The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction

KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of Drosophila melanogaster KNL1 (Spc105) has never been shown to bind MTs or to recruit PP1. Furthermore, the phosphoregulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in D. melanogaster. Here, these apparent discrepancies are resolved using in vitro and cell-based assays. A phosphoregulatory circuit that utilizes Aurora B kinase promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag and deletion/chimera mutants are used to define the interplay of MT and PP1 binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction.

10.1091/mbc.E21-06-0307-T

1059-1524

Audett, M.R.

9019031b-6c62-44ba-a12c-a970a2bc3834

Johnson, E.L.

d2f46649-9ac7-4cea-a0c3-f71a344ce6b2

McGory, J.M.

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Barcelos, D.M.

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Szalai, E.O.

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Przewloka, M.R.

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Maresca, T.J.

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1 January 2022

Audett, M.R.

9019031b-6c62-44ba-a12c-a970a2bc3834

Johnson, E.L.

d2f46649-9ac7-4cea-a0c3-f71a344ce6b2

McGory, J.M.

e0fe921a-516a-4748-84c8-a2b47f6fb770

Barcelos, D.M.

3f9f658f-a6b8-45c4-9313-73b81c6494de

Szalai, E.O.

0d87d8ba-9df6-462b-8cad-0e2bae937ba0

Przewloka, M.R.

9b25e73c-ec15-43df-a5a4-ac9574bb20ab

Maresca, T.J.

2f9aa770-02f5-4e2b-8526-4f6adb1edbc7

Audett, M.R., Johnson, E.L., McGory, J.M., Barcelos, D.M., Szalai, E.O., Przewloka, M.R. and Maresca, T.J. (2022) The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction. Molecular Biology of the Cell, 33 (1), [33:ar1]. (doi:10.1091/mbc.E21-06-0307-T).

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Accepted/In Press date: 22 October 2021

Published date: 1 January 2022

Additional Information: Funding Information: We thank Alex Pratt for generating a number of plasmids utilized in this study. We also acknowledge Sue Biggins (Fred Hutchinson Cancer Research Center) for sharing the Sli15-Ipl1 DNA construct, Iain Cheeseman (Whitehead Institute–MIT) for sharing Ipl1 protein, and Pat Wadsworth (University of Massachusetts, Amherst [UMass Am-herst]) for sharing constructs and insights. We are grateful to Vikash Verma, Jennifer Le, and all members of the Maresca lab (UMass Amherst) past and present for sharing many insightful scientific conversations. The photoactivation data were gathered in the Light Microscopy Facility and Nikon Center of Excellence at the Institute for Applied Life Sciences, UMass Amherst, with support from the Massachusetts Life Sciences Center. Thank you also to Jim Chambers (UMass Amherst) for help with the photoactivation experiments. This work was supported by a National Institutes of Health (NIH) grant (GM107026) to T.J.M. and by an NIH T32 training grant that supported M.R.A. (GM108556) and J.M.M. (GFM135096) as fellows in the UMass Biotechnology Training Program. M.R.P. is supported by Wellcome Trust grant 208908/Z/17/Z, and E.O.S. was supported by the Gerald Kerkut Charitable Trust and the University of Southampton Global Partnerships Award (Global Research Initiator Scheme). Funding Information: We thank Alex Pratt for generating a number of plasmids utilized in this study. We also acknowledge Sue Biggins (Fred Hutchinson Cancer Research Center) for sharing the Sli15-Ipl1 DNA construct, Iain Cheeseman (Whitehead Institute–MIT) for sharing Ipl1 protein, and Pat Wadsworth (University of Massachusetts, Amherst [UMass Amherst]) for sharing constructs and insights. We are grateful to Vikash Verma, Jennifer Le, and all members of the Maresca lab (UMass Amherst) past and present for sharing many insightful scientific conversations. The photoactivation data were gathered in the Light Microscopy Facility and Nikon Center of Excellence at the Institute for Applied Life Sciences, UMass Amherst, with support from the Massachusetts Life Sciences Center. Thank you also to Jim Chambers (UMass Amherst) for help with the photoactivation experiments. This work was supported by a National Institutes of Health (NIH) grant (GM107026) to T.J.M. and by an NIH T32 training grant that supported M.R.A. (GM108556) and J.M.M. (GFM135096) as fellows in the UMass Biotechnology Training Program. M.R.P. is supported by Wellcome Trust grant 208908/Z/17/Z, and E.O.S. was supported by the Gerald Kerkut Charitable Trust and the University of Southampton Global Partnerships Award (Global Research Initiator Scheme). Publisher Copyright: © 2022 Audett et al.

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