234 research outputs found
Enhancing the resolution of cohesin dynamics in meiosis
Meiosis is a specialized form of cell division in which one diploid mother cell is converted into four haploid daughter cells. Cohesin is a multi-protein complex, providing cohesion to replicated chromosomes. During meiosis, cohesin is removed from chromosomes in two steps. First, it is proteolytically cleaved from chromosome arms in anaphase I, whereas cohesin in the vicinity of the centromere is protected from cleavage. This pericentromeric cohesin is then removed in anaphase II. This stepwise loss of cohesin is part of the current model of meiotic chromosome segregation. Evidence for this kind of cohesin dynamics came originally from immunofluorescence experiments with very limited spatial resolution.
A new workflow was established by combining a novel synchronization system for budding yeast meiosis with a calibrated and optimized ChIP-Seq protocol. This workflow allows resolving the cohesin dynamics in the course of the two meiotic divisions with unprecedented temporal and spatial resolution.
With this new experimental system, we confirmed the existence of two cohesin fractions on chromosomes, a protected and an unprotected fraction. Contrary to the current model, we detected both fractions in the region around the centromere. This indicates that the distinction between arm cohesin and pericentromeric cohesin is not identical to the classification into unprotected cohesin and protected cohesin. These results suggest that the mechanism of protection is not only determined by the localization of the cohesin protein complex. Additionally, we discovered significant differences in the cohesin protection activity among individual chromosomes.
The protein Sgo1 is required for the centromeric protection of cohesin. Sgo1 was analyzed directly with the new workflow, and we generated novel insights into the loading of the protection machinery onto chromosomes and the establishment of centromeric protection in meiosis. The protection machinery is loaded onto chromosomes in a cohesin-dependent mechanism, and a novel model of a dynamic three-step loading mechanism of the protection machinery is presented. This model explains how the cells are able to provide a robust and reliable protection to cohesin located in very diverse patterns on different chromosomes. Moreover, the model suggests a new function of the protein Sgo1 in centromeric protection.
A last result is that the polo-like kinase of budding yeast, Cdc5, is involved in regulating the levels of the protection machinery, which are loaded onto chromosomes
Probing Control of Glucose Feeding in Cultivation of <em>Saccharomyces cerevisiae</em>
In order to maximize the biomass yield in fed-batch cultivations of Saccharomyces cerevisiae, a pulse feeding strategy originally developed by Mats Åkesson at the department of automatic con-trol (Åkesson, 1999), was implemented. The controller was intended to keep the specific glucose uptake rate, qs, below the critical specific glucose uptake rate qs crit , to avoid over-flow metabolism. Simulations, made to see if the method worked, and to find optimal working conditions, were done before the experiments. A robust PID controller was developed in order to regulate the dis-solved oxygen tension at 30%, by changing the stirrer speed. Two different commercial strains of yeast (from Jästbolaget AB, Sweden) were used: the so-called blue yeast, for ordinary doughs, and the so-called red yeast, for sweet doughs. Glucose was the carbon source in the cultivations. The specific glucose uptake rate, qs, was controlled by the feed rate. If an up pulse in the feed rate resulted in a decrease of the DOT, below a certain point, the feed rate was increased in proportion to .DOT. If qs exceeded qs crit no decrease in DOT would be seen, and therefore the feed rate was decreased. When the stirrer speed was close to its maximum value, a safety net in the regulator prevented further feed rate increase. With the implemented control strategy growth was fully respirative. This was shown by high bio-mass yield values, low glucose and ethanol concentrations during the fed-batch cultivations, and also by a RQ, close to 1.08, throughout the fed-batch experiments. In some of the fed batch ex-periments, indications of synchronization of the culture could be seen. The µ values were slightly lower than expected. Therefore one may suspect that qs never reached qs crit , before the feed rate was decreased due to saturation in the oxygen transfer. In order to im-prove the method, parameters like pulse length and initial cell mass concentration will have to be adjusted
Novel Formulation and Application of Model Predictive Control.
Model predictive control (MPC) has been extensively studied in academia and widely accepted in industry. This research has focused on the novel formulation of model predictive controllers for systems that can be decomposed according to their nonlinearity properties and several novel MPC applications including bioreactors modeled by population balance equations (PBE), gas pipeline networks, and cryogenic distillation columns. Two applications from air separation industries are studied. A representative gas pipeline network is modeled based on first principles. The full-order model is ill-conditioned, and reduced-order models are constructed using time-scale decomposition arguments. A linear model predictive control (LMPC) strategy is then developed based on the reduced-order model. The second application is a cryogenic distillation column. A low-order dynamic model based on nonlinear wave theory is developed by tracking the movement of the wave front. The low-order model is compared to a first-principles model developed with the commercial simulator HYSYS.Plant. On-line model adaptation is proposed to overcome the most restrictive modeling assumption. Extensions for multiple column modeling and nonlinear model predictive control (NMPC) also are discussed. The third application is a continuous yeast bioreactor. The autonomous oscillations phenomenon is modeled by coupling PBE model of the cell mass distribution to the rate limiting substrate mass balance. A controller design model is obtained by linearizing and temporally discretizing the ODES derived from spatial discretization of the PBE model. The MPC controller regulate the discretized cell number distribution by manipulating the dilution rate and the feed substrate concentration. A novel plant-wide control strategy is developed based on integration of LMPC and NMPC. It is motivated by the fact that most plants that can be decomposed into approximately linear subsystems and highly nonlinear subsystems. LMPCs and NMPCs are applied to the respective subsystems. A sequential solution algorithm is developed to minimize the amount of unknown information in the MPC design. Three coordination approaches are developed to reduce the amount of information unavailable due to the sequential MPC solution of the coupled subsystems and applied to a reaction/separation process. Furthermore, a multi-rate approach is developed to exploit time-scale differences in the subsystems
Mitotic checkpoint inactivation at anaphase onset
The mitotic checkpoint prevents chromosome segregation until all chromosomes have reached bi-polar orientation and come under tension on the mitotic spindle. Once this is achieved, the protease separase is activated to cleave the chromosomal cohesin complex and trigger
anaphase. Cohesin cleavage releases tension between sister chromatids,
however the mitotic checkpoint fails to respond to this apparent tension
defect. The aim of this study was to understand why the mitotic
checkpoint remains silent when sisters lose tension due to cohesin
cleavage in anaphase.
We showed in budding yeast that loss of sister chromatid cohesion
at anaphase onset could re-activate the mitotic checkpoint. This is
normally prevented by separase-dependent activation of the Cdc14
phosphatase. Cdc14 in turn downregulates the mitotic checkpoint by
dephosphorylation of Sli15/INCENP, part of the conserved Aurora B
kinase complex and proposed tension sensor at the kinetochores.
Consequent relocation of Sli15/INCENP from centromeres to the central
spindle during anaphase is a distinctive feature of the Aurora B kinase
complex. Our results imply the existence of a conserved mechanism of
mitotic checkpoint inactivation in anaphase. Dephosphorylation of
Sli15/INCENP and its spatial separation from kinetochores prevent the
checkpoint from re-engaging when tension between sister chromatids is
lost in anaphase
Investigating the kinetochore complex in Schizosaccharomyces pombe using advanced fluorescence microscopy techniques
Major insights into various biological processes and structures could be achieved using fluorescence
microscopy, which is a non-invasive, live- and fixed cell compatible, high contrast imaging technique.
Here, the molecule of interest can be specifically labeled with a fluorescent marker using a multitude of
different labeling techniques, best suited for the individual biological research question. The location
of the molecule of interest can be deduced from the emitted fluorescent signal of the marker due to
their immediate proximity.
However, structures smaller than the diffraction limit of light of about 200 nm can not be resolved
by conventional fluorescence microscopy. Other techniques, such as e.g. electron microscopy
(EM) or X-ray crystallography allow for higher resolutions, but lack the target specific read-out
or are not compatible with in vivo studies. Nevertheless, by utilizing advanced optical components
and illumination patterns and designing fluorophores with tightly controllable photophysics,
the diffraction limit of light could be circumvented leading to improved resolutions. From these
super-resolution techniques, only single-molecule localization microscopy (SMLM) allows for a
quantitative analysis of the target molecule due to the spatiotemporal detection of the fluorescent marker.
The segregation of sister-chromatids to the corresponding daughter cells is a vital and irreversible
process, which needs to be tightly regulated. Here, a multi-protein complex called the kinetochore
(KT), which serves as a force-sensing linker between the centromere in chromosomes and kinetochore
microtubules (kMTs) originating from the spindle pole body (SPB), plays a pivotal role as errors
in this process lead to aneuploidy or cell death. Thus, understanding the architecture and regulation
of this complex is essential. However, even though certain subcomplexes of the KT could be
resolved by EM or X-ray crystallography in vitro, the full KT nanostructure was not resolved in vivo yet.
Hence in chapter 2, the in vivo nanoscale structure of the fission yeast KT complex was investigated
using SMLM. The fission yeast Schizosaccharomyces pombe was used as the model organism of
choice, due to its small regional centromeres. It acts as an intermediate between the point centromere
in the budding yeast Saccharomyces cerevisiae, on which only one KT assembles, and the larger
regional centromeres in humans. To investigate the KT in fission yeast, a structure smaller than the
diffraction limit of light, different SMLM imaging and labeling strategies in microbes were developed
or applied fitting this research question. However, for the creation of the KT map at least two different
super-resolved targets are required: one reference protein at the centromere and one protein of interest
(POI) a time in the KT complex. As no combination of commonly used photoswitchable organic dyes
for SMLM proved to be applicable in fission yeast, the focus was shifted towards photoactivatable
and - convertable fluorescent proteins (FPs) as alternative fluorescent markers. Knowing this, the KT
structure was investigated using a multi-color SMLM approach based on FPs utilizing an orthogonal
sequential illumination pattern and a KT protein database was generated. Developing novel image
analysis tools and controls allowed for the extraction of intra- KT distances and POI copy numbers.
Based on these parameters, first conclusions on the structure, preferred KT assembly pathways and
stoichiometries were drawn and a model of the fission yeast KT was proposed.
Finally, to investigate the KT structure in even greater detail, a new imaging technique combining
expansion microscopy (ExM) and SMLM, termed single-molecule expansion microscopy in fission
yeast (SExY) was developed in chapter 3, which increases the imaging resolution of SMLM by the
corresponding expansion factor (EF) of the sample. For this, the fixed sample was first embedded
in a hydrogel and then expanded upon incubation in aqueous media. To achieve an even expansion,
the proteins were covalently linked to the gel mesh and obstacles like protein connections, cell
walls or membranes were dissolved in homogenization steps prior to expansion. Then, the sample
was imaged using the SMLM based imaging technique photoactivated localization microscopy
(PALM), which lead to single-digit nanometer resolutions. Since KT proteins are low abundant,
we optimized for an increase in protein retention yield, which we could improve by half compared
to the initial protocol. We also optimized for an isotropic expansion of the sample, which we
controlled by determining the EFs of different cell organelles and the distribution of cytosolic FPs
compared to non-expanded cells. With the final SExY protocol at hand we were than able to visualize
KT proteins as well as other nuclear targets in vivo at a single digit nanometer range for the first time
Rad51 에 의한 DNA 이중가닥 절단 수선과 유전자 재조합 기전 연구
학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2015. 2. 황덕수.Embryonic stem cells (ESCs) are pluripotent and self-renewing cells that originate from inner cell mass of blastocyst. ESCs should have ability to divide and grow indefinitely while sustaining their pluripotency. To preserve their self-renewal ability and faithful DNA replication responsible for genomic stability, ESCs have developed powerful machineries to preserve genomic integrity distinguished from differentiated cells, but they are not fully elucidated yet. Therefore, the suppression of mutations against DNA damage in ESCs is essential for the maintenance of genomic integrity as well as cell proliferation and inheritance of genetic trait.
Homologous recombination (HR) is one of the key processes to maintain genomic integrity against DNA replication stress. Rad51 is an important protein of HR in all eukaryotes and its functions are homology search and strand invasion. Here, I investigated that Rad51 preserves G2/M transition to regulate cell cycle progression and the level of Rad51 is a reflective of high percentage of S phase in ESCs.
ESCs exhibit prominent populations of S-phase cells compared with differentiated somatic cells. Different from many somatic cells that express Rad51 protein in cell cycle-dependent manner, Rad51 in ESCs is constitutively expressed independent of each cell cycle phases and its level is extremely higher than somatic cells. Unlike its continuously elevated protein level, the formation of Rad51 foci increased as cells enter S-phase, and decreased as cells prepare their division. The foci formation tendency is consistent with γH2AX, the marker of DNA double-strand breaks (DSBs). Also, Rad51 is entirely dissociated from chromosome during mitosis. Rad51 knockdown induces the phosphorylation of Chk1, the sign for DNA damage checkpoint activation. The FACS analysis showed that the populations of G2/M phases are accumulated and BrdU incorporation is reduced in Rad51-knockdown cells. In conclusion, HR activity of Rad51 is essential to repair spontaneously occurred DSBs, which are caused by rapid and frequent DNA replication events.
Meiosis includes a complex progression of chromosomal events which results in the physical connection of homologous chromosomes. During meiosis, cohesin complexes physically hold sister chromatids together, they are required for DSB repair and faithful chromosome segregation. Rec8 is a key component of the meiotic cohesin complex, which regulates sister chromatid cohesion and recombination between homologous chromosomes. DNA physical analysis of recombination in yeast mutant strains that Rec8 phosphorylation sites were mutated to alanines reveals a general principle: Rec8 phosphorylation is required for the timely and efficient progression of recombination at DSB-to-double Holliday Junction (dHJ) transition in the stage of homologous partner choice with the first DSB end releasing. I demonstrate that Rec8 phosphorylation does not affect for the homologous partner choice but is required for latter stages of crossover (CO)-designated meiotic recombination. Further, elimination of Mek1 kinase, which impedes checkpoint activation, relieves the meiotic progress delay caused by Rec8 deletion or Rec8 phosphorylation-defective alleles. The obtained results point to a general logic for the relationship between Rec8 and Mek1 kinase that involve in recombination progression and regulatory surveillance during meiosis.ABSTRACT i
TABLE OF CONTENTS iv
LIST OF FIGURES vii
LIST OF TABLES x
LIST OF ABBREVIATIONS xi
CHAPTER 1. Rad51 preserves G2/M transition in mouse embryonic stem cells to regulate cell cycle progression 1
Abstract 2
Introduction 4
Materials and Methods 9
Cell culture 9
Cell cycle synchronization and FACS analysis 10
BrdU FACS analysis 11
RNA interference 11
Immunofluorescence 12
Extract preparation and immunoblotting analysis 13
Results 15
Rad51 protein is expressed constantly throughout the cell cycle in mESCs 15
Rad51 specifically localizes on S-phase chromosomes in mESCs 22
Rad51 localizes inside the replication factory in mESCs 35
Rad51 depletion causes a proliferation defect in mESCs 40
Proliferation delay in Rad51-depleted mESCs is due to the activation of the DNA damage checkpoint 45
Rad51 depletion does not disturb DNA replication in mESCs 51
Discussion 57
CHAPTER 2. Rec8 phosphorylation mediates crossover-designated recombination and regulatory surveillance in meiosis 64
Abstract 65
Introduction 66
Materials and Methods 72
Yeast strains 72
Meiotic time courses 72
Meiotic progression analysis 73
DNA physical analysis 74
Immunoblotting 75
Chromosome spreading and immunofluorescence 76
Spore viability test 77
Results 78
Characterization of Rec8 phosphorylation 78
DNA physical analysis system used for studying meiotic recombination 78
Rec8 phosphorylation is dispensable for timely and efficient DSB formation 87
Rec8 phosphorylation is critical for timely progression of the DSB into SEI 93
Rec8 phosphorylation mediates efficient progression of CO-designated DSBs 98
Rec8 phosphorylation functions in ensuring CO-fated recombinational interaction coordinating with Zip1 99
Mek1 activation and Rec8 phosphorylation are dispensable for recombinational progression 102
Rec8 phosphorylation is responsible for recombinational progression in the absence of sister chromatids 120
Discussion 125
REFERENCES 134
ABSTRACT IN KOREAN 147
ACKNOWLEDGEMENTS 150Docto
Process Oscillations in Continuous Ethanol Fermentation with Saccharomyces cerevisiae
Based on ethanol fermentation kinetics and bioreactor engineering theory, a system composed of a continuously stirred tank reactor (CSTR) and three tubular bioreactors in series was established for continuous very high gravity (VHG) ethanol fermentation with Saccharomyces cerevisiae. Sustainable oscillations of residual glucose, ethanol, and biomass characterized by long oscillation periods and large oscillation amplitudes were observed when a VHG medium containing 280 g/L glucose was fed into the CSTR at a dilution rate of 0.027 h1. Mechanistic analysis indicated that the oscillations are due to ethanol inhibition and the lag response of yeast cells to ethanol inhibition.
A high gravity (HG) medium containing 200 g/L glucose and a low gravity (LG) medium containing 120 g/L glucose were fed into the CSTR at the same dilution rate as that for the VHG medium, so that the impact of residual glucose and ethanol concentrations on the oscillations could be studied. The oscillations were not significantly affected when the HG medium was used, and residual glucose decreased significantly, but ethanol maintained at the same level, indicating that residual glucose was not the main factor triggering the oscillations. However, the oscillations disappeared after the LG medium was fed and ethanol concentration decreased to 58.2 g/L. Furthermore, when the LG medium was supplemented with 30 g/L ethanol to achieve the same level of ethanol in the fermentation system as that achieved under the HG condition, the steady state observed for the original LG medium was interrupted, and the oscillations observed under the HG condition occurred. The steady state was gradually restored after the original LG medium replaced the modified one. These experimental results confirmed that ethanol, whether produced by yeast cells during fermentation or externally added into a fermentation system, can trigger oscillations once its concentration approaches to a criterion.
The impact of dilution rate on oscillations was also studied. It was found that oscillations occurred at certain dilution rate ranges for the two yeast strains. Since ethanol production is tightly coupled with yeast cell growth, it was speculated that the impact of the dilution rate on the oscillations is due to the synchronization of the mother and daughter cell growth rhythms. The difference in the oscillation profiles exhibited by the two yeast strains is due to their difference in ethanol tolerance.
For more practical conditions, the behavior of continuous ethanol fermentation was studied using a self-flocculating industrial yeast strain and corn flour hydrolysate medium in a simulated tanks-in-series fermentation system. Amplified oscillations observed at the dilution rate of 0.12 h1 were postulated to be due to the synchronization of the two yeast cell populations generated by the continuous inoculation from the seed tank upstream of the fermentation system, which was partly validated by oscillation attenuation after the seed tank was removed from the fermentation system. The two populations consisted of the newly inoculated yeast cells and the yeast cells already adapted to the fermentation environment.
Oscillations increased residual sugar at the end of the fermentation, and correspondingly, decreased the ethanol yield, indicating the need for attenuation strategies. When the tubular bioreactors were packed with ½” Intalox ceramic saddles, not only was their ethanol fermentation performance improved, but effective oscillation attenuation was also achieved. The oscillation attenuation was postulated to be due to the alleviation of backmixing in the packed tubular bioreactors as well as the yeast cell immobilization role of the packing.
The residence time distribution analysis indicated that the mixing performance of the packed tubular bioreactors was close to a CSTR model for both residual glucose and ethanol, and the assumed backmixing alleviation could not be achieved. The impact of yeast cell immobilization was further studied using several different packing materials. Improvement in ethanol fermentation performance as well as oscillation attenuation was achieved for the wood chips, as well as the Intalox ceramic saddles, but not for the porous polyurethane particles, nor the steel Raschig rings. Analysis for the immobilized yeast cells indicated that high viability was the mechanistic reason for the improvement of the ethanol fermentation performance as well as the attenuation of the oscillations.
A dynamic model was developed by incorporating the lag response of yeast cells to ethanol inhibition into the pseudo-steady state kinetic model, and dynamic simulation was performed, with good results. This not only provides a basis for developing process intervention strategies to minimize oscillations, but also theoretically support the mechanistic hypothesis for the oscillations
Enhancing the resolution of cohesin dynamics in meiosis
Meiosis is a specialized form of cell division in which one diploid mother cell is converted into four haploid daughter cells. Cohesin is a multi-protein complex, providing cohesion to replicated chromosomes. During meiosis, cohesin is removed from chromosomes in two steps. First, it is proteolytically cleaved from chromosome arms in anaphase I, whereas cohesin in the vicinity of the centromere is protected from cleavage. This pericentromeric cohesin is then removed in anaphase II. This stepwise loss of cohesin is part of the current model of meiotic chromosome segregation. Evidence for this kind of cohesin dynamics came originally from immunofluorescence experiments with very limited spatial resolution.
A new workflow was established by combining a novel synchronization system for budding yeast meiosis with a calibrated and optimized ChIP-Seq protocol. This workflow allows resolving the cohesin dynamics in the course of the two meiotic divisions with unprecedented temporal and spatial resolution.
With this new experimental system, we confirmed the existence of two cohesin fractions on chromosomes, a protected and an unprotected fraction. Contrary to the current model, we detected both fractions in the region around the centromere. This indicates that the distinction between arm cohesin and pericentromeric cohesin is not identical to the classification into unprotected cohesin and protected cohesin. These results suggest that the mechanism of protection is not only determined by the localization of the cohesin protein complex. Additionally, we discovered significant differences in the cohesin protection activity among individual chromosomes.
The protein Sgo1 is required for the centromeric protection of cohesin. Sgo1 was analyzed directly with the new workflow, and we generated novel insights into the loading of the protection machinery onto chromosomes and the establishment of centromeric protection in meiosis. The protection machinery is loaded onto chromosomes in a cohesin-dependent mechanism, and a novel model of a dynamic three-step loading mechanism of the protection machinery is presented. This model explains how the cells are able to provide a robust and reliable protection to cohesin located in very diverse patterns on different chromosomes. Moreover, the model suggests a new function of the protein Sgo1 in centromeric protection.
A last result is that the polo-like kinase of budding yeast, Cdc5, is involved in regulating the levels of the protection machinery, which are loaded onto chromosomes
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