Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables

Abstract

Microtubules and actin filaments interact and cooperate in many processes in eukaryotic cells, but the functional implications of such interactions are not well understood. In the yeast Saccharomyces cerevisiae, both cytoplasmic microtubules and actin filaments are needed for spindle orientation. In addition, this process requires the type V myosin protein Myo2, the microtubule end-binding protein Bim1, and Kar9. Here, we show that fusing Bim1 to the tail of the Myo2 is sufficient to orient spindles in the absence of Kar9, suggesting that the role of Kar9 is to link Myo2 to Bim1. In addition, we show that Myo2 localizes to the plus ends of cytoplasmic microtubules, and that the rate of movement of these cytoplasmic microtubules to the bud neck depends on the intrinsic velocity of Myo2 along actin filaments. These results support a model for spindle orientation in which a Myo2-Kar9-Bim1 complex transports microtubule ends along polarized actin cables. We also present data suggesting that a similar process plays a role in orienting cytoplasmic microtubules in mating yeast cells.

Figure 1.

MYO2–BIM1 fusion compensates for the spindle orientation defect in kar9Δ cells. (A and B) Microtubule immunofluorescence was used to determine the localization of preanaphase spindles. (A) Examples of spindle localization in wild-type, kar9Δ, and kar9Δ MYO2–BIM1 cells. Bar, 2 μm. (B) The percentage of cells with spindles located in each of four different regions of budded cells. (C and D) A MYO2–BIM1::URA3 kar9Δ::LEU2 strain was crossed to a dyn1Δ::HIS3 strain. The resulting diploid was sporulated, and 46 tetrads were dissected. The genotype of meiotic segregants was determined by scoring for the linked auxotrophic markers. The genotype of inviable spores could be inferred in all cases by assuming 2:2 segregation of markers. (C) Growth of meiotic segregants from five tetrads. kar9Δ dyn1Δ strains (indicated by arrows) formed microcolonies that did not grow further. kar9Δ dyn1Δ MYO2–BIM1 strains (indicated by arrowheads) were viable, and grew as well as wild-type cells. (D) Viability of spores of each genotype obtained from the cross.

Figure 2.

MYO2–BIM1 fusion compensates for the microtubule orientation defect in α-factor–treated kar9Δ cells. (A) Examples of an oriented microtubule that extends into the mating projection tip and a misoriented microtubule that does not extend into the projection tip of α-factor–treated cells. The projection tip (indicated by the dotted line) was defined by drawing a line perpendicular to the long axis of the cell and one quarter of the cell's length away from the end of the projection. Microtubules were visualized by immunofluorescence microscopy. Bar, 2 μm. (B) The percentage of MYO2 and conditional myo2 mutant yeast cells with microtubules oriented toward the projection tip was determined before (black bars) and after a 5-min shift to the restrictive temperature of 35°C (gray bars). 100 cells were counted in each case. (C) The percentage of wild-type, KAR9MYO2, and kar9Δ MYO2 cells with microtubules oriented toward the projection tip. 50 cells were counted in each case.

Figure 3.

The rate of cytoplasmic microtubule movement depends of the intrinsic velocity of Myo2. (A and B) Fluorescence images of live cells containing GFP–Tub1. Time intervals (in seconds) between images are indicated in the top left of each image. White arrowhead tracks movement of microtubule end; open arrowhead is a fiduciary marker near the bud neck. Bar, 2 μm. (A) Cells containing wild-type Myo2 with six IQ repeats. Microtubule in cell on left begins movement at 0.0 s; microtubule in cell on right begins movement at 11.2 s (see Video 1). (B) Cell containing a mutant Myo2 lacking IQ repeats (see Video 2). (C) Scatter plot of cytoplasmic microtubule tip velocities. Each black square corresponds to an individual measurement (11 measurements for 0 IQ and 12 for 6 IQ). Gray dots indicate average velocities.

Figure 4.

Myo2 is associated with the tips of cytoplasmic microtubules. (A) Immunofluorescence microscopy of MYO2–BIM1 cells using anti-Tub2 and anti-Myo2 antibodies. (B–D) Fluorescence images of live cells containing (B) CFP–Tub1 and Myo2–GFP, (C) GFP–Tub1, and (D) GFP–Tub1 and Myo2–GFP. (E) Plot of average fluorescence intensities along cytoplasmic microtubules, starting from just off the plus ends, in GFP–TUB1 cells, GFP–TUB1 MYO2–GFP cells, and GFP–TUB1 MYO2–GFP kar9Δ cells. Intensities along 20 microtubules were measured for each cell type. Bars, 2 μm.

Figure 5.

Model for cytoplasmic microtubule orientation in budding yeast. Cytoplasmic microtubules arise from the spindle pole body and extend toward the mother cell cortex. Kar9 interacts with the microtubule end–binding protein Bim1, and with the tail of Myo2. Movement of Myo2 along polarized actin filaments transports the end of the microtubule to the bud neck. Further myosin-dependent movement or microtubule shortening could then serve to draw the spindle toward the bud.