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. 2014 Nov;4(11):140163.
doi: 10.1098/rsob.140163.

Targeting the INCENP IN-box-Aurora B interaction to inhibit CPC activity in vivo

Florence H Gohard  1 , Daniel J St-Cyr  2 , Mike Tyers  3 , William C Earnshaw  4
Affiliations

Targeting the INCENP IN-box-Aurora B interaction to inhibit CPC activity in vivo

Florence H Gohard et al. Open Biol. 2014 Nov.

Abstract

The chromosome passenger complex (CPC) is an essential regulator of mitosis and cytokinesis. The CPC consists of Aurora B kinase, inner centromere protein (INCENP), and the targeting subunits survivin and borealin/Dasra B. INCENP is a scaffolding subunit for the CPC and activates Aurora B via its conserved IN-box domain. We show that overexpression of soluble IN-box in HeLa cells affects endogenous CPC localization and produces a significant increase in multinucleated and micronucleated cells consistent with CPC loss of function. The dominant-negative effect of soluble IN-box expression depends on residues corresponding to hINCENP W845 and/or F881, suggesting that these are essential for Aurora B binding in vivo. We then screened a targeted library of small (five to nine residues long) circular peptide (CP) IN-box fragments generated using split intein circular ligation of proteins and peptides (SICLOPPS) methodology. We identified a number of CPs that caused modest but reproducible increases in rates of multinucleated and micronucleated cells. Our results provide proof of concept that inhibition of the Aurora B-IN-box interaction is a viable strategy for interfering with CPC function in vivo.

Keywords: Aurora B; INCENP; chromosomal passenger complex; cyclic peptide; cytokinesis; mitosis.

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Figures

Figure 1.
Figure 1.
Expression and processing of SICLOPPS in HeLa cells. (a) Translation of SICLOPPS constructs produces a linear precursor that undergoes post-translational splicing to yield a linear product and a circularized peptide or protein (adapted from [30]). (b) Experimental timeline for testing SICLOPPS expression by transient transfection in this study. (c) Immunoblot detection of the SICLOPPS linear precursor (P) and the faster migrating linear product (L).
Figure 2.
Figure 2.
Soluble IN-box expression impairs CPC function. (a) Representation of the CPC highlighting the location of the IN-box (adapted from [6]). (b) Sequence of the soluble IN-box fragment inserted into the SICLOPPS construct containing IN-box residues (green) flanked by native Ssp DnaE extein residues (black). Boxed regions indicate location of mutations made to yield IN-boxW845G, IN-boxF881A, IN-boxdbl and IN-boxAAA constructs. (c) Representative micrographs of DAPI and rhodamine phalloidin-stained puromycin-selected cells transiently expressing soluble IN-boxWT constructs for 48 h. Dotted outlines indicate cells with nuclear morphological aberrations. (d) Quantification of abnormal nuclei frequency (ANF) in the same samples as the previous panel as well as for an Aurora B kinase-dead control. (e) Quantitative western blot detection of the SICLOPPS linear precursor (P) and the faster migrating linear product (L) in samples treated as in (c). Unspecific bands are marked with an asterisk. (f) Quantification of the ANF elicited by SICLOPPSAA IN-box mutant constructs under identical conditions to those outlined in (c). (for (c) and (f): n = 3; more than 1000 cells per replicate; error bars: ±s.e.m.; *** indicates a significance of p < 0.001 between the sample and empty vector control as determined using the χ²-test).
Figure 3.
Figure 3.
Soluble IN-box expression causes Aurora B mislocalization. (a) Representative micrographs showing the localization of Aurora B in puromycin-selected mitotic HeLa cells transiently transfected with either SICLOPPSAA IN-boxWT or an empty vector control 48 h after transfection. DNA, the mitotic spindle and centromeres were detected using DAPI, anti-tubulin and ACA, respectively. (b) Quantification of Aurora B mislocalization during mitotic phases and cytokinesis in samples treated as in the previous two panels. (n = 3; more than 100 cells per replicate). Cells in which Aurora B was clearly visible in the cellular region matching canonical CPC localization [1] were scored as ‘normal’. Faint Aurora B accumulation in the correct region was scored as ‘partial’, as was clear signal only present in part of the region where it should be expected. All other cells were scored as ‘mislocalized’. Coloured boxes have been added around the merged images in panel A to show how these representative cells would be scored. (c) Distribution of cells in different stages of mitosis and cytokinesis in puromycin-selected HeLa cells transiently expressing SICLOPPSAA IN-box constructs for 48 h. (n = 3; more than 100 cells per replicate; error bars: ±s.e.m.; *** indicates a significance of p < 0.001 between the sample and empty vector control).
Figure 4.
Figure 4.
SICLOPPS library design and initial screen. (a) Upper half: conservation of the INCENP IN-box region used to design the library across a range of model organisms. Arrows highlight the residues equivalent to hINCENP W845 and F881. Lower half: coverage of the IN-box core and sub-libraries generated for this study. Each SICLOPPS construct was designed so that the circularized region consisted of an invariant cysteine residue followed by n variable residues based directly on the INCENP sequence. (b) Heat-map indicating the number of preliminary hits found that encompass a given IN-box residue. (c) Ranked and aligned sequences of the preliminary hits identified in the initial library screen comparing the ANF in puromycin-selected samples transiently expressing library constructs for 48 h to that of cells transfected with an empty vector. hINCENP residue numbers are indicated above the sequence of the IN-box fragment; the aligned constructs beneath the sequence are ranked in descending order. Horizontal dotted lines indicate standard deviation thresholds above the ANF found in samples transfected with an empty vector control.
Figure 5.
Figure 5.
(a) Reproducibility and processing dependency of the hits identified in the initial screen. Reproducibility indicates that, when assayed in triplicate, the ANF increase elicited by a given processing-competent SICLOPPS construct was significantly different from that of an empty vector control (p < 0.05, n = 3, Mann–Whitney U test). Processing dependency means that the SICLOPPSAA did not have a significant effect on ANF increase relative to the same control (p > 0.05, n = 3, Mann–Whitney U test). (b) ANF increase of the SICLOPPSWT and SICLOPPSAA versions of reproducible and processing dependent library constructs (n ≥ 3; error bars: ±s.e.m.; *, ** and *** indicate a significances of p < 0.05, <0.01 and <0.001, respectively, between the sample and empty vector control using Mann–Whitney U test). (c) Quantitative western blot detection of SICLOPPS processing in selected processing-competent library constructs. The extent of processing, indicated beneath each lane, is a measure of linear product (L) band intensity divided by the sum of the product and the precursor (P) bands. (d) Correlation between the extent of processing and the ANF elicited of selected IN-box library constructs, as measured by quantitative western blotting and fluorescence microscopy, respectively.
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