Poleward Tubulin Flux in Spindles: Regulation and Function in Mitotic Cells -- Buster et al. 18 (8): 3094 Data Supplement - Supplemental Materials -- Molecular Biology of the Cell

Poleward Tubulin Flux in Spindles: Regulation and Function in Mitotic Cells
Mol. Biol. Cell Buster et al. 18: 3094

Supplemental Materials

This article contains the following supporting material:

Supplemental Movie 1 - Photobleach tracking in a mitotic S2 cell constitutively expressing eGFP-α-tubulin. Measurements are made of the distances between the bleach marks on prominent microtubule fibers and the spindle pole. Flux rates were calculated by tracking the change in bleach mark position through movie frames. This cell had been treated with control dsRNA. Display rate: 10 frames per second; 21 secs of recording time per sec of movie display time.
Supplemental Movie 2 - Flux in a photobleached S2 cell (expressing eGFP-α-tubulin) following Mast RNAi. Compared to control RNAi cells, the bleach marks move polewards at a significantly reduced rate. Display rate: 10 frames per second; 32 secs of recording time per sec of movie display time.
Supplemental Movie 3 - Flux in a photobleached S2 cell (expressing eGFP-α-tubulin) following KLP67A RNAi. Compared to control RNAi cells, the bleach marks move polewards at a significantly increased rate. Display rate: 10 frames per second; 32 secs of recording time per sec of movie display time.
Supplemental Movie 4 - Flux in a photobleached S2 cell (expressing eGFP-a-tubulin) following Msps RNAi. The flux rate in these cells is dramatically decreased, as shown by the essentially stationary bleach mark. Display rate: 10 frames per second; 37 secs of recording time per sec of movie display time.
Supplemental Movie 5 - Prometaphase-to-metaphase S2 cell stably transfected with Msps-eGFP. Distinct spots of Msps appear to localize to kinetochores. Display rate: 10 frames per second; 185 secs of recording time per sec of movie display time.
Supplemental Movie 6 - Anaphase S2 cell stably expressing Msps-eGFP. Msps remains associated with centrosomes and the presumptive kinetochores throughout anaphase. Display rate: 6 frames per second; 56 secs of recording time per sec of movie display time.
Supplemental Movie 7 - Flux in a photobleached S2 cell (expressing eGFP-α-tubulin) following Msps/KLP10A co-RNAi. The flux rate in these cells remains very low, as shown by the essentially stationary bleach mark in this treated cell. The flickering brightness is due to contrast/brightness adjustments of individual frames to keep the bleach mark visible. Display rate: 10 frames per second; 35 secs of recording time per sec of movie display time.
Supplemental Movie 8 - Flux in a photobleached S2 cell (expressing eGFP-α-tubulin) following Msps/KLP59C co-RNAi. The flux rate in these cells remains very low, as shown by the essentially stationary bleach mark in this treated cell. We have not found a co-RNAi treatment that rescues the decreased flux resulting from diminished Msps, suggesting that Msps activity is essential for flux. Display rate: 10 frames per second; 33 secs of recording time per sec of movie display time.
Supplemental Movie 9 - Anaphase in a photobleached S2 cell (expressing eGFP-α-tubulin) following control RNAi. The flux rate can be measured from the poleward velocities of the bleach marks, and chromatid-to-pole rates can be measured from the poleward velocities of the chromosomes. Green, eGFP-tagged microtubules; red, Hoechst-stained chromosomes. Display rate: 10 frames per second; 30 secs of recording time per sec of movie display time.
Supplemental Figure 1 - dsRNA interference (RNAi) in S2 cells.
(A) Western blots of lysates from S2 cells treated by RNAi to knock-down the indicated proteins. Samples were taken on days 0 (just prior to the initial dsRNA application), 3, and 7 of the dsRNA application period. α-Tubulin immunoblotting was used to evaluate sample loading. See Methods for details of KLP59C knock-down analysis.
(B) Target proteins are knocked-down when co-RNAi treatments are used to diminish expression of two different targets simultaneously. Cell lysates were collected at the end (day 7) of the RNAi treatment and Western blotted to evaluate knock-down efficiency.
(C) Percent knock-down of target proteins following RNAi. ND, not determined; KLP, percent knock-down of the indicated kinesin-like protein.
(D) Sequences of PCR primers used to generate DNA templates for in vitro transcription of target-specific dsRNA.
Supplemental Figure 2 - Average flux rates for all treatments.
To make comparisons more convenient, all treatments are shown on a single graph, including the treatments presented in Figures 1 and 2. Rates are from spindles before anaphase onset. Statistically significant differences (P
Supplemental Figure 3 - Fluorescence recovery after photobleaching.
(A) Selected plots of fluorescence recovery following photobleaching of either plus- or minus-ends of spindle microtubules in eGFP-α-tubulin expressing S2 cells. The RNAi treatments are indicated above the panels.
(B) Percent recovery of fluorescence following photobleaching for all treatments. Light bars, microtubule minus-ends; black bars, plus-ends. Only one treatment (Msps/KLP10A co-RNAi) is statistically different than the control (for both ends). Error bars, +SD. Numbers within bars, N.
Supplemental Figure 4 - Immunolocalization of target proteins in mitotic S2 cells.
(A) Immunolocalization of the indicated proteins (red) following RNAi of the indicated target proteins (left). Spindles are oriented vertically. Microtubules, green. Scale bar, 5 µms.
(B) KLP67A immunolocalization during mitosis. After metaphase, KLP67A redistributes from kinetochores to central spindle microtubules (red arrowhead), but KLP67A frequently remains visible on kinetochores during anaphase A (yellow arrowheads).
Supplemental Figure 5 - Flux is not required for chromosome congression, tension, or spindle length. Results of all treatments.
(A) Congression evaluated as centromere displacement from the spindle equator as a percent of spindle length.
(A') Displacement is presented as the distance (µms) between centromere and spindle equator, instead of as a percent of spindle length as in (A). For this graph only, data were analyzed with the Kolmogorov-Smirnov (non-parametric and distribution free) test. For each spindle, the displacement distances (µms) of the anti-Cid stained centromeres from the spindle equator were averaged. Spindle averages were normally used for analysis to avoid weighting the datasets towards spindles with higher numbers of measurable kinetochores. For two co-RNAi treatments (Mast/10A and Mast/67A), too few spindles were found to permit analysis using spindle averages. In these two cases, datasets of all the individual displacement measurements were used for analysis.
(B) Results of all RNAi treatments and colchicine on the inter-kinetochore distance. The Mast/KLP67A co-RNAi treatment (§) was not statistically analyzed (N=2).
(C) Results of all RNAi treatments on spindle length.