Figure 5

The number of astral MTs associated with old and new SPBs is increased in γtub-Y362E cells. (a) Representative metaphase spindles (two SPBs 0.8 to 2 µm apart) in wild-type (WT) and γtub-Y362E cells. MTs are tagged with Venus-Tub1 (shown in green) and SPBs tagged with Spc42-Cerulean (shown in red). Corresponding explanatory schematics for astral MT organization are shown to the right. (b,c) Astral MT count for old and new SPBs in wild-type (b) and γtub-Y362E (c) cells. (d) Percentage of total time both SPBs have astral MTs in WT and γtub-Y362E cells. (e) Proposed mechanism for loss of alignment in γtub-Y362E cells. (1) During normal spindle alignment, Kar9-Bim1 complexes are loaded onto astral MTs and track the plus end. Kar9 binds to the type 5 myosin Myo2 which guides the astral MT plus end to the bud by walking on actin cables that originate at the bud cortex, while astral MTs projecting into the mother cell, regardless of their association with Kar9-Bim complexes, are short lived. (2 and 4) Astral MTs terminating in the bud exert a “pulling” force, via Kar9 or dynein, (3) astral MTs terminating in the mother are capable of “pushing” the spindle away from the mother cell cortex. An increased in the number of astral MTs in γtub-Y362E cells results in uncoordinated pushing and pulling forces (via Kar9 or dynein) at both SPBs, resulting in failure to achieve or maintain spindle alignment.