A study on premature segregation of unreplicated chromosomes 4

Chapter4 Regulation of Spindle Elongation by
Cdc34
4.1 Background
In the previous chapter, we have explored the various reasons responsible for
premature nuclear division in Cdc6 depleted cells. It has long been thought that
precocious segregation of chromosomes is due to premature onset of mitosis.
However, recent evidence suggests that premature chromosome segregation can occur
without the onset of mitosis, APC activation, cohesion cleavage or biorientation of
kinetochores.
The DNA replication checkpoint has been reported to thwart untimely
chromosome segregation not by inhibiting mitotic entry but by directly regulating
spindle dynamics and by preventing replication fork collapse to allow duplication of
centromeric DNA and hence kinetochore bi-orientation (Krishnan et al. 2004). To be
more precise, the critical effectors of DNA replication checkpoint, Mec1 and Rad53,
(orthologues of human ATM/ATR- and Chk2-like kinases, respectively) prevent
precocious chromosome segregation by suppressing the accumulation of spindle
elongation effectors such as Cin8 and Stu2, thus precluding premature induction of
spindle elongation during early S phase (Krishnan et al. 2004) (Krishnan et al. 2005).
A recent study in yeast suggests that concerted action by two prominent
kinases Cdk1 and polo (Cdc5) are required to fully inactivate Cdh1, an activator of
the E3 ubiquitin ligase APC (anaphase promoting complex) (Crasta et al. 2008).
APC
Cdh1
is essential for proteolytic destruction of microtubule associated proteins
such as Cin8, Kip1 and Ase1. Therefore, Cdh1 inactivation is required to halt the
destruction of these proteins to facilitate spindle assembly and spindle elongation.
These findings helped uncover an additional mechanism by which DNA damage
checkpoint prevents premature chromosome segregation (Zhang et al. 2009). It was
shown that activation of the DNA damage checkpoint leads to inactivation of Cdc5
polo kinase via Rad53-mediated phosphorylation (Zhang et al. 2009). Thus,
inactivation of both Cdk1 and Cdc5 by the checkpoint prevents Cdh1 inactivation,

which in turn continues to mediate the destruction of spindle elongation-inducing
proteins Cin8, Kip1 and Ase1. Thus these studies envisaged that the DNA damage
checkpoint, in addition to suppressing cohesin cleavage, maintains APC
Cdh1
in an
active state to restrain spindle extension until the damaged chromosomes are repaired.
Hence, both DNA replication and DNA damage checkpoints can prevent premature
chromosome segregation by restricting the accumulation of microtubule associated
proteins such as Cin8, Kip1, Ase1 and Stu2. The exact mechanism as to how DNA
replication checkpoint promotes destabilization of the microtubule associated proteins
remains unclear. The activated DNA damage checkpoint leads to the perturbation of
Cdh1/Cdk1/polo/MAPs control circuit; Cdh1 is maintained in an active state and
spindle is restrained from prematurely elongating (Crasta et al. 2008). Since cells
lacking Cdc6 also encountered premature spindle elongation resulting from an
accumulation of MAPs, it is possible that Cdc6 (putative G1-M checkpoint
component) is able to directly regulate spindle elongation by targeting the Cdh1-
Cdk1/polo/MAPs control circuit. This notion would be consistent with Cdc6’s role in
the inactivation of Cdk1. This chapter explores the relationship between Cdc6 and
spindle dynamics.
4.2 Results
4.2.1 Premature segregation of unreplicated chromosomes in cells
lacking Cdc7 and Cdc45
In the preceding chapter, we have documented that Cdc6 deficient cells fail to initiate
DNA replication and proceed to undergo what-appears-to-be premature mitosis,
causing a reductional anaphase. We also noted that the premature nuclear division is
accompanied by high abundance of microtubule associated proteins such as Cin8,
Kip1 and Ase1. Hence, it is possible that Cdc6 is involved in the regulation of
effectors of spindle elongation and spindle dynamics. However, before the role of
Cdc6 in spindle elongation is investigated, it is necessary to ascertain if premature
chromosome segregation is a characteristic associated specifically with cdc6 mutant

or it is a common characteristic of cells that can undergo START but are unable to
initiate S phase. To test this notion, we monitored spindle behaviour in cells lacking
Cdc7 and Cdc45. Cdc7 is a serine/threonine kinase essential for DNA replication and
requires Dbf4 for its activity (hence the term Ddk for Dbf-dependent kinase) (Bousset
et al. 1998) (Jares et al. 2000). Cdc45 is required for the initiation and elongation step
of DNA replication (Zou et al. 1997). The CDC45 gene was shown to genetically
interact with components of replication factors such as MCM and ORC. We
constructed cdc7Δ GAL-CDC7 (US5582) and cdc45Δ GAL-CDC45 (US5585) strains
for this study. Both strains were first arrested in metaphase by growth in galactose
medium containing nocodazole. Cells were then transferred to YEPD for 30 minutes
to allow depletion of Cdc7 and Cdc45 before they were released into YEPD medium
containing α-factor to synchronize them in G1 phase. Once the cultures were
uniformly arrested in G1, α-factor was removed and cells were released into YEPD
medium at 25˚C and samples taken every 20 minutes to score the spindle lengths. As
shown in Figure 15, cells lacking Cdc7 arrested in G1 with 1N DNA but proceeded to
elongate the spindles from 80 minutes onwards with spindle length exceeding 4µ m.
Similarly, cells lacking Cdc45 also arrested in G1 with a large bud and unreplicated
chromosomes but proceeded to extend the spindle from 80 minutes onwards with
spindle length exceeding 4µm (Figure 16). Thus, cells defective in Cdc7 or Cdc45
traverse START, assemble a bud and fail to initiate DNA replication, but prematurely
segregate the unreplicated chromosomes despite the presence of a functional CDC6.
One interpretation of these results is that Cdc6, Cdc7 and Cdc45 are all involved in
preventing untimely spindle elongation and segregation of unreplicated chromosomes.
Alternatively, it is not inconceivable that premature segregation is not specifically due
to the lack of Cdc6, Cdc7 or Cdc45 functions but is a common characteristic of cells
that have traversed START but fail to initiate DNA replication. In other words, the
premature partitioning of the chromosomes is regulated by some unknown
mechanism and the mutations in genes such as CDC6, CDC7 and CDC45 only allow
manifestation of this underlying regulation because of their common inability to
initiate DNA replication.

Glu NOC
Glu a
Glu 60min
Glu 120min
Glu 180min
Nomarski
DAPI Anti-tubulin

Time (mins)
0
20
40
60
80
100
120
0 20 40 60 80 100 120 140 160 180
0-2 um
2-4 um
>4 um
1N 2N
Figure 15. Premature segregation of unreplicated chromosomes in cells lacking
Cdc7
We constructed cdc77 strain for this study. The strain was first arrested

in metaphase by growth in galactose medium containing nocodazole. Cells were then

transferred to YEPD for 30 minutes to allow depletion of Cdc7 before they were

released into YEPD medium containing -factor to synchronize them in G1 phase.

Once the cultures were uniformly arrested in G1, -factor was removed and cells were

released into YEPD medium at 25C and samples taken every 20 minutes to score the

spindle lengths and for FACS analysis
% cells
short spindle
(0-2 mm)
medium spindle
(2-4 mm)
long spindle
(>4 mm)
Spindle Length

Nomarski
DAPI Anti-tubulin

0
20
40
60
80
100
120
0 20 40 60 80 100 120 140 160 180
0-2 um
2-4 um
>4 um

Glu NOC
Glu a
Glu 60min
Glu 120min
Glu 180min
Figure 16. Premature segregation of unreplicated chromosomes in cells lack-
ing Cdc45
 strain was first arrested in metaphase by growth in galactose
medium containing nocodazole. Cells were then transferred to YEPD for 30 min-
utes to allow depletion of Cdc45 before they were released into YEPD medium
            


and for FACS analysis.
 2N
short spindle
(0-2 mm)
medium spindle
(2-4 mm)
long spindle
(>4 mm)
% cells
Time (mins)
Spindle Length
4.2.2 Depletion of Cdc6 in cdc34-1 cells fails to promote spindle
assembly or spindle elongation
To directly address the possibility of a role for CDC6 in spindle regulation, we utilize
cdc34-1 cells which traverse START and duplicate their centrosomes but can neither
initiate S phase nor assemble a short spindle because the inter SPB bridge remains
unbroken. If Cdc6 is involved in regulating spindle biogenesis and dynamics, cdc34-

1 cells lacking Cdc6 is expected to promote spindle assembly or spindle elongation.
For this experiment, we constructed cdc34-1 (US6005) and cdc34-1 cdc6Δ MET-
CDC6 (US7012) strains expressing GFP-tagged spindle pole body component Spc42
to ascertain spindle length in both strains. To ensure Cdc6 is degraded completely,
both strains were arrested in nocodazole supplemented with methionine to repress
CDC6 transcription. These cells were then released into methionine medium (+Met)
at non-permissive temperature of 36˚C. Samples from both cdc34-1 and cdc34-1
cdc6Δ MET-CDC6 cells were collected at 180 minutes and analyzed by
immunofluorescence microscopy. As shown in Figure 17, 100% of both cdc34-1 and
cdc34-1 cdc6Δ MET-CDC6 strains exhibit one Spc42-GFP dot indicating that the
duplicated SPBs, have not separated and no spindle was assembled. In addition,
immunofluorescence staining of 180 minutes sample showed no sign of spindle
formation or spindle elongation. These results imply that Cdc6 does not influence
spindle biogenesis significantly.

Nomarski
DAPI Spc42GFP
cdc34-1
Nomarski
DAPI Anti-tubulin
Nomarski
DAPI Anti-tubulin
Nomarski
DAPI Spc42GFP
cdc34-1

MET-CDC6

100%
no spindle

100%
no spindle
100%
no spindle
100%
no spindle
Figure 17. Depletion of Cdc6 in cdc34-1 cells fails to promote spindle assembly
or spindle elongation.
cdc34-1     


  CDC6     

cdc34-1-

4.2.3 Ectopic expression of Sic1 and Cdh1 prevent premature
spindle elongation in Cdc6 depleted cells
In Chapter 3, we provided evidence that premature spindle elongation in cells lacking
Cdc6 was due to the accumulation of microtubule associated proteins such as Cin8,
Kip1 and Ase1. Since it has been shown previously that APC
Cdh1
is responsible for
the ubiquitination and proteasome-mediated degradation of Cin8, Kip1 and Ase1, we
therefore considered the possibility that Cdh1 inactivation may be relevant to Ase1,
Cin8 and Kip1 stability in Cdc6 depleted G1 arrested cells. If this is true, then over-
expression of Cdh1 would be expected to promote degradation of spindle elongation
effectors such as Cin8 in Cdc6 depleted cells. To test this, we arrested two separate
cultures of cdc6Δ MET-CDC6 GAL-HA3-CDH1 CIN8-HA
3
(US7022) in

+Met+glucose medium (to repress transcription of CDC6) containing nocodazole.
Subsequently, both cultures were released from metaphase arrest into +Met medium
containing α-factor to ensure that the cells were arrested in G1. One culture was then
released into +Met+glucose medium at room temperature to inhibit over-expression
of Cdh1. Another culture was released into medium containing Raffinose+Galactose
to drive over-expression of GAL-HA3-CDH1. As shown in Figure 18A, Cdh1
expression was apparent from 80 minutes after the release. As Cdh1 over-expression
peaked from 120 minutes onwards, Cin8 was concurrently degraded from 120
minutes. The unstable Cin8 was not present in sufficient amounts to allow Cdc6
deficient cells to elongate their spindles, resulting in the phenotype observed: cells
with short spindles (Figure 18A). In contrast, in the control culture where Cdh1 was
not over-expressed, Cin8 remained stable. The results of this experiment imply that
Cdh1 may be inactive in Cdc6 depleted cells, leading to the accumulation of proteins
such as Cin8 and, thus, premature spindle elongation.
It is known that Sic1 is responsible for inhibition of Cdk1/Clb5, 6 kinases in
G1 phase (prior to S phase onset) (Barberis et al. 2005). Moreover, active Cdk1 is
also responsible for inactivating Cdh1 via phosphorylation at multiple sites (Crasta et
al. 2008). Therefore if Cdk1 activity is inhibited, Cdh1 will remain active. To further
verify that premature spindle elongation in Cdc6 depleted cells is a result of Cdh1
inactivation (with accumulation of microtubule associated proteins such as Cin8), we
expressed non-degradable version of Sic1 in Cdc6 depleted cells. We first arrested
cdc6Δ MET-CDC6 GAL-ndSIC1 (US7013) strain in nocodazole containing medium
supplemented with methionine to repress CDC6 transcription. Cells were then
released into α-factor containing medium to resynchronize cells in G1. The cells were
subsequently released into medium containing Raffinose and Galactose to facilitate
over-expression of non-degradable Sic1. Samples were taken at 160 minutes to
monitor the presence of long spindles. Almost 100% of the cells failed to elongate
their spindles and arrested in G1 with large buds and short spindles (Figure 18B).
This is similar to the previous experiment where Cdh1 over-expression in cells
lacking Cdc6 led to arrest with short spindles. Clearly, Cdh1 inactivation is

mandatory for premature spindle elongation in Cdc6 deficient cells. This also
suggests that the Sic1 degradation step must be tightly regulated since untimely
degradation of Sic1 promotes Cdk1-mediated inactivation of Cdh1, accumulation of
Cin8 and Ase1 and hence premature spindle elongation. If SCF-mediated degradation
of Sic1 is an essential step in determining the fate of spindle in Cdc6 depleted cells,
then there is a strong possibility that SCF-component Cdc34 is important in the
regulation of spindle elongation.

Nomarski
DAPI Anti-tubulin

3

3
Nomarski
DAPI Anti-tubulin

100%
short spindles
100%
short spindles
RG at 25˚C 160min
RG at 25˚C 160min

3

3

160
140

120
100
80
60
40
20

25˚C
+met Glu
180
+met NOC

3

3

160
140
120
100
80
60
40
20

25˚C
+met RG
180
+met NOC
Cin8-HA

3
Cdh1-HA
3
G6PD
G6PD
Cdh1-HA
3
Cin8-HA
3
Figure 18. Ectopic expression of Sic1 and Cdh1 prevent premature spindle elongation in
Cdc6 depleted cells.
A. Two separate cultures of 
3

3
were arrested
in +Met+glucose medium (to repress transcription of ) containing nocodazole . Subse-
quently, both cultures were released from metaphase arrest into +Met medium containing
                
+Met+glucose medium at room temperature to inhibit over-expression of Cdh1. Another culture
was released into medium containing raffinose+galactose to drive over-expression of 
3
. Samples were collected for immunofluorescence and Western Blot analysis.
B.  strain was arrested in nocodazole containing medium
supplemented with methionine to repress  transcription. Cells were then released into

medium containing raffinose and galactose to facilitate over-expression of non-degradable Sic1.
Samples were taken at 160 minutes for immunofluorescence analysis.
A
B

4.2.4 cdc34 and cdc34 cdc6 mutant cells can assemble short bipolar
spindles in the absence of Cdh1 or Sic1 but fail to elongate them
Once cells have traversed START, Cdc34 function becomes critical for G1/S
transition due to its involvement in Sic1 degradation. Cells deficient in Cdc34
function are unable to initiate S phase and arrest in G1 phase (post START) with a
bud and duplicated SPBs but are unable to assemble a spindle in accordance with the
regulatory scheme shown in figure below. In contrast, cells lacking Cdc6 are also
unable to undergo S phase and arrest in G1 phase (post START) but with a
prematurely extended spindle. This raises the possibility of Cdc34’s involvement in
regulating spindle elongation since Cdc34 is functional in cells lacking Cdc6.
Previous experimental data had shown that ectopic expression of Sic1 and Cdh1 are
sufficient to prevent premature spindle elongation in cells lacking Cdc6. This is
suggestive of a role for Cdc34 in Sic1 degradation. With Sic1 degraded, Cdk1 can
inactivate Cdh1; cells prematurely elongate the spindle mirroring that seen in Cdc6
deficient cells. We tested this notion by depleting Sic1 or Cdh1 independently in cells
deficient in Cdc34 and investigate if it is sufficient to permit spindle assembly and
spindle elongation.
We treated cdc34-1 cdh1Δ (US7015), cdc34-1 cdc6Δ MET-CDC6 cdh1Δ
(US6016) and cdc6Δ MET-CDC6 sic1Δ (US5690) cells with nocodazole containing
+Met medium to deplete Cdc6. Subsequently, these cells were released into medium
containing methionine at 36˚C to inactivate Cdc34. As expected these cells arrested
with multiple buds in G1 phase in the absence of DNA replication.
Immunofluorescence staining of the spindles revealed that these cells are capable of
assembling short spindles but are unable to extend them (Figure 19). This suggests
that both Sic1 degradation and Cdh1 inactivation are critical steps in promoting
assembly of short spindles, however, neither event alone can promote spindle
elongation in Cdc6 deficient cells. These experiments also strengthen the notion that
spindle elongation seen in Cdc6 deficient cells is not due to the lack of Cdc6 function
per se. The results also imply that Cdc34 (E2 enzyme), acting in synergy with SCF
and Cdc4 (F-box protein) to degrade Sic1, not only regulates G1-S transition but also

facilitates Cdh1 inactivation which leads to the stabilization of microtubule associated
proteins and sets up the context for the assembly of a short spindle.


Nomarski
DAPI Anti-tubulin

Nomarski
DAPI Anti-tubulin

Nomarski
DAPI Anti-tubulin

100%
100%
100%
short spindles
short spindles
short spindles
36˚C 180min
36˚C 180min
36˚C 180min
Figure 19. cdc34 and cdc34 cdc6 mutant cells can assemble short bipolar spin-
dles in the absence of Cdh1 or Sic1 but fail to elongate them.
,  and 
cells were arrested in +Met medium containing nocodazole to deplete Cdc6. Sub-

inactivate Cdc34. Samples were collected at 180 minutes for immunofluorescence
analysis.
4.2.5 Sic1 degradation promotes Cdh1 inactivation and short spindle

assembly
Before returning to the relationship between Cdc34, microtubule associated proteins
and spindle elongation in Cdc6 deficient cells, we take a short detour to explore the
connection between Sic1 and Cdh1 in the context of the regulatory scheme outlined in
Figure 20 below, since it is important for the regulation of spindle dynamics in Cdc6
mutant.



Figure 20: Regulatory scheme outlining the connection between Sic1 and Cdh1
for the regulation of spindle dynamics in cdc6 mutant.

SCF/Cdc34

Sic1

Cdk1/Clb

APC
Cdh
1
MAPs
Short

Spindl
e
S Phase


The question we intended to address is whether Sic inactivation indeed leads to Cdk1-

mediated inactivation of Cdh1. The active Cdk1 has been shown to phosphorylate
Cdh1 on S16, S42, T157 and T173 residues creating polo-box binding domains
recognized by Cdc5 (yeast Polo-like kinase). This leads to the recruitment of Cdc5 to
Cdh1 and additional phosphorylation on S125 and S259 residues (Crasta et al. 2008).
Thus, multiple phosphorylations by these two kinases are required to inactivate Cdh1
completely. To confirm the notion that Sic1 degradation promotes Cdk1-mediated
inactivation of Cdh1, we monitored the phosphorylation status of Cdh1 in cdc34-1
(US5677) and cdc34-1 sic1Δ (US5722) cells expressing HA
3
-CDH1 from its native
locus. Cells were synchronized in G1 phase using α-factor and then released into
YEPD medium at 36˚C. As expected, Cdh1 immunoprecipitated from cells deficient
in Cdc34 showed no hyper-phosphorylation (i.e. Sic 1 active, Cdk1 inactive, Cdh1
active), whereas it is highly phosphorylated in Cdc34 deficient cells lacking Sic1
(Figure 21). Treatment with calf intestinal alkaline phosphatase (CIP) causes higher
molecular weight, slower mobility bands to disappear suggesting that their lower
mobility is indeed due to phosphorylation (Figure 21). As mentioned earlier, cdc34-1
cells arrest in G1 with large buds, unreplicated DNA and no bipolar spindle.
However cdc34-1 sic1Δ cells arrested in G2 with large buds and short spindles (data
not shown). These observations support the notion that Cdh1 inactivation and short
spindle assembly is tightly connected with Sic1 degradation mediated by Cdc34. In
the following section we make use of these observations and use SIC1 or CDH1
deletion to allow cdc34-1 mutant cells to assemble a short spindle and explore the
cellular requirements for spindle elongation.
-
-
+
+
CIP
cdc34-1 CDH1-HA

3

3

3
cdc34-1 CDH1-HA
3
Cdh1-HA
3

Figure 21. Sic1 degradation promotes Cdh1 inactivation and
short spindle assembly
To confirm the notion that Sic1 degradation promotes Cdk1-mediated
inactivation of Cdh1, we monitored the phosphorylation status of
Cdh1 in cdc34-1 and cells expressing CDH1-HA
3
from

          
collected for immunoprecipitation and Western Blot analysis.

4.2.6 Ectopic expression of microtubule associated proteins induces
spindle elongation in Cdc34 deficient cells devoid of Cdh1
The experiments described in the preceding sections suggest that premature spindle
elongation in Cdc6 deficient cells is a consequence of the loss of regulation of
microtubule associated proteins such as Ase1, Cin8 and Kip1. If so, then
overexpression of Ase1, Cin8 and Kip1 would be expected to induce spindle
elongation in cdc34-1 cdh1Δ and cdc34-1 cdc6Δ MET-CDC6 cdh1Δ cells, given that
they are able to assemble short spindles (Figure 19). To test this possibility, cdc34-1
cdc6Δ MET-CDC6 cdh1Δ cells carrying GAL-CIN8 (US7024) or GAL-KIP1

(US7026) and cdc34-1 cdh1Δ cells carrying GAL-ASE1 (US7028) were first
synchronized in G2-M by nocodazole treatment in raffinose medium supplemented
with methionine to fully repress CDC6 transcription. These cells were then
subjected to second synchronization in the subsequent G1 phase by α-factor
treatment in medium containing raffinose and methionine to ensure complete
depletion of Cdc6. The cells were pre-induced with galactose for 1 hour to express
Cin8, Kip1 or Ase1 and then released into medium containing raffinose and
galactose at 34˚C. As a control, cdc34-1 (US1688) and cdc34-1 cdc6Δ MET-CDC6
cdh1Δ (US7016) cells without GAL constructs were treated identically. As expected,
cdc34-1 cells arrest with no spindle whereas cdc34-1 cdh1Δ and cdc34-1 cdc6Δ
MET-CDC6 cdh1Δ cells arrest with short spindles, (Figure 19 and 22) confirming the
previous findings that Cdh1 inactivation promotes stability of microtubule associated
proteins leading to short spindle assembly in Cdc34 deficient cells. However, over-
expression of Ase1 in cdc34-1 cdh1Δ and Cin8 or Kip1 separately in cdc34-1 cdc6Δ
MET-CDC6 cdh1Δ cells allowed ~60% of the cells to arrest with long spindles,
indicating that over-expression of these proteins could induce spindle elongation
(Figure 22 and 23). These findings suggest that microtubule associated proteins are
the limiting factors preventing spindle elongation in cdc34-1 cdh1Δ, cdc34-1 cdc6Δ
MET-CDC6 cdh1Δ and cdc34-1 cdc6Δ MET-CDC6 sic1Δ cells (Figure 19).
Additionally, since only 60% of the cells are able to elongate their spindles, these
results also suggest that the over-expressed microtubule associated proteins remain
unstable in Cdc34 deficient cells despite the absence of active Cdh1. If so, then
Cdc34 may have an additional novel role in regulating stability of microtubule
associated proteins.
To test this notion, we also treated cdc34-1 cells carrying GAL- ASE1
(US7027), GAL-CIN8 (US7023) and GAL-KIP1 (US7025) as described above. We
found that overexpression of microtubule associated proteins can only induce
approximately 30% of the cells to assemble short spindles but cannot elicit spindle
elongation (Figure 22 and 23). These observations are consistent with our hypothesis
that microtubule associated proteins become more unstable in cdc34-1 cells when

Cdh1 is fully activated.
Nomarski
DAPI Anti-tubulin
cdc34-1

Nomarski
DAPI Anti-tubulin
cdc34-1 GALCIN8

Nomarski
DAPI Anti-tubulin

GALCIN8

Nomarski
DAPI Anti-tubulin



30% short spindles
70% no spindle
62% long spindles
38% short spindles
100% no spindle
100% short spindles
Figure 22. Ectopic expression of microtubule associated proteins induces spindle
elongation in Cdc34 deficient cells devoid of Cdh1.
cdc34-1 GAL-CIN8



           

       
       





Nomarski
DAPI Anti-tubulin
cdc34-1 GALKIP1

Nomarski
DAPI Anti-tubulin

GALKIP1

Nomarski
DAPI Anti-tubulin


Nomarski
DAPI Anti-tubulin


20% short spindles
80% no spindle
28% short spindles
72% no spindle

58% long spindles
42% short spindles
60% long spindles
40% short spindles
Figure 23. Ectopic expression of microtubule associated proteins induces spindle
elongation in Cdc34 deficient cells devoid of Cdh1.
The cdc34-1GAL-KIP1 
cdc34-1

            
-


           

4.2.7 Cdh1 resistant microtubule associated proteins cannot induce
complete spindle elongation in cdc34-1 and cdc34-1 cdh1Δ cells
Since over-expression of Ase1, Cin8 and Kip1 cannot induce spindle elongation in
100% of the cdc34-1 cdh1Δ double mutant cells, it is possible that these proteins are
unstable even in the absence of Cdh1. We test this possibility by treating cdc34-1 and
cdc34-1 cdh1Δ cells carrying non-degradable (nd) Cin8 (US7018 and US7020) or
Ase1 (US7019 and US7021) with α-factor to synchronize them in G1 phase.
Subsequently, they were released into YEPD medium at 36˚C to inactivate Cdc34
function and samples were taken at 240 minutes for spindle length analysis. As
shown in Figure 24, expression of ndCin8 can only induce short spindle assembly in
16% of cdc34-1 cells and long spindle in 25% of cdc34-1 cdh1Δ cells. Similarly,
expression of ndAse1 also induces short spindle assembly in 15% of cdc34-1 cells
and long spindle in 20% of cdc34-1 cdh1Δ cells (Figure 25). This strongly suggests
that Cdh1 resistant microtubule associated proteins such as ndAse1 and ndCin8 are
still unstable in Cdc34 deficient cells, since only very small proportion of the cells can

elongate their spindles. Hence these results are consistent with our proposed novel
role involving Cdc34 in regulating the stability of microtubule associated proteins in
G1 phase.
Nomarski
DAPI Anti-tubulin

Nomarski
DAPI Anti-tubulin


36˚C 240min
25% long spindles
75% short spindles
16% short spindles
84% no spindle
36˚C 240min
Figure 24. Cdh1 resistant microtubule associated proteins cannot induce
complete spindle elongation in cdc34-1 and  cells.
We treated cdc34-1 and   cells carrying non-degradable


