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Coupling of growth and sliding velocity

of microtubules in fission yeast mitotic


spindles
Research proposal

Koen Hoogendoorn 941124359110


Supervisors : Marcel Janson & Hannie van der Honing
Chair group: Cell biology
January 2015
Bsc Biology, Cell and molecular biology

Koen Hoogendoorn

January 2015

Table of Contents
1.

Introduction ........................................................................................................................ 3

2.

Approach, analysis, presentation results ............................................................................ 5


2.1 Yeast crosses and treatments to be analysed .................................................................... 5
2.2 Imaging and image analysis ............................................................................................. 5
2.3 Estimation of the amount of data ..................................................................................... 5

3.

Project planning.................................................................................................................. 6
3.1 Work scheme .................................................................................................................... 6
3.2 Important dates ................................................................................................................. 6

4.

Backup strategies ................................................................................................................ 6


4.1 Failed crosses ................................................................................................................... 6

5.

References .......................................................................................................................... 7

Summary
Microtubules are part of the cytoskeleton in eukaryotic cells, and they facilitate a vast array of
important processes in the cell. One of these functions is segregation of the chromosomes
during cell division. At the start of this process, a structure made out of microtubules, called a
spindle, is formed between the two spindle pole bodies (SPBs, functional equivalents of the
centrosomes in animal cells) in the cell nucleus of Schizosaccharomyces pombe. This
structure pushes the two SPBs away from each other while pulling the chromosomes towards
the SPBs to allow for successful cell division. The velocity at which the SPBs are pushed
away from each other is known to be influenced by motor proteins and other microtubule
associated proteins (MAPs) that bind to the antiparallel microtubules in the middle of the
spindle. The aim of this experiment will be to find out if this velocity is also influenced by the
growth rate of the microtubules in the spindle. This will be done by analysing the velocity at
which the SPBs move in yeast cells under influence of varying concentrations of the
microtubule depolymerizing drug MBC. Another yeast strain has been created where alp14, a
gene that stabilises microtubules, has been knocked down. This strain will also be analysed to
compare results with. This analysis will contribute to the understanding of the exact
mechanism of the presumed coupling and give more insight into the precise regulation of
mitosis.

Koen Hoogendoorn

January 2015

1. Introduction
Schizosaccharomyces pombe is a widely studied yeast species. These unicellular organisms
only grow at their tips, and divide into two identical daughter cells through medial fission..
These properties make S.pombe very useful for research in the field of cell division. The cell
cycle of S.pombe can be divided into two parts (figure 1). The vegetative cycle, which is
active under favourable conditions. In this cycle the cells propagate through mitosis, and thus
will create exact copies of themselves.

Figure 1: Cell cycle of S.pombe with a clear distinction between the generative (left) and the vegetative (right) cycle.
(Source: Pombenet, http://www-bcf.usc.edu/~forsburg/main4.html)

If the living conditions of the cells worsen, and nutrients become scarce, they will switch to
the generative cycle [1]. In this cycle, sporulation and genetic recombination will take place.
The spores formed by this process are much more resistant to unfavourable conditions, and
will survive until more favourable conditions arise. In this experiment, only the M-phase of
the vegetative cycle will be examined. In this phase, the actual mitosis takes place. [2]. At the
onset of mitosis, the interphase bundles are degenerated, and a spindle is formed. In the
spindle, three different types of microtubules are formed. Astral microtubules, which emanate
from the SPBs position the spindle into the right orientation within the cell. Kinetochore
microtubules attach to the chromosomes at the kinetochore regions to later pull them to the
edges of the nucleus. The type of microtubules that will be important to this experiment are
the polar microtubules. These microtubules form an antiparallel bundle consisting of about 25
microtubules, originating from SPBs, interdigitate in the middle of the nucleus (figure 2) [3].

Figure 2: Simplified structure of the mitotic spindle. Chromosomes and kinetochore microtubules are not shown.

Koen Hoogendoorn

January 2015

The creation and maintenance of the mitotic spindle is strictly regulated by various proteins
[4-6]. At the start of the anaphase, the regulatory protein Ase1p is recruited to the overlap [6],
and forms the midzone. Ase1p acts as a scaffolding protein and recruits more MAPs to the
midzone to further regulate spindle elongation. The Cls1p protein for example stabilises the
microtubules at the edge of the midzone [4], ensuring that the overlap is maintained
throughout the anaphase.
In the midzone, plus end directed kinesins bind to the antiparallel microtubules and push them
in opposite directions, which, in combination with microtubule growth causes the spindle to
elongate to allow for proper separation of the genetic content of the cell. Even though the
genome of S.pombe has nine kinesins and a single dynein, only one of these motor proteins,
klp9+, has a large influence on the elongation speed of the spindle [2]. If the gene coding for
this protein is disabled, the elongation speed is drastically lowered. Other kinesins also have
an influence on mitosis, but this cannot be observed by looking at spindle elongation
velocities [7, 8]. In addition to the motor proteins, regulatory MAPs can be disabled or
upregulated to study their effects on spindle elongation. For example, it has been shown that
the amount of Ase1p directly correlates with the length of the midzone [9].
Another factor that is expected to influence spindle elongation velocity is the growth rate of
the microtubules in the spindle. The spindle will never be able to expand faster than the
microtubule growth rate, since the interdigitating microtubules are prevented from sliding
apart [5]. It is possible to inhibit microtubule growth by adding the microtubule
depolymerising drug methyl-2-benzimidazole-carbamate (MBC), and look at the effects of the
drug on the spindle elongation velocity. Furthermore, a mutant that will be used in this
experiment to influence the spindle elongation is Alp14. The gene encoding for the alp14
protein, homolog of XMAP215 in Xenopus [10], has been disrupted so no functional protein
will be created. Since alp14 is a microtubule stabilising protein, the mutant will have a
negatively influence on the microtubule growth rate. Earlier data suggests that the growth rate
of microtubules indeed influences the elongation velocity of the spindle [9].
The main goal of this experiment will be to prove or disprove the existence of a coupling
between the microtubule growth rate and the sliding speed of the spindle mentioned in the
section above. To reach this goal, the spindles of fission yeast will be observed during
anaphase B since this is when the most rapid elongation takes place. The spindles will be
observed while under the influence of different concentrations of MCB. The Alp14 strain
will also be used to acquire more conclusive data. When placing this experiment in the bigger
picture, it will help to contribute to the understanding of the underlying mechanism of the
coupling, and give further insight into the regulatory processes of mitosis.

Koen Hoogendoorn

January 2015

2. Approach, analysis, presentation results


2.1 Yeast crosses and treatments to be analysed
In the experiment, two crosses of S.pombe resulting from three different strains will be
analysed. In both cases the transgenic strain GFP-Ase1 Ase1 (JT154) will be used to be able
to observe the localisation of the Ase1 protein. The first cross will be made with transgenic
line Sid4-RFP (JT133) which will fluorescently label the SPBs. The other cross will be made
with transgenic line Alp14 (PT203) to inhibit microtubule growth. These crosses have
already been conducted, and viable colonies have already been selected and grown on plates.
Of these colonies, only cells that are near the end of the metaphase or the start of the anaphase
at the moment of imaging will be used since that is the moment the spindle is formed and
elongating.
Using the GFP-Ase1 Ase1 x Sid4-RFP cross, the influence of four concentrations (0, 10, 25
and 50 g/ml) of the drug MBC, which causes lower microtubule growth rates, on the sliding
speed will be tested.
The GFP-Ase1 Ase1 x Alp14 cross will be imaged without further treatment, and is
expected to produce results similar to the cells that are affected by MBC.

2.2 Imaging and image analysis


The imaging of the fluorescently labelled components in the cell will be performed on a
Roper Spinning Disk Confocal microscope. The fast imaging rate and high resolution are
ideal for monitoring small processes and movements in high detail and large quantities. For
each cell to be analysed, a timelapse of 60 minutes will be made with intervals between
images of 20 seconds. To detect both fluorophores, two lasers with wavelengths of 491 and
561 nm will be used to excite GFP and RFP respectively and emission filters for the
corresponding fluorophores will be used. A z-series of 2.5m in six steps will have to be
conducted as well, since the SPBs will not necessarily be in the same two-dimensional plane.
This z-series will be combined into a maximum projection using the capture software before
it is used for analysis with ImageJ.
In ImageJ, the timelapse of the moving SPBs will be analysed frame by frame to determine
elongation velocities. These velocities will be stored in an excel file, and different groups will
be compared using the appropriate statistical methods.

2.3 Estimation of the amount of data


For every group, the aim is to acquire at least twenty viable cells. This will result in 100
velocities. The midzone lengths will also be measured for future research. The analysis should
not take too much time if everything goes according to plan. In that case, there will be a more
than sufficient time available for imaging. In the period it takes for the microscope to make
one timelapse, it should be possible to at least create the kymograph of the previous timelapse.
If this is not the case, the two re-exam weeks can be used to catch up on the analysis, and
possibly the start of writing the report.

Koen Hoogendoorn

January 2015

3. Project planning
The project will essentially consist of three parts. Imaging, data processing and writing.
Throughout the entire project, I will read literature that is related to the project, and try to
apply the information from that literature to my project. In week two, I will start with imaging
the cells. If the colonies that were started the Friday before turn out right, I could start that
Monday. Imaging will take approximately 2-3 weeks, and it should be possible to start the
analysis simultaneously (or with a slight delay). The weeks in the re-examination period will
be used to get back on track if work doesnt progress as quickly as hoped for.

3.1 Work scheme


Activity-Week
1 2 3 4 re-exams
Reading literature
x x x x x
x
Writing proposal
x x
Imaging /processing
with capture program
x x x
Analysis with imageJ/excel
x x x
x
Writing report
x
x

5 6 7 8
x x x x

x x x x

3.2 Important dates


12-1-15: Hand in research plan (draft)
13-1-15: Feedback on research plan
15-1-15: Hand in research plan (final)
16-1-15: Present research plan
27-2-15: Hand in report (draft)
02-3-15: Feedback on report
11-3-15: Hand in report (final)
13-3-15: Present report

4. Backup strategies
4.1 Failed crosses
The amount of viable colonies acquired from the pre made crosses is not very high, and there
is still a chance that these colonies contain the endogenous Ase1 gene. If a PCR reaction
proves this to be the case, results obtained from these crosses will not be as reliable as hoped
for. Two new crosses have been initiated as a backup in case the colonies turn out to be
inviable. If this does not result in viable colonies, other strains will have to be used. For
example, a strain with only Ase1-GFP Ase1 could still be used, since localisation of the
SPBs can also be inferred from the location of the fluorescent Ase1. This does however result
in less pretty images.

Koen Hoogendoorn

January 2015

5. References
1. Forsburg SL (2001) Growth and Manipulation of S. pombe. Current Protocols in Molecular
Biology. John Wiley & Sons, Inc.
2. Fu C, Ward JJ, Loiodice I, Velve-Casquillas G, Nedelec FJ, et al. (2009) PhosphoRegulated Interaction between Kinesin-6 Klp9p and Microtubule Bundler Ase1p Promotes
Spindle Elongation. Developmental Cell 17: 257-267.
3. Khodjakov A, La Terra S and Chang F (2004) Laser Microsurgery in Fission Yeast: Role
of the Mitotic Spindle Midzone in Anaphase B. Current Biology 14: 1330-1340.
4. Bratman SV and Chang F (2007) Stabilization of Overlapping Microtubules by Fission
Yeast CLASP. Developmental Cell 13: 812-827.
5. Braun M, Lansky Z, Fink G, Ruhnow F, Diez S, et al. (2011) Adaptive braking by Ase1
prevents overlapping microtubules from sliding completely apart. Nature Cell Biology 13:
1259-1264.
6. Loodice I, Staub J, Setty TG, Nguyen N-PT, Paoletti A, et al. (2005) Ase1p Organizes
Antiparallel Microtubule Arrays during Interphase and Mitosis in Fission Yeast. Molecular
Biology of the Cell 16: 1756-1768.
7. Troxell CL, Sweezy MA, West RR, Reed KD, Carson BD, et al. (2001) pkl1 +andklp2 +:
Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both
Mitosis and Meiosis. Molecular Biology of the Cell 12: 3476-3488.
8. West RR, Malmstrom T and McIntosh JR (2002) Kinesins klp5+ and klp6+ are required for
normal chromosome movement in mitosis. Journal of Cell Science 115: 931-940.
9. Teapal J, Sjollema J and Janson ME (2014) Microtubule-based positioning mechanisms.
Cell biology. Wageningen: Wageningen University & Research centre. pp. 71-91.
10. Garcia MA, Vardy L, Koonrugsa N and Toda T (2001) Fission yeast chTOG/XMAP215
homologue Alp14 connects mitotic spindles with the kinetochore and is a component of the
Mad2dependent spindle checkpoint. 3389-3401 p.