GHENT UNIVERSITY UNIVERSIDAD COMPLUTENSE DE MADRID …
Transcript of GHENT UNIVERSITY UNIVERSIDAD COMPLUTENSE DE MADRID …
Masterthesis performed at
GHENT UNIVERSITY UNIVERSIDAD COMPLUTENSE DE MADRID
FACULTY OF PHARMACEUTICAL SCIENCES FACULTY OF PHARMACY
Department of Pharmaceutical Analysis Department Of Microbiology II
Laboratory of Pharmaceutical Microbiology Unit Four
Academic year 2011-2012
The RSC chromatin remodeling complex and transcription through the cell wall integrity pathway in Saccharomyces cerevisiae
Lisa FLORIN
First Master In Drug Development
Promoter
Prof. Dr. T. Coenye
Co-promoter
Prof. Dr. J. Arroyo
Commissioners
Prof. Dr. H. Nelis
Dr. I. Vandecandelaere
Masterthesis performed at
GHENT UNIVERSITY UNIVERSIDAD COMPLUTENSE DE MADRID
FACULTY OF PHARMACEUTICAL SCIENCES FACULTY OF PHARMACY
Department of Pharmaceutical Analysis Department Of Microbiology II
Laboratory of Pharmaceutical Microbiology Unit Four
Academic year 2011-2012
The RSC chromatin remodeling complex and transcription through the cell wall integrity pathway in Saccharomyces cerevisiae
Lisa FLORIN
First Master In Drug Development
Promoter
Prof. Dr. T. Coenye
Co-promoter
Prof. Dr. J. Arroyo
Commissioners
Prof. Dr. H. Nelis
Dr. I. Vandecandelaere
COPYRIGHT
"The author and the promoters give the authorization to consult and to copy parts of this
thesis for personal use only. Any other use is limited by the laws of copyright, especially
concerning the obligation to refer to the source whenever results from this thesis are cited."
May 23, 2012
Co-promoter Promoter Author
Prof. Dr. Javier Arroyo Prof. Dr. Tom Coenye Lisa Florin
SUMMARY
ATP-dependent chromatin remodeling complexes, such as the RSC complex, affect
chromatin by disruption of the nucleosome structure, thereby making DNA accessible for
transcription. Mitogen-activated protein kinase (MAPK) pathways react and adapt to
changes, extracellular or intracellular, and make use of chromatin remodeling complexes to
regulate transcriptional responses. There are indications that the RSC complex could be
involved in the transcriptional respons regulated by the yeast cell wall integrity pathway
(CWI) to modify the nucleosomes at stress-induced genes in response to cell wall stress. The
central MAPK in the CWI pathway is Slt2, which activates mainly the Rlm1 transcription
factor. The complication about studying this relationship is that some of the subunits of the
RSC complex are essential for cell viability, thus “degron strains” are used. These act as the
wild type at 24°C and as a conventional mutant at 37°C, by degradation of the Rsc subunit.
Here, investigation was done to confirm the functionality of the degron strains, to set
up experimental conditions for studying the relationship between the RSC complex and the
CWI pathway and the assumption about the participation of the RSC complex was
strengthened. Experiments were mainly done with two strains: W303-1A (WT) and rsc8deg.
Although the rsc8deg strain at 24°C had a slightly slower cell growth and different cell
morphology and the Slt2 basal activation levels were slightly higher in comparison with WT,
the Rsc8 subunit was still functional. At 37°C, Rsc8 degradation occurred fast and after 1h
functional Rsc8 was strongly diminished. Because of heat stress at 37°C, the Slt2 MAPK
pathway was activated in both strains, but equally strong, indicating that Rsc8 does not
influence the activation of the CWI pathway. This is a requirement for comparing results
concerning gene expression at targeted genes. The expression of MLP1, one of the genes
targeted by Rlm1, was lower 37°C than at 24°C for rsc8deg . This suggests that the RSC
complex is involved in regulating transcriptional responses in the CWI pathway under stress.
MAPK pathways are very well conserved between all eukaryotes. Dysfunctions in
MAPK pathways are often the cause of human diseases, so elucidating the molecular
mechanisms by studying the dynamics of this pathways in yeast can help to discover new
therapeutic targets.
SAMENVATTING
ATP-afhankelijke chromatine remodelerende complexen, zoals het RSC complex, werken in
op het chromatine door vervorming van het nucleosoom en maken hierdoor het DNA
toegankelijk voor transcriptie. Mitogeen-geactiveerde proteïne kinase (MAPK) pathways
voelen intracellulaire of extracellulaire veranderingen en reageren daarop. Ze recruteren
chromatine remodelerende complexen om de transcriptionele respons te regelen. Er zijn
indicaties dat het RSC complex in Saccharomyces cerevisiae gebruikt wordt door de celwand
integrititeitspathway (CWI) om nucleosomen aan stress-geinduceerde genen te modificeren
als antwoord op stress gevoeld aan de celwand. Het centrale MAPK in de CWI pathway is Slt,
wat voornamelijk de Rlm1 transcriptie factor activeert. De moeilijkheid bij het bestuderen
van het RSC complex is dat sommige subunits noodzakelijk zijn voor het overleven van de
cel. Daarom worden “degron stammen” gebruikt: deze gedragen zich als wild type (WT) bij
24°C maar als conventionele mutant bij 37°C, wanneer de Rsc subunit afgebroken wordt.
Onderzoek met WT en rsc8deg werd verricht om de werking van de degron stammen
te bevestigen en om experimentele condities op te stellen. Ook de veronderstelling van de
betrokkenheid van het RSC complex in de CWI pathway werd versterkt. Hoewel rsc8deg een
tragere groei en een verschillende cel morfologie had bij 24°C en de basale activatie van Slt2
hoger lag dan bij de WT, was de Rsc8 subunit bij deze temperatuur nog steeds functioneel.
Bij 37°C degradeerde de Rsc8 subunit snel, binnen het uur waren de functionele Rsc8
niveaus heel laag. Als antwoord op stress veroorzaakt door hitte bij 37°C was de Slt2 MAPK
pathway geactiveerd, even sterk in beide stammen. Dit houdt in dat Rsc8 de activiteit van de
Slt2 MAPK pathway niet beïnvloedt, wat een nodige voorwaarde is voor verder onderzoek.
De expressie van MLP1, één van de genen onder controle van Rlm1, was lager bij 37°C dan
24°C voor rsc8deg . Dit suggereert dat het RSC complex betrokken is in het reguleren van de
transcriptionele respons in de CWI pathway onder invloed van hittestress.
De MAPK pathways zijn heel sterk geconserveerd in alle eukaryoten. Abnormaliteiten
in deze pathways in mensen veroorzaken vaak ziekten. De moleculaire mechanismen van
deze pathways ophelderen via het bestuderen van de dynamiek in gistcellen kan helpen om
nieuwe therapeutische drugtargets te ontdekken.
ACKNOWLEDGEMENT
I would like to make use of this opportunity to thank all the people who helped me to bring
this thesis to a successful end.
First of all, I want to thank my promoter Prof. Dr. T. Coenye for giving me the chance to
experience this amazing Erasmus adventure and for correcting this work with care. A special
thanks also to my co-promoter Prof. Dr. J. Arroyo for welcoming me in his lab, for handing me
my thesis subject and for revising my work.
Furthermore, I would like to express my appreciation to Belén Sanz for assisting my with the
experiments, for her daily enthusiasm, for her endless patience and for kindly answering my
many questions.
Finally, also a word of thanks to all the laboratory workers of unit four for creating an
excellent work atmosphere and for their willingness to stand my by with advise.
TABLE OF CONTENTS
1. INTRODUCTION ............................................................................................................................... 1
1.1. ENVIRONMENTAL STRESS ....................................................................................................... 1
1.2. MAPK PATHWAYS .................................................................................................................... 1
1.2.1. Mammalian MAPK pathways .......................................................................................... 3
1.2.2. Yeast MAPK pathways ..................................................................................................... 4
1.3. CELL WALL INTEGRITY PATHWAY IN SACCHAROMYCES CEREVISIEAE .................................... 5
1.3.1. Properties of the cell wall ................................................................................................ 5
1.3.2. Cell Wall Integrity pathway ............................................................................................. 5
1.3.3. Activation of the CWI pathway ........................................................................................ 8
1.4. ATP-DEPENDENT CHROMATIN REMODELING COMPLEXES .................................................... 9
1.4.1. Chromatin structure and transcription ........................................................................... 9
1.4.2. Families and mechanism of action of chromatin remodeling complexes ..................... 10
1.4.3. The remodels the structure of chromatin complex ...................................................... 12
1.5. MAPK PATHWAYS AND REMODELING COMPLEXES .............................................................. 13
2. OBJECTIVES .................................................................................................................................... 15
3. MATERIALS AND METHODS .......................................................................................................... 17
3.1. YEAST STRAINS AND GROWTH CONDITIONS ........................................................................ 17
3.1.1. Saccharomyces cerevisiae strains .................................................................................. 17
3.1.2. Growth conditions ......................................................................................................... 19
3.2. WESTERN BLOTTING .............................................................................................................. 21
3.2.1. Growing and recovery of cells ....................................................................................... 21
3.2.2. Cell lysis and preparation of yeast extract .................................................................... 22
3.2.3. Preparing and loading acrylamide gel ........................................................................... 24
3.2.4. Transfer of proteins from gel to nitrocellulose membrane .......................................... 26
3.2.5. Immunodetection .......................................................................................................... 26
3.3. PLATE SENSIBILITY ................................................................................................................. 28
3.3.1. Materials ........................................................................................................................ 28
3.3.2. Methods ........................................................................................................................ 28
3.4. GROWTH EXPERIMENT DEGRON STRAINS ............................................................................ 29
3.4.1. Materials ........................................................................................................................ 29
3.4.2. Methods ........................................................................................................................ 29
3.5. CELL VIABILITY: FLUORESCENCE MICROSCOPY ..................................................................... 29
3.5.1. Materials ........................................................................................................................ 29
3.5.2. Methods ........................................................................................................................ 29
3.6. β-GALACTOSIDASE ASSAY ...................................................................................................... 30
3.6.1. Transformation of yeast cells ........................................................................................ 30
3.6.2. β-galactosidase activity ................................................................................................. 31
4. RESULTS ......................................................................................................................................... 35
4.1. CELL VIABILITY ....................................................................................................................... 35
4.1.1. Growth curves ............................................................................................................... 35
4.1.2. Cell viability with fluorescence microscopy .................................................................. 37
4.2. PLATE SENSIBILITY ON YPD .................................................................................................... 41
4.3. DEGRADATION OF THE RSC COMPLEX .................................................................................. 42
4.4. ACTIVATION OF CWI PATHWAY ............................................................................................ 43
4.5. MLP1 TRANSCRIPTIONAL ACTIVATION .................................................................................. 44
5. DISCUSSION ................................................................................................................................... 47
6. CONCLUSIONS ............................................................................................................................... 50
7. LITERATURE LIST ............................................................................................................................ 51
ABBREVIATIONS
ATP Adenosine triphosphate
CWI Cell wall integrity
DHFR Dihydrofolate reductase
DNA Deoxyribonucleic acid
GEF Guanosine nucleotide exchange factor
HA Human influenza hemagglutinin
MAPK(KK) Mitogen-activated protein kinase (kinase kinase)
OD Optical density
PBS Phosphate-buffered saline
PMSF Phenylmethylsulphonyl fluoride
PSA Ammonium persulphate
TEMED Tetramethylenediamine
RSC Remodels the structure of chromatin
SC Synthetic medium complete without uracil
SCG Synthetic medium complete without uracil with galactose
SWI/SNF Switch/sucrose non-fermentable
SDS Sodium dodecyl sulphate
TBS Tris-buffered saline
TTBS Tween tris-buffered saline
WT Wild type W303-1A
YPD Yeast peptone dextrose
YPG Yeast peptone galactose
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1. INTRODUCTION
1.1. ENVIRONMENTAL STRESS
A cell in his environment is constantly at danger to be suddenly submitted to some
kind of stress. In this case, stress can be defined as any suboptimal growth condition (change
in nutrient provision, temperature, osmolarity, pH…) or any substance that diminishes the
cell viability (Congo Red, Calco-Fluor White, Caspofungin, Zymolyase, caffeine…). When the
extracellular surroundings of the cell change, its intracellular milieu is not in balance
anymore. In order to survive, the cell needs to react as soon as possible. Different pathways
in the cell are activated in purpose to guarantee its survival and to maintain the integrity of
the cell. The sort of organism, its natural environment and its present physiological condition
are factors that determine the adaptive response. Multicellular organisms are much more
protected against extracellular stress than unicellular organisms, due to their ability of
intracellular homeostasis which makes that extracellular alterations in the environment are
more efficiently buffered. However, the cascades that are activated in stress conditions are
very well conserved between all eukaryotes. These signaling pathways are called after their
central elements, mitogen-activated protein kinases (MAPKs), also called stress-activated
protein kinases (de Nadal et al., 2011).
1.2. MAPK PATHWAYS
The function of the mitogen-activated protein kinase pathways is to react and adapt
to changes, intracellular or extracellular. The MAPK pathways consist of sensing complexes,
either fixed on the plasma membrane or intracellular, which put in motion a signal
transduction system that eventually leads to an accurate dynamic outcome. In this way they
monitor several important cell processes, such as proliferation, differentiation, apoptosis,
motility and response to various stresses. The sensors are activated by a huge array of
stimuli, including hormones, neurotransmitters, changes in heat or osmolarity, drugs,
pheromones, growth factors, cell cycle signals... Signals from different inputs are integrated
and on this basis certain elements are triggered (Qi & Elion, 2005).
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Every MAPK pathway in a cell consists of the same basic system, a linear chain of
serine/threonine kinases. The MAP kinase kinase kinase (MAPKKK, MAP3K or MEKK)
phosphorylates the MAP kinase kinase (MAPKK, MAP2K or MEK) which in turn
phosphorylates the MAP kinase (MAPK). Sometimes there is a MAP kinase kinase kinase
kinase (MAPKKKK), which can be bound to the plasma membrane and in this way pass down
signals to the MAPKKK. Also G-proteins can be involved in the activation of the MAPK
cascade (Qi & Elion, 2005).
The MAPK is the central element in the entire pathway and puts in motion numerous
processes by means of phosphorylation of proteins. This kinase localizes to various
subcellular structures, such as the microtubuli, the active cytoskeleton, the endoplasmic
reticulum, the mitochondria and the Golgi apparatus. Furthermore, the MAPK can
translocate from the cytoplasm to the nucleus to modify its nuclear targets, mostly
transcription factors (Hancock, 2010).
This leads in general to two types of responses. The post-translational modification of
the appropriate proteins is very fast and initiates the earliest adaptations in answer to the
detected stress agent or to a physiological process. This adjustment is however not sufficient
to survive. A slower, but a very important part of the response to stress, is the
transcriptional activation of genes. Each stress agent has its own characteristical pattern of
activated genes, which can be seen as a transcriptional fingerprint. On the other side there is
a set of genes that respond every time the cell suffers from stress, independent of the kind
of stress. This is known as the environmental stress response (de Nadal, 2011).
All the activated cascades taken together, post-translational modification as well as
gene expression, lead to the ultimate adaptation of the cell. However, after a certain time
the cell returns to its basic state, even if the stimulus still exists. This is important to
guarantee the survival of the cell. When the MAPKs are stimulated continuously the cell
growth slows down or stops, due to induction of apoptosis-like responses and cell cycle
delays (Yaakov et al., 2003). The main regulators of the MAPK pathways are protein
phosphatases that dephosphorylate the protein kinases. The release of the phosphate is
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recognized by a trafficking system in the cell that can translocate the deactivated kinase back
to the cytosol, where it is now ready to be activated again (Molina et al., 2010).
MAP kinases also influence gene expression in another way, besides phosphorylation:
by binding to promoter sequences on the chromatin as a part of the transcription complex.
Moreover the MAPK can be attached to coding regions of stress-responsive genes, indicating
it is an elongation factor as well as an initiation factor. These kinases also influence other
components of the general transcription machinery, such as the RNA polymerase II (Alepuz
et al., 2003). These mechanisms indicate that the MAPKs may act in general as chromatin-
associated enzymes (Pokholok et al., 2006).
Although there are different MAPK pathways in one cell, there is a relative separation
between all these cascades, the interaction between elements of the different MAPK
pathways is limited. This is because of the assistance of scaffold proteins. These proteins are
involved in MAPK signaling but do not necessarily have a kinase function. They act as a
support, an anchorage for the kinase proteins. In this way, they avoid undesirable crosstalk
(Hancock, 2010).
As mentioned before, each eukaryotic organism uses the MAPK pathways. The signal
transduction chains are more conserved than the sensing or effector mechanisms. A number
of MAPKs are preserved from yeast to mammals. Studying the dynamics of stress responses
in relative simple cells like yeast could help to comprehend the molecular mechanisms of
MAPK pathways in mammals and how mammalian cells apply their transcription capacity to
act as fast and effective as possible in response to adverse circumstances. Saccharomyces
cerevisiae yeast cells are broadly used as a model eukaryotic organism due to their simple
growth conditions (Widmann et al., 1999).
1.2.1. Mammalian MAPK pathways
The functionality of MAPK pathways in mammalian cells is difficult to study, because
the cell lines require far more complex media, grow slower and the different pathways are
more complicated so that it is harder to reveal the basic mechanisms. The facts about
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mammalian MAPK pathways were mostly discovered from genetic research on Drosphila
melanogaster, the common fruit fly, and Caenorhabditis elegans, a roundworm. In
mammalians, five families of MAPKs are revealed. The extracellular signal-regulated kinases
(ERK1 and ERK2) influence proliferation, differentiation and meiosis. Jun N-terminal kinases
(JNK1, JNK2 and JNK3) are activated in neural inflammation, apoptosis, development and
several stress conditions. Typical physical and chemical stresses, like osmotic shock or heat
shock, trigger the p38 MAPK kinases. These are also stimulated by cytokines such as
interleukin-1 and tumor necrosis factor and play thus a significant role in asthma and
autoimmunity. There is not much known yet about ERK3/ERK4 and ERK5 families (Qi, M.S. &
Elion, E.A., 2005).
The MAPK pathways in humans are of great importance. Abnormalities in these
strictly regulated signaling pathways provoke severe maladies. Many neurodegenerative
diseases find their origin in an atypical element of a MAPK pathway, as was seen with
Alzheimer, Parkinson and amyotrophic lateral sclerosis. Furthermore dysfunctional MAPK
pathways are implicated in the development of diabetes and several types of cancer.
Therefore, elucidating the mechanics of these pathways could help discovering new
therapeutic drug targets (Kim & Choi, 2010).
1.2.2. Yeast MAPK pathways
Concerning MAPK pathways, a lot of research has been done on Saccharomyces
cerevisiae yeast cells. In this yeast, all five MAPK pathways were genetically defined. Fus3
and Kss1 pathways are corresponding with the mammalian ERK1/ERK2 pathway and
regulate the fusing of two organisms for copulation when mating pheromones are sensed in
the environment. The yeast Hog1 pathway is most comparable to p38 mammalian MAPK
pathway and modulates osmolarity. The other three MAPK pathways are distinct from the
mammalian pathways. Slt2, also called Mpk1, is the central protein in the cell wall integrity
pathway, which organizes cell integrity and budding. Finally, Smk1 regulates sporulation. The
MAPK pathways in yeast are not necessary for cell survival, but they do play important roles.
Fus3, Kss1 and Smk1 are activated in physiological conditions, while Hog1 and Slt2 are
activated upon stress (Qi, M.S. & Elion, E.A., 2005).
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1.3. CELL WALL INTEGRITY PATHWAY IN SACCHAROMYCES CEREVISIEAE
The purpose of the cell wall integrity (CWI) pathway is to detect cell wall stress and
pass this information down for a fast adaptation of the cell. It is activated in circumstances
where the cell wall stability and thus the integrity of the cell is at risk. This can occur during
normal growth conditions or because of an upcoming environmental challenge. The CWI
pathway is known to be stimulated after oxidative and heat stress, low and high pH, cell-wall
interfering compounds and DNA damage. When the cell wall fails to protect the cell from the
changing environment, the yeast dies (Levin, 2011).
1.3.1. Properties of the cell wall
The cell wall of the Saccharomyces cerevisiae is strong and elastic and represents
almost 10-25% of the cell mass, necessary to support its many functions. It is indispensable
for the maintenance of cell shape and cell integrity. It also serves as an anchor for cell wall
proteins. Furthermore, the yeast cell wall needs to constantly remodel itself in function of
the cell cycle progression. Finally, it needs to withstand a rapid changing environment. To
meet all these functions, the yeast cell wall is constantly remodeled in a highly regulated
way by several pathways, of which the most important is the cell wall integrity pathway. The
cell wall consists of two layers: the inner layer exists mainly of β-1,3- and β-1,6-glucan
polymers and chitin, the outer layer is a network of glycoproteins. The structure of the yeast
cell wall is not the same in every place, thinner in some places and thicker in others: it is said
that the cell wall has a polarized structure (Cid et al., 1995).
1.3.2. Cell Wall Integrity pathway
The CWI pathway contains five cell-surface sensors with a similar structure of which
the main plasma membrane proteins are Wsc1 and Mid2. The localization of these
mechanosensors varies: concentrated at sites of polarized growth or uniformly distributed
over the plasma membrane. Alongside these extracellular sensors, the CWI pathway has
another, intracellular, source of information: signals coming from the cell cycle (Rodicio &
Heinisch, 2010). The cell surface sensors transmit the stress to the Rho1 GTPase through
three guanosine nucleotide exchange factors (GEFs): Rom1, Rom2 and Tus1. GEFs regulate
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the Rho1 GTPase together with GTPase activating proteins (GAPs) (Schmidt et al., 2002;
Yoshida et al., 2009). Also phosphoinositides have a role in this process: they help activating
Rho1 and recruit its effectors to the plasma membrane.
Rho1 controls several effectors which together coordinate the response of the cell,
mostly working on the cell wall. Rho1 activates the β-1,3-glucan synthase and the β-1,6-
glucan synthase, enzymes operational at the cell wall. Bni1 and Bnr1 regulate actin filament
assembly and Sec3 is a component of the exocyst, a protein complex that is involved in
vesicle trafficking. Skn7 is a transcription factor for a set of stress-induced genes. The most
important and best studied effector however is the protein kinase C1 (Pkc1). Ultimately,
there is an augmentation of β-glucan and chitin polymers in the cell wall, an increase of
several cell wall proteins and a relocalisation of different proteins which pertain to cell wall
construction machinery. Also the coherence between the different cell wall proteins is
changed (Levin, 2011).
The Pkc1 protein kinase is a homolog of the mammalian protein kinase C. This protein
kinase has several substrates but its main role is the regulation of the Mpk1/Slt2 MAPK
cascade, one of the five MAPK signaling pathways in yeast. It serves as an amplification of
Figure 1.1. Construction of the cell wall integrity pathway in Saccharomyces cerevisiae. The central Rho1 GTPase is triggered by cell surface sensors and cell cycle stimuli through GEFs and GAPs. In answer to this, Rho1 activates an array of effectors.
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the cell surface signal passed on to Rho1. Pkc1 phosphorylates Bck1, a MAPKKK which in turn
phosphorylates Mkk1 and Mkk2 (2 similar MAPKKs). These serve to phosphorylate the MAP
kinase Slt2, also called Mpk1 (Levin, 2011).
Slt2 associates with its targets and regulators. It mainly activates two transcription
factors by phosphorylation: Swi4/6 and Rlm1. Swi4/6 is a dimeric regulator of transcription
of a specific subset of stress-induced genes. Rlm1 has most of the genes, generally encoding
for cell wall proteins or proteins involved in cell wall synthesis, induced by CWI signaling
under its control. Moreover, the greatest part of the transcriptional activation response
induced by Congo Red or heat shock depends on this transcription factor (Jung & Levin,
1999). Rlm1 is related to the mammalian transcription factor MEF2 and shares a similar
DNA-binding specificity in vitro (Dodou & Treisman, 1997).
One of the induced genes under control of the Rlm1 transcription factor is MLP1,
whose expression product Mlp1 is a pseudokinase paralog of Slt2/Mpk1. It can activate
Swi4/6 for transcription in a non-catalytic manner, a way that does not involve protein
phosphorylation. It was seen that protein levels of Mlp1 raised a hundredfold in presence of
Figure 1.2. Activation of the Slt2 MAPK pathway, a branch of the CWI pathway in Saccharomyces cerevisiae. Pkc1, activated by Rho1, puts into motion the MAPK pathway. Mpk1 activates the transcription factors Rlm1 and Swi4/6 by phosphorylation and Rlm1 in turn induces gene expression of MLP1. Mlp1 regulates the Swi4/6 in a non-catalytic manner.
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cell wall stress. This characteristic can be used in a β-galactosidase assay (see materials and
methods 3.6.).
1.3.3. Activation of the CWI pathway
The CWI pathway is activated by substances or conditions that compromise the cell
wall stability. The response of the cell depends on the actual kind of stress the cell is
submitted to. Measuring the amount of Slt2/Mpk1 in the cell gives an indication for the
strength of activation of the CWI pathway.
One of the stressing agents is heat stress. The response to elevated temperature is
delayed until 20 min and is maximal after 30 min, which would mean that the cell doesn’t
detect the surrounding heat itself, but rather a secondary response to this heat. Slt2 is
activated from 37°C, but the heat stress is a lot more pronounced at 39°C. The conclusions
concerning the duration of Slt2 activation are not unanimous: some results showed that the
activation is continuous (Zarzov et al., 1996), while other experiments illustrated that the
level of activity returned to basic levels after two hours (Guo et al., 2009).
A sudden downturn in the extracellular osmolarity asks for an adaptation to a
stronger cell wall that prevents the cell swallowing too much water. The answer is very fast
but transient. This is in contrast to the reaction to hyper-osmotic shock, which induces a
delayed response. However, the main adaptation to this last type of stress emanates from
the Hog1 pathway (Rodríguez-Peña et al., 2010).
Apart from stress factors against which the cell needs to protect itself, also normal
physiological processes that require a change in cell wall structure can stimulate the CWI
pathway. One example are mating pheromones: when the cell sense these proteins, it gets
stimuli to stop the cell cycle and form a mating projection (site where the contact between
the two cells initiate) towards the other sensed cell. Therefore, the cell needs to remodel its
shape (morphogenesis) and the cell wall is triggered to reform its construction (Elion, 2000).
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Cell wall interfering compounds are chemical agents that provoke cell wall stress and
therefore induce the cell wall integrity pathway. Congo Red and Calco-Fluor White are chitin
antagonists. The echinocandins like Caspofungin are a class of substances that can be used
as antifungal drugs because they inhibit the β-1,3-glucan synthase of the target cell wall.
Zymolyase is an enzyme that causes cell wall lysis. Also caffeine can induce this pathway.
Drugs similar to rapamycine depolarize the actin cytoskeleton by making its conformation
more uniformly spread over the cell. Also actin antagonists and drugs that cause
endoplasmatic reticulum stress provoke the initiation of the CWI pathway. Some of this
drugs are used to activate the CWI pathway in a well controlled way for studying the
dynamics of this pathway (Levin, 2011).
1.4. ATP-DEPENDENT CHROMATIN REMODELING COMPLEXES
1.4.1. Chromatin structure and transcription
In physiological conditions, the deoxyribonucleic acid (DNA) is tightly packed in the
nucleus of the eukaryotic cell in a form called chromatin. That way all the DNA can be stored
efficiently. The basic repeating unit of this DNA packaging is a nucleosome. It consists of 147
basepairs of the double DNA strands curled around an octamer of histone proteins
(Kornberg & Lorch, 1999). This packaging affects all DNA related processes, including
transcription, replication, recombination and DNA-repair, due to the inaccessibility of the
DNA. For these processes, the DNA strands have to be available for the different
transcription regulators and enzymes, nucleases and restriction enzymes (enzymes that
recognize a specific nucleotide sequence and cut the double DNA strands in that place). In
normal conditions, the DNA is too closely packed in nucleosomes to physically interact with
these protein complexes. The chromatin structure needs therefore to be locally loosened,
opened (Cockerill, 2011).
The nucleosomes act as a gene repressor: only the genes which are provided with
positive regulatory mechanisms can be transcribed, the expression of all the other genes in
eukaryotes is prevented. First it was thought that the local histones need to be fully
detached from the associated promoter DNA (Elgin S. C., 1988). Later this theory was
adapted thanks to the discovery of immunoprecipitation, a technique that determines the
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particular DNA binding site of a specific nuclear protein. Now there was evidence that the
histones are not fully detached from the DNA double strands but still remain associated,
although in a modified form so that the DNA is accessible for nucleases and transcription
factors (Paranjape et al., 1994).
The process of nucleosome-DNA disruption is called chromatin remodeling, which is
indispensable for efficient initiation of the transcription at the promoters. These conclusions
were first found for Saccharomyces cerevisiae yeast cells. Later it was demonstrated that
these findings are relevant for all eukaryotic cells and consequently chromatin remodeling is
a fundamental aspect of eukaryotic regulation of transcription (Lee et al., 2004).
Chromatin remodeling is done by the cooperation of histone modifying enzymes and
adenine triphosphate-dependent chromatin remodeling complexes. First, the histone
modifying enzymes (histone acyltransferases and histone deacytelases) recognize specific
sequences at the nucleosome histone N-terminal tails and covalently mark them (covalent
post-translational modifications of histone proteins) (Strahl & Allis, 2000). These changes are
identified by the ATP-dependent chromatin remodeling complexes, whereafter the
complexes dissemble and reassemble the chromatin structure so that it is now accessible for
interaction with transcription factors and other proteins. They couple these reactions to the
hydrolysis of adenine triphosphate (ATP), to provide their energy (Saha et al, 2006).
1.4.2. Families and mechanism of action of chromatin remodeling complexes
ATP-dependent chromatin remodeling complexes are large multisubunit complexes
and most of them have additional functions above their remodeling activities. As a general
attribute, these all have a common remodeling mechanism and an ATPase subunit which
belongs to protein superfamily SWI2/SNF2. The complexes can be divided into four families,
depending on the identity of the ATPase subunit: SWI2/SNF2 group, imitation SWI (ISWI)
group, CHD family and the INO80 family. These individual families are conserved from yeast
to humans (Bao & Shen, 2007). An eukaryotic cell contains more than one type of complex.
The confirmation for interaction with DNA was found by isolating the SWI/SNF and RSC
11
complexes (Cairns et al., 1994) and by demonstrating that these complexes make the DNA
more susceptible for nucleases (DNase-hypersensitive sites) (Lorch et al., 1998).
To the SWI2/SNF2 (SWItch/Sucrose Non-Fermentable) group belong the following
complexes: the yeast SWI/SNF complex, the yeast RSC complex, the Drosophila Brahma
complex and the human BRM and BRG1 complexes, all of them highly conserved in terms of
subunit composition. To this group belong the following complexes:. The SWI/SNF complex
was the first remodeling complex discovered. It is strongly related to the RSC complex
(Vignali et al, 2011).
Up till now the mechanisms of action of chromatin remodeling are not fully clarified.
The interactions with the nucleosomes depend greatly upon the sort of complex. The
process can be divided into several steps. First, the modifying complexes need to recognize
and bind a specific gene sequence (Figure 1.3. A). This step occurs independent of ATP (Lorch
et al., 1998).
After binding, the remodeling
complex disrupts the nucleosome
structure in an ATP-dependent way (Figure
1.3. B). The SWI/SNF and RSC complexes
disrupt the nucleosome cores in a more
or less stable way, so that the disturbance
keeps existing after removal of ATP and
the remodeling complex (Côté et al.,
1998). Furthermore these complexes can
reverse the disruption to its original state.
Thus, the SWI/SNF and RSC complexes
function as catalysts of the inter-
conversion between two nucleosome
conformations (Schnitzler et al.,1998).
Figure 1.3. Mechanism of chromatin remodeling by ATP-dependent chromatin remodeling complexes (Vignali et al., 2000)
12
The remodeling of chromatin is the logic consequence of the nucleosome-DNA
disruption, it is a stable adjustment in the chromatin structure and in this state it is possible
for the transcription factors to associate with the promoter of the gene sequence that needs
to be transcribed. In this phase, DNA-hypersensitive sites on the targeted gene sequence
arise, due to the loss of protection by nucleosomes. The outcome of the disruption caused
by the ATP-dependent chromatin remodeling complexes is not always the same, but
depends on the promoter or enhancer (Vignali et al., 2000.)
The nucleosome rearrangement during the chromatin remodeling can occur by two
different mechanisms (Figure 1.3. C): the histones implicated in the relocation can be
transferred to another DNA segment (trans) or they can be transported along the same DNA
segment to another position (cis), but this requests the attendance of histone acceptors. For
SWI/SNF and RSC complexes, it is know that they are able to do both (Vignali et al., 2000.)
The ATP-dependent chromatin remodeling complexes possess promoter specificity in
vivo, but not in vitro when the complex is added in excess with respect to the nucleosomes.
This makes it easier to study their functions. Recently, there is a clarification what causes the
promoter specificity: it would reside in the promoter nucleosomes, rather than in the
remodeling complexes (Lorch et al., 2011).
1.4.3. The remodels the structure of chromatin complex
The ‘remodels the structure of chromatin’ (RSC) complex consists of 17 subunits
(Rsc1, Rsc2, Rsc3, Rsc4, Rsc6, Rsc8, Rsc9, Lob7, Arp7, Arp9, Sfh1, Sth1, Htl1, Rtt102, Rsc12,
Rsc30 and Rsc58), many of them homologous to subunits of the SWI2/SNF2 complex and
three completely identical (http://www.yeastgenome.org). The RSC complex was initially
identified by these identical subunits. The biochemical activities of both complexes based on
the shared catalytic ATPase subunit are similar, but the RSC complex has distinct non-
catalytic roles (Du et al., 1998).
The amounts of RSC complex found in yeast cells are far higher than of SWI/SNF. In
fact, the RSC complex is the most abundant complex of all the ATP-dependent chromatin
13
remodeling complexes. It contains subunits that are essential for cell viability, whereas none
of the proteins of the SWI2/SNF2 is vital for survival. This implicates that it is not possible to
investigate the functions of the RSC complex by using rscΔ mutants, because the cultures
would die. Instead, degron mutant strains are used (see materials and methods 3.1.1.).
The RSC complex plays a part in many different biological processes: the complex has
about 700 physiological targets. Some of its most important functions are listed here. It plays
a key role in the double strand break repair: when both strands of the double helix are
damaged, the repair machinery needs to be able to approach the damaged DNA, so the RSC
complex opens the structure (Chai et al., 2005). It may be necessary that the cellular RSC
complex levels are high to overcome this damage (Koyama et al., 2002). During segregation
of the chromosomes in the yeast cell core, this complex is needed at the kinetochore for
accurate chromosome transmission (Huang et al., 2004). The RSC complex has a function in
regulation of the cell cycle. More specific, this complex is necessary for the ploidy
maintenance of the yeast cell, which means guarding that the cell maintains the same
amount of copies of its chromosomes. Furthermore the RSC complex controls the G1/S-
phase transition (Campsteijn et al., 2007). It was seen in vitro that this chromatin remodeling
complex is necessary for efficient transcription elongation with RNA polymerase II (Zhang et
al., 2006).
1.5. MAPK PATHWAYS AND REMODELING COMPLEXES
ATP-dependent chromatin remodeling complexes have been shown to be recruited
by MAPK pathways to alter nucleosome positions at targeted genes. In this process different
remodeling complexes often collaborate with each other and with histone modifying
enzymes. The Hog1 kinase, when activated upon osmostress, induces the RSC complex, the
SWI/SNF complex and the SAGA complex (a histone modifying enzyme) to regulate
transcriptional responses (Proft & Struhl, 2002; Mas et al., 2009). Very recently, it was
discovered that the Rlm1 transcription factor, activated by the Slt2 MAPK of the CWI
pathway, mediates transcriptional initiation at stress-induced genes together with the
SWI/SNF remodeling complex (Sanz et al., 2012).
14
There is evidence that the RSC complex, next to the SWI/SNF complex, has a function
in the CWI pathway. A co-operation between the RSC complex, the SWI/SNF complex and
the ISWI complex in nucleosome displacement at promoters of heat shock induced genes
has been reported (Erkina et al., 2010). Moreover, essential rsc mutant strains appeared to
be hypersensitive to several cell wall stress agents: temperature, DNA damage and
microtubule depolymerization (Wilson et al., 2005). There is in any case a relation between
genes targeted by the RSC complex and genes induced in stress conditions. This indicates
that the induced changes in the RSC complex localization by stress correlate with the
transcription induction/repression of certain genes. (Damelin et al., 2002).
All these data suggest that the RSC complex might be recruited by the CWI pathway
to regulate transcriptional responses at stress-induced genes in presence of cell wall stress.
In this work, these presumptions are strengthened and the basis for refining further research
is given.
15
2. OBJECTIVES
Mitogen-activated protein kinase (MAPK) pathways, very highly conserved in all
eukaryotes, are signal transduction pathways activated by various physiological mediators as
well as a huge array of environmental stresses. The central elements in these pathways, the
MAPKs, make use of ATP-dependent chromatin remodeling complexes to locally disrupt the
nucleosome-DNA structure at targeted genes. This loosening of the normally very dense
chromatin structure makes the genes more accessible for transcriptional regulators and
enzymes so that effective transcription initiation is possible.
In Saccharomyces cerevisiae the CWI pathway is activated in response to situations that
compromise the cell integrity which leads to activation of a central Slt2 MAP kinase. Slt2
mainly activates the Rlm1 transcription factor which regulates transcription of the genes
induced in the adaptive response. The main objective is to evaluate the possible role of the
‘remodels the structure of chromatin’ (RSC) complex, one of the ATP-dependent chromatin
remodeling complexes, in the cell wall integrity (CWI) pathway, in the yeast Saccharomyces
cerevisiae, in the regulation of transcriptional responses to cell wall stress. This objective was
approached by comparing a conditional mutant rsc8deg strain, carrying a degron in the
essential Rsc8 subunits of the RSC complex, with the W303-1A wild type strain.
The principle of degron strains is that they behave as a wild type strain at room
temperature (24°C) but as a mutant strain at elevated temperature (37°C). So, a
characterization of the cell viability of the rsc degron strains under both set of conditions is
useful to obtain a general idea on the behavior of these cells. The growth rate of the
different mutants is compared to that of the wild type (WT) and the cell shape and cell
viability is observed by fluorescence microscopy. Moreover, to be able to use these rsc
degron strains it needs to be guaranteed that degradation of the essential Rsc subunits is
carried out at 37°C. This can be confirmed with western blotting.
The difference in gene expression, mediated by the CWI pathway, in response to stress
in the degron strains with a non functional RSC complex and the WT cannot be compared if
the RSC complex has a direct influence on the Slt2 MAPK pathway. Moreover, the first
16
option to stress the cells is with a cell wall interfering compound, namely Congo Red. As the
degradation of the RSC complex only starts at 37°C and this temperature is also a stress
factor, it needs to be researched if it is possible to use Congo Red in these circumstances.
More specific, to be able to dispense the stress on the cells with Congo Red, a time needs to
be found where there is degradation of the essential subunits of the RSC complex but where
there is no CWI activation. Therefore, the degree and duration of Slt2 activation in WT and
rsc8deg are investigated with western blotting. To know how resistant the WT is against
Congo Red stress, the sensibility of this strain against this drug is tested by a phenotypical
analysis
The ultimate objective is to examine if the RSC complex influences gene expression
induced by the cell wall integrity pathway in answer to stress. A first impression of the
involvement of this complex can be given by using a MLP1-lacZ reporter system performing a
β-galactosidase assay. This test gives the degree of mRNA levels of the MLP1 gene, a gene
under the influence of Rlm1 that is induced by stimulation of the Slt2 pathway under stress
conditions.
17
3. MATERIALS AND METHODS
3.1. YEAST STRAINS AND GROWTH CONDITIONS
3.1.1. Saccharomyces cerevisiae strains
With exception of the slt2Δ strain, all the strains used were descendants of the
W303-1A wild type of the yeast Saccharomyces cerevisiae (see table 3.1.). The degron in
each rscdeg mutant concerned an essential subunit of the RSC complex.
Most experiments were performed with W303-1A wild type and with one mutant
strain, namely rsc8deg. This strain was picked randomly from the collection of rscdeg mutants
because it was expected that all of these acted similarly.
Table 3.1. Saccharomyces cerevisiae yeast strains used and their source: wild type, rsctd
mutants and slt2Δ mutant
Strain Genotype Source
W303-1A MATa; ade2-1; ura3-1; leu2-3; 112; trp1-1; his3-11; 15 can1-
100
Euroscarf
YN 182 MATa; Pgal1-10::UBR1-HIS3; Pcup1-degron::rsc8-TRP1 Campsteijn et
al., 2007
YN 220 MATa; Pgal1-10::UBR1-HIS3; Pcup1-degron::sfh1-TRP1 Campsteijn et
al., 2007
YN 321 MATa; Pgal1-10::UBR1-HIS3; Pcup1-degron::rsc9-TRP1 Campsteijn et
al., 2007
SLT2Δ MAta; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; SLT2::KanMX4 Euroscarf
As mentioned in the introduction, some subunits of the RSC complex are essential for
cell viability, thus accordingly it is not possible to use conventional mutant strains for the
experiments. In this work, rsc degron strains are used as alternative.
Degron strains are a type of conditional mutants that are important tools in the
analysis of essential genes. This type of cells act as the corresponding wild type under
permissive conditions and as the mutant under non-permissive or restrictive conditions.
18
Under these circumstances, the targeted protein loses its function very fast by applying
conditional degradation, based on the N-end rule. The N-end rule states that the in-vivo half
life of a certain protein in a cell is related to the amino acid situated at its N-terminal end. A
protein degrades faster if it carries a more destabilizing residue at the N-end. In eukaryotes,
the ubiquitin protein plays an important part in the N-end rule pathway by covalently fusing
with the target protein. Hereby, ubiquitin puts in motion a pathway which results in the
degradation of the ubiquitin-protein conjugate. For Saccharomyces cerevisiae, the N-
terminal destabilizing residues are arginine, histidine and lysine (Varshavsky, 1997).
Degrons or degradation signals are parts of a protein that induce metabolic
instability. It consist at least of two essential compounds: the N-terminal residue and one or
more internal lysine residues. The destabilizing N-terminal residue of the protein is called N-
degron. This residue binds directly to the recognizing protein of the N-end pathway, Ubr1.
Ubr1 forms a complex with a second protein, Ubc2, which contains an ubiquitin. This
complex, attached to the N-terminal end of the target protein, mediates the formation of a
polyubiquitin-chain at the internal lysine. This polyubiquitin-chain is in turn recognized by
the 26S proteasome, which degrades the polyubiquitin-target protein conjugate
(Varshavsky, 1997).
This N-end rule pathway is used to produce degron mutants. The targeted gene is
fused with a mutant form of the dihydrofolate reductase (DHFR) gene. The mutant DHFR
protein contains a N-terminal destabilizing arginine residue. This gene in turn is fused to a
gene which encodes for ubiquitin. So when the gene sequence is transcribed, the following
fusion protein is made: (N-terminal) ubiquitin – DHFR – target protein (C-terminal). The
ubiquitin is cleaved by Ubp, but the fusion protein remains stable. This is because the
internal lysine in the mutant DHFR is protected by neighboring residues, thus it is not visible
for the Ubr1-Ubc2 complex and the fusion protein continues existing at room temperature
(24°C). Elevated temperature (37°C) induces the degradation. The three-dimensional
conformation of DHFR changes lightly, whereby the internal lysine residue now becomes
visible for the Ubr1-Ubc2 complex, which puts the degradation process in motion (Figure
3.1.). A mutant gene based on this principle, is represented as genedeg (Dohmen, 2006).
19
The conditional degradation of the target protein is only activated in a medium with
galactose because the UBR1 gene was placed behind a galactose-inducible PGAL1 promoter.
Only when galactose is present in the medium, the expression of UBR1 and consequently the
degradation of the target protein is possible (Dohmen, 2006).
The difference with conventional mutants is that with this type of mutants there is
still a low expression of functional proteins enough to guarantee the cell viability, although
the cell growth is reduced. So using this degron strains permits to investigate the
consequences of a non functional RSC subunit without compromising the cell viability.
3.1.2. Growth conditions
3.1.2.1. Materials
Culture media
Yeast extract Peptone Dextrose (YPD)
- Peptone 20 g/L (Cultimed, Madrid, Spain)
- Yeast extract 10 g/L (Pronadisa, Madrid, Spain)
- Dextrose 20 g/L (Merck, Darmstadt, Germany)
For Yeast extract Peptone Galactose (YPG), 20 g/L galactose (Duchefa Biochemie,
Haarlem, The Netherlands) instead of glucose was added .
Synthetic Complete medium without uracil (SC)
Figure 3.1. Degradation process of target protein: After cleaving of the ubiquitin, the Ubr1/Ubc2 complex binds the N-terminal arginine residue. At 24°C, the internal lysine is inaccessible as scaffold for the poly-ubiquitin chain and the target protein remains stable. At 37°C, the 3D conformation of the target protein changes and degradation by the 26S proteasome is possible
20
- Yeast nitrogen base 1,7 g/L (Pronadisa, Madrid, Spain)
- Dextrose 20 g/L (Merck, Darmstadt, Germany)
- Ammonium sulphate 5 g/L (Panreac, Madrid, Spain)
- Synthetic Complete (Kaiser) Drop-out –URA (Hunstanton, England)
For Synthetic Complete medium without uracil but with galactose instead of dextrose
(SC-GAL), 20 g/L galactose was added.
For solid media, 20 g/L of European bacterial agar was added. Liquid and solid media
were autoclaved at 121°C during 15 minutes to kill all microorganisms present. For plates
with Congo Red (Merck, Darmstadt, Germany), a Congo Red solution in water was prepared
beforehand so that the final concentration of Congo Red in YPD was 25 μg/mL, 50 μg/mL or
100 μg/mL.
Equipment
- Presoclave 30 (P Selecta, Barcelona, Spain)
- Incubator 24°C/30°C/37°C Unitron Infors AG (Bottmingen, Switzerland)
- Ovens 30°C - 37°C (Heraeus, Hanau, Germany)
- Spectrophotometer Ultrospec 3300 pro Amersham Bioscience (Amersham, UK)
- Pipettes Pipetman Gilson (Middleton, USA) and sterilized pipette tips (Sorenson
BioScience, Salt Lake City, US)
- Falcon tubes BD Biosciences FalconTM (California, USA)
- Eppendorf tubes (Hamburg, Germany)
3.1.2.2. Methods
For routine cultures, yeast cells were preinoculated in sterile conditions in the
adequate liquid medium. Following media were used: yeast peptone dextrose (YPD), yeast
peptone galactose (YPG), synthetic complete medium without uracil (SC) and synthetic
complete medium without uracil with galactose instead of glucose (SCG). Cultures were
grown overnight at 24°C and 200 rpm. Following day, the optical density (OD) was measured
at 600 nm in a tenfold dilution in distilled water. The optical density is a way to determine
the amount of cells in the culture. For these experiments, the cells needed to be in the
21
exponential phase (OD 0,4 – 1). Thus cultures were refreshed to 0,2 – 0,3 OD and grown at
24°C for 2h30min. Next, the cultures were divided into two parts. One part was allowed to
continue growing under the same conditions (24°C) while the other one was shifted to 37°C.
Cells were collected at different times by centrifugation.
Whenever needed, strains were plated on YPD plates, grown for two days at 30°C
and afterwards stored at 4°C. This way, the yeast did not grow and colonies could later be
used for preinoculating or for transferring to another plate.
3.2. WESTERN BLOTTING
Western blotting is an analytical technique that is used to detect specific proteins in a
sample. The samples contain denatured proteins, which are separated during a gel
electrophoresis. Subsequently, the proteins are transferred from the gel to a membrane. The
proteins can then be visualized with different techniques.
3.2.1. Growing and recovery of cells
3.2.1.1. Materials
- Eppendorf Centrifuge 5810R (Hamburg, Germany)
- Liquid N2 or solid carbon dioxide dry ice
3.2.1.2. Methods
One colony of each strain, W303 and rsc8deg, was preinoculated in 120 mL of YPG.
The cultures were grown as described under growth conditions. At every timepoint, the
optical density of the cells was measured, 20 to 25 mL of each culture was collected in
Falcon flasks and the same amount of ice was added to end cell activity. The samples were
centrifuged 3 minutes at 2700 rpm at 4°C and the supernatant was removed . The cell
samples were frozen by immersing the pellets in liquid N2 or in solid carbon dioxide and
stored at -80°C.
22
3.2.2. Cell lysis and preparation of yeast extract
3.2.2.1. Materials
Reagents
- Lysis buffer 1L:
NaF: 2,1 g (Sigma-Aldrich, Madrid, Spain)
Na2H2P2O7: 2,3 g (Sigma-Aldrich, Madrid, Spain)
β-glycerol: 10,8 g (Sigma-Aldrich, Madrid, Spain)
Sodium orthovanadate: 0,18 g (Sigma-Aldrich, Madrid, Spain)
Triton X-100: 10 mL (PlusOne Fisher Scientific, Madrid, Spain)
Sodium dodecyl sulphate (SDS) 10%: 10 mL (Duchefa, Haarlem, The Netherlands)
Glycerol 10%: 100 mL (Panreac, Madrid, Spain)
Tris pH7,5 1M: 50 mL (Duchefa, Haarlem, The Netherlands)
NP40 10: 10 mL (Fluka Biochemika, Buchs, Switzerland)
NaCl 5M: 30 mL (Panreac, Madrid, Spain)
EDTA 0,5M pH8: 10 mL (Panreac, Madrid, Spain)
Distilled water: 775 mL
- Phenylmethylsulphonyl fluoride (PMSF) 0,1M
PSMF: 171,2 mg (Sigma-Aldrich, Madrid, Spain)
Isopropanol: 10 mL (Panreac, Madrid, Spain)
- Protease inhibitors cocktail solution:
1 tablet protease inhibitor cocktail Complete Mini EDTA-free (Roche, Mannher,
Germany)
1,5 mL distilled water
- Loading buffer
Tris-HCl 40 mM pH6,8 (Duchefa, Haarlem, The Netherlands)
Bromofenol blue 2% (Panreac, Madrid, Spain)
10 % SDS (Duchefa, Haarlem, The Netherlands)
33% glycerol (Panreac, Madrid, Spain)
20% β-mercaptoethanol (Panreac, Madrid, Spain)
Equipment
23
- Glass beads 1mm, Sartorium Stedim Biotech (Goettingen, Germany)
- Heraeus Fresco 21 centrifuge Thermo electron corporation (Hanau, Germany)
- FastPrep 24 MPTM Biomedicals (Illkirch Cedex, France)
- Spectrophotometer Beckman Coulter DU 640 (Barcelona, Spain)
- AccublockTM Labnet International Digital Dry Bath (Edison, USA)
3.2.2.2. Methods
To avoid protein degradation the samples were kept on ice. A combination of cell
breaking mixture and mechanical stress made the cells burst open and release their
proteins. The cell breaking mix consisted of lysis buffer to solubilize the proteins,
phenylmethylsulfonyl fluoride (PMSF, a serine protease inhibitor) and a cocktail of protease
inhibitors to prevent that the proteins present in the sample are digested by the released
enzymes. To each sample, 200 – 300 μL of mixture was added, depending on the size of the
pellet. The cell pellet was resuspended in the cell breaking mix and 1mm Ø glass beads were
added to promote the breaking of cells.
The samples were subjected to a very high speed during two cycles of 35 seconds at
5,5 m/s in the FastPrep homogenizer with two minutes rest in ice in between. To separate
the protein extract from cell debris, the cells were centrifuged 15 minutes at 4°C at a speed
of 13000 rpm. Now the proteins were present in the supernatant, while the cell waste was
founded in the pellet. After using, the protein extract could be stored at -80°C for later use
when necessary.
To equilibrate the protein amount in each sample, protein concentrations were
measured in a 1/200 dilution in water by UV spectroscopy at 280 nm The concentration
needed to be in the 0,2 and 0,8 μg/μL range (lineair dynamic range). When not, a greater
dilution was made. The protein concentration in the electrophoresis samples was set at 50
μg/μL in a total volume of 50 μL. For each sample was calculated how much protein extract
was needed and distilled water was added to obtain a final volume of 40 μL. Finally, 10 μL of
loading buffer was added and samples were heated at 100°C during 3 min to denature the
24
proteins. When a concentration of 50 μg/μL could not be achieved because the protein
concentration was too low, more sample was injected in the gel.
3.2.3. Preparing and loading acrylamide gel
3.2.3.1. Materials
Reagents
- Acrylamide 30% (National diagnostics, Madrid, Spain)
- N,N-methylenebisacrylamide 2% (National diagnostics, Madrid, Spain)
- TrisCl pH8,8 1M % (Duchefa, Haarlem, The Netherlands)
- TrisCl pH6,8 1M % (Duchefa, Haarlem, The Netherlands)
- Distilled water
- SDS 10% (Duchefa, Haarlem, The Netherlands)
- Ammonium persulphate (PSA) 10% (Bio-Rad, Barcelona, Spain)
- Tetramethyleendiamide (TEMED) (Bio-Rad, Barcelona, Spain)
- Precision Plus ProteinTM All Blue Standards Bio-Rad (Barcelona, Spain)
- Electrophoresis buffer:
Tris: 3,0285 g/L (Duchefa, Haarlem, The Netherlands)
Glycine: 14,46 g/L (Duchefa, Haarlem, The Netherlands)
- SDS: 1 g/L (Duchefa, Haarlem, The Netherlands)
Equipment
- Gel elektroforese: Biorad Powerpac 300 (Barcelona, Spain)
3.2.3.2. Methods
The Sodium Dodecyl Sulphate PolyAcrylamide Gel Elektrophoresis (SDS-PAGE) was
applied, which utilizes a polyacrylamide gel. For this type of gel, the polymer is formed by
acrylamide and N,N-methylenebisacrylamide. The polymerization is only initiated by
addition of ammonium persulphate (PSA) and tetramethylethylenediamide (TEMED). The
separation of the proteins by molecular weight depends upon the size of the pores made in
the gel. This pore size can be changed by changing the concentration of acrylamide and
bisacrylamide in the gel. The pore size decreases with an increasing percentage of total
25
acrylamide (acrylamide + biacrylamide). Thus, the percentage of acrylamide in the gel
determines the rate of migration and the grade of separation of the proteins.
The electrophoresis gel consisted of two parts and was made between two glass
plates. The resolving gel had a total acrylamide concentration of 8% and was meant to
separate the proteins. The top 2 cm consisted of stacking gel which had a different
composition in order to make wells to load the samples into. For reagent quantities used for
both gels, see table 3.2. TEMED and PSA needed to be added at the last moment because
they initiated the polymerization. After the resolving gel was polymerized, the stacking gel
was added on top and a comb, which made 10 wells into the gel, was placed upon.
Table 3.2. Gel components and quantities used for resolving gel 8% and stacking gel 5% acrylamide
Gel components RESOLVING GEL (mL) STACKING GEL (mL)
Acrylamide 30% 1,6 0,325
Bisacrylamide 0,64 0,135
TrisCl pH8,8 1M 2,25 /
TrisCl pH6,8 1M / 0,25
Distilled water 1,41 1,25
SDS 10% 0,06 0,02
PSA 10% 0,03 0,02
TEMED 0,008 0,002
Once polymerized, the gel could be loaded. The comb was removed and the glass
plates were placed in a plastic container, which was filled with electrophoresis buffer. The
first well was filled with 2 μL of protein ladder which contained a mixture of proteins with
known molecular weights. Next wells were filled with 10 μL of each sample. The
electrophoresis was run under 170 – 185 Volts. The blue loading buffer marked how far the
electrophoresis was advanced. The electrophoresis was turned off when the blue line
reached the bottom of the glasses.
26
3.2.4. Transfer of proteins from gel to nitrocellulose membrane
3.2.4.1. Materials
Reagents
Transfer mix
- 80 % transfer buffer
Tris 5,8 g/L (Duchefa Biochemie, Haarlem, The Netherlands)
Glycine 2,5 g/L (Duchefa Biochemie, Haarlem, The Netherlands)
SDS 0,37 g/L (Duchefa, Haarlem, The Netherlands)
- 20% ethanol (Sigma-Aldrich, Madrid, Spain)
Equipment
- Nitrocellulose membrane GE Healthcare HybondTM ECL (Amersham, UK)
- Whatmann papers and sponges
- Transfer system: Biorad Powerpac 300 (Barcelona, Spain)
3.2.4.2. Methods
After electrophoresis was finished, proteins were localized on different places on the
gel depending on their molecular weight. To analyze the results, the proteins were
transferred to a nitrocellulose membrane. The gel was covered with a membrane and
Whatmann papers and sponges covered each side. This was placed in a plastic holder, which
in turn was put in container. An ice cube was added to avoid the temperature rising too high.
The container was filled with transfer buffer and the transfer was hold during one hour at
100 volts.
3.2.5. Immunodetection
3.2.5.1. Materials
Reagents
- Ponceau red (Panreac, Madrid, Spain)
- TTBS:
50 mL TBS 10x (BD Biosciences, California, USA)
27
0,5 mL Tween 20 (Duchefa Biochemie, Haarlem, The Netherlands)
950 mL distilled water
- Skimmed powder milk (Asturiana, Sierra de Granda, Spain)
- Visualizing mix for 1 membrane
Western blot detection reagents 1 & 2: 0,5 mL (GE Healthcare ECL Prime, Amersham,
UK)
H2O2: 2 μL (Panreac, Madrid, Spain)
- Anti-fosfo p42/44 MAPK (Thr202/Tyr204) (Cell Signaling, Ba rcelona, Spain)
- Anti-actin (ICN Biomedicals, Irvine, US)
- Anti-HA (Covance, Madrid, Spain)
- Anti-mousse, IgG-HRP (Amersham Pharmacia Biotech, Amersham, UK)
- Anti-rabbit IgG-HRP (Amersham Pharmacia Biotech, Amersham, UK)
- Developing solution: 750 mL distilled water, 175 mL developer (Kodak X-anat EX II,
Rochester, USA)
- Fixative reagent: 750 mL distilled water, 250 mL fix (Kodak X-anat EX II, Rochester,
USA)
Equipment
- Plastic bags
- Platform shaker Heidolph Rotamax 120 ( Schwabach, Germany)
- Film cassette (Kodak X-Omatic, Rochester, USA) and film paper (GE Healthcare,
Amersham, UK)
3.2.5.2. Methods
To check if transfer was carried out successfully and if proteins levels in each sample
were more or less equal, membranes could be submersed in ponceau red, which makes
proteins visible. After using, the membranes were washed with tween tris-buffered saline
(TTBS).
Specific proteins were visualized by incubating the nitrocellulose membrane with
antibodies. Primary antibodies bind these proteins and in turn these antibodies are
28
recognized by secondary antibodies which carry a reporter. When a substrate is added, the
reporter causes chemiluminescence of that substrate. To block all non-specific binding sites
on proteins, the membrane was submersed in a 5% solution of milk in TTBS for 30 min to 1h
on a moving plate. Hereafter, the membrane was incubated with the primary antibody
solution (5 μL of antibody in 4 mL TTBS and 1 mL milk) during 1h30. Next, the membranes
were washed with TTBS during 30 minutes, refreshing the liquid at least 3 times. Then the
secondary antibody was added (1,67 μL of antibody in 4 mL TTBS and 1 mL milk) by
incubation during 45 minutes. Afterwards, the membranes were washed once again.
The protein bands were visualized by developing a film in a dark room. This process is
called enhanced chemiluminescence (ECL). The membranes were put in a cassette made for
developing and a visualizing mix was sprinkled upon the membranes. The final steps of this
procedure were carried out in the dark room. Film paper was placed in the cassette on top
of the membranes, exposed for a certain time and afterwards submersed in a developing
solution until a signal became visible. The film was washed with water and subsequently
placed in a fixative reagens. After washing with water the film was now developed and
specific protein bands were visible.
3.3. PLATE SENSIBILITY
3.3.1. Materials
- YPD plates
- YPD + Congo Red plates 25 μg/mL, 50 μg/mL and 100 μg/mL
- Sterile 96 well plate (Greiner Bio-One, Frickenhousen, Germany)
3.3.2. Methods
One colony of the each strain was preinoculated in 20 mL YPD and grown overnight
at 24°C. Next day, the cultures were refreshed at 0,2 OD and grown for 2h30. Hereafter, OD
was measured once more and an eppendorf was prepared with 1 mL culture at an OD of 0,2.
1/5 dilutions were made in a sterile 96 well plate. The first well was filled with 200 μL of the
culture obtained from the eppendorf, the following four wells contained 160 μL YPD and 40
μL of culture from the previous well.
29
From each well, 4 μL was transferred to a plate, either YPD or YPD with a Congo Red
concentration 100 μg/mL, 50 μg/mL and 25 μg/mL. All plates were made in duplicate: one of
each was incubated at 30°C and the other at 37°C. Growth was scored after four days.
3.4. GROWTH EXPERIMENT DEGRON STRAINS
3.4.1. Materials
See materials 3.1.3.1.
3.4.2. Methods
One colony of each strain was preinoculated in 20 mL and grown overnight at 24°C.
Following day, OD’s were measured at 600 nm and cultures were refreshed at 0,1 OD in a
volume of 30 mL. After two hours, cultures were separated: 15 mL of the culture was grown
at 24°C and the other 15 mL at 37°C. Cultures were measured every two hours, starting at
this time.
3.5. CELL VIABILITY: FLUORESCENCE MICROSCOPY
3.5.1. Materials
Reagents
- Phosphate-buffered saline (PBS) (BD Biosciences, California, USA)
- Propidium iodide (Sigma Aldrich, Madrid, Spain)
Equipment
- Heraeus Fresco 21 centrifuge Thermo electron corporation
- Fluorescence microscope Nikon eclipse TE2000-U (Amstelveen, The Netherlands)
3.5.2. Methods
Using fluorescence microscopy on a cell sample stained with propidium iodide is
useful to observe the amount of dead cells in that sample. Propidium iodide is a fluorescent
marker that can be used in cell viability assays. It reacts with nucleic acids in the cell, which
30
gives the cell its fluorescence. In normal conditions the lipid membrane double layer is
impenetrable for this compound and the cell is invisible in the fluorescent mode of the
microscope. However, when the cell is dead and its cellular material accessible, it can bind
the DNA and RNA and the cell becomes visible.
Cells from WT and rsc8deg mutant were recovered at 1h, 2h and 3h, both at 24°C as at
37°C. 3mL culture was taken, centrifuged 2 min at 3500 rpm at 4°C and resuspended in 1 mL
TBS. Samples needed to be kept on ice at all times. 1 μL propidium iodide was added and
samples rested 3 min protected against light. Next, cells were washed three times with 1 mL
PBS, centrifugating at the same conditions and eliminating the supernatant. Finally the cells
were resuspended in 50 μL PBS. The samples were now ready to examine with the
fluorescence microscope. First, representative photos were taken from the normal modus of
the microscope. Subsequently this position was held but the modus was switched to
fluorescence and now only the dead cells were seen.
3.6. β-GALACTOSIDASE ASSAY
The β-galactosidase assay is used to measure the relative degree of transcription of a
certain gene. In this test, the transcriptional activity of the MLP1 gene was measured. This
gene is highly activated at cell wall stress conditions and can in this light be used as an
indicator of degree of activation of the CWI pathway under stress conditions. The yeast cells
were transformed with a plasmid that contains the promoter region of the gene MLP1 fused
to lacZ gene. When transcription factors trigger the transcription of this gene, the enzyme β-
galactosidase is formed. The amount of this enzyme is then a measure for the strength of
transcription of the targeted gene
3.6.1. Transformation of yeast cells
3.6.1.1. Materials
Reagents
- Polyethylene glycol (PEG) (Duchefa Biochemie, Haarlem, The Netherlands)
- Lithium acetate (Fluka Biochemika, Buchs, Switzerland)
- Plasmid MLP1-LacZ (Garcia et al., 2009)
31
Equipment
- AccublockTM Labnet International Digital Dry Bath (Edison, USA)
- Centrifuge Hermle Z 200 A (Wehingen, Germany)
- Petri plates SC
3.6.1.2. Methods
For transformation, cultures were grown overnight in YPD and OD was measured to
ensure that the yeasts were in the exponential phase (OD 0,4-1). 10 mL to 15 mL of culture
were taken, centrifuged 2 min at 2500 rpm, the cells were resuspended in the little medium
that remained and passed on to eppendorf tubes. These tubes were centrifuged during 2
min at 5000 rpm. The pellet was resuspended in 100 μL of a mixture of 80 μL PEG 50% and
20 μL lithium acetate 1M. After vortexing, 2 μL of the required plasmid, containing 0,6 μg
DNA was added and the tubes were vortexed again. Next, the cells needed to rest 15 min at
room temperature and 20 min at 42°C by occasionally agitating. Finally, the tubes were
centrifuged during 1 min at 5000 rpm and the pellet was resuspended in 100 μL sterile
water.
Most of the cells were now transformed with the plasmid. To pick and grow only
these cells that received the plasmid, the cultures were plated on a selective medium: a
minimal medium that only contains the most essential nutrients but without uracil. WT and
rsc8deg cells without plasmid cannot grow without taking up this nucleic acid from its
environment because they posses a defect URA3 gene. On the other hand the transformed
yeasts have a functional URA3 gene on their plasmids that encode for an enzyme that can
make uracil out of a precursor, allowing them to grow on this medium. Growth was scored
after two days at 30°C. Four colonies of each strain were then transferred to a new plate and
also grown for two days.
3.6.2. β-galactosidase activity
3.6.2.1. Materials
Reagents
32
- breaking buffer
Tris-Cl pH8 1M: 0,1L/L (Panreac, Madrid, Spain)
DTT 0,5M: 2 mL/L (Duchefa Biochemie, Haarlem, The Netherlands)
Glycerol 100%: 0,2L/L (Panreac, Madrid, Spain)
Water milli-Q: 0,7L/L (Millipore, Billerica, USA)
- PMSF 0,1M (Fluka Biochemika, Buchs, Switzerland)
- Bradford reagent (Bio-Rad, Barcelona, Spain)
- Bovine serum albumin (Sigma-Aldrich, Madrid, Spain)
- Z buffer
Na2HPO4.7H2O: 16,1 g/L (Panreac, Madrid, Spain)
NaH2PO4.2H2O: 6,24 g/L (Panreac, Madrid, Spain)
KCl: 0,75 g/L (Merck, Darmstadt, Germany)
MgSO4.7H20: 0,246 g/L (Panreac, Madrid, Spain)
Add water until final volume of 1L and adjust the pH to 7
β-mercaptoethanol: 2,7 μL/mL, added before experiment (Panreac, Madrid, Spain)
- Na2CO3 1M (Panreac, Madrid, Spain)
Equipment
- Centrifuge Z206A (Hermle LaborTechnik, Wehingen, Germany)
- Glass beads 1mm, Sartorium Stedim Biotech (Goettingen, Germany)
- 96 well microtiter plate (Greiner Bio-One, Frickenhousen, Germany)
- Microplate reader Model 680 (Bio-Rad, Hercules, CA)
3.6.2.2. Methods
The growing and recovery of cells for this experiment was very similar to western
blotting (see 3.2.1.), with the slight alteration that 10 to 15 mL cultures were obtained and
centrifuged 2 minutes at 2750 rpm. Pellets were kept at -80°C and could be used another
day. The supernatant was removed by centrifuging 2 min at 5000 rpm.
To measure the β-galactosidase activity, the cells needed to be broken first. This was
done by resuspending the cells in 150-250 μL breaking buffer and adding 5 μL PMSF 0,1M.
33
1mm Ø glass beads were added. The mechanical disruption was performed as described in
the western blotting assay (see 3.2.2.)
The Bradford protein assay is a spectroscopic analytical procedure used to measure
the protein concentration. In a 96 well plate all reactions were prepared in duplicate:
samples were diluted in water and 40 μL Bradford reagent was added (see Figure 3.3.) The
reference protein in the standard series consisted of bovine serum albumin (BSA) in a
concentration of 10 mg/mL. There was waited 20 minutes and the absorbancy at 595 nm
was measured.
The β-galactosidase reaction itself was carried out in a 96 well plate. Every well was
filled with 70 μL Z buffer with β-mercaptoethanol and 10 μL sample was added. The blank
contained 10 μL breaking buffer instead. Every sample was carried out in triplicate. To start
the reaction, 20 μL ONPG was added to each well. This is a substrate for the β-galactosidase
and when clieved by this enzyme, the reaction product ortho-nitrofenol is yellow. The
reaction was incubated at 30°C during 15 minutes. Then the reaction was stopped by adding
50 μL Na2CO3. The absorbance of ortho-nitrofenol was measured at 415 nm.
The protein concentration in the samples was calculated by linear regression of
standard series obtained from ½ dilutions of a 83,3 μg/mL bovine serum albumin solution.
The mean value of two measured OD’s at 595 nm of every sample was used, also for the
standard series. There is a linear relation between the measured optical densities at 595 nm
Figure 3.3. Preparing samples and standard series for measuring protein concentrations using Bradford reaction.
34
and the protein concentration. The actual protein concentration was 67 times higher than
the measured concentration in the 96 well plate. The final concentration was expressed in
mg protein/mL solution.
For every sample, the mean absorbance at 415 nm was taken for the three values of
the absorbance for the β-galactosidase product ortho-nitrofenol. These measurements
depended on the amount of protein and the reaction time. The activity of the β-
galactosidase enzyme was calculated from the formula below.
With: OD415 optical density of ortho-nitrofenol at 415 nm
Vtot total volume in each well, here 0,15 mL
0,0045 optical density of an 1 nmol/mL solution of o-nitrofenol
C protein concentration in mg/mL
V sample volume used in reaction in mL, here 0,01 mL
t reaction time (min)
So, the specific activity of β-galactosidase is expressed in the amount nanomoles
ortho-nitrofenol per mg protein and per min of exposure time. The higher this activity, the
more the MLP1 gene was transcribed.
35
4. RESULTS
4.1. CELL VIABILITY
4.1.1. Growth curves
This assay was done to find out how the mutant behaved under permissive and
restrictive conditions, namely at 24°C and 37°C, in terms of cell growth. Most experiments
were done with only two strains: the WT and rsc8deg. It is expected that the mutant grows
more or less equally fast as the WT at room temperature, while it grows slower at elevated
temperature because the degradation of the essential Rsc8 subunit of the RSC complex has
implications on cell viability. However, from optical densities measured when recovering
cells for other experiments it could be derived that the rsc8deg mutant strain was growing
slower than the WT under both set of conditions. On this basis it is suspected that the
mutant does not behave entirely as a WT under permissive conditions at 24°C.
For this reason it was decided to compare the growth of this mutant with two other
rscdeg mutants, namely rsc9deg and sfh1deg, to know if there are significant differences. This
experiment was carried out 2 times. A representative data set for each strain is sown in the
Figures 4.1. – 4.4.
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
1,1
1,2
1,3
0 2 4 6
op
tica
l de
nsi
ty 6
00
nm
time (h)
Growth of W303-1A in function of time
W303-1A - 24°C
W303-1A - 37°C
Figure 4.1. Growth of W303-1A in function of time in YPG at 24°C and 37°C
36
In Figure 4.1. it is seen that the WT grows much faster at 37°C than at 24°C. Higher
temperature stimulates the growth of the yeast cells. At 37°C there is already some cell wall
stress provoked by this elevated temperature that triggers the CWI pathway, but not yet
enough to compromise the cell growth of the yeast. The heat stress is much more significant
at 39°C. The YPG medium affects the growth of the WT strain. In general, Saccharomyces
cerevisiae yeast cells grow slower in YPG than in YPD and the activation of the CWI pathway
in this medium is reduced.
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
1,1
1,2
1,3
0 1 2 3 4 5 6
op
tica
l de
nsi
ty 6
00
nm
time (h)
Growth of rsc9deg in function of time
rsc9 - 24°C
rsc9 - 37°C
Figure 4.2. Growth of rsc8 degron mutant in function of time in YPG at 24°C and 37°C
Figure 4.2. Growth of rsc8 degron mutant in function of time in YPG at 24°C and 37°C
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
1,1
1,2
1,3
0 1 2 3 4 5 6
op
tica
l de
nsi
ty 6
00
nm
time (h)
Growth of rsc8deg in function of time
rsc8 - 24°C
rsc8 - 37°C
Figure 4.3. Growth of rsc9 degron mutant in function of time in YPG at 24°C and 37°C
37
As can be derived from Figures 4.2. – 4.4., the growth of the different degron
mutants was very similar. This indicates that, concerning cell growth, there is no significant
difference between the different mutant strains, so the rsc8deg strain is further utilized.
The growth of all mutants at 37°C is very slow, which was expected and indicates that
induction of the degron leads to loss of cell viability. However it is seen that also at 24°C the
growth of the mutant strains is compromised. At 24°C the Rsc essential subunit is not
degraded so the functionality of the RSC complex should be the same between WT and
degron mutants. There are probably other small differences between the degron mutant
strains and the WT that cause such a difference in growth.
4.1.2. Cell viability with fluorescence microscopy
A possible explanation for the fact that the mutant cells grew slower than expected
at 24°C, was that the mutant cells only lived for a few hours and started dying from then.
Therefore, the proportion living/dead cells at one, two and three hours after refreshing of
cultures of WT and rsc8deg was examined with fluorescence microscopy. As explained in
materials and methods, the fluorescent marker propidium iodide can only bind nucleic acids
when the cells are dead and thus only these cells can be made visible in the fluorescent
mode of the microscope. See Figures shown below (Figure 4.5-4.9) for the results.
Figure 4.4. Growth of sfh1 degron mutant in function of time in YPG at 24°C and 37°C
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
1,1
1,2
1,3
0 1 2 3 4 5 6
op
tica
l de
nsi
ty 6
00
nm
time (h)
Growth of sfh1deg in function of time
sfh1 - 24°C
sfh1 - 37°C
38
Figure 4.5. Representative light microscope images of W303-1A and rsc8deg
cells (left) and the corresponding fluorescence microscope images of the dead cells (right) after incubating the cells 1hour at 24°C.
Figure 4.6. Representative light microscope images of W303-1A and rsc8deg
cells (left) and the corresponding fluorescence microscope images of the dead cells (right) after incubating the cells 1hour at 37°C.
39
Figure 4.7. Representative light microscope images of W303-1A and rsc8deg
cells (left) and the corresponding fluorescence microscope images of the dead cells (right) after incubating the cells 2 hours at 24°C.
Figure 4.8. Representative light microscope images of W303-1A and rsc8deg
cells (left) and the corresponding fluorescence microscope images of the dead cells (right) after incubating the cells 2 hours at 37°C.
40
Figure 4.8. Representative light microscope images of W303-1A and rsc8deg
cells (left) and the corresponding fluorescence microscope images of the dead cells (right) after incubating the cells 3 hours at 24°C.
Figure 4.9. Representative light microscope images of W303-1A and rsc8deg
cells (left) and the corresponding fluorescence microscope images of the dead cells (right) after incubating the cells 3 hours at 37°C.
41
There was no significant difference in the proportion living/dead cells between WT
and rsc8deg. Also the quantity of fluorescent cells between strains at 24°C and 37°C was
similar. So, the hypothesis mentioned above could not be confirmed on basis of these
results. In general, the samples from the wild type contained more cells than the samples
from the mutant which could be expected on basis of the differences in cell growth.
Although the cells from the mutant strain did not die, they were somewhat bigger
and slightly more irregular in terms of cell shape than the wild type. This was noticed from
the strains both at 24°C as at 37°C, already from the first hour. This strengthens the
suspicions that the there are small genotypic differences between WT and degron strains
which cause the phenotypical differences observed.
4.2. PLATE SENSIBILITY ON YPD
If Congo Red stress will be used as stress agent to examine the involvement of the
RSC complex in the CWI pathway for transcription of genes, then it has to be tested first how
sensible the WT is to this drug.
This experiment was done with W303-1A, slt2Δ and rsc8deg, but as YPD was used
instead of YPG, the RSC complex in the last strain was functional. Slt2Δ was used as a control,
to check if the selected media at the different temperatures had their expected effect.
Also on YPD the rsc8degstrain grew slightly less than WT at 24°C and 37°C. The growth
of slt2Δ was reduced at 37°C. This can be explained as following: heat stress requires
activation of the CWI pathway and since the Slt2 mutant protein is not functional, the signal
transduction pathway is interrupted and the subsequent activation of the right proteins is
absent. However, the applied heat stress was not enough to actually kill the cells. The cell
viability would be a great deal less in presence of heat stress at 39°C.
The growth of all three strains in presence of Congo Red at concentrations of 100
μg/mL, 50 μg/mL and 25 μg/mL was almost non-existent. This suggests that the W303-1A
42
wild type is very sensible to stress from Congo Red. These results make it difficult to use
Congo Red as a stress factor on the yeast cells.
4.3. DEGRADATION OF THE RSC COMPLEX
Before starting to investigate the consequences of a non functional RSC complex in
mutant cells in the presence of cell wall stress, it was needed to make sure that these strains
functioned as expected. The rsc8deg strain had to act as the WT at room temperature,
meaning that the RSC complex is functional. At 37°C, it had to act as a mutant, meaning that
the targeted essential Rsc8 subunit is degraded and the RSC complex is non functional. This
experiment was carried out seven times in total by western blotting.
HA is an epitope present in the Rsc8 subunit of the rsc8deg strain. However, RSC8 in
the WT doesn’t contain this epitope. HA can be detected by the anti-HA antibody, produced
in mice. This antibody is in his turn recognized by the anti-mouse antibody, which has a
visualizing reporter. At room temperature, the Rsc8 subunit in the mutant strain is functional
and thus the anti-HA antibody can bind to its target and protein bands are visible at the
corresponding molecular weight. At 37°C however, the complex is degraded and the
antibody cannot find its epitope anymore, thus there may just very thin bands visible,
indicating that there is still a minimal amount of RSC complex to continue cell survival.
The intensity of the protein bands per se doesn’t say much, as it is possible that the
proteins levels in each sample were not similar. So anti-actin antibody was added to see how
much actin was present in each sample. Actin is a general protein that is abundant in each
Figure 4.10. Growth of dilutions of W303, Slt2 and Rsc8 strains on YPD plates at growth optimum 30°C (left) and 37°C (right)
43
celltype and acts as a representation of the amount of protein present in the sample. Thus
when interpreting the results, the amount of actin must always be taken into account.
As can be derived from Figure 4.11., the RSC complex is functional at 24°C, but starts
degrading soon after the cells feel the elevated temperature. After one hour at 37°C, levels
of functional RSC complex were already much lower and after two hours almost non-
existent. However, other experiments with other timepoints were performed and in one of
these experiments it was seen that after 30 min the degradation of the Rsc8 subunit was not
yet fully completed. As expected, there were never protein bands visible in the wild type.
It can be concluded that the RSC complex is non functional at 37°C after one hour of
incubation at 27°C and that rsc8deg can therefore be used as a mutant rsc strain.
4.4. ACTIVATION OF CWI PATHWAY
The cell wall integrity pathway is activated in response to various cell wall stresses,
including heat stress. By stressing the cells, the CWI pathway is induced and the Slt2 MAPK
pathway is stimulated, which in turn activates the Rlm1 transcription factor to start
transcription initiation. The central question in this work is if the RSC complex is also
recruited by the Slt2 MAP kinase pathway to open chromatin structure to make it accessible
for DNA binding proteins such as transcription factors and RNA polymerase II. To be able to
examine this, the RSC complex may not have an intrinsic influence on the Slt2 MAPK
pathway but the degree of activation needs to be equal to the WT.
Furthermore, as the goal was to research by stressing the cells with Congo Red, there
was sought after a timepoint where the RSC complex is non functional, but where there is no
influence of the elevated temperature anymore in terms of activation of the CWI pathway.
Figure 4.11. Western blotting: HA and actin protein bands at 24°C and 37°C of the rsc8deg
strain
44
As referred to in the introduction, previous experiments found opposite results concerning
the duration of the stimulation of the CWI pathway in response to heat stress. One
experiment had as conclusion that the activation of this pathway was temporary and that
the activation of the Slt2 MAPK returned back to its basic state after two hours (Guo et al.,
2009), while others found that this signaling pathway was activated during the whole period
of applied heat stress (Zarzov et al., 1996).
The activation of the CWI pathway in WT and rsc8deg was measured by detecting the
amounts of phosphorylated MAP kinase Slt2 with western blotting. An epitope in this kinase
was detected by the anti-fosfo p42/44 MAPK antibody, produced in rabbits, which in turn
was recognized by the anti-rabbit antibody. This experiment was repeated seven times on
times.
At every similar experiment carried out, there was always activation during the whole
period of heat stress at 37°C (see Figure 4.12.). Also after 30 min, activation of the CWI
pathway could be seen. This is in consensus with what was stated in the introduction,
namely that the activation of the CWI pathway is fully achieved after 30 min.
In rsc8deg strain, there was also a very high basal phosphorylation level of Slt2 at 24°C.
The difference between the intensity of the protein bands of Slt2 between 0 to 3 hours and 4
hours for 24°C can be explained by looking at the actin. There is more actin at 4 hours than
at previous timepoints, so also more Stl2-P is expected. The difference of Slt2 activation
between WT and rsc8deg points again the difference in basic state out.
4.5. MLP1 TRANSCRIPTIONAL ACTIVATION
Figure 4.12. Western blotting: Slt2-P and actin protein bands at 24°C and 37°C of wild type (left) and rsc8deg
mutant (right)
45
With the β-galactosidase test, a first impression can be obtained about the role of the
RSC complex in transcriptional response in the CWI pathway in response to stress. The
activity of the β-galactosidase is a measure for the degree of transcription of the MLP1 gene
and thus for the amount of mRNA of MLP1 in the cell. Upon heat stress, the CWI pathway is
activated and Slt2 phoshorylates the transcription factor Rlm1. One of the target genes of
Rlm1 is MLP1. Normally in stress conditions, MLP1 is expressed a hundredfold higher than
under vegetative growth conditions.
As seen in Figure 4.13., only the β-galactosidase specific activity after 2 hours was
plotted, the results from previous timepoints are not interesting because the transcription of
the stress-induced genes lag behind the Slt2 activation. It is not because there is visible
phosphorylation of Slt2 after 30 min and 1 h, that also the gene transcription at the targeted
genes is already initiated. The gene transcription response is always slower than the post-
translational modification of proteins. So before 2 h , the transcription initiation is not yet
sufficiently advanced to be able to draw conclusions out of the obtained results.
Figure 4.13 shows that the transcription of MLP1 in WT is more than doubled at 37°C
in comparison to 24°C. This is logic because there is no heat stress present at 24°C. At 37°C
however, heat stress activates the Slt2 MAPK pathway, which leads to phosphorylation of
Slt2. This in turn phosphorylates the Rlm1 transcription factor. This factor moves to the
targeted genes, such as MLP1, and activates transcription at the promoters.
Figure 4.13. β-galactosidase assay of W303-1A and rsc8deg
grown in SCG
0
10
20
30
40
50
60
70
2h
β-g
alac
tosi
das
e u
nit
s
(nm
ole
s/m
in.m
g p
rote
in)
time of recovery cells
β-galactosidase activity in function of time
W303-1A-24°C
W303-1A-37°C
rsc8-24°C
rsc8-37°C
46
In the Rsc8 mutant at 24°C, the MLP1 transcription is quite low because there is no
expected cell wall stress. The MLP1 transcription activity is much less at 37°C, which
indicates that the transcription in general of induced genes is less. The second time this
experiment was repeated, the results were analogous. These results suggest that the
transcriptional activation of MLP1 by heat stress depends on Rsc8. This experiment was
carried out a second time with similar results.
Because both strains grew even slower in SCG and few cells were obtained from
these experiments, the following design was to grow the cells overnight in SC with glucose
instead of galactose. Indeed, next day the measured optical density was higher and cultures
were refreshed with YPG. This means that the mutant cells only had two hours to adapt to
the galactose. In these two hours, enough copies of the Ubr1 enzyme needed to be
synthesized for degradation of the Rsc8 subunits at 37°C. Although more cells were
obtained, this turned out to be too little time for Rsc8 degradation because protein bands
for HA were still visible after 2 hours and, less, after 3 hours.
47
5. DISCUSSION
ATP-dependent chromatin remodeling complexes were already described to be
implicated in nucleosome reorganization at promoters of stress-responsive genes. Different
remodeling complexes need to interact with each other for correct expression of these
genes. Also for histone modifying enzymes it was shown that they are required for activation
of transcription in stress responses. This is illustrated with the Hog1 pathway: the Hog1
MAPK arranges the recruitment of SWI/SNF as remodeling complex and of SAGA complex
and Rpd3 as histone modifying enzymes (de Nadal et al., 2011). For the CWI pathway, only
the SWI/SNF complex has been discovered as a regulator of transcription (Sanz et al., 2012),
but possibly the RSC complex is also recruited in this pathway.
Previously, a screening test was done where the sensibility of all mutants carrying non
essential genes in Saccharomyces cerevisiae was tested to Caspofungin, Zymolyase and
Congo Red. Multiple non essential rsc mutants appeared to be hypersensitive to these drugs.
In another assay mutants were transformed with a plasmid carrying a reporter system which
consisted of the MLP1 promoter and a gene that encoded for resistance against a antibiotic.
The mutants were grown in a medium with this antibiotic and one of the three cell wall
interfering compounds. Only mutants who could induce proper transcriptional responses
were able to survive. That way it was discovered that two of the non essential RSC subunits
were necessary for transcription activities at these stresses. These two subunits were then
tested if the mutants had a higher level of Slt2 activation but this was not the case, meaning
that these subunits don’t interfere with Slt2 activation. (Garcia & Botet, manuscript in
preparation).
A first problem in the use of the rsc degron strains is their slow growth. This implicates
that the research is much more time-consuming because before starting every experiment it
takes more time for the cells to reach the good optical density. Moreover, less cells are
recovered, which makes their signals lower and sometimes more difficult to interpret. Even
though the rsc degron strains should act as a WT at 24°C, it was noticed that this was not
entirely the case. Differences were observed concerning cell growth, cell morphology and
basic state of the Slt2 activation. A possible explanation is that the cell wall of the mutant
48
strain is more affected by stress, which has as consequences that there is a constantly higher
basic activation level of the Slt2 MAPK pathway, a slower cell growth and more irregular cell
shape. This is however in contrast with the lower levels of MLP1 gene measured in the β-
galactosidase assay at 24°C, because that means a lower Slt2 phosphorylation.
It was the original intention to find a certain time after refreshing the cultures where, at
37°C, there is no functional RSC complex and no visible activation of Slt2. At that time, Congo
Red could be added as a cell wall stress agent. This way, the consequences of a non
functional RSC complex in the presence of this cell wall stress agent could be investigated
without the interference of heat stress. It is preferred to examine the relation between the
RSC complex and the CWI pathway by stressing the cells with a specific chemical stress
agent, such as Congo Red or Zymolyase. The stress caused by these cell wall interfering
compounds is more accurate and more easy to manage than heat stress. However it was
found that Slt2 is activated continuously at high temperature. Moreover, the W303-1A wild
type appeared to be very sensitive to Congo Red as there was negligible growth on YPD
plates with Congo Red concentration of 100 μg/mL, 50 μg/mL and 25 μg/mL. These data
suggest that it is maybe better to work in the future with heat stress as actual stress agent.
The intensity of Slt2-P protein bands under heat stress at 37°C is more or less equal in
WT and rsc8td, indicating that the activation of the CWI pathway is similar between strains
with functional and non functional RSC subunits. Thus the RSC complex does not influence
the functionality of the MAPK cascade itself. This is important towards further research,
because it is not possible to compare the consequences of cell wall stress when the strength
of stimulation of the CWI pathway is unequal in both strains. To quantify the western blot
results, western blot densitometry could be done. In this technique, the density of all
proteins bands is measured and Slt2 activation is expressed mathematically, which facilitates
the comparison of the results.
The results from the β-galactosidase assay showed that the MLP1 gene expression is
lower in Rsc8 at 37°C in comparison with 24°C. This could suggest that the RSC complex is
involved in the transcription initiation of MLP1. Hypothetical, if the RSC complex is recruited
by Slt2 in the CWI pathway, than this ATP-dependent chromatin remodeling complex locally
49
opens the chromatin structure at the promoters of stress-induced genes so that
transcription factors and RNA polymerase II can bind to these gene sequences and start
initiation of transcription. A non functional RSC complex cannot be recruited to the
promoters of these stress-induced genes to remodel the nucleosome-DNA interaction of the
chromatin so transcription cannot be initiated efficiently.
In previous research on the implication of the related SWI/SNF complex in the CWI
pathway, it was found that the transcription factor Rlm1 binds with this complex to direct
which genes need to be remodeled (Sanz et al., 2012). In this light it could be interesting to
investigate if the Rlm1 interaction is similar with the RSC complex and if chromatin
remodeling by the RSC complex is required for cell wall stress transcriptional response. The
interaction between Rlm1 and RSC complex can be studied by in vitro and in vivo co-
precipitation assays. The nucleosome reassembly by the RSC complex could be investigated
by comparing histone occupancy at the MLP1 gene in wild type and different rscdeg strains. If
indeed this complex is implicated in nucleosome rearrangement, than the histone occupancy
should be lower on certain regions of this gene.
To confirm the influence of rsc mutants on transcriptional responses in conditions of cell
wall stress and to elucidate the mechanisms of how the Slt2 MAP kinase recruits the RSC
complex to the stress-induced genes, more experiments need to be done. The mRNA of
stress-induced genes could be purified and the levels be compared between WT and degron
strains. If the RSC complex is involved in transcription initiation, the mRNA levels of these
genes should be lower in the rscdeg strains. By carrying out a microarray analysis where all
RNA is analyzed, all stress induced and repressed genes in presence of cell wall stress can be
compared between WT and rscdeg. This allows to investigate which genes specifically
experience a reduced expression when no functional RSC complex is present.
Furthermore chromatin immunoprecipitation assays could be useful to detect the
occupation sites of subunits of the RSC complex at stress-induced genes. In these
experiments antibodies are used to target specifically the Rsc subunit so that afterwards the
chromatin-protein complex can be isolated and quantified with the aid of qRT-PCR.
50
6. CONCLUSIONS
The rsc8deg strain used in this work behaved more or less as the W303-1A wild type
strain at 24°C. The Rsc8 essential subunit of the RSC complex was functional under these
conditions, but the growth of these cells was slower than expected. Confirmation for slight
differences in genotype with the WT was found in the results of microscopy, where the
rsc8deg cells had a bigger and more irregular morphology, and in the results from western
blotting, where the basal Slt2 activation was higher. However, the degradation of the Rsc8
subunit started at 37°C and functional Rsc8 levels were clearly diminished after 1 h and
stayed very low during further time. Thus at 37°C the rsc8deg strain acts as a conventional
mutant strain and this strain can be used for further research.
The strength of Slt2 activation at 37°C was similar with WT and rsc8deg, meaning that the
Rsc8 subunit does not interfere with the activation of the CWI pathway. Thus, the potential
difference in gene expression between both strains is completely due to the non functional
Rsc8 subunit and not to an unequal activation of the Rlm1 transcription factor caused by this
subunit.
As the Slt2 activation at 37°C was of equal strength after different hours, there was no
timepoint where Congo Red could be added as a stress agent without influence of the heat
stress. Moreover, WT cells seemed very sensible to Congo Red, which impedes working with
this cell wall perturbing compound. It is preferred to use heat stress.
The MLP1 degree of transcription was lower at 37°C in rsc8deg cells than at 24°C.
These results suggest that the transcriptional activation of the gene is dependent on the
Rsc8 subunit as chromatin remodeler: it locally disrupts the nucleosome structure at MLP1
so that Rlm1 can access the MLP1 promoter to initiate transcription.
To confirm these assumptions, further tests can be done, such as gene expression
analysis, chromatin immunoprecipitation, histone occupancy and co-precipitation assays
between Rsc subunits and the Rlm1 transcription factor.
51
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