Inhibitor ‐ 3 ensures bipolar mitotic spindle attachment by limiting association of SDS22 with kinetochore ‐ bound protein phosphatase ‐ 1 by Annika Eiteneuer, Jonas Seiler, Matthias Weith, Monique Beullens, Bart Lesage, Veronica Krenn, Andrea Musacchio, Mathieu Bollen, and Hemmo Meyer EMBO J. Volume 33(22): November 18, 2014 ©2014 by European Molecular Biology Organization

Inhibitor ‐ 3 (I3) and SDS22 are required for proper chromosome alignment and progression through mitosis Efficiency of siRNA ‐ mediated depletion of indicated PP1 ‐ interacting proteins shown by Western blot. p97 was probed as a loading control. Annika Eiteneuer et al. EMBO J. 2014;33: ©2014 by European Molecular Biology Organization

Depletion of SDS22 and I3 affect PP1 activity at the kinetochore AIncreased Aurora B autophosphorylation upon SDS22 or I3 depletion. Annika Eiteneuer et al. EMBO J. 2014;33: ©2014 by European Molecular Biology Organization

Depletion of SDS22 and I3 does not affect the amount of PP1γ at the kinetochore PP1γ localization is not affected by SDS22 or I3 depletion. Annika Eiteneuer et al. EMBO J. 2014;33: ©2014 by European Molecular Biology Organization

Depletion of I3 induces re ‐ localization of SDS22 to kinetochores Neither SDS22 nor I3 colocalize with PP1γ at metaphase kinetochores under control conditions. Annika Eiteneuer et al. EMBO J. 2014;33: ©2014 by European Molecular Biology Organization

I3 limits association of SDS22 with KNL1 ‐ bound PP1 SDS22 localizes to the outer kinetochore upon I3 depletion (see also Supplementary Fig S4). Annika Eiteneuer et al. EMBO J. 2014;33: ©2014 by European Molecular Biology Organization

Forced localization of SDS22 to kinetochores by overexpression inhibits PP1 ‐ mediated dephosphorylation of Aurora B ASDS22 recruitment to kinetochores depends on PP1 binding. Annika Eiteneuer et al. EMBO J. 2014;33: ©2014 by European Molecular Biology Organization

Binding of SDS22 to PP1 inhibits dephosphorylation of Aurora B in vitro APP1 specifically dephosphorylates Aurora B pT232. Annika Eiteneuer et al. EMBO J. 2014;33: ©2014 by European Molecular Biology Organization

Depletion of SDS22 or I3 interferes with inactivation of Aurora B at centromeres and chromosome segregation during anaphase A, BI3 depletion induces SDS22 localization to kinetochores also in anaphase. Annika Eiteneuer et al. EMBO J. 2014;33: ©2014 by European Molecular Biology Organization

Model figure for the role of SDS22 and I3 in the regulation of kinetochore ‐ bound PP1PP1 dynamically exchanges between a KNL1 ‐ bound state at the kinetochore and a SDS22 ‐ I3 ‐ bound state in solution. Annika Eiteneuer et al. EMBO J. 2014;33: ©2014 by European Molecular Biology Organization