Figure 5.

Expression of TUB1-828 during G1 α-factor arrest results in unattached microtubules and inhibited spindle assembly upon release from α-factor arrest. (A and B) GFP-labeled microtubules in strain DBY9580, containing either pTS210 (GAL1p-vector), pRB2785 (GAL1p-TUB1), or pRB2949 (GAL1p-TUB1-828, also shown with differential interference contrast), grown in raffinose and arrested with α-factor then subjected to galactose for 2 h. In B, cells were released from α-factor arrest by washing into glucose medium and incubated for 90 min. Arrows indicate free microtubules not attached to the presumed spindle pole body. Videos 5A_TUB1.mov (GAL1p-TUB1) and 5A_828.mov (GAL1p-TUB1-828) illustrate microtubule dynamics under these conditions and movement of the free microtubules in A, 5B_828.mov (GAL1p-TUB1-828) shows cells in B. Bar, 5 μm. (C) Distribution of microtubule structures and cell shapes in cultures 90 min after release from α-factor arrest shown in B. Drawings above each column indicate presence of bud and appearance of microtubules. Aberrant microtubule bundles and unattached microtubules indicated in the four columns on the right. (D) Anti-tubulin antibodies reveal unattached microtubules and aberrant microtubule bundles in cells expressing TUB1-828 in the absence of GFP::TUB1. Strain DBY9585, containing plasmid pRB2949 (GAL1p-TUB1-828), was arrested with α-factor in raffinose, subjected to galactose for 2 h (top row) then washed into galactose medium and grown for 3.5 h (bottom row). Control strains DBY9583 and DBY9584, containing plasmids pTS210 (GAL1p-vector) and pRB2785 (GAL1p-TUB1), appeared normal as in A and B (our unpublished data). Bar, 3 μm.