Spc24 and Stu2 Promote Spindle Integrity When DNA Replication Is Stalled -- Ma et al. 18 (8): 2805 Data Supplement - Supplemental Material -- Molecular Biology of the Cell

Spc24 and Stu2 Promote Spindle Integrity When DNA Replication Is Stalled
Mol. Biol. Cell Ma et al. 18: 2805

Supplemental Material

This article contains the following supporting material:

Supplemental Figure 1 - Ndc80 is mislocalized in spc24-9 mutants. (A) Ndc80-VFP Spc29-CFP wild type (SPC24) and spc24-9 cells were grown at 25ºC to log phase, then incubated at 37ºC for three hours, fixed in 70% ethanol and imaged. Four types of Ndc80 localization patterns were detected in spc24-9 mutants: Type I: Wild type Ndc80, 44% of cells; Type II: Ndc80 localized to one SPB (14% of cells); Type III: Multiple Ndc80 foci (20% of cells); Type IV: Little or no Ndc80-VFP signal (22% of cells). (B) Ndc80-VFP Spc29-CFP wild type (SPC24) and spc24-9 cells were grown at 25ºC to log phase, then incubated at 30ºC in 0.2M HU for three hours, fixed in 70% ethanol and imaged. In spc24-9 cells, Ndc80-VFP localization either resembled wild type cells (20% of cells) or Ndc80-VFP levels were very low and barely detectable (80% of cells). In the overlay, green is VFP signal and red is CFP. Scale bar is 2 μm for all images.
Supplemental Figure 2 - Stu2ΔN-VFP localization overlaps with endogenous Stu2-CFP. Wild type (SPC24) and spc24-9 strains carrying Stu2ΔN-VFP on a 2μ plasmid and endogenous Stu2-CFP were either grown at 30ºC to log phase or incubated in 0.2M HU for three hours, fixed in 70% ethanol and imaged. In the overlay, green is VFP signal and red is CFP. Scale bar is 2 μm for all images
Supplemental Figure 3 - Time-lapse analysis of Stu2-VFP. Stu2-VFP Spc29-CFP cells were imaged using time-lapse microscopy at 30ºC as described in the Materials and Methods. Stu2-VFP is represented in greyscale and Spc29-CFP in red. Arrows in Image 10,b point to Stu2-VFP bilobed foci on the nuclear side of Spc29-CFP. Arrows in Image 0, c point to Stu2-VFP localization on the central spindle (cs) and poles (p).
Supplemental Figure 4 - Overexpression of STU2ΔN does not affect wild type spindle length in an unperturbed cell cycle. Vector control (pRS326) and STU2ΔN were expressed in cells carrying Stu2VFP Spc29CFP. Cells were grown to log phase at 30°C, arrested with α -factor for 1.5hours at 30ºC, released to 30ºC and time points taken every 15min. Spindle length was calculated as described in the Materials and Methods. Thirty cells were counted for each time point.
Supplemental Figure 5 - spc24-9 mutants display spindle expansion early in the cell cycle but a delay in anaphase. Wild type (SPC24) and spc24-9 cells carrying Stu2VFP Spc29CFP were grown at 25ºC to log phase, incubated with α -factor for 1hour at 25ºC, resuspended in prewarmed 37ºC media with new α -factor and incubated for 2 hours at 37ºC. Cells were then released from the G1 arrest into 37ºC and time points taken every 10min. Spindle length was measured by calculating the distance from Spc29-CFP foci to Spc29-CFP foci. Fifty cells were counted per time point.
Supplemental Figure 6 - spc24-9 mutants accelerate through S phase and delay at anaphase at restrictive temperature. Wild type (SPC24) and spc24-9 cells were grown at 25ºC to early log phase, arrested in G1 phase with pheromone, released to 37ºC and time points taken every 15 min. DNA content was measured by flow cytometry as described in the Materials and Methods.
Supplemental Table 1