Article Figures & Tables
Figures
- Fig. 1.
Conditions for assembly and dynamics of MTs in lysate. (A) Two examples of kymographs showing GFP-Tub1 (green) assembly from rhodamine-labeled MT seeds (magenta) and in lysates from each stage in the cell cycle. Minus ends are on the left, plus ends on the right. Time on the y-axis, 5 s pixel−1, totaling 10 min, starting from the top. (B) Kymographs of S phase lysate without additional ATP (left) and with 0.9 mM ATP. (C) Effect of culturing and grinding conditions on MT behavior in S phase-arrested lysate. (D) Western blot probing for levels of tubulin and Mcd1, a nuclear protein, in S phase lysates. Total lysate (T) and lysate cleared by ultracentrifugation (C) were analyzed. The first row of numbers indicates the percentage of Mcd1 present in the cleared lysate compared with the total lysate, normalized to the 4 l/medium vial lane. The second row of numbers indicates the percent of GFP-Tub1 present in the cleared lysate compared with the total lysate, normalized to the 4 l/medium vial lane. (E) MT growth profiles in S phase lysates based on culture volume and milling vial size. (F) MT growth profiles for lysates by cell cycle. The percentage of time spent in phases for the entire population of MTs is compared across stages of the cell cycle. MT measurements were pooled from three lysates generated from independent harvests of cells. MT counts for each condition are listed in Table 1.
- Fig. 2.
MT activity in mixed lysates is titratable. (A) MT growth profiles for mixed lysates. The 1:3 S:G1 lysate lacks any MT activity. (B) Growth rates of MTs in mixed lysates compared with wild-type lysates arrested in S phase. (C) MT shrinkage rates in S phase-arrested and mixed lysates. None are statistically different from the others, with the exception of the 1:3 S:G1 lysate. MT counts for each condition were 9:1=30, 3:1=25, 1:1=30. **P<0.01, ***P<0.001 and ****P<0.0001.
- Fig. 3.
MAPs dynamically associate with MTs in lysates. (A) Kymograph of GFP-Tub1 (green) with Bim1-TagRFP-T and rhodamine-labeled seeds (magenta). Bim1-TagRFP-T is along MTs and decorates the plus end during growth and can be present on shrinking ends. (B) Fields of MTs in flow-through experiments. Dynamic MTs in S phase lysate were arrested and some began to shrink back to the seeds after flow through of G1 lysate. (C) In the reciprocal experiment, there were no MTs growing from seeds after 10 min in G1 lysate. At 20 min after flow-through of S phase lysate, MTs had grown and were dynamic. (D) Kymograph of S phase-arrested lysate expressing Bim1-TagRFP-T being replaced with S phase-arrested lysate with untagged Bim1 (white line).
- Fig. 4.
MT dynamics depend upon the cell cycle and motor activity in lysates. (A) Kymographs of kip3Δ, KAR3-AID and kip3Δ KAR3-AID lysates made from cells that were treated with auxin (KAR3-AID genotype cells) and were either asynchronous or arrested in S phase, metaphase or anaphase. Rhodamine-labeled seeds in magenta and GFP-Tub1 in green. (B,C,D) Growth rates, shrinkage rates and growth profiles for lysates made from cells with these genotypes in S phase (B), metaphase (C) and anaphase (D). MT measurements were pooled from three lysates generated from independent harvests of cells. MT counts for each condition are listed in Table 2. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.
- Fig. 5.
Kar3 binding partners alter MT polymerization activity. (A) Fields of rhodamine-labeled seeds (magenta) and GFP-Tub1 (green) using asynchronous cik1Δ and vik1Δ lysates. Scale bars: 2 μm. (B) MT growth profiles for KAR3-AID, cik1Δ and vik1Δ lysates from asynchronous cultures. KAR3-9myc-AID data from asynchronous lysate is presented again for comparison. No MT polymerization was observed in three separate cik1Δ lysates. Measurements for MTs in vik1Δ lysates were pooled from two lysates. A total of 62 MTs were counted.
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