PP2A is essential for maintaining centriole engagement in mitosis

figure 24. The recruitment of PP2A to centrosomes depends on Sgo1.

(A,B) PP2A-binding deficient Sgo1 A2 (A) and C2 (B) variants still localize to the centrosomes.

(A) Expression of Myc-Sgo1 A2, A2^N61I or A2Y57A, K62A

was induced with doxycycline (dox) for 48 h in stable Hek293 Flp-In T-REx cells. 24 h before fixation, cells were transfected with SGO1 siRNA. Cells were pre-extracted prior to fixation and centrin 2 (centrosomal marker), Myc (Sgo1 isoforms) and DNA (Hoechst 33342) were visualized by IFM.

(B) Cells inducibly expressing Myc-Sgo1 C2, C2^N61I or C2Y57A, K62A

were treated as described in (A).

Centrin 2 (centrosomal marker), Myc (Sgo1 isoforms) and DNA (Hoechst 33342) were visualized by IFM.

(C,D) PP2A-binding deficient Sgo1 A2 (C) and C2 (D) variants do not recruit PP2A to centrosomes.

Cells were treated as described in (A) and stained for centrin 2, PP2A-C and DNA (Hoechst 33342).

Scale bars: 5 µm.

2.4.2. PP2A is essential for maintaining centriole engagement in mitosis

If the protection of centriole engagement depends on Sgo1-dependent recruitment of PP2A, then PP2A-binding deficient Sgo1 variants should not be able to prevent the premature centriole disengagement caused by Sgo1 depletion. And indeed, the Sgo1 N61I and Y57A, K62A mutants were unable to prevent premature centriole disengagement in the absence of endogenous Sgo1 (figure 25). These results strongly suggest that Sgo1’s function as a recruitment factor for PP2A is conserved between centromeres and centrosomes.

centrin 2 Myc merge centrin 2 Myc

centrin 2 PP2A C mergecentrin 2

PP2A C DNA

centrin 2 PP2A C mergecentrin 2 PP2A C

centrin 2 Myc mergecentrin 2 Myc

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figure 25. PP2A binding-deficient variants of Sgo1 A2 and C2 fail to rescue the premature centriole disengagement caused by Sgo1 depletion.

Transgenic Hek293 cell lines inducibly expressing siRNA-resistant wild type (WT) Myc-Sgo1 A2/C2 or PP2A binding-deficient variants thereof (Y57A, K62A or N61I) were treated as described in figure 10A, before being analyzed by immunoblotting (A) and centrosome isolation followed by IFM using centrin 2 and C-Nap1 antibodies (B).

(A) Transgene expression was analyzed by Myc immunoblots. Immunodetection of α-tubulin served as loading control.

(B) Quantification of centriole disengagement. Each column represents averages of three independent experiments (circles, 100 centrosomes each). The amount of centriole disengagement of + dox cells was normalized to - dox cells (set to 100%).

To corroborate these results, I tested, whether artificially tethering PP2A to centrosomes can bypass the need for Sgo1 to protect centriole engagement.

Therefore, I fused the open reading frame (ORF) of PP2A-B’α to an extended version of the CTS and used this construct to generate Hek293 cell lines stably, but inducibly, expressing the fusion protein. Upon doxycycline addition, this protein readily localized to the centrosomes (figure 26A). Crucially, assessment of centriole engagement status revealed that PP2A-B’α-CTS indeed suppressed premature centriole disengagement by 30%, while uninduced cells were not able to mitigate the Sgo1 depletion phenotype (figure 26B and C and data not shown).

A

Sgo1 A2^Y57A,K62A A2^N61I C2 C2^Y57A,K62A C2^N61I

rel. centriole disengagement [%]

RESULTS

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figure 26. PP2A is the actual effector protecting centrosome engagement.

(A) Schematic view of chimeric protein consisting of PP2A-B’α and the C-terminus of Sgo1 A2 (aa 493–561) drawn to scale. A transgenic cell line inducibly expressing Myc-PP2A-B’α-CTS was treated with dox to induce transgene expression for 2 days. To enrich mitotic cells, cells were first arrested in S-phase using thymidine for 20 h and then released from the arrest 11 h prior to pre-extraction and fixation. Centrin 2, Myc and DNA (Hoechst 33342) were visualized by IFM. Scale bar: 5 µm.

(B,C) Artificial recruitment of PP2A to the centrosome prevents premature centriole disengagement caused by Sgo1 depletion. The transgenic Hek293 cell line inducibly expressing Myc-PP2A-B’α was treated as described in figure 10A, before being analyzed by immunoblotting (B) and centrosome isolation followed by IFM using centrin 2 and C-Nap1 antibodies (C).

(B) Transgene expression was analyzed by a Myc immunoblot. Detection of topoisomerase IIα (topo) seved as loading control.

(C) Quantification of centriole disengagement. Each column represents averages of three independent experiments (circles, 100 centrosomes each). The amount of centriole disengagement of + dox cells was normalized to - dox cells (set to 100%).

To exclude the possibility that the observed rescue effect was caused by the higher level of PP2A rather than its tethering to the centrosome, I expressed Myc-tagged PP2A-B’α, PP2A-B’α-CTS and PP2A-B’α-CTS^AAA in Hek293T cells (figure 27B).

Additionally, the cells were treated with nocodazole to investigate, if the localization and function of PP2A and its chimeric variants depend on microtubules. IFM revealed that only PP2A-B’α-CTS localized to the centrosomes in a Sgo1-depletion background, while the ILY to AAA variant and WT PP2A-B’α did not (figure 27A).

Consistent with its localization, only PP2A-B’α-CTS partially suppressed premature

CTS

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centriole disengagement by (30%), while neither the corresponding ILY to AAA variant nor WT PP2A-B’α rescued the Sgo1 depletion phenotype (figure 27C). Thus, Sgo1’s centrosomal function lies in its ability to recruit PP2A, which then acts as the actual effector for the protection of centriole engagement.

figure 27. Artificial recruitment of PP2A to the centrosome rescues premature centriole disengagement caused by Sgo1 knockdown in the absence of microtubules.

(A) A PP2A-B'α-CTS fusion protein is artificially directed to centrosomes. Myc-tagged PP2A-B’α, PP2A-B’α-CTS and PP2A-B’α-CTSAAA were transiently expressed in Hek293T cells for 48 h. To prevent recruitment via Sgo1, cells were depleted of endogenous Sgo1 by transfection of siRNA 24 h later. Microtubules were depolymerized by addition of nocodazole 16 h prior to pre-extraction and fixation. Centrin 2, Myc (PP2A variants) and DNA (Hoechst 33342) were visualized by IFM.

(B) Hek293T cells were transfected with plasmids encoding Myc-tagged PP2A-B’α, PP2A-B’α-CTS or PP2A-B’α-CTS^AAA and treated as described in figure 10A but finally arrested with nocodazole instead of taxol before being analyzed by immunoblotting.

(C) IFM on isolated centrosomes from (B). Each column represents averages of three independent experiments (circles, 100 centrosomes each).

C B

A

α-tubulin Myc

– WT CTS CTS^AAA Myc-PP2A B’α

0 20 40 60 80 100 120

- wt -CTS -CTS^AAA

rel. centriole disengagement [%]

PP2A B‘α Myc-PP2A WT

Myc-PP2A-CTS

Myc-PP2A-CTS^AAA

DNA Myc centrin 2 merge Myc

centrin 2

RESULTS

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