A study on premature segregation of unreplicated chromosomes 5

Chapter 5 A search for Cdc34-mediated up- or down-
regulated proteins that mediate spindle
elongation

5.1 Background
As described above, we have observed that Cdc34 can induce premature spindle elongation
by regulating spindle dynamics. Although both cdc34-1 and cdc6Δ MET-CDC6 cells arrest
in G1 phase and cannot undergo S phase, they both demonstrate very different spindle
phenotypes. cdc34-1 mutant (with functional Cdc6) cannot assemble a spindle and
duplicated spindle poles remain connected by a bridge; however cdc6Δ MET-CDC6 cells
(with functional Cdc34) not only assemble a spindle but also untimely extend it to cause
segregation of unreplicated chromosomes. This raised the possibility that Cdc34 plays a
central role in inducing spindle elongation. In Chapter 3, we have presented evidence
suggesting that Cdc34 stimulates spindle elongation by stabilizing microtubule associated
proteins. To elucidate the nature of Cdc34’s role in spindle extension, we employed a
strategy which involved labeling of cells with stable amino acid isotopes (SILAC) (Ong et al.
2002) which would help to identify proteins that are up- or down-regulated through
quantitative mass spectrometry (de Godoy et al. 2008). SILAC is a metabolic labeling
method that utilizes a cell’s machinery to incorporate exogenous heavy isotopes of amino
acid residues into expressed proteins. This labeling strategy facilitates subsequent mass
spectrometric analysis to differentiate between peptide signals from labeled or unlabeled
source.

5.2 Results
5.2.1 Cdc34 promotes up-regulation of the polo-like kinase Cdc5 during
premature spindle elongation

For SILAC mass spectrometry, cdc34-1 cdc6Δ MET-CDC6 lys1Δ strain (US7048) was used.
Two overnight cultures were grown; one in medium supplemented with normal unlabelled
lysine (refer to Section 2.2.15 for more details) and the other in medium supplemented with

labeled H-lysine. The next day, both cultures were diluted and synchronized in mitosis using
nocodazole and methionine was added to repress and deplete CDC6. Subsequently, the
unlabelled lysine cells were released into medium supplemented with methionine at 36ºC.
Three hours later, these cells were collected for protein extraction. In contrast, the labeled H-
lysine cells were released into medium containing methionine at 24˚C for three hours. Cells
were again collected for the preparation of total protein extracts. Subsequently, both ‘total
protein extracts’ from 36˚C and 24˚C samples were subjected to mass spectrometric analysis
as described in Section Error! Reference source not found
As shown in Figure 33A, the spectra produced by mass spectrometry analysis
identified 2781 proteins. Of these, 187 were up-regulated, 2390 were unchanged and 204
were down-regulated. Cdc5, which was up-regulated two fold upon restoration of Cdc34
function, specifically drew our attention because when overexpressed, Cdc5 has been shown
to promote, spindle formation in spindle assembly-defective cdc28Y19E cells. Additionally,
Cdc5 is also reported to play a synergistic role with Cdk1 to phosphorylate Cdh1 at multiple
sites leading to its inactivation (Crasta et al. 2008). This allows accumulation of microtubule
associated proteins and, consequently spindle formation.

Up-regulated Proteins:
Protein
IDs
Protein Names
Gene
Names
Molecular
Weight (kDa)
Ratio H/L
Normalized
Ratio H/L
Significance
YJR090C'

SCF'E3'ubiquitin'
ligase'complex'F<
box'protein'Grr1'

GRR1

132.73'

18.714'

1.04E<19'

YMR001C'

Cell'cycle'
serine/threonine<
protein'kinase'
Cdc5'

CDC5'

81.03'

2.6913'

0.0020991'

Down-regulated Proteins:

Protein
IDs
Protein Names
Gene
Names
Molecular
Weight (kDa)
Ratio H/L
Normalized
Ratio H/L
Significance
YGL216W'

Kinesin<like'
protein'Kip3'

KIP3'

91.089'

0.51741'

0.044283'


Table 6: Cdc34 mediated Up- or Down-regulated proteins with functions relevant to
SCF and spindle dynamics.

To confirm the above observations, we attempted to monitor the stability of Cdc5 in
the absence and presence of Cdc34 function. We treated two separate cultures of cdc34-1
cdc6Δ MET-CDC6 cdh1Δ cells (US6938) with nocodazole to arrest them in mitosis in
medium supplemented with methionine at 24˚C to repress CDC6 transcription and to ensure
complete Cdc6 depletion. Subsequently, both cultures were released into medium containing
methionine at 36˚C to inactivate Cdc34 and arrest at Cdc34-execution point until 210 minutes.
At this juncture, one culture was kept in 34˚C while the other was shifted to 24˚C to allow
restoration of Cdc34 function. Samples were taken every 30 minutes for Western Blot
analysis. Similar to microtubule associated proteins such as Ase1 and Cin8, Cdc5 is unstable
in the absence of Cdc34, even when CDH1 is deleted. This suggests that, Cdc5 may be

targeted for proteolysis via the ubiquitin-independent pathway. Upon restoration of Cdc34,
Cdc5 is stabilized (Figure 33B).



90
60
30
210
180
150
120
60
30
+met NOC
-met cyc
120
150

90
+met 36˚C +met 36˚C
90
60
30
210
180
150
120
60
30
+met NOC
-met cyc
120
150
90
+met 36˚C +met 24˚C
Peptide sequence: FFTTQICGAIK
∆m/z=4.01
G6PD
Cdc5
G6PD
Cdc5
Two-fold
increase in Cdc5
B
A
Figure 33. Cdc34 promotes up-regulation of the polo-like kinase Cdc5 during premature
spindle elongation
A. For SILAC mass spectrometry,  strain was used. Two

overnight cultures were grown; one in medium supplemented with normal unlabelled lysine
(refer to Section 2.2.15 for more details) and the other in medium supplemented with labeled
H-lysine. The next day, both cultures were diluted and synchronized in mitosis using nocoda-
zole and methionine was added to repress and deplete . Subsequently, the unlabelled
lysine cells were released into medium supplemented with methionine at 36ºC. Three hours
later, these cells were collected for protein extraction. In contrast, the labelled H-lysine cells

collected for the preparation of total protein extracts. Subsequently, both ‘total protein extracts’
    
Section 2.2.15. As shown is the spectrum of Cdc5 produced by mass spectrometry analysis.
B. To confirm the above observations, we attempted to monitor the stability of Cdc5 in the
absence and presence of Cdc34 function. We treated two separate cultures of 
 cells with nocodazole to arrest them in mitosis in medium supplemented
 transcription and to ensure complete Cdc6 depletion.
-


Samples were taken every 30 minutes for Western Blot analysis.


5.2.2 Ectopic expression of Cdc5 can induce spindle formation and
elongation
Our initial investigations in cdc34-1 and cdc34-1 cdc6Δ MET-CDC6 cdh1Δ cells have
provided evidence that ectopic expression of Ase1, Cin8 and Kip1 can induce spindle
formation and spindle elongation in a large majority of cells, although not in all cells. Since
Cdc5 stabilization is mediated by Cdc34, similar to that observed for microtubule associated
proteins, and its stabilization coincides with spindle elongation, we tested if Cdc5 over-
expression can affect the fate of spindles. To test this possibility, cdc34-1 and cdc34-1cdh1Δ
cells each carrying GAL-CDC5 and GAL-CDC5 (N209A-kinase dead) (US7044, US7046,
US7045 and US7047) were first synchronized in G2-M by nocodazole treatment in raffinose

medium supplemented with methionine. These cells were then subjected to second
synchronization in the subsequent G1 phase by α-factor treatment in medium containing
raffinose and methionine. The cells were pre-induced with galactose for 1 hour to express
Cdc5 or kinase-dead Cdc5 and then released into medium containing raffinose and galactose
at 34˚C. As shown in Figure 34, over-expression of Cdc5 allowed 30% of cdc34-1 cells to
form short spindles at non-permissive temperature. Moreover, over-expression of Cdc5 can
induce spindle elongation in 60% of cdc34-1 cdh1Δ cells. It should be noted that these cells
can assemble short spindles without Cdc5 over-expression due to lack of Cdh1 function. As
a control, we have overexpressed kinase-dead Cdc5 and have observed that kinase-dead Cdc5
fails to promote any spindle formation or spindle elongation. The data suggests that Cdc5
can contribute to the regulation of spindle dynamics and can promote spindle elongation.

Nomarski
DAPI Anti-tubulin
cdc34-1 GALCDC5(N209A)

Nomarski
DAPI Anti-tubulin


Nomarski
DAPI Anti-tubulin
cdc34-1 GALCDC5

Nomarski
DAPI Anti-tubulin


100% no spindles
70% no spindles

40% short spindles
30% short spindles
60% long spindles
100% short spindles
Figure 34. Ectopic expression of Cdc5 can induce spindle formation and
elongation.
           
-

cdc34-1GAL-CDC5
GAL-CDC5-


      

-


5.2.3 Cdc5 is unstable in the absence of Cdc34 function
Our previous investigations showed that Ase1 and Cin8 are highly unstable in cdc34-1 cells
compared to that in cdc6Δ cells. This explains the inability of cdc34-1 cells to assemble a
spindle. Both Ase1 and Cin8 become more stable when CDH1 is deleted in cdc34-1 cells
and they contribute to short spindle assembly. Since Cdc5 overexpression cannot induce
spindle formation and spindle elongation in all the cdc34-1 and cdc34-1 cdh1Δ cells, it is
possible that Cdc5 is in low abundance in Cdc34 deficient cells due to enhanced proteolysis.
To test this, cdc34-1, cdc6Δ MET-CDC6, cdc34-1 cdh1Δ and cdc6Δ MET-CDC6 cdh1Δ cells
carrying myc
6
-tagged CDC5 under the control of the GAL1 promoter (US7044, US7046,
US7049 and US7050) were grown overnight in medium lacking methionine. The overnight

cultures were washed and inoculated into raffinose and galactose medium supplemented with
methionine at 36˚C. Cells were kept in this medium for 2 hours to pre-induce Cdc5 and then
released into their respective non-permissive conditions in glucose medium with 0.1mg/ml
cycloheximide (to inhibit de novo synthesis of Cdc5) and the fate of the protein pulse was
monitored. As shown in Figure 35, protein pulse was not detected in cdc34-1 cells indicating
that Cdc5 is extremely unstable. However, Cdc5 was remarkably stable in cdc6Δ cells,
correlating with premature spindle elongation. Moreover, Cdc5 was more stable in cdc34-1
cdh1Δ cells as compared to cdc34-1 cells (Figure 35, lower panels). This suggests that Cdc5
may be degraded via an ubiquitin-independent pathway in the absence of Cdc34. Overall,
Cdc5 protein pulse appeared most stable in cdc6Δ cells due to the presence of Cdc34. This
strongly supports the notion that Cdc34 is involved in regulating the stability of spindle
elongation effectors such as Ase1, Cin8 and Cdc5.

cdc34-1 GALCDC5-myc
6
100
80
60
40
20
-MET cyc
+MET Glu cycloheximide
36ºC

100
80
60
40
20
-MET cyc


+MET Glu cycloheximide
36ºC


6

6

100
80
60
40
20
-MET cyc
+MET Glu cycloheximide 36ºC

120

6

6
100
80
60
40
20
-MET cyc
+MET Glu cycloheximide 36ºC


120

6

6


Cdc34 -
Cdc34 +
(Cdc34 +)
(Cdc34 -)
Figure 35. Cdc5 is unstable in the absence of Cdc34 function.

cdc34-1-
cdc34-1
         
myc
6
CDC5GAL1
 


-




5.3 Discussion
Using SILAC mass spectrometry, we have identified a few up- or down-regulated proteins
with functions closely related to spindle elongation upon restoration of Cdc34 function.

Amongst those proteins, Cdc5 seems to be a plausible candidate as it has been reported to
play a synergistic role with Cdk1 to inactivate Cdh1, thus allowing the accumulation and up-
regulation of microtubule associated proteins such as Cin8 and Kip1. These proteins
presumably generate a ‘sliding force’ to induce spindle elongation regardless of which cell
cycle stage the cells are in. Cdc34 appears to be also responsible for stabilization and up-
regulation of Cdc5. Once stabilized in G1 phase, Cdc5 can contribute to spindle elongation
in cells that fail to undergo S phase. This is because prolonged delay in G1 phase will allow
Cdc5 to accumulate; consequently Cdh1 is inactivated, thus resulting in the accumulation of
microtubule associated proteins and triggering premature spindle elongation. Our mass
spectrometric analysis has also identified Kip3 as one the proteins that is, unlike Cdc5, down-
regulated upon the restoration of Cdc34 function. Kip3, a kinesin-8 family member,
undergoes plus-end directed motility and can perform plus-end microtubule depolymerization
(Du et al.), (Walczak 2006), (Gupta et al. 2006). It is possible that Kip3 is subjected to
negative regulation by Cdc5. This may contribute to premature spindle elongation due to
stabilization of the spindle dynamics.
Polo-like kinases (e.g. Cdc5) are evolutionarily conserved proteins that belong to a
subfamily of Ser/Thr protein kinases. In the budding yeast, polo-like kinase Cdc5 is well
known for its multiple roles in mitosis (Lee et al. 2005). During mitosis, Cdc5 regulates
transcription of mitotic genes via phosphorylation of Ndd1, a subunit of the Mcm1-Fkh2-
Ndd1 transcription factor (Darieva et al. 2006). In anaphase, Cdc5 phosphorylates the
subunits of condensin to promote chromosome condensation (St-Pierre et al. 2009). In
addition, Cdc5 mediated phosphorylation during metaphase to anaphase transition leads to

cleavage of cohesins by separase (Alexandru et al. 2001). Cdc5 has also been implicated in
regulation of APC/C and in the degradation of cyclin B during anaphase (Charles et al. 1998;
Shirayama et al. 1998). As a component of the FEAR pathway, Cdc5 mediates the release of
Cdc14 phosphatase in early anaphase through phosphorylation (Visintin et al. 2003; Rahal et
al. 2008). Furthermore, Cdc5 phosphorylates Bfa1, the negative regulator of the MEN
pathway to promote mitotic exit (Hu et al. 2001). During cytokinesis, Cdc5 is also involved
in Rho1 activation and contractile actin ring (CAR) assembly (Yoshida et al. 2006).

In cultured mammalian cells, Plk1 facilitate the establishment of a centrosomal
scaffold for microtubule nucleation by phosphorylating and displacing the centrosomal
protein, Nlp (Casenghi et al. 2003). Plk1 also appears to regulate microtubule dynamics by
either positively or negatively regulation of various microtubule associated components.
While Plk1 has been reported to phosphorylate and diminish the microtubule-stabilizing
activity of TCTP (Yarm 2002), Plx1 was reported to stabilize microtubules via negative
regulation of microtubule-destabilizing protein, Stathmin/Op18 in Xenopus (Budde et al.
2001).
Thus polo kinase(s) participate in a myriad of cellular functions. While polo kinase’s
role in the regulation of spindle dynamics has long been known in vertebrate cells, its
involvement in aspects of spindle biogenesis in budding yeast has emerged only in the last
few years. The present study specifically underscores its role in the premature spindle
elongation which can lead to extreme genomic instability in cells that are either delayed or
failed completely to initiate DNA replication. Cdc34 emerges in these investigations as the
master regulator of spindle dynamics in G1 phase by promoting stability of microtubule
associated proteins and Cdc5 to induce premature spindle elongation should cell cycle
committed (post START) cells fail to execute S phase.

Chapter6 Perspective and future directions
Since 19
th
century, abnormal chromosome number – also known as aneuploidy – has been
recognized as a near ubiquitous feature of human cancers. Study of colorectal cancers has
shown that approximately 85% display aneuploidy errors, and contain cells with an average
of 60 to 90 chromosomes (Pellman 2001). Moreover, high clinical grades tumours are
closely associated with greater degrees of aneuploidy. In normal cells, the constancy of
chromosome number is maintained through a highly coordinated progression through the cell
division cycle. The cellular coordination is imposed by checkpoint controls to ensure that, if
a certain event is executed erroneously or interrupted, the initiation of the subsequent phase
of the cell cycle is delayed. Budding yeast Saccharomyces s cerevisiae has served as an

excellent model to uncover the regulatory circuits underlying these controls. While
replication, DNA damage and spindle assembly checkpoints have been extensively
investigated, G1-M checkpoint has remained under explored. In this study we have
attempted to uncover the mechanism underlying the G1-M checkpoint; deficiency of which
leads to premature segregation of the unreplicated chromosomes; a phenotype that represents
an extreme form of chromosome instability.
An Overview and the Emerging Model
It has long been thought that precocious segregation of chromosomes in checkpoint deficient
mutants is due to a premature entry into mitosis. However, recent evidence suggests that, at
least in case of the replication checkpoint deficient mutants, premature segregation of
unreplicated chromosomes is not due to premature entry into mitosis but because spindle
dynamics is deregulated (Krishnan et al. 2004). Tight regulation of the spindle dynamics is
particularly necessary in the early part of the division cycle when chromosomes have not
attained biorientation and so are not able to resist the tendency of the spindle to elongate.

Cells that are unable to initiate DNA replication (cdc6, cdc7 and cdc45 mutants for instance)
represent the danger posed by the spindle to unreplicated chromosomes. It has long been
thought that precocious segregation of unreplicated chromosomes in cdc6 mutants is due to
premature entry into mitosis. This has led to the postulation of a G1-M checkpoint of which
Cdc6 is considered to be a prominent component (Toyn et al. 1995). However, the exact
nature of this checkpoint and Cdc6’s role in it has remained a mystery.
So far, Cdc6 is the only known component of the G1-M checkpoint. Although unable
to initiate S phase, Cdc6 deficient cells undergo precocious partitioning of the unreplicated
chromosomes. Therefore, Cdc6 has been ascribed the role of a checkpoint protein whose
function is to prevent onset of mitosis when cells fail to initiate DNA replication. In a
somewhat extended version of this notion, the signals emerging from the pre-replication
complex activates Cdc6’s checkpoint function which prevents premature initiation of mitosis
(hence, segregation of unreplicated chromosomes) (Piatti et al. 1995). This model is
conceptually attractive as it suggests a long-range coordination control that prevents cell
cycle division-committed G1 cells from initiating mitosis prematurely. Our study

documented in this thesis brings this entire notion of G1/M checkpoint into question. First,
our results show that premature segregation of chromosomes in Cdc6 deficient cells does not
result from untimely onset of mitosis. We also find that premature segregation of
unreplicated chromosomes is a phenotype exhibited not only by Cdc6 deficient cells but also
other mutants (cdc7Δ and cdc45Δ) that fail to initiate DNA replication, despite their ability to
assemble a pre-replication complex. This suggested that untimely segregation of unreplicated
chromosomes is perhaps a common property of G1 cells that traverse START but are unable
to initiate DNA replication.
However, cdc34 mutant appears to be an exception to this tentative rule. Cells
deficient in Cdc34 function traverse START and are unable to initiate DNA replication but

yet do not undergo precocious chromosome segregation. It can be argued that this is so
because Cdc6 is functional in cdc34 mutant and prevents untimely partitioning of
chromosomes. The fact that cdc34 cdc6Δ double mutant also fails to segregate chromosomes
precociously implies that it is not the deficiency of Cdc6 per se that causes untimely
chromosome segregation. This is in fact not surprising because cdc34 cdc6Δ mutant cells are
unable to assemble a spindle that can mediate chromosome segregation. Short spindle
assembly can be induced in the cdc34 cdc6Δ double mutant by deletion of CDH1 (which
controls the stability of microtubule binding proteins Cin8, Kip1 and Ase1). Surprisingly,
cdc34 cdc6Δ cdh1Δ triple mutant assembles a short spindle yet is unable to extend it to
segregate unreplicated chromosomes. These observations together with the previous report
that premature chromosome segregation in replication checkpoint deficient mutants is due to
the deregulation of spindle dynamics (Krishnan et al. 2004) prompted us to direct our efforts
to the exploration of spindle regulation in cdc34 cdc6Δ cells.
Once cells traverse START in late G1 , they are irreversibly committed to undertake the
progression through the division cycle and exhibit two conspicuous morphological features
before initiation of S phase: emergence of a bud and duplicated spindle poles connected by an
inter SPB bridge. The short spindle assembly requires breaking of the bridge that connects
the duplicated SPBs. It has been previously shown that plus-end directed kinesin motors
Cin8 (homologue of Eg5) and Kip1 and microtubule associated protein Ase1 mediate SPB

separation by breaking the bridge, thus leading to short spindle assembly (Crasta et al. 2006).
Like mitotic cyclins, Cin8, Kip1 and Ase1 are targeted for proteolytic destruction by E3
ubiquitin ligase APC
Cdh1
(anaphase promoting complex activated by Cdh1) that is active
during G1 through to S phase. To be able to separate SPBs and assemble a short spindle, it is
imperative for cells to accumulate these microtubule associated proteins. The premature
extension of the spindle in G1 can be viewed not as an unscheduled entry into mitosis per se

but as a disconnection of the spindle cycle from the rest of the cell cycle machinery. Under
normal circumstances, short spindle forms only at the end of S phase. However, the failure to
initiate S phase causes an extended stay in G1 phase. This results in the accumulation of
elongation-conducive microtubule binding proteins (Cin8, Kip1, Ase1) and eventually
premature extension of the spindle. Since at this stage, chromosomes are unreplicated,
kinetochore duplication, bi-orientation and cohesins are unable to resist the dramatic spindle
extension, as they collectively do during normal S phase, G2 and metaphase, hence spindles
form and prematurely elongate (i.e. premature chromosome segregation). Thus initiation of
DNA replication that allows duplication of kinetochores, chromosome biorientation and
tethering of chromosomes by cohesins provide a major deterrent to premature spindle
extension. As our results suggest, the mechanics of MAPs accumulation in G1 is controlled
by a complex regulatory network. Previous studies had shown that Cdh1 is a potent inhibitor
of spindle assembly in that the expression of a constitutively active allele (cdh1-m11A)
causes cells to arrest in G2 with unseparated SPBs (Crasta et al. 2006). Failure to break the
intra-SPB bridge can be attributed to very low levels of Cin8, Kip1and Ase1. Further
analysis revealed that Cdc28/Cdk1-Clb kinase and Cdc5 acted in synergy to fully inactivate
Cdh1 via phosphorylation at multiple sites during normal cell cycle to allow accumulation of
MAPs and the assembly of a short spindle (Crasta et al. 2008). As noted before, deletion of
CDH1 in cdc34-1 cdc6Δ cells only allows biogenesis of short spindles, but spindle elongation
is still prohibited. However, restoration of Cdc34 function results in further accumulation of
Cin8 and Ase1, triggering spindle elongation and premature chromosome segregation. This

suggests that the limiting factor that prevents spindle elongation is the further accumulation
of microtubule associated proteins. These results also imply that the MAPs are targeted for
destruction not only by Cdh1 but also by another machinery that is negatively regulated by
Cdc34 (or SCF by implication).

Based on our observations, we propose that Cdc34 has a dual role in mediating
premature spindle elongation in cells that fail to undergo S phase (Figure 36). Firstly, Cdc34
catalyzes degradation of Sic1 with the help of Cdc28-Cln kinase, leading to the activation of
Cdc28/Clb kinase required for Cdh1 inactivation. This results in accumulation of
microtubule associated proteins to an extent such that only biogenesis of short spindles is
permitted. Cdc34 also appears to be involved in the stabilization and up-regulation of
microtubule associated proteins such as Ase1 and Cin8, and Cdc5 by protecting them against
the yet-to-be-identified degradation pathway. This further augments the accumulation of
Ase1 and Cin8 to a sufficiently high level that is necessary to provide sufficient force to
elongate the spindle. The up-regulated Cdc5 also works in synergy with Cdk1 to enhance the
inactivation of Cdh1. In addition, Cdc5 is also required for proper spindle dynamics and
growth (Park et al. 2008). Intriguingly, Cdc5 can phosphorylate several spindle pole body
(SPB) or spindle associated proteins, in vitro, raising the possibility that Cdc5 regulates
spindle functions via phosphorylation of these proteins directly. Cdc5 has also been
implicated in negatively regulating microtubule-destabilizing proteins (Park et al. 2008).
Hence, yeast microtubule destabilizing protein Kip3 may also participate in this regulatory
circuit.



Figure 36. The emerging model proposed that Cdc34 has a dual role in mediating
premature spindle elongation in cells that fail to undergo S phase.
START
Unreplicated DNA
Protein X

Ubiquitin – independent
Degradation pathway









Sic 1




P
P
P
P
Cdc28 / Cln




Degradation by SCF

(Cdc34 as a component)

Cdh 1
Cin 8, Kip 1, Ase 1,
Cdc 5









Cdc28/Clb activation

Finally, this regulatory scheme suggests that Cdc34, and not Cdc6, is a key player in the
premature spindle elongation and untimely segregation of unreplicated chromosomes. Cdc6
does not directly participate in this process; instead, its deficiency prevents the initiation of
DNA replication and allows the manifestation of the underlying circuitry. Cells that have
traversed START and completed the ‘Cdc34-execution point’ also set in motion the spindle
assembly and elongation machinery. It is through the initiation of DNA replication (via
kinetochore duplication, cohesin-loading and biorientation) that cells restrain spindle
elongation and avoid premature chromosome segregation. In other words, cells that have
traversed START and completed Cdc34 execution step must initiate DNA replication or face
extreme chromosomal instability.
Future Directions
While this study has contributed to a better understanding of premature spindle elongation
and has introduced a new perspective where both spindle cycle and cell cycle require precise
and timely coordination especially in cells with unreplicated chromosomes, much remains to
be done in order to achieve a full understanding of the mechanisms underlying the entire

regulatory network that safeguards genomic integrity by avoiding premature spindle
elongation. Our experimental outcomes, together with mass spectrometric analysis, suggest
that Cdc34 is the most upstream master regulator of spindle dynamics. We postulate that
Cdc34 catalyzes short spindle biogenesis by targeting Cdh1 inactivation involving a number
of regulators and simultaneously boosts the abundance of microtubule associated proteins by
protecting them against ubiquitin-independent degradation pathway. Cdc34 also promotes
stability of Cdc5 that plays an important role in complete inactivation of Cdh1 and negative
regulation of microtubule depolymerization factors such as Kip3. In order to strengthen this
hypothesis, it is imperative to investigate the following:

(i) Since Cdc34 is component of the SCF complex, it will be necessary to identify the
participating F-box proteins, which determines the substrate specificity of the SCF complex
in the present context. This will help in the identification of proteins whose destruction is
required for the stabilization of both microtubule associated proteins and Cdc5 polo kinase.
(ii) Since Chinese hamster ovary cells can assemble a mitotic spindle and progress
through M-phase with unreplicated chromosomes, it will be interesting to determine if Cdc34
function is required for the unscheduled progression into mitosis. A positive answer to this
query may suggest the conservation across species of the regulatory scheme we have
proposed.
(iii) As Kip3 was identified as one of the proteins down-regulated during premature
spindle elongation, it will be useful to determine if the Cdc34-mediated accumulation of
Cdc5 is responsible for the negative regulation of Kip3.
(iv) It will be interesting to ascertain if the function we have ascribed to Cdc34 in
inducing premature spindle elongation is via its requirement for the activity of SCF complex
or independent of SCF.
In closing, this study has attempted to attain a better understanding of the regulation of
spindle dynamics in G1 and its coordination with S phase initiation in Saccharomyces
cerevisiae. It is hoped that by gaining further insights into the spindle biogenesis and spindle
elongation process regulated by Cdc34, Cdks, Polo kinases, APC
Cdh1

, our understanding of
how proliferating cells manage to segregate their chromosomes with high fidelity between
daughter cells will be enhanced. Insights gained from the studies on model organisms may
eventually be useful in understanding the cell cycle coordination and chromosome stability in
vertebrate cells.


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