Table S1. Aspergillus nidulans strains used in this study Strain Genotype Derivation

Transcription

1 Supplemental Material De Souza et al., 211 Table S1. Aspergillus nidulans strains used in this study Strain Genotype Derivation CDS295 pyrg89; pyroa4; pyrg Af ::son promotor::gfp-son nup98/nup96 ; chaa1 This study CDS36 pyrg89 pabaa1; mad2aδ::pyrg; nup188-gfp::pyrg Af fwa1 This study CDS391 pyrg89 pabaa1; wa3; sld937 bub3 nup49-gfp::pyrg Af This study CDS436 pyrg89 ya2; arg2::gpdp::stua C-term-DsRedT4-arg (NLS-DsRed); This study pyroa4; nup49-gfp::pyrg Af ; fwa1 CDS437 pyrg89; arg2::gpdp::stua C-term-DsRedT4-arg (NLS-DsRed); This study pyroa4; mad2aδ::pyrg; nup49-gfp::pyrg Af ; fwa1 CDS453 pyrg89 pabaa1; nimt23 cdc25 ; nup188-gfp::pyrg Af fwa1 This study CDS465 pyrg89; pyroa4; pyrg Af ::son promotor::gfp-son nup98/nup96 ; This study mad2aδ::pyrg; chaa1 CDS466 pyrg89 pabaa1; wa3; mlp1-gfp::pyrg Af ; mad2aδ::pyrg; (se15 1 ) This study CDS487 ya2 pabaa1 (pyrg89 1 ); ndc8-mcherry::pyrg Af ; mad2a-gfp::pyrg Af ; (De Souza et al, 29) chaa1 CDS526 pyrg89; arg2; nup49-mcherry::pyrg Af choa1; GFP-tubA chaa1 (De Souza et al, 29) CDS532 pabaa1 pyrg89; nime Cyclin -GFP::pyrG Af ; nup49-mcherry::pyrg Af ; chaa1 (De Souza et al, 29) CDS544 pyrg89 ya2; arg2 ndc8-mcherry::pyrg Af ; nime Cyclin -GFP::pyrG Af ; (De Souza et al, 29) chaa1 (nira14 1 ) CDS561 pyrg89; wa3; arg2; mlp1-gfp::pyrg Af ; nup49-mcherry::pyrg Af ; fwa1 (De Souza et al, 29) se15 (nira14 1 ) CDS564 pyrg89; wa3 ; arg2 ndc8-mcherry::pyrg Af ; mlp1-gfp::pyrg Af ; fwa1 (De Souza et al, 29) chaa1 se15 (nira14 1 ) CDS565 pyrg89; wa3; ran-gap-gfp::pyrg Af ; pyroa4; nup49-cherry::pyrg Af This study CDS578 pyrg89; arg2 ndc8-mcherry::pyrg Af ; chaa1 fwa1 mad1-gfp::pyrg Af (De Souza et al, 29) (nira14 1 ) CDS64 pyrg89; pyroa4; nup49-mcherry::pyrg Af ; chaa1 mad1-gfp::pyrg Af (De Souza et al, 29) CDS631 pyrg89; arg2; nup49- mcherry::pyrg Af ; mad1δ::pyroa Af GFP-tubA This study chaa1 CDS637 pyrg89; arg2; nup49-mcherry::pyrg Af ; fwa1 (nira14 1 ) (autonomously This study replicating plasmid pcds-51kd containing pyr4::son promotor::gfp(z) 2 -NES and AMA-1) CDS648 (pyrg89 1 ); nimt23 cdc25 nime cyclin -HA; arg2; scc1-gfp::pyrg Af ; nup49- This study mcherry::pyrg Af ; fwa1 (nira14 1 ) CDS843 pyrg89; wa3; pyroa4; nup49-cr::pyroa Af ; bop1-gfp::ribo Af (ribo2 1 ) This study CDS998 pyrg89; wa3 cyclin -GFP::pyroA Af cdk1::cdk1f::pyr4+; arg2; nup49- This study mcherry::pyroa Af ; (fwa1 1 chaa1 1 se15 1 nira14 1 ) CDS112 pyrg89; wa3 cyclin -GFP::pyrG Af ; bime7; mad1-mcherry::pyrg Af ; This study (fwa1 1 nira14 1 chaa1 1 ) CDS117 (ya1 1 ); wa3; arg2; bime7; nup49-mcherry::pyroa Af ; mad1- This study GFP::pyroA Af (fwa1 1 ) CDS138 pyrg89; bub1-gfp-stag::pyrg Af wa3; pyroa4; arg2; nup49- This study mcherry::pyroa Af ; fwa1 (nira14 1 chaa1 1 ) CDS141 pyrg89 (ya1 1 ); wa3 bub1-gfp-stag::pyrg Af ; arg2; (pyroa4 1 ); bime7; This study nup49-mcherry::pyroa Af ; (fwa1 1 chaa1 1 se15 1 nira14 1 ) SO594 pyrg89; wa3; arg2; nup49-gfp::pyrg Af ; fwa1 (nira14 1 ) (Osmani et al, 26) SO599 ya2 (pyrg89 1 ); wa3; arg2; nup188-gfp::pyrg Af ; fwa1 (Osmani et al, 26) hcds23 (pyrg89; wa3; nkuaδ::arg; -GFP::ribo AF ; (ribo2 1 fwa1 1 nira14 1 )) / (pyrg89; wa3; nkuaδ::arg; -GFP::ribo AF ; benaδ::pyrg Af (ribo2 1 fwa1 1 nira14 1 )) This study 1

2 De Souza et al., In some strains we have not confirmed all nutritional or color markers which could be covered by, or be to recessive to, other markers in the strain. Af genes from Aspergillus fumigatus used for complementation of the corresponding A. nidulans nutritional mutations. Supplementary Figure S1 Mitotic exit and NPC reassembly are delayed by SAC activation but occur after a defined period of time without checkpoint satisfaction. enomyl was used at 2.4 μg/ml to prevent microtubule and spindle formation. (A) Time lapse imaging of -GFP in wild type or mad2δ cells transiting mitosis with or without spindle function. Kymographs show each time course on the same time scale. In the absence of microtubules, spindle formation does not occur and NPC reformation is delayed in a mad2 dependent manner although NPCs eventually reform around a single undivided nucleus after 47.6± 8.9 min (n = 1). () ar graph showing the mad2 dependent delay in mitotic exit and NPC reassembly of, Nup188, Nup98 and Mlp1 when cells enter mitosis without spindle function (n 3). Note that as happens in a normal mitosis (De Souza et al, 29), during release from SAC arrest without spindle function, Mlp1 reassembly to NPCs occurs after reassembly of. (C) Kymographs showing mitotic dispersal and return of -GFP and NLS-DsRed to nuclei in wild type and mad2δ cells transiting mitosis without spindle function. (D) Kymograph showing the status of RanGAP-GFP in comparison to -mcherry during mitosis without spindle function. RanGAP is cytoplasmic during interphase but can enter nuclei when NPCs are partially disassembled during mitosis (De Souza et al, 24). Scale bars, 5 µm. 2

3 De Souza et al., 211 Supplementary Figure S2 Cyclin is degraded in a temporally and spatially regulated manner during mitotic exit. (A) Cyclin -GFP together with -mcherry during exit from a normal mitosis. () Graph showing quantification of levels (red) and Cyclin levels in the nucleus (green) and cytoplasm (grey). Note that degradation of nuclear Cyclin occurs before the degradation of the cytoplasmic pool. Imaging in this figure was carried out at 32 o C. Scale bar, 5 μm. Supplementary Figure S3 Cyclin is maintained in the kinetochore/sp region during SAC arrest but is rapidly degraded during exit from SAC arrest. (A) Cyclin -GFP together with the Ndc8-mCherry kinetochore marker in a cell transiting mitotic SAC arrest without spindle function for the experiment shown in Figure 4C. () Graph showing quantification of Ndc8 levels (red) and Cyclin levels in the nucleus (green) and cytoplasm (grey). Imaging in this figure was carried out at 32 o C. Scale bar, 5 μm. Supplementary Figure S4 Cell cycle oscillations without spindle function. (A) Time lapse images of a benaδ cell following -GFP. The periods of mitotic arrest are indicated by the gold arrows. () Kymograph representation of the experiment shown in (A). (C) ar graph showing the duration of interphase and mitotic arrest in cells which undergo two consecutive cell cycles. Indicated are wild type or benaδ cells germinated without benomyl and wild type cells germinated in the presence of 2.4 μg/ml benomyl. Imaging in this figure was carried out at 32 o C. Scale bars, 5 μm. 3

4 De Souza et al., 211 Supplementary Figure S5 Cell cycle oscillations of SAC activation and inactivation without spindle function. Mad2-GFP together with -mcherry in a cell undergoing two cell cycles without spindle function. Mad2 cycles from NPCs to kinetochores, demonstrating that the SAC is on (gold arrows), and back to NPCs showing that the SAC is turned off. Imaging in this figure was carried out at 32 o C. Scale bars, 5 μm. Supplementary Figure S6 Cell cycle oscillations of localized Cyclin accumulation and degradation in the absence of spindle function. (A) Time lapse images of the experiment shown in Figure 6C. Shown are -mcherry, Cyclin -GFP (in false color) and the merged images for a wild type cell undergoing multiple cell cycles in benomyl without spindle function. The periods of SAC arrest are indicated by the gold arrows. () Graph showing nuclear levels of -mcherry which increase during the time course (red). The horizontal line indicates the periods of interphase (blue) and mitotic SAC arrest (gold). Imaging in this figure was carried out at 32 o C. Scale bars, 5 μm. Supplementary Figure S7 The mitotic nucleolar disassembly and reassembly cycle occurs during each SIME when cells undergo multiple cell cycles without spindles. (A-C) From the same experiment showing -mcherry together with the op1-gfp nucleolar marker in a benomyl treated cell which undergoes two cell cycles without spindles at 32 o C. (A) Kymographs of the complete time course. The period of the mitotic SAC arrest is indicated by vertical gold lines. ( and C) Time lapse images and pixel intensity profiles for the periods of SIME indicated in (A). Frames in which the old nucleolus (o) and new nucleolus (n) are apparent are indicated with an asterisk. (D) A 4

5 De Souza et al., 211 field of cells which have undergone two cell cycles without spindles. Note that cells have undergone polarized growth and generally have an enlarged nucleus containing a single nucleolus. Scale bars, 5 μm. Supplementary Figure S8 Cdk1F mutants undergo a benomyl induced mitotic arrest for a similar period of time as wild type cells. (A) Cyclin -GFP together with the - mcherry during transit through a benomyl induced SAC arrest in a Cdk1F mutant which cannot undergo inhibitory tyrosine phosphorylation (Ye et al, 1996). () Graph showing quantification of Cyclin levels in the nucleus (green) and cytoplasm (grey). The period of the SAC arrest is indicated by low nuclear levels (red). Note that in Cdk1F mutants Cyclin levels continue to increase during SAC arrest before Cyclin is rapidly degraded prior to reassembly. (C) Graph showing the period of benomyl induced mitotic arrest in Cdk1F mutants and wild type cells (n 29). Imaging in this figure was carried out at 32 o C. Scale bar, 5 μm. Supplementary Figure S9 Reactivation of the SAC does not occur when mitotic exit is blocked. (A) Imaging of -mcherry together with Mad1-GFP in a cell entering mitosis in the presence of benomyl at the bime7 APC1 non-permissive temperature of 42 o C. disassembles from NPCs during mitotic entry and remains dispersed during the bime7 APC1 mitotic arrest. Mad1-GFP locates to kinetochores for 37.4± 8.3 min (n=17) before dispersing from kinetochores indicating that the SAC has been inactivated. Although cells remain arrested in mitosis in the presence of benomyl, Mad1 does not return to kinetochores. This indicates that while the SAC can be inactivated without mitotic exit, preventing mitotic exit prevents additional rounds of SAC reactivation. Scale bar, 5 μm. () Kymograph representations of the experiment shown in (A). 5

6 De Souza et al., 211 References De Souza CP, Hashmi S, Nayak T, Oakley, Osmani SA. (29) Mlp1 acts as a mitotic scaffold to spatially regulate spindle assembly checkpoint proteins in Aspergillus nidulans. Mol iol Cell 2: De Souza CP, Osmani AH, Hashmi S, Osmani SA. (24) Partial nuclear pore complex disassembly during closed mitosis in Aspergillus nidulans. Curr iol 14: Osmani AH, Davies J, Liu HL, Nile A, Osmani SA. (26) Systematic deletion and mitotic localization of the nuclear pore complex proteins of Aspergillus nidulans. Mol iol Cell 17: Ye XS, Fincher RR, Tang A, O'Donnell K, Osmani SA. (1996) Two S-phase checkpoint systems, one involving the function of both IME and Tyr15 phosphorylation of p34 cdc2, inhibit NIMA and prevent premature mitosis. EMO J 15:

7 De Souza et al., 211 A Wild type 1' -GFP Wild type + en mad2 5' 1' mad2 + en 1' Time of mitotic Nup dispersal (min) Wild type Wild type + en mad2 mad2 + en 2' 1' 2' 2' Nup1881 Nup98 Mlp1 3' 15' 3' 3' C 4' 2' 4' 4' Wild Type + en NLS Merge mad2 + en NLS Merge 2 min 5' 25' 5' 5' 6' 45' 3' 6' 6' 2 min 5' 7' 35' 7' 7' D 55' Wild type + en Ran-GAP Merge 8' 4' 8' 8' Kymographs Wild type Wild type + en mad2 mad2 + en 2 min 2 min Supplementary Figure S1 7

8 A De Souza et al., 211 ' 1' 2' 3' Cyclin (False color) Cyclin + Relative levels Time (min) Nuclear Cyclin Cytplasmic Cyclin Nuclear Supplementary Figure S2 A Relative levels enomyl ' ' ' Time (min) Nuclear Cyclin Cytplasmic Cyclin Ndc8 Ndc8 Cyclin (False color) Cyclin + Ndc8 24' 48' 72' 24' 48' 72' 24' 48' 72' Supplementary Figure S3 8

9 A De Souza et al., 211 benaδ ' 6' 12' 18' 24' 3' 3 min M M benaδ C Time (Min) WT WT+en benaδ (5) (15) (5) Interphase WT interphas e WT Con mitosis 2 Mitosis Supplementary Figure S4 9

10 enomyl ' 6' 12' 18' 24' 3' 36' De Souza et al., 211 Mad2 ' 6' 12' 18' 24' 3' 36' Mad2 + ' 6' 12' 18' 24' 3' 36' Supplementary Figure S5 1

11 De Souza et al., 211 A enomyl -mcherry ' 6' 12' 18' 24' 3' 36' 42' Cyclin -GFP (False Color) ' 6' 12' 18' 24' 3' 36' 42' Cyclin -GFP + -mcherry ' 6' 12' 18' 24' 3' 36' Relative Nuclear levels 42' Time (Min) Supplementary Figure S6 11

12 De Souza et al., 211 A enomyl op1 Merge D M SIME I DIC op1 Time M SIME II SIME I op1 + op1 C SIME II op1 + op1 ' ' M M 1' 1' * * o * * 2' 2' o n n Supplementary Figure S7 12

13 A ' Cdk1F + enomyl De Souza et al., ' 48' 72' ' Cyclin (False color) 24' 48' 72' ' Cyclin + 24' 48' 72' C Relative levels Time (min) Nuclear Cyclin Cyto Cyclin Nuclear Mitotic dispersal (min) WT Cdk1F Supplementary Figure S8 13

14 A ' APC1 bime7 + enomyl De Souza et al., Mad1 6' 12' 18' 24' + Mad1 Mad1 G2 SAC on 3 min APC1 bime7 Mitotic Arrest + enomyl Supplementary Figure S9 14