error bars who standard error of the mean of three replicates. (C) Anc-GK2PID is capable of orienting the mitotic spindle.
Figure 2—figure supplement 2. Robustness of functional inferences about ancestral proteins to uncertainty about the sequence reconstruction.

Figure 2—figure supplement 2.

For each of Anc-gkdup and Anc-GK1PID, an alternate reconstruction (Alt-All) was synthesized, containing the next-best amino acid state at all sites with multiple plausible states ((PP>0.2). The enzyme activity of Anc-gkdup in a coupled enzymatic assay for cofactor turnover (panel A) and the Pins-binding activity of Anc_GK1PID (panel B) in a fluorescence anisotropy assay are shown for both maximum likelihood and Alt-All reconstructions. Affinities and maximal velocities differ quantitatively, but the presence/absence of each property is robust to incorporation of uncertainty about the ancestral sequence (see Figure 1F).