The Mitotic Exit Network Mob1p-Dbf2p Kinase Complex Localizes to the Nucleus and Regulates Passenger Protein Localization -- Stoepel et al. 16 (12): 5465 Data Supplement - Supplemental Materials -- Molecular Biology of the Cell

The Mitotic Exit Network Mob1p-Dbf2p Kinase Complex Localizes to the Nucleus and Regulates Passenger Protein Localization
Mol. Biol. Cell Stoepel et al. 16: 5465

Supplemental Materials

This article contains the following supporting material:

Supplemental Figures S1-S5 - Supplement Figure S1: Mob1p is required for Cdc14p release from the nucleolus. Cdc14p-GFP localization in late anaphase-arrested mob1-95 cells. Asynchronously growing FLY558 were shifted to non-permissive temperature (34°C) for 2.5 hours and analyzed by differential interference contrast (DIC, left panels) and fluorescence microscopy (right panels). Cdc14p-GFP remains sequestered in the nucleolus in mob1-95 cells. Similar results were obtained with mob1-77 cells (data not shown).
Supplement Figure S2: Truncated Mob1p localizes to the anaphase spindle. Asynchronously growing haploid GFPMob1Δ132 Nic96-RFP cells (FLY2055) were analyzed by fluorescence microscopy. Mob1p was detectable on an axial structure that is distinct from the nucleoplasmic bridge within anaphase cells.
Supplement Figure S3: Mob1Δ78 colocalizes with kinetochore proteins. Asynchronously growing cells expressing truncated Mob1Δ78-YFP and Slk19p-CFP (FLY2076) were analyzed by fluorescence microscopy. Mob1Δ78-YFP shows a partial co-localization with Slk19p-CFP during anaphase (see arrowheads).
Supplement Figure S4: The chromosome passenger proteins, Ipl1p, Sli15p and Bir1p are not maintained on spindle midzones in mob1-77 cells. mob1-77 cells were blocked and released in metaphase using NDZ, as described in the text. Upon release from NDZ, the percentage of cells that lacked Ipl1p-GFP, Bir1p-GFP and Sli15p-GFP on the spindle midzone were scored over time (minutes). T=0 corresponds to the time when cell were released from the metaphase block.
Supplement Figure S5: Truncated Mob1p localizes to nuclei independently of Ipl1p and Mps1p. A) Localization of GFP-Mob1Δ78 and GFP-Mob1Δ132 in conditional ipl1-321 cells. Asynchronously growing cells were shifted to restrictive temperature (37°C) for 2-3 hours. Truncated GFP-Mob1 was expressed in ipl1-321 (SBY322) via low copy plasmids (pRS314-GFP-Mob1Δ78, pRS314-GFP-Mob1Δ132). B) GFP-Mob1Δ78 localizes to nuclei and SPBs (arrowheads) conditional mps1-1 cells. Asynchronously growing cells (were synchronized in G1 and released to restrictive temperature (37°C) for 2-3 hours. GFP-Mob1Δ78 was expressed in mps1-1 cells (FLY060) via low copy plasmids (pRS314-GFP-Mob1Δ78). Similar results were obtained for GFP-Mob1Δ132 (data not shown)
Supplemental Movie 1 - Wild type GFP-Mob1 localizes to the nucleus during anaphase Time-lapse movies of merged optical sections of three mitotic GFP-Mob1 cells (FLY331). Note the brief appearance of nuclear fluorescence near the SPBs of the middle cell. 6 x 0.2 um optical sections of GFP-Mob1 cells were captured every 3 minutes and merged to a single plane. The merged images for each timepoint were contrast enhanced and presented as a Quicktime movie.
Supplemental Movie 2 - GFP- Mob1Δ78 time-lapse Time-lapse movie of anaphase GFP-Mob1Δ78 cells (FLY1874). Note the recruitment of Mob1Δ78 to the SPBs during early anaphase (left cell).
Supplemental Movie 3 - GFP-Mob1Δ132 time-lapse Time-lapse movie of anaphase GFP-Mob1Δ132 cell (FLY1875).
Supplemental Movie 4 - GFP-Mob1Δ132 time-lapse Time-lapse movie of late anaphase GFP-Mob1Δ132 cell (left). The cell on the right may be checkpoint-arrested in metaphase.