Constitutive dynein activity in She1 mutants reveals differences in microtubule attachment at the yeast spindle pole body

Abstract

The organization of microtubules is determined in most cells by a microtubule-organizing center, which nucleates microtubule assembly and anchors their minus ends. In Saccharomyces cerevisiae cells lacking She1, cytoplasmic microtubules detach from the spindle pole body at high rates. Increased rates of detachment depend on dynein activity, supporting previous evidence that She1 inhibits dynein. Detachment rates are higher in G1 than in metaphase cells, and we show that this is primarily due to differences in the strengths of microtubule attachment to the spindle pole body during these stages of the cell cycle. The minus ends of detached microtubules are stabilized by the presence of γ-tubulin and Spc72, a protein that tethers the γ-tubulin complex to the spindle pole body. A Spc72-Kar1 fusion protein suppresses detachment in G1 cells, indicating that the interaction between these two proteins is critical to microtubule anchoring. Overexpression of She1 inhibits the loading of dynactin components, but not dynein, onto microtubule plus ends. In addition, She1 binds directly to microtubules in vitro, so it may compete with dynactin for access to microtubules. Overall, these results indicate that inhibition of dynein activity by She1 is important to prevent excessive detachment of cytoplasmic microtubules, particularly in G1 cells.

FIGURE 1:

she1∆ increases the rate of cytoplasmic microtubule detachment from the SPB. (A) Time-lapse images of a G1-arrested she1Δ cell expressing GFP-Tub1. The yellow arrowheads point to the plus end and the green arrowheads point to the minus end of a cytoplasmic microtubule that detaches from the SPB. Each frame advances 10 s. Scale bar, 5 μm. See Supplemental Video S1. (B) Rates of cytoplasmic microtubule detachment in wild-type (WT; CUY2015 and CUY2018), she1Δ (CUY2016 and CUY2019), nip100Δ (CUY1991 and CUY2033), and nip100Δ she1Δ (CUY2017 and CUY2034) cells. AS, asynchronous cells; G1, G1-arrested cells; M, metaphase-arrested cells. Data are given in Supplemental Table S1.

FIGURE 2:

(A) In wild-type (KAR1) G1 cells, Spc72 binds to Kar1 and microtubules are nucleated from the half-bridge. Graph shows cytoplasmic microtubule detachment rate in KAR1 (CUY2015) and KAR1 she1∆ (CUY2016) G1 cells. (B) The kar1-∆15 mutation eliminates the Spc72-binding site on Kar1; therefore, microtubules nucleate from the outer plaque in G1 kar1-∆15 cells. Graph shows microtubule detachment rate in kar1-∆15 (CUY2008) and kar1-∆15 she1∆ (CUY2009) G1 cells. (C) In wild-type (KAR1) metaphase cells, Spc72 binds to Nud1, and microtubules are nucleated from the outer plaque. Graph shows microtubule detachment rate in KAR1 (CUY2018) and KAR1 she1∆ (CUY2019) metaphase cells. (D) The kar1-∆15 mutation does not affect the binding of Spc72 to Nud1; therefore, microtubule attachment to the outer plaque at metaphase is not altered. Graph shows cytoplasmic microtubule detachment rate in kar1-∆15 (CUY2020) and kar1-∆15 she1∆ (CUY2021) metaphase cells. G1, G1-arrested cells; M, metaphase-arrested cells. Data are given in Supplemental Table S1.

FIGURE 3:

Detached cytoplasmic microtubules contain γ-tubulin and Spc72. (A) Detached cytoplasmic microtubules in G1-arrested she1Δ cells expressing mCherry-Tub1 and GFP-Tub4 (left, CUY2028) or mCherry-Tub1 and Spc72-GFP (right, CUY2037). Arrowheads indicate GFP signal at the minus ends of detached microtubules. Stars indicate SPBs. Scale bar, 2 μm. (B) Graphs of microtubule lengths after detachment from the SPB. Time 0 is defined as the frame when the microtubule detaches. Green points indicate the presence of GFP-Tub4 at the minus end; black points indicate that the GFP signal is not detected. (C) Diagram shows that cytoplasmic microtubules nucleate from the half-bridge in cells expressing the Spc72–Kar1 fusion protein and from the outer plaque in cells expressing the Spc72-Cnm67 fusion protein in both G1 and metaphase cells. Key to proteins as in Figure 2. (D) Rate of cytoplasmic microtubule detachment in wild-type (WT; CUY2015 and CUY2018), she1Δ (CUY2016 and CUY2019), SPC72-KAR1 (CUY2010 and CUY2025), SPC72-KAR1 she1Δ (CUY2011 and CUY2035), SPC72-CNM67 (CUY2022 and CUY2024), and SPC72-CNM67 she1Δ (CUY2023 and CUY2030) cells. G1, G1-arrested cells; M, metaphase-arrested cells. Data are given in Supplemental Table S1.

FIGURE 4:

Overexpression of She1 inhibits localization but not assembly of the dynactin complex. Cells contained a SHE1 construct expressed from the GAL1/10 promoter on a plasmid. SHE1 was overexpressed by shifting cells to galactose-containing medium for 4 h. (A) Quantification of spindle misorientation. Midanaphase spindles (3–6 μm in length) were scored as properly oriented if the spindle spanned the bud neck and misoriented if the spindle resided entirely within the mother cell body. Wild type (WT), CUY1972 containing pCUB1263; dyn1Δ, CUY1930 containing pCUB1288; nip100Δ, CUY1991 containing pCUB1299. (B) Localization of dynein and dynactin proteins at microtubule plus ends and the SPB. Dynein and dynactin proteins were tagged with GFP and microtubules with mCherry-Tub1. Nip100-GFP, CUY2055 containing pCUB1293; Arp1-GFP, CUY2056 containing pCUB1293; Jnm1-GFP, CUY2057 containing pCUB1293; Dyn1-GFP, CUY2040 containing pCUB1299. White arrowheads indicate GFP signal on the ends of cytoplasmic microtubules; yellow arrowheads indicate cytoplasmic microtubule ends with no GFP signal. Scale bar, 2 μm. (C) Quantification of dynein and dynactin protein localization taken from images like those shown in B. (D) Coimmunoprecipitation of dynactin proteins. Cell lysates were incubated with anti-HA affinity gel. Precipitated material was run on SDS–PAGE and blotted for with either anti-HA (left) or anti-Myc (right) antibodies. Top row, Arp-HA Nip100-Myc (MY8960 containing pXX3; second row, Nip100-HA Ldb18-Myc (CUY1933 containing pXX3); third row, Jnm1-HA Arp1-Myc (CUY1936 containing pCUB1263); bottom row, Jnm1-HA Nip100-Myc (CUY1938 containing pCUB1263).

FIGURE 5:

She1 associates with microtubules. (A) Lysate from cells expressing She1-13Myc (CUY1865) was incubated with various amounts of preassembled microtubules, the mixture centrifuged, and the supernatant (S) and pellet (P) fractions analyzed by Western blotting using anti-Myc (top) and anti-tubulin (bottom). Molar amounts of tubulin in microtubules used in each experiment are indicated above the lanes. (B) As in A, except that purified GST-She1 was used instead of cell lysates. Anti-GST antibody was used to visualize GST-She1.