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

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
Molecular biology of the cell (Impact Factor: 4.47). 04/2012; 23(12):2319-26. DOI: 10.1091/mbc.E12-03-0223
Source: PubMed
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.

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Available from: Zane Bergman
Volume 23 June 15, 2012 2319
M BoC | ARTICLE
Constitutive dynein activity in she1 mutants
reveals differences in microtubule attachment
at the yeast spindle pole body
Zane J. Bergman*, Xue Xia, I. Alexandra Amaro
, and Tim C. Huffaker
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853
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 spin-
dle pole body at high rates. Increased rates of detachment depend on dynein activity, sup-
porting 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 pre-
vent excessive detachment of cytoplasmic microtubules, particularly in G1 cells.
INTRODUCTION
Proper function of microtubules depends on their correct organiza-
tion within cells. In most cells, microtubules are organized by the
microtubule-organizing center (MTOC), which nucleates microtu-
bule assembly. Microtubule plus ends extend outward from the
MTOC, creating a polarized array of microtubules that the cell uses
for the directional transport of vesicles, organelles, and chromo-
somes (reviewed in Desai and Mitchison, 1997). Because many of
these transport events involve the movement of large cargoes, they
must generate considerable force. For example, in yeast, single
microtubules are used to pull the nucleus toward the bud neck and
chromosomes toward the spindle poles (O’Toole
et al., 1999). Thus
the anchoring of microtubules to the MTOC must be strong enough
to withstand these forces.
The MTOC in the budding yeast, Saccharomyces cerevisiae, is
the spindle pole body (SPB), an 0.5-GDa structure that is embed-
ded in the nuclear membrane (reviewed in Jaspersen and Winey,
2004). The SPB is a trilaminar, disk-shaped structure: a central plaque
spans the nuclear envelope, an outer plaque faces the cytoplasm,
and an inner plaque faces the nucleoplasm. Early in the cell cycle, a
structure termed the half-bridge is adjacent to one side of the cen-
tral plaque. Subsequent SPB duplication produces side-by-side
SPBs that are separated by a bridge that is twice the size of the half-
bridge. SPB separation severs the bridge, and SPBs with their as-
sociated half-bridges move to opposite sides of the nuclear enve-
lope to form the spindle poles.
The γ-tubulin complex nucleates microtubules at the SPB
(Marschall
et al., 1996; Spang et al., 1996). In yeast, the γ-tubulin
complex contains the γ-tubulin protein Tub4, Spc97, and Spc98
(Knop and Schiebel, 1997). In the cytoplasm, this complex is linked
to the SPB through Spc72 (Knop and Schiebel, 1998). In G1 cells,
Spc72 is primarily unphosphorylated and binds to Kar1, which is
located in the half-bridge. As cells enter S phase and proceed
Monitoring Editor
Kerry S. Bloom
University of North Carolina
Received: Mar 20, 2012
Revised: Apr 19, 2012
Accepted: Apr 19, 2012
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E12-03-0223) on April 25, 2012.
Present addresses: *Department of Biology, San Francisco State University, San
Francisco, CA 94132;
Vybion, Ithaca, NY 14850.
Address correspondence to: Tim Huffaker (tch4@cornell.edu).
© 2012 Bergman
et al. This article is distributed by The American Society for Cell
Biology under license from the author(s). Two months after publication it is avail-
able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB
®
,” “The American Society for Cell Biology
®
,” and “Molecular Biology of
the Cell
®
” are registered trademarks of The American Society of Cell Biology.
Abbreviations used: GFP, green fluorescent protein; MTOC, microtubule-organiz-
ing center; SPB, spindle pole body.
http://www.molbiolcell.org/content/suppl/2012/04/23/mbc.E12-03-0223.DC1.html
Supplemental Material can be found at:
Page 1
2320 | Z. J. Bergman et al. Molecular Biology of the Cell
respectively, in G1 than in metaphase. In G1 and metaphase cells,
microtubule detachment is 15- and 10-fold more frequent, respec-
tively, in she1 cells than in wild-type cells.
Woodruff
et al. (2009) reported that She1 limits dynein activity to
anaphase by inhibiting the recruitment of dynactin to cytoplasmic
microtubule ends at other points in the cell cycle. This result sug-
gests that the increased microtubule detachment observed in she1
cells is likely due to untimely dynein activity. To test this possibility,
we measured microtubule detachment in cells lacking the dynactin
complex protein Nip100, which is essential for dynein activity. Mi-
crotubule detachment rates in she1 nip100 cells were even less
than those in wild-type cells for asynchronous, G1, and metaphase
populations (Figure 1B). Thus the increased frequency of microtu-
bule detachment in she1 cells depends on dynein activity.
Detachment rate depends on the site of cytoplasmic
microtubule anchorage
We were curious as to why the microtubule detachment rate
differed between G1 and metaphase. In cycling cells, cytoplasmic
microtubules originate from both the outer plaque and half-bridge
through mitosis, Spc72 becomes phosphorylated and binds prefer-
entially to Nud1, a component of the outer plaque (Pereira et al.,
1999). Therefore, cytoplasmic microtubules primarily arise from the
half-bridge during G1 and from the outer plaque during mitosis.
The primary role of cytoplasmic microtubules in yeast is to orient
the spindle. Two pathways contribute to spindle orientation. Early in
the cell cycle a complex of Bim1, Kar9, and Myo2 associates with
the plus end of the cytoplasmic microtubule (Korinek
et al., 2000;
Lee
et al., 2000; Yin et al., 2000). Myo2 is a myosin V protein that
translocates along polarized actin cables into the bud, thereby pull-
ing the microtubule and its attached SPB toward the bud neck (Yin
et al., 2000; Hwang et al., 2003). This event places the metaphase
spindle adjacent to the bud neck. Later in the cell cycle, a second
pathway involving dynein ensures that the elongating spindle passes
through the bud neck and into the bud (Moore
et al., 2009). During
anaphase, dynactin is recruited to the cytoplasmic microtubule plus
end, where it activates dynein. Dynein then interacts with Num1, a
protein bound to the bud cortex. Here the minus end–directed mo-
tor activity of dynein reels in the cytoplasmic microtubule, thereby
pulling the attached SPB into the bud (Farkasovsky and Küntzel,
1995; Heil-Chapdelaine
et al., 2000; Lee et al., 2005; Markus and
Lee, 2011).
To ensure the proper timing of dynein function, its activity is re-
stricted to a small portion of the cell cycle around anaphase (re-
viewed in Moore et al., 2009). Dynein is found on cytoplasmic micro-
tubules during the majority of the cell cycle, so the timing of dynein
activity is believed to depend on the loading of the dynactin com-
plex, which becomes enriched at cytoplasmic microtubule plus ends
during anaphase (Woodruff et al., 2009). She1 inhibits the loading of
the dynactin complex onto microtubule ends at other stages of the
cell cycle (Woodruff
et al., 2009). In this study, we show that She1
activity is important to prevent high rates of cytoplasmic microtu-
bule detachment from the SPB. We characterize the molecular na-
ture of these detachment events, show that their frequency depends
on the way in which cytoplasmic microtubules are anchored to the
SPB, and propose a model for She1 action.
RESULTS
Detachment of cytoplasmic microtubules from the SBP in
she1 mutants depends on the cell cycle and dynein activity
We noticed that cytoplasmic microtubules in she1 cells frequently
detached from their anchor point at the SPB and moved freely around
the cell periphery before depolymerizing (Figure 1A and Supplemen-
tal Video S1). Similar cytoplasmic microtubule detachment from
the SPB was previously observed in cells containing cnm67 or
SPC72
stu2
mutations, which affect the integrity of the SPB outer
plaque (Hoepfner
et al., 2000; Usui et al., 2003). We quantified this
effect by determining the fraction of cytoplasmic microtubules that
detach from the SPB per minute. In asynchronously growing wild-
type cells only 0.02% of microtubules detach (Figure 1B). In contrast,
in asynchronously growing she1 cells 0.7% of microtubules detach.
Further observation of microtubule detachment in asynchronous
cultures revealed that the majority of these events occurred in cells
that were growing early in the cell cycle, before the formation of a
bipolar spindle. To measure this difference, we created uniform
populations of cells by arresting them either in G1, by exposure to
α-factor, or in metaphase, by depletion of Cdc20. During G1 arrest,
0.1% of microtubules detach in wild-type cells and 1.5% of microtu-
bules detach in she1 cells (Figure 1B). During metaphase arrest,
0.02% of microtubules detach in wild-type cells and 0.2% of micro-
tubules detach in she1 cells. Thus, in wild-type and she1 cells
microtubule detachment is five- and eightfold more frequent,
FIGURE 1: she1 increases the rate of cytoplasmic microtubule
detachment from the SPB. (A) Time-lapse images of a G1-arrested
she1 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), she1 (CUY2016 and CUY2019), nip100 (CUY1991
and CUY2033), and nip100 she1 (CUY2017 and CUY2034) cells. AS,
asynchronous cells; G1, G1-arrested cells; M, metaphase-arrested
cells. Data are given in Supplemental Table S1.
AS G1 M
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
nim/gnihcated sTMc %
A
B
WT
she1
nip100
nip100she1
t = 0 st = 10 t = 20 t = 30 t = 40
t = 50 t = 60 t = 70 t = 80 t = 90
t = 100t = 110t = 120t = 130t = 140
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Volume 23 June 15, 2012 Detachment of microtubules in she1 cells | 2321
microtubule numbers, we observed a significant number of cyto-
plasmic microtubules in kar1-
15 and kar1-15 she1 cells that are
not attached to the SPB (0.5 ± 0.1 and 0.6 ± 0.1 per cell, respec-
tively). Although these might have detached from the SPB, it seems
more likely they are nucleated by cytoplasmic aggregates of Spc72
that fail to bind to the SPB (Vallen et al., 1992; Pereira et al., 1999).
The kar1-
15 mutation also increased the rate of cytoplasmic micro-
tubule detachment threefold above that observed in wild-type cells.
Although we do not know the mechanism of this increased rate of
release, we refer to it as spontaneous release because it does not
depend on dynein activity; introducing the nip100 mutation does
not lower cytoplasmic microtubule detachment in kar1-
15 cells
(Supplemental Table S1).
Using the kar1-15 mutation, we could measure the effect of
she1 on microtubule detachment from both the half-bridge (she1
cells) and the outer plaque (kar1-15 she1 cells) at the same point
in the cell cycle (G1). In G1 kar1-
15 she1 cells, the cytoplasmic
microtubule detachment rate is nearly fourfold lower than in she1
cells (Figure 2, A and B). This rate is similar to the spontaneous re-
lease rate observed in kar1-15 cells, indicating that she1 has rela-
tively little effect in kar1-15 cells. The nip100 mutation does not
lower microtubule detachment in kar-15 she1 cells, indicating
that this residual release is not due to dynein activity (Supplemental
Table S1). Thus microtubule attachment to the outer plaque, even
under suboptimal conditions, is stronger than the normal microtu-
bule attachment to the half-bridge.
Detached microtubules possess γ-tubulin and Spc72
We next set out to establish the place at which cytoplasmic micro-
tubule detachment occurs. The first aim was to find whether
cytoplasmic microtubules broke somewhere along the length of
during the early portion of the cell cycle but extend exclusively from
the outer plaque once the spindle has formed (Byers and Goetsch,
1975; O’Toole
et al., 1999; Pereira et al., 1999). In cells arrested in
G1 by treatment with α-factor, cytoplasmic microtubules originate
only from the half-bridge (Figure 2A). Hence, cells arrested in G1 by
exposure to α-factor and in metaphase by depletion of Cdc20 con-
tain cytoplasmic microtubules that originate only from the half-
bridge and outer plaque, respectively. Because cytoplasmic micro-
tubules originate from two alternate locations in G1 and metaphase
cells, detachment rates could reflect differences in the strengths of
attachment of microtubules to the SPB. An alternative explanation is
that the amount of force pulling on cytoplasmic microtubules could
differ during G1 and metaphase. To distinguish between these two
possibilities, we wanted to compare detachment rates from these
two locations in the same cell cycle state. To this end, we created a
strain in which cytoplasmic microtubules are nucleated from the
outer plaque even when arrested in G1. The kar1-
15 mutation de-
letes the portion of Kar1 that binds Spc72 and thus eliminates cyto-
plasmic microtubule nucleation from the half-bridge (Vallen
et al.,
1992; Pereira
et al., 1999). In metaphase cells, when cytoplasmic
microtubules normally nucleate from the outer plaque, we predicted
that the kar1-
15 mutation should have little effect on cytoplasmic
microtubule detachment, and this is what we observed for SHE1
and she1 cells (Figure 2, C and D).
In G1 cells, we observed about half the normal number of cyto-
plasmic microtubules in kar1-15 and kar1-15 she1 cells (2.1 mi-
crotubules per wild-type cell and 1.0 microtubule per kar1-15 cell).
These cytoplasmic microtubules presumably arise from the outer
plaque where Spc72 binds to Nud1. At this stage of the cell cycle,
when Spc72 binding to Kar1 is normally favored, binding to Nud1 is
not optimal (Pereira
et al., 1999). In addition to reduced cytoplasmic
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.
A
B
G1 M
Tub1
Tub4
Tub2
Spc97/98
Spc72
Cnm67 Kar1
Nud1
KAR1 KAR1
kar1-15 kar1-15
she1
she1
she1
she1
SHE1
SHE1
SHE1
SHE1
0
0.3
0.6
0.9
1.2
1.5
1.8
C
D
% cMTs detaching/min
0
0.3
0.6
0.9
1.2
1.5
1.8
% cMTs detaching/min
0
0.3
0.6
0.9
1.2
1.5
1.8
% cMTs detaching/min
0
0.3
0.6
0.9
1.2
1.5
1.8
% cMTs detaching/min
Page 3
2322 | Z. J. Bergman et al. Molecular Biology of the Cell
she1 cells in G1 expressing mCherry-Tub1 and green fluorescent
protein (GFP)–Tub4 (Figure 3A). Of the 72 cytoplasmic microtu-
bules observed detaching from the SPB, 62 (86%) had visible GFP-
Tub4 at the end detaching from the SPB. We also imaged cyto-
plasmic microtubule detachment in she1 cells in G1 expressing
mCherry-Tub1 and Spc72-GFP (Figure 3A). Spc72-GFP was ob-
served on the ends of 21 of 26 (81%) detached cytoplasmic micro-
tubules. On the other hand, we never observed Spc42 on the ends
of detached cytoplasmic microtubules in cells expressing GFP-
Tub1 and Spc42-mRFP. This result was expected, since Spc42 is in
the central core of the SPB. In summary, these results indicate that
detached cytoplasmic microtubules contain γ-tubulin and Spc72 at
their minus ends, indicating that the break must occur somewhere
on the SPB-proximal side of Spc72.
We also measured the lifetime of 10 detached cytoplasmic micro-
tubules in she1 cells expressing mCherry-Tub1 and GFP-Tub4; 4 of
these are plotted in Figure 3B. The GFP-Tub4 decoration remained
on the minus ends of detached microtubules for 69 ± 38 s. During
this time some microtubules elongated, some shortened, and some
remained fairly constant in length. After loss of the GFP-Tub4 deco-
ration, microtubules inevitably shortened and disappeared in 17 ±
7 s. Thus the minus ends of detached cytoplasmic microtubules ap-
pear to be stabilized initially against depolymerization by the pres-
ence of a cap of γ-tubulin and associated proteins. Loss of this cap
results in rapid depolymerization of the microtubule.
We next examined whether the break point in G1 cells was be-
tween Spc72 and Kar1. Spc72
1-276
–Kar1
192-433
is a fusion protein that
combines the γ-tubulin complex–binding portion of Spc72 and the
half-bridge-binding region of Kar1 (Pereira
et al., 1999). This con-
struct was expressed in a strain lacking the native Spc72 and Kar1
proteins, so that the only source of these proteins is the fusion pro-
tein (Figure 3C). Thus all cytoplasmic microtubules in these cells are
anchored at the half-bridge through the Spc72–Kar1 fusion protein.
Addition of the Spc72–Kar1 fusion protein lowers the rates of cyto-
plasmic microtubule detachment twofold in wild-type cells and
11-fold in she1 cells (Figure 3D). Because the fusion protein sub-
stantially reduces cytoplasmic microtubule detachment, we con-
clude that the interaction between Spc72 and Kar1 is the linkage
that is normally broken during this process in G1 cells.
We used a second fusion protein to examine whether the
break point in metaphase cells was between Spc72 and Cnm67.
Spc72
1-276
–Cnm67
1-581
is a fusion protein that combines the Tub4-
binding region of Spc72 and the outer plaque–binding portion of
Cnm67 (Gruneberg
et al., 2000). This fusion protein bypasses the
need for Nud1 that normally bridges these two proteins. Cells
expressing the Spc72–Cnm67 fusion lack the native Spc72, so the
only source of cytoplasmic microtubule anchoring is through the
fusion protein located in the outer plaque (Figure 3C). The pres-
ence of the Spc72–Cnm67 fusion protein did not reduce the rate
of cytoplasmic microtubule detachment; in fact detachment rates
rose fivefold in wild-type cells and 1.3-fold in she1 cells (Figure
3D). Thus we cannot conclude that it is the linkage between
Spc72 and Cnm67 that is normally broken during cytoplasmic mi-
crotubule detachment in metaphase cells.
Of interest, the Spc72–Kar1 fusion did not reduce cytoplasmic
microtubule detachment rates in metaphase-arrested cells; in fact
these rates rose 17-fold in wild-type and twofold in she1 cells
(Figure 3D). Thus, even the enhanced stability provided by this fu-
sion protein at the half-bridge is still less than that provided by the
normal outer plaque connection in metaphase cells. In addition, the
Spc72–Cnm67 fusion reduced the rate of cytoplasmic microtubule
detachment in G1 cells: twofold for wild-type cells and ninefold
the polymer or whether they were pulled intact from the SPB. If the
latter were true, it might be possible to observe anchoring pro-
teins from the γ-tubulin complex or even the SPB on the minus
ends of detached cytoplasmic microtubules. We initially imaged
FIGURE 3: Detached cytoplasmic microtubules contain γ-tubulin and
Spc72. (A) Detached cytoplasmic microtubules in G1-arrested she1
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), she1
(CUY2016 and CUY2019), SPC72-KAR1 (CUY2010 and CUY2025),
SPC72-KAR1 she1 (CUY2011 and CUY2035), SPC72-CNM67
(CUY2022 and CUY2024), and SPC72-CNM67 she1 (CUY2023 and
CUY2030) cells. G1, G1-arrested cells; M, metaphase-arrested cells.
Data are given in Supplemental Table S1.
GFP-Tub4 Spc72-GFP
mCh-Tub1Merge
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
G1 M
WT
she1 SPC72-CNM67
SPC72-CNM67 she1
SPC72-KAR1
SPC72-KAR1 she1
A
nim/gnihcated sTMc %
B
C
Spc72-Kar1Spc72-Cnm67
D
020406080100 120140 160
Time since detachment (sec)
0
3
1
2
MT Length (µm)
GFP
0
3
1
2
0
3
1
2
0
3
1
2
Page 4
Volume 23 June 15, 2012 Detachment of microtubules in she1 cells | 2323
caused by inhibiting dynein activity—specifically, the elongation of
anaphase spindles within the mother cell. To test this, we imaged
cells expressing She1 from the highly efficient GAL1/10 promoter.
We quantified the fraction of midanaphase spindles (between 3 and
6 μm) that were incorrectly oriented with both spindle poles in the
mother cell. Cells overexpressing She1 had a level of spindle misori-
entation similar to that in dyn1 and nip100 cells (Figure 4A).
Overexpressing She1 in dyn1 or nip100 cells did not increase
their frequency of spindle misorientation, indicating that the She1
acts through the dynein pathway.
To investigate the mechanism of She1 inhibition of dynein activ-
ity, we overexpressed She1 in cells expressing GFP-tagged dynac-
tin complex proteins and mCherry-Tub1 and scored cells for GFP
signal at cytoplasmic microtubule plus ends and the SPB. For cells
for she1 cells (Figure 3D). This indicates that even the weakened
outer plaque connection provided by this fusion protein is still
stronger than the normal half-bridge connection. Both of these re-
sults support the conclusion of the preceding section that the con-
nection of cytoplasmic microtubules to the outer plaque is stronger
than that at the half-bridge.
Overexpression of She1 inhibits dynactin complex
localization to microtubule ends
Our observation that excessive cytoplasmic microtubule detach-
ment in she1 cells depends on dynein activity is consistent with
previous work suggesting that She1 negatively regulates dynein
activity (Woodruff
et al., 2009). Thus we hypothesized that over-
expression of She1 would produce a phenotype similar to that
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; dyn1, CUY1930 containing
pCUB1288; nip100, 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).
Glu
Gal
WT dyn1 nip100
20
40
60
80
mis-oriented spindles (%)
B
GluGal
GluGal
GluGal
Nip100-GFP
Arp1-GFP
Jnm1-GFP
A
mCh-Tub1 MergeGFP
GluGal
Dyn1-GFP
0
Nip100 Jnm1Arp1 Dyn1
Glu Gal Glu Gal Glu Gal Glu Gal
0
20
40
60
80
100
% cells with spindle
SPB only MT plus ends only
MT plus ends & SPB
C
HA MYC
Glu GluGal
Gal
Arp10
Nip100
Jnm1
Jnm1
Ldb18
Arp1
Nip100
Nip100
D
Properly
oriented
Mis-oriented
mCh-Tub1 MergeGFP
Page 5
2324 | Z. J. Bergman et al. Molecular Biology of the Cell
tween She1 and microtubules could be direct or could be mediated
by another protein in the cell extract. To determine whether She1
can bind directly to microtubules, we purified GST-She1 from bacte-
rial cells and incubated it with Taxol-stabilized microtubules. GST-
She1 bound to microtubules with an apparent dissociation constant
of 1 μM (Figure 5B). Thus She1 has the ability to bind to microtu-
bules directly and with an affinity similar to that of other microtu-
bule-associated proteins (Blake-Hodek
et al., 2010).
DISCUSSION
We found that in cells lacking She1, cytoplasmic microtubules de-
tach from the SPB at greatly elevated rates. This phenotype has also
been noted by Markus
et al. (2011). Microtubule detachment re-
quires dynein activity; simultaneous loss of the dynactin protein
Nip100 completely eliminates the she1 effect. This result agrees
with previous reports indicating that She1 is a negative regulator of
dynein function. This conclusion is also supported by our data show-
ing that overexpression of She1 produces a defect in spindle orien-
tation identical to that caused by loss of dynein or Nip100.
To determine how She1 inhibits dynein activity, we examined the
localization of dynein and dynactin complex proteins in cells overex-
pressing She1. Whereas She1 overexpression has little effect on dy-
nein localization, it greatly reduces the localization of dynactin pro-
teins to cytoplasmic microtubule plus ends. This agrees with previous
reports that loss of She1 allows dynactin components to localize to
cytoplasmic microtubule plus ends at earlier points in the cell cycle
(Woodruff
et al., 2009) and in greater number (Markus et al., 2011).
She1 overexpression does not affect the integrity of the dynactin
complex, supporting the previous suggestion that She1 interferes
with the interaction between dynein and dynactin (Woodruff
et al.,
2009; Markus
et al., 2011). However, She1 did not coprecipitate with
the dynactin complex, indicating that any interaction is transient.
She1 was shown previously to associate with cytoplasmic microtu-
bules during G1 and preanaphase, when dynein is inactive, but not
during anaphase, when dynein is active (Woodruff et al., 2009). Our
results show that She1 can bind directly to microtubules, leading to
the possibility that She1 inhibits dynein activity by competing with
dynactin for access to microtubules.
Although dynein activity is down-regulated during G1, it is still
the major cause of microtubule detachment during this portion of
the cell cycle; loss of Nip100 causes a fourfold decrease in microtu-
bule detachment in G1 cells. On the other hand, Myo2, the type V
myosin protein that is responsible for directing cytoplasmic microtu-
bules to the bud in the early stages of the cell cycle and might
therefore be expected to play a role in microtubule detachment in
G1 cells, has relatively little effect on microtubule detachment. Even
if we attribute all of the microtubule detachment in nip100 cells to
Myo2 activity, it still amounts to only 25% of the rate in wild-type
cells. In G1 cells lacking She1, which may contain maximal dynein
activity, 95% of microtubule detachment can be attributed to dynein
activity (as determined by comparing detachment in she1 vs.
she1 nip100 cells). These results likely reflect the fact that dynein
pulls harder on cytoplasmic microtubules than Myo2; in vitro studies
show that cytoplasmic dynein generates more than twice the force
of myosin V (Gennerich
et al., 2007; Mehta et al., 1999).
Although dynein pulls on the SPB-distal plus ends of cytoplasmic
microtubules, the breakage event occurs at the SPB. Detached mi-
crotubules always appear to be full length and most contain γ-tubulin
and Spc72 at their minus ends. Because Spc72 binds Spc97 and
Spc98 but not Tub4 (Knop and Schiebel, 1998), we assume that the
entire γ-tubulin complex is present. Of interest, the γ-tubulin com-
plex appears sufficient to stabilize the minus ends of detached
expressing Nip100-GFP, Arp1-GFP, and Jnm1-GFP, She1 overex-
pression decreased the number of cells with plus-end labeling
(nine-, seven-, and eightfold, respectively) and increased the num-
ber of cells with labeling only at the SPB (seven-, 17-, and fourfold,
respectively; Figure 4, B and C). The overall number of cells that
displayed a GFP signal was relatively unchanged. Overexpression
of She1 did not substantially affect the localization of Dyn1-GFP
(Figure 4, B and C).
One possible mechanism by which She1 could inhibit dynactin
loading onto microtubule plus ends is by interfering with dynactin
assembly. To test this possibility, we overexpressed She1-GFP and
assessed the interaction between dynactin complex proteins by
coimmunoprecipitation. Binding between Arp10-3HA and Nip100-
13Myc, Nip100-3HA and Ldb18-13Myc, Jnm1-3HA and Arp1-
13Myc, and Jnm1-3HA and Nip100-13Myc was not disrupted in
cells overexpressing She1-GFP (Figure 4D). Thus, She1 does not ap-
pear to interfere with dynactin assembly. In addition, She1-GFP was
not found in any of the immunoprecipitates, indicating that overex-
pression of She1-GFP does not disturb dynactin localization through
direct interaction with its subunits (Supplemental Figure S1).
She1 associates directly with microtubules
As previously reported, She1-GFP localizes to mitotic spindles, cyto-
plasmic microtubules, and a ring structure at the bud neck (Wong
et al., 2007; Woodruff et al., 2009). We assessed She1’s ability to
associate with microtubules by incubating whole-cell extracts of a
strain expressing She1-13Myc with preassembled, Taxol-stabilized
bovine microtubules. The microtubules were then spun down and
the amount of She1-13Myc in the pellet and supernatant compared.
She1-13Myc bound to microtubules in a microtubule concentration–
dependent manner (Figure 5A). The apparent dissociation constant,
equal to the concentration of polymerized tubulin required to
cosediment half of the She1-13Myc, is 2 μM. This association be-
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.
A
B
SP
SSS
SSS
PPP
PPP
MYC
Tubulin
Tubulin
GST
0 µM1µM2µM5µM
0 µM1µM3µM
Page 6
Volume 23 June 15, 2012 Detachment of microtubules in she1 cells | 2325
Microscopy and image analysis
Images were obtained using a spinning disk confocal system as pre-
viously described (Huang and Huffaker, 2006). All images are maxi-
mum-intensity projections of z-series stacks (z = 0.7 μm). Analysis
was performed using ImageJ (National Institutes of Health, Bethesda,
MD). The mean values and standard deviations of microtubule de-
tachment were determined using the Poisson approximation to the
binomial distribution.
Microtubule cosedimentation assays
For assays using whole-cell extracts, log-phase yeast cells express-
ing She1-13Myc (CUY1865) were centrifuged for 5 min at 2000 rpm,
washed with lysis buffer (80 mM 1,4-piperazinediethanesulfonic
acid [PIPES], 1 mM ethylene glycol tetraacetic acid [EGTA], 1 mM
MgSO
4
, 5% glycerol, 100 mM KCl, 0.25% Brij-35, 2 mM dithio-
threitol [DTT], pH 6.8), and resuspended in lysis buffer containing
20 μM Taxol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leu-
peptin, and 10 μg/ml pepstatin. Cells were lysed in a bead beater,
and lysates were precleared by centrifugation at 350,000 × g for
5 min at 4°C. Tubulin (Cytoskeleton, Denver, CO) was diluted to
1 mg/ml in PEM-DGT (100 mM PIPES, 2 mM EGTA, 1 mM MgCl
2
,
20 μM Taxol, 1 mM GTP, and 4 mM DTT) and precleared by cen-
trifugation at 150,000 × g for 5 min at C. The supernatant was
incubated at 37°C for 20 min to allow microtubule polymerization,
followed by centrifugation at 35,000 × g for 20 min at room tem-
perature. The microtubule pellet was resuspended in PEM-DGT to
a final concentration of 5 mg/ml. Various amounts of microtubules
were added to 20 μl of precleared yeast cell lysate and incubated
for 20 min on ice. Samples were centrifuged at 175,000 × g for
10 min at 4°C. Supernatant and pellet fractions were analyzed by
SDS–PAGE and Western blotting using 9E10 anti-Myc (Covance,
Princeton, NJ) and DM1α anti-tubulin (Sigma-Aldrich, St. Louis,
MO) antibodies.
For assays using purified protein, She1–glutathione S-transferase
(GST) was expressed in BL21 Escherichia coli cells containing plas-
mid pXX6. Cells were grown at 26°C in 1 l of Luria–Bertani broth
containing 50 μg/ml kanamycin and then induced by adding 50 μM
isopropyl-β-d-thiogalactoside for 4 h. Cells were then spun down,
washed with cold phosphate-buffered saline (PBS), and resuspended
in 8.5 ml of cold PBS containing EDTA-free protease inhibitors
(Roche, Basel, Switzerland). Cells were lysed by the addition of 10 μl
of 100 mg/ml lysozyme for 30 min, followed by sonication. Triton
X-100 was added to 1% and the mixture rocked at 4°C for 20 min.
Cell debris was pelleted at 12,000 × g for 25 min at 4°C. The super-
natant was incubated with glutathione–Sepharose beads for 2.5 h at
4°C. Beads were then pelleted, washed four times with cold PBS,
resuspended in 350 μl of 80 mM reduced glutathione in PBS (pH 7),
and rocked for 16 h at 4°C. Purified GST-She1 was then obtained
by adding the mixture to a minichromatography column (Bio-Rad,
Hercules, CA) and spinning at 5000 × g for 2 min. GST-She1 was
incubated with assembled microtubules at room temperature for
5 min and then spun at 175,000 × g for 40 min. Supernatant and
pellet fractions were analyzed by SDS–PAGE and Western blotting
using anti-GST antibodies.
Coimmunoprecipitations
Coimmunoprecipitations were performed as described previously
(Wolyniak
et al., 2006), except that anti-hemagglutinin (HA) affinity
gel or anti-Myc affinity gel (Sigma-Aldrich) was used to precipitate
epitope-tagged proteins from cell lysates. Western blots were
probed with either 9E10 anti-Myc or HA.11 anti-HA antibodies
(Covance).
microtubules, because these microtubules disappear rapidly after
loss of this structure. In G1-arrested cells, Spc72 is primarily unphos-
phorylated and binds to Kar1 in the half-bridge. A Spc72–Kar1 pro-
tein fusion greatly reduces microtubule detachment in G1 cells, in-
dicating that the Spc72–Kar1 interaction is the weak link at the
half-bridge. In metaphase cells, Spc72 is phosphorylated and binds
to Nud1, which in turn binds Cnm67, in the outer plaque. However,
fusing Spc72 to Cnm67 did not reduce microtubule detachment
from the outer plaque. This could be taken as evidence that the
Spc72 attachment is not the weak link in the outer plaque. On the
other hand, one could simply argue that the Spc72–Cnm67 fusion
damages the outer plaque sufficiently to allow microtubule detach-
ment by an aberrant mechanism.
Microtubule detachment rates are higher in G1 cells than in
M-phase cells. Three lines of evidence indicate that this difference is
due to a weaker microtubule attachment to the SPB half-bridge,
rather than a stronger pulling force, in G1 cells. 1) We used the
kar1-15 mutation to restrict microtubule nucleation to the outer
plaque. This allowed us to measure the effect of she1 on microtu-
bule detachment from both the half-bridge (she1 cells) and the
outer plaque (kar1-15 she1 cells) at the same point in the cell cy-
cle (G1). The microtubule detachment rate from the half-bridge was
fourfold higher than from the outer plaque. 2) We used the Spc72–
Kar1 protein fusion to direct microtubule attachment to the half-
bridge. This strengthens microtubule attachment to the half-bridge,
but even this strengthened attachment is weaker than normal outer
plaque attachment. Similarly, we used the Spc72-Cnm67 fusion pro-
tein to direct microtubule attachment to the outer plaque. This
weakens microtubule attachment at the outer plaque, but even this
weakened attachment is stronger than the normal half-bridge at-
tachment. 3) If we assume that dynein is maximally active in she1
cells, then the pulling force should be equivalent in G1 and M phase
she1 cells; however, microtubule detachment is eightfold more fre-
quent in G1 cells. Thus we conclude that microtubule attachments
to the outer plaque are stronger than those at the half-bridge.
It is not entirely clear why yeast nucleate cytoplasmic microtu-
bules from two distinct locations on the SPB. Nucleation from both
sites is not essential for vegetative growth; both the Spc72–Kar1
fusion, which confines nucleation to the half-bridge, and the Spc72–
Cnm67 fusion, which confines nucleation to the outer plaque, allow
cells to grow (Pereira et al., 1999; Gruneberg et al., 2000). However,
microtubules coming from the half-bridge are essential for karyo-
gamy (Conde and Fink, 1976; Pereira
et al., 1999) and, because
mating occurs only between cells arrested in G1 by mating factors,
it follows that the half-bridge would be the predominant site of mi-
crotubule nucleation in cycling G1 cells. The switch to the outer
plaque as cells enter mitosis may be made in part to withstand the
pulling forces exerted by dynein, which is most active in the later
portions of the cell cycle.
MATERIALS AND METHODS
Yeast strains and plasmids
Yeast strains used in this study are S288C or derivatives and are
listed in Supplemental Table S2. Plasmids used are listed in Supple-
mental Table S3.
MATa cells were arrested in G1 by adding 3 μg/ml α-factor to the
medium for 3 h. Cells containing P
MET3
-CDC20-3HA were arrested
in metaphase by adding 20 μg/ml methionine to the medium for
3 h. She1 was overexpressed in cells containing P
GAL1/10
-SHE1,
P
GAL1/10
-SHE1-GFP, or P
GAL1/10
-SHE1-13MYC by growing cultures
to log phase in 2% raffinose medium for several generations and
then shifting them to 2% galactose medium for 4–6 h.
Page 7
2326 | Z. J. Bergman et al. Molecular Biology of the Cell
ACKNOWLEDGMENTS
We thank Mark Rose (Princeton University, Princeton, NJ), Elmar
Schiebel (Zentrum für Molekulare Biologie der Universität Heidel-
berg, Heidelberg, Germany), and Dean Dawson (Oklahoma Medical
Research Foundation, Oklahoma City, OK) for yeast strains and plas-
mids. This work was supported by National Institutes of Health
Grant GM40479 (to T.C.H.). I.A.A. was funded by National Institutes
of Health Predoctoral Grant GM073576.
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Page 8
SUPPLEMENTAL MATERIALS
Constitutive Dynein Activity in she1 Mutants Reveals Differences in Microtubule
Attachment at the Yeast Spindle Pole Body
Zane J. Bergman, Xue Xia, I. Alexandra Amaro and Tim C. Huffaker
Table S1. Rates of cytoplasmic microtubule detachment!
Genotype
Cell Cycle
Phase
% Detachment
Microtubules
Observed
Wild type
AS
0.021 ± 0.011
1893
Wild type
G1
0.116 ± 0.026
1640
Wild type
M
0.023 ± 0.023*
426
she1Δ
AS
0.699 ± 0.062
1701
she1Δ
G1
1.536 ± 0.090
1593
she1Δ
M
0.194 ± 0.051
721
nip100Δ
AS
0.027 ± 0.011
2191
nip100Δ
G1
0.032 ± 0.018
940
nip100Δ
M
0.008 ± 0.008*
1243
nip100Δ she1Δ
AS
0.014 ± 0.007
2801
nip100Δ she1Δ
G1
0.068 ± 0.028
884
nip100Δ she1Δ
M
0.013 ± 0.009
1519
kar1-Δ15
AS
0.082 ± 0.041
488
kar1-Δ15
G1
0.351 ± 0.057
1023
kar1-Δ15
M
0.014 ± 0.014*
721
kar1-Δ15 she1Δ
AS
0.361 ± 0.098
358
kar1-Δ15 she1Δ
G1
0.329 ± 0.056
1000
kar1-Δ15 she1Δ
M
0.301 ± 0.063
728
kar1-Δ15 nip100Δ
AS
0.020 ± 0.020*
495
kar1-Δ15 nip100Δ
G1
0.635 ± 0.114
455
kar1-Δ15 nip100Δ
M
0.023 ± 0.023*
436
kar1-Δ15 nip100Δ she1Δ
AS
0.014 ± 0.014*
708
kar1-Δ15 nip100Δ she1Δ
G1
0.465 ± 0.081
665
kar1-Δ15 nip100Δ she1Δ
M
0.018 ± 0.018*
545
SPC72-KAR1
AS
0.242 ± 0.044
1238
SPC72-KAR1
G1
0.049 ± 0.013
2846
SPC72-KAR1
M
0.389 ± 0.071
744
SPC72-KAR1 she1Δ
AS
0.347 ± 0.071
660
SPC72-KAR1 she1Δ
G1
0.145 ± 0.051
549
SPC72-KAR1 she1Δ
M
0.347 ± 0.078
546
SPC72-CNM67
AS
0.036 ± 0.018
1098
SPC72-CNM67
G1
0.050 ± 0.016
1816
SPC72-CNM67
M
0.121 ± 0.043
658
SPC72-CNM67 she1Δ
AS
0.121 ± 0.049
493
SPC72-CNM67 she1Δ
G1
0.174 ± 0.044
862
SPC72-CNM67 she1Δ
M
0.248 ± 0.087
320
AS, asynchronous cells; G1, G1-arrested cells; M, metaphase-arrested cells.
* Denotes best approximation for genotypes with no observed detachment events.
!
Page 9
!
2
Table S2. Yeast strains
Strain
Genotype
CUY1865
MATa ade2-101 his3-Δ200 leu2-3 112 ura3-52 SHE1-13MYC::HIS3MX
CUY1930
MATa his3Δ0 leu2Δ0 met15Δ0 ura3Δ0 dyn1Δ::kanMX P
TUB1
-GFP-TUB1::LEU2
CUY1933
MATα ade2Δ his3-Δ200 leu2Δ ura3-52 LDB18-13MYC::HIS3MX
NIP100-3HA::kanMX
CUY1936
MATa ade2Δ his3 leu2 lys2-801 met15Δ0 JNM1-3HA::kanMX
ARP1-13MYC::kanMX
CUY1938
MATα his3 leu2 lys2-801 met15Δ0 ura3 JNM1-3HA::kanMX
NIP100-13MYC::kanMX
CUY1972
MATa his3-Δ200 leu2-3 112 lys2-801 ura3-52 P
HIS3
-mCherry-TUB1::URA3
CUY1990
MATa his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 arp1Δ::kanMX P
TUB1
-GFP-TUB1::URA3
CUY1991
MATa his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 nip100Δ::kanMX P
TUB1
-GFP-TUB1::URA3
CUY2008
MATa kar1-Δ15 ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
P
TUB1
-GFP-TUB1::URA3
CUY2009
MATa kar1-Δ15 ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
P
TUB1
-GFP-TUB1::URA3 she1Δ::HIS3MX
CUY2010
MATa ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
SPC72
1-276
-KAR1
192-433
::LEU2 kar1Δ ::HIS3 spc72Δ::kanMX P
TUB1
-GFP-
TUB1::URA3
CUY2011
MATa ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
SPC72
1-276
-KAR1
192-433
::LEU2 kar1Δ ::HIS3 spc72Δ::kanMX she1Δ ::TRP1
P
TUB1
-GFP-TUB1::URA3
CUY2015
MATa his3-Δ200 leu2-3 112 lys2-801 trp1-1 ura3-52 P
TUB1
-GFP-TUB1::URA3
CUY2016
MATa his3-Δ200 leu2-3 112 lys2-801 trp1-1 ura3-52 P
TUB1
-GFP-TUB1::URA3
she1Δ::HIS3MX
CUY2017
MATa his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 nip100Δ::kanMX P
TUB1
-GFP-TUB1::URA3
she1 Δ::HIS3MX
CUY2018
MATα his3- Δ200 leu2-3 112 lys2-801 trp1-1 ura3-52 P
TUB1
-GFP-TUB1::URA3
P
MET3
-3HA-CDC20::TRP1
CUY2019
MATα his3- Δ200 leu2-3 112 lys2-801 trp1-1 ura3-52 P
TUB1
-GFP-TUB1::URA3
P
MET3
-3HA-CDC20::TRP1 she1Δ::HIS3MX
CUY2020
MATa kar1-Δ15 ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
P
TUB1
-GFP-TUB1::URA3 P
MET3
-3HA-CDC20::TRP1
CUY2021
MATa kar1-Δ15 ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
P
TUB1
-GFP-TUB1::URA3 she1Δ::HIS3MX P
MET3
-3HA-CDC20::TRP1
CUY2022
MATa ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
P
SPC72
-SPC72
1-276
-CNM67
1-581
::URA3 spc72Δ::HIS3MX P
TUB1
-GFP-TUB1::LEU2
CUY2023
MATa ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
P
SPC72
-SPC72
1-276
-CNM67
1-581
::URA3 spc72Δ::HIS3MX P
TUB1
-GFP-TUB1::LEU2
she1Δ::kanMX
CUY2024
MATa ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
P
SPC72
-SPC72
1-276
-CNM67
1-581
::URA3 spc72Δ::HIS3MX P
TUB1
-GFP-TUB1::LEU2
P
MET3
-3HA-CDC20::TRP1
CUY2025
MATa ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
SPC72
1-276
-KAR1
192-433
::LEU2 kar1Δ ::HIS3 spc72Δ::kanMX P
TUB1
-GFP-
TUB1::URA3 P
MET3
-3HA-CDC20::TRP1
CUY2027
MATa his3-Δ200 leu2-3 112 lys2-801 trp1-1 ura3-52 P
TUB1
-GFP-TUB1::URA3
she1Δ::HIS3MX kar3Δ::TRP1
CUY2028
MATa ade2-101 his3-Δ200 lys2-801 ura3-52 she1Δ::kanMX GFP-TUB4::HISMX6
P
HIS3
-mCherry-TUB1::URA3
Page 10
!
3
CUY2030
MATa ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
P
SPC72
-SPC72
1-276
-CNM67
1-581
::URA3 spc72Δ::HIS3MX she1Δ::kanMX
P
TUB1
-GFP-TUB1::LEU2 P
MET3
-3HA-CDC20::TRP1
CUY2033
MATa his3-Δ200 leu2-3 112 trp1-1 ura3-52 nip100Δ::kanMX
P
TUB1
-GFP-TUB1::URA3 P
MET3
-3HA-CDC20::TRP1
CUY2034
MATa his3-Δ200 leu2-3 112 trp1-1 ura3-52 nip100Δ::kanMX she1Δ::HIS5
P
TUB1
-GFP-TUB1::URA3 P
MET3
-3HA-CDC20::TRP1
CUY2035
MATa ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
SPC72
1-276
-KAR1
192-433
::LEU2 kar1Δ ::HIS3 spc72Δ::NAT
R
she1Δ::kanMX
P
TUB1
-GFP-TUB1::URA3 P
MET3
-3HA-CDC20::TRP1
CUY2037
MATa his3-Δ200 leu2-3 112 lys2-801 ura3-52 she1Δ::HIS3MX SPC72-
GFP::kanMX P
HIS3
-mCherry-TUB1::URA3
CUY2039
MATa his3-Δ200 leu2-3 112 lys2-801 ura3-52 she1Δ::kanMX
SPC97-GFP:: HIS3MX P
HIS3
-mCherry-TUB1::URA3
CUY2040
MATα his3-Δ200 leu2-3 112 lys2-801 trp1-Δ63 ura3-52 DYN1-3GFP::TRP1
P
HIS3
-mCherry-TUB1::URA3
CUY2041
MATa ade2-101 his3-Δ200 leu2-3 112 trp1-1 ura3-52 STU2-3GFP::TRP1
P
HIS3
-mCherry-TUB1::URA3
CUY2045
MATa his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 cnm67Δ::kanMX P
TUB1
-GFP-TUB1::LEU2
CUY2046
MATa his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 cnm67Δ::kanMX she1Δ::HIS3MX
P
TUB1
-GFP-TUB1::URA3
CUY2055
MATα his3-Δ200 leu2-3 112 lys2-801 ura3-52 NIP100-GFP::HIS3MX
P
HIS3
-mCherry-TUB1::URA3
CUY2056
MATα his3-Δ200 leu2-3 112 lys2-801 ura3-52 ARP1-GFP::HIS3MX
P
HIS3
-mCherry-TUB1::URA3
CUY2057
MATα his3-Δ200 leu2-3 112 lys2-801 ura3-52 JNM1-GFP::HIS3MX
P
HIS3
-mCherry-TUB1::URA3
CUY2062
MATa kar1-Δ15 ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
nip100Δ::kanMX P
TUB1
-GFP-TUB1::URA3
CUY2063
MATa kar1-Δ15 ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
nip100Δ::kanMX she1Δ::HIS3MX P
TUB1
-GFP-TUB1::URA3
CUY2064
MATa kar1-Δ15 ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
nip100Δ::kanMX P
TUB1
-GFP-TUB1::URA3 P
MET3
-3HA-CDC20::TRP1
CUY2065
MATa kar1-Δ15 ade2-101 his3-Δ200 leu2Δ1 lys2-801 trp1-Δ63 ura3-52
nip100Δ::kanMX she1Δ::HIS3MX P
TUB1
-GFP-TUB1::URA3
P
MET3
-3HA-CDC20::TRP1
MY8960
MAT
α
ade2 ade3 his3-200 leu2-1 lys2-801 trp1-63::FUR1-TRP1 ura3-52
NIP100-13MYC::kanMX ARP10-3HA::kanMX
All strains were constructed in this lab, except for MY8960, which came from the Rose lab
(Princeton University)
!
! !
Page 11
!
4
Table S3. Plasmids!
Plasmid
Type
Relevant markers
pXX3
YCp
Amp
R
URA3 P
GAL1/10
-SHE1-GFP
pXX6
YCp
Kan
R
GST-TEV-SHE1
pXX23
YIp
Amp
R
URA3 P
GAL1/10
-SHE1
pCUB1263
YCp
Amp
R
HIS3 P
GAL1/10
-SHE1-GFP
pCUB1288
YCp
Amp
R
URA3 P
GAL1/10
-SHE1-13MYC
pCUB1293
YCp
Amp
R
LEU2 P
GAL1/10
-SHE1
pCUB1299
YCp
Amp
R
HIS3 P
GAL1/10
-SHE1-13MYC
pKN109
YIp
Amp
R
TRP1 P
MET3
-3HA-CDC20
Figure S1. She1-GFP does not precipitate with dynactin complex proteins. Protocol is as
described for Figure 4D, except that the blot was probed with anti-GFP antibody. The two left-
hand lanes show She1-GFP in 50 µg of whole cell extracts (WCE) from uninduced (Glu) and
induced (Gal) cells (CUY1933 containing pXX3). The two right-hand lanes show She1-GFP in
material precipitated with anti-HA affinity gel from induced Nip100-3HA (CUY1933 containing
pXX3) and Jnm1-3HA (CUY1936 containing pCUB1263) cells.
Page 12
  • Source
    • "The late pathway is initiated when the yeast-specific dynein inhibitor She1 (sensitivity to high expression) is removed from astral microtubules at the metaphase–anaphase transition (Woodruff et al., 2009). She1 appears to act specifically by preventing recruitment of dynactin to microtubules (Bergman et al., 2012) and by inhibiting dynein motility (Markus et al., 2012). In the late pathway, cytoplasmic dynein is targeted to "
    [Show abstract] [Hide abstract] ABSTRACT: Accurate positioning of spindles is essential for asymmetric mitotic and meiotic cell divisions that are crucial for animal development and oocyte maturation, respectively. The predominant model for spindle positioning, termed "cortical pulling," involves attachment of the microtubule-based motor cytoplasmic dynein to the cortex, where it exerts a pulling force on microtubules that extend from the spindle poles to the cell cortex, thereby displacing the spindle. Recent studies have addressed important details of the cortical pulling mechanism and have revealed alternative mechanisms that may be used when microtubules do not extend from the spindle to the cortex.
    Preview · Article · Jan 2013 · The Journal of Cell Biology
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    [Show abstract] [Hide abstract] ABSTRACT: Microtubules and microtubule-associated proteins are fundamental for multiple cellular processes including mitosis and intracellular motility, but the factors that control MAPs are poorly understood. In this report, two MAPs, the CLIP-170 homolog Bik1p and the Lis1 homolog Pac1p, interact with several proteins in the sumoylation pathway. Bik1p and Pac1p interact with Smt3p the yeast SUMO, Ubc9p an E2, and Nfi1p an E3. Bik1p interacts directly with SUMO in vitro and overexpression of Smt3p and Bik1p results in its in vivo sumoylation. Modified Pac1p is observed when the SUMO protease Ulp1p is inactivated. Both ubiquitin and Smt3p copurify with Pac1p. In contrast to ubiquitination, sumoylation does not directly tag the substrate for degradation. However, SUMO-Targeted Ubiquitin Ligases (STUbLs) can recognize a sumoylated substrate and promote its degradation via ubiquitination and the proteasome. Both Pac1p and Bik1p interact with the STUbL Nis1p-Ris1p and the protease Wss1p. Strains deleted for RIS1 or WSS1 accumulate Pac1p conjugates. This suggests a novel model in which the abundance of these MAPs may be regulated via STUbLs. Pac1p modification is also altered by Kar9p and the dynein-regulator, She1p. This work has implications for the regulation of dynein's interaction with various cargoes, including its off-loading to the cortex.
    Full-text · Article · Oct 2012 · Molecular biology of the cell
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    [Show abstract] [Hide abstract] ABSTRACT: Background: Cytoplasmic dynein motility along microtubules is critical for diverse cellular processes ranging from vesicular transport to nuclear envelope breakdown to mitotic spindle alignment. In yeast, we have proposed a regulated-offloading model to explain how dynein motility drives microtubule sliding along the cortex, powering transport of the nucleus into the mother-bud neck [1, 2]: the dynein regulator She1 limits dynein offloading by gating the recruitment of dynactin to the astral microtubule plus end, a prerequisite for offloading to the cortex. However, whether She1 subsequently affects cortically anchored dynein activity during microtubule sliding is unclear. Results: Using single-molecule motility assays, we show that She1 strongly inhibits dynein movement along microtubules, acting directly on the motor domain in a manner independent of dynactin. She1 has no effect on the motility of either Kip2, a kinesin that utilizes the same microtubule track as dynein, or human kinesin-1, demonstrating the specificity of She1 for the dynein motor. At single-molecule resolution, She1 binds tightly to and exhibits diffusional behavior along microtubules. Diffusive She1 collides with and pauses motile dynein motors, prolonging their attachment to the microtubule. Furthermore, Aurora B/Ipl1 directly phosphorylates She1, and this modification appears to enhance the diffusive behavior of She1 along microtubules and its potency against dynein. In cells, She1 dampens productive microtubule-cortex interactions specifically in the mother compartment, polarizing spindle movements toward the bud cell. Conclusions: Our data reveal how inhibitory microtubule-associated proteins selectively regulate motor activity to achieve unidirectional nuclear transport and demonstrate a direct link between cell-cycle machinery and dynein pathway activity.
    Preview · Article · Nov 2012 · Current biology: CB
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