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. 2013 Sep;195(1):159-70.
doi: 10.1534/genetics.113.152728. Epub 2013 Jul 5.

Coupling unbiased mutagenesis to high-throughput DNA sequencing uncovers functional domains in the Ndc80 kinetochore protein of Saccharomyces cerevisiae

Jerry F Tien  1 , Kimberly K Fong, Neil T Umbreit, Celia Payen, Alex Zelter, Charles L Asbury, Maitreya J Dunham, Trisha N Davis
Affiliations

Coupling unbiased mutagenesis to high-throughput DNA sequencing uncovers functional domains in the Ndc80 kinetochore protein of Saccharomyces cerevisiae

Jerry F Tien et al. Genetics. 2013 Sep.

Abstract

During mitosis, kinetochores physically link chromosomes to the dynamic ends of spindle microtubules. This linkage depends on the Ndc80 complex, a conserved and essential microtubule-binding component of the kinetochore. As a member of the complex, the Ndc80 protein forms microtubule attachments through a calponin homology domain. Ndc80 is also required for recruiting other components to the kinetochore and responding to mitotic regulatory signals. While the calponin homology domain has been the focus of biochemical and structural characterization, the function of the remainder of Ndc80 is poorly understood. Here, we utilized a new approach that couples high-throughput sequencing to a saturating linker-scanning mutagenesis screen in Saccharomyces cerevisiae. We identified domains in previously uncharacterized regions of Ndc80 that are essential for its function in vivo. We show that a helical hairpin adjacent to the calponin homology domain influences microtubule binding by the complex. Furthermore, a mutation in this hairpin abolishes the ability of the Dam1 complex to strengthen microtubule attachments made by the Ndc80 complex. Finally, we defined a C-terminal segment of Ndc80 required for tetramerization of the Ndc80 complex in vivo. This unbiased mutagenesis approach can be generally applied to genes in S. cerevisiae to identify functional properties and domains.

Keywords: Hec1; Illumina; coiled coil; total internal reflection fluorescence (TIRF).

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Figures

Figure 1
Figure 1
The Ndc80 complex contains Ndc80, Nuf2, Spc24, and Spc25.
Figure 2
Figure 2
Workflow of the linker-scanning mutagenesis screen. (A) An Ndc80 transposition library, containing 15-bp insertions at random locations, was created using MuA transposition. (B) The transposition library was screened in S. cerevisiae using a red/white plasmid shuffle system. Lethal insertions that disrupt the function of Ndc80 appear as nonsectoring red colonies in the screen. From these red colonies, plasmids were isolated and the positions of the lethal insertions were determined by Illumina sequencing.
Figure 3
Figure 3
Lethal insertions were found in distinct clusters in NDC80. (A) Bar diagram showing the positions of notable structural features in Ndc80. (B) Probability of coiled-coil formation as predicted by Paircoil2 (McDonnell et al. 2006). (C) Similarity of Ndc80 protein sequences between select fungal species (see Materials and Methods). (D) Left: the insertion density for the transposition library (blue line) and from the red colonies (red bars) is shown along NDC80. Insertion density is plotted as the number of unique insertion sites within a 21-bp window. Arrows indicate the positions of the representative insertions characterized. Right: from the insertion density plot, the distributions of insertion densities for the transposition library (blue line) and from the red colonies (red bars) are shown. (E) Immunoblot of cell lysate showing that lethal insertion mutants (GFP tagged) are expressed in vivo over the endogenous Ndc80 (untagged). (*) Nonspecific band from the α-Ndc80 antibody. Act1 serves as a loading control. (F) Coomassie-stained gel of recombinant Ndc80 complex containing lethal insertions. Recombinant Ndc80 complexes were purified by affinity chromatography and gel filtration. Mutant Ndc80 complexes migrated similarly to the wild-type complex in the gel filtration column and were collected at the same elution volume. The band for Ndc80 containing the ins1148 mutation reproducibly migrates higher than the wild-type protein.
Figure 4
Figure 4
The ability of the Dam1 complex to enhance binding of Ndc80 complexes to microtubules is abrogated by a lethal insertion in the helical hairpin. (A) Four lethal insertion clusters (red) are mapped onto homologous regions of the human Ndc80 (blue) crystal structure (PDB 2VE7; Ciferri et al. 2008). Lethal insertion clusters are labeled based on their representative insertion. The structure is illustrated with the UCSF Chimera package (Pettersen et al. 2004). (B) Residence time distributions of GFP-tagged Ndc80 complex on microtubules fit with single exponentials (dashed lines) to determine the dissociation rate constants, koff. Top: wild-type Ndc80 complex in the absence (black, n = 622) or presence (red, n = 252) of Dam1 complex. Bottom: ins839 Ndc80 complex in the absence (black, n = 374) or presence (red, n = 357) of Dam1 complex. (C) Plots of mean-squared displacement (MSD) ± SEM vs. time lag for the binding events in B. Linear fits to the data (dashed lines) were used to determine the diffusion constant, D.
Figure 5
Figure 5
A C-terminal segment of Ndc80 controls tetramerization of the Ndc80 complex. (A) Sample images of GFP–Ndc80 lethal insertion mutants colocalized with Nuf2–mCherry at kinetochores in vivo. Endogenous Ndc80 is untagged. Scale bar, 5 µm. (B) The fluorescence intensity of GFP and mCherry are quantified and shown. Intensity is plotted as the mean of fluorescence signal—mean of background signal. Error bars represent SEM. The total numbers of mCherry spots quantified are: wild type (n = 686), ins506 (n = 788), ins656 (n = 760), ins839 (n = 913), ins940 (n = 643), ins1148 (n = 372), ins1687 (n = 576), and ins1957 (n = 389). (C) Immunoprecipitation (IP) of GFP–Ndc80 lethal insertion mutants, and untagged endogenous Ndc80, with Nuf2-TAP or Spc24–TAP. (*) Nonspecific band from the α-Ndc80 antibody. On the immunoblots, input is 0.5% of the clarified lysate and TAP IP is 10% of the total volume.
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