The “primary sensor” that detects heat or changes in the environment of the cell and activates the two stress-transduction systems has still not been fully recognised. The increased transcription of HSPs occurs due to these changes when HSFs bind to HSE in the promoter region. Stress-response proteins (similar to HSPs) are also transcribed when Msn2/4 binds to STREs (Figure1). The accumulation of these proteins is observed because of HS response. Recently, several findings seem to have identified the plasma membrane, particularly the fatty acids, to play an important role in the induction of the HS response (Mansilla et al, 2004; Chatterjee et al, 2000; Chatterjee et al, 1997; Carratu et al, 1996). The findings in this project will demonstrate how the membrane and its lipid composition seem to respond and counteract stress, thus providing evidence that the membrane is the “primary sensor” and explain its influence on the transient nature of the response.
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4.1.1 Effect of KCl on HS response
Results showed that when S.cerevisiae cells were subjected to normal heat shock at various temperatures, maximal heat shock induction occurred at 38oC with 0.016µg-1 enzyme activity (Figure 9). In addition, a subsequent decline in the induction was observed followed by a slight increase at 42oC. The cells are able to grow up to 42oC after which the cells undergo cell death and this is clearly seen in figure 9 by the decline in activity, from 0.01µg-1 to 0.001µg-1. These results provided a baseline for comparison with the results of the heat shock with KCl. Under osmotic stress, in the presence of 0.5M KCl heat shock, the maximal induction increased to 42oC with 0.008µg-1 enzyme activity (Figure 10) and in the presence of 1M KCl heat shock, the maximal induction increased even more to a lethal temperature of 45oC with 0.009µg-1 enzyme activity (Figure 11). During the exposure to 0.5M KCl, a pattern that correlated with the controls can be seen; however, there was a slight increase in the β-galactosidase activity at 47oC (0.004µg-1) compared to the control (0.003µg-1). Similarly, in the presence of 1.0M KCl, there was a decrease in the β-gal activity at 42oC and an increase in the activity at 47oC (0.007µg-1) compared to the control (0.003µg-1).
The findings observed could be explained using the knowledge of osmosis on the yeast cell membrane. Potassium is a major cellular cation (K+) that is present in high concentrations inside the cell and determines the cell volume, turgor and cytoplasmic ionic strength (Mulet et al, 1999). They play an essential role within eukaryotic cells, thus should be maintained within its concentration range to avoid toxicity. The normal intracellular concentration of K+ in S.cerevisiae ranges from 200-300mM (Barreto et al, 2012) therefore; increasing the external concentration can subject the cell to hyperosmotic stress. Exposing S.cerevisiae cells to KCl concentrations 0.5M and 1M increased the solute concentration outside the cell, causing water to move out of the cell and subsequently leads to cell shrinkage via the movement of the plasma membrane away from the cell wall (plasmolysis) and decrease in the cellular volume. This causes the cell membrane to become more compact, resulting in the reorganisation and close association of the membrane proteins and lipids (Salari et al, 2013).
This compaction of the lipids and proteins may be a possible reason for the increased tolerance of cells when exposed to high temperatures. The compact cell membrane may require an even higher temperature (thermal stress) to induce HS response, compared to cells that do not have compact membranes. The late onset of HS response to lethal temperatures can result in an increase in the resistance to physiological changes hence, prolonging the cellular degradation. This reason may explain the findings that are illustrated in the results, which show an increase in the maximal HS induction temperature when exposed to 0.5M and 1M of KCl compared to normal maximal HS temperature. Increasing the concentration of KCl, from 0M to 0.5M to 1.0M, resulted in an increase in the compactability and therefore, an increase (and shift) in the maximal HS induction temperature from 38oC to 42oC to 45oC (Figures 10 and 11). In Figure 10 and 11, it clearly shows a large enzyme activity (0.007µg-1) at 47oC compared to the control (0.003µg-1); this may be due to the increase in ion concentration outside that increases compactability of the membrane providing more tolerance to heat. Therefore, stimulates a late onset to HS response. β-galactosidase activity expression coincides with this explanation, as the intensity of the yellow is more rapid during the maximal induction temperatures. As shown in figures 10 and 11, overall β-galactosidase activity was reduced significantly, this may be due to low levels of HSPs. Thus, less β-galactosidase are transcribed because of increased tolerance to the heat in the precedence of KCl.
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Deeper analyses into the molecular aspects of S.cerevisiae cells provide a greater understanding of the many interactions that takes place in order to elicit the HS response. Yeast responds to salt stress via KCl in two different ways: osmotic stress and ion toxicity. However, KCl has no toxicity (Garcia et al, 1997) or has minimal toxicity compared to other salt ions such as Na+ and Li+, which inhibits specific metabolic pathways (Nasser & El-Moghaz, 2010). It is observed that the increased levels the ions may result in some disruption to various enzyme activities and disturb the hydrophobic-electrostatic balance that maintains the protein structure (Serrano, 1996).
S.cerevisiae cells respond to hyperosmolarity by rapidly removing their intracellular water, which leads to cell shrinkage and loss of turgor (Mager and Siderius, 2002), also cytoskeleton collapses; leading to the depolarisation of actin patches (HolubáÅ™ová et al, 2000). These changes stimulate signalling pathways, which include mechanoreceptors (stretch-activated channels), high-osmolality glycerol (HOG) mitogen activated protein (MAP) kinase pathway and protein kinase A (PKA) (Mulet et al, 1999). Recent studies show that during salt stress, dynamic post-translational response arises, where a rapid decrease in plasma membrane proteins occurs due to the internalisation of the proteins. This affects the membrane morphology and ionic homeostasis and ensures survival of the cell (Szopinska et al., 2011).
Under osmotic stress, S.cerevisiae elicits both specific and general stress response, particularly to hyperosmotic stress (Alonso-Monge et al, 2001). The specific response pathway that reacts to changes in the osmolality is the HOG-MAP pathway. The plasma membranes of S.cerevisiae cells contain a histidine kinase sensor, Sln1p and the Sho1p sensor that monitor osmotic changes especially to ion concentration. When turgor pressure is affected, the Sln1p sensor becomes inactive, this activates the MAP kinase pathway (PKC MAP kinase route), and phosphorylates the MAP kinase Hog1 (Klipp et al, 2005 and Alonso-Monge et al, 2001). This stimulates the production and accumulation of glycerol, essential for yeast cells. Glycerol is also accumulated via the closure of the Fps1p channel and by increasing the expression of GPD1 and GPP2 genes, which codes for the enzymes needed for the production of glycerol (Alonso-Monge et al, 2001). The accumulation of glycerol internally is an adaptation technique that leads to morphological changes such as invagination of the plasma proteins (Szopinska et al., 2011). It also allows the cells to regain turgor (Mager and Siderius, 2002 and Klipp et al, 2005), take up water from the environment and restore the cell size (Hohmann, 2002 and Alonso-Monge et al, 2001). Cellular integrity is maintained and the cytoskeleton is repaired (Mager and Siderius, 2002).
The general stress triggers the expression of stress-induced genes, which is transcribed after the binding of Msn2 and Msn4 transcription factors to the stress transcriptional regulatory elements (STRE’s), in the promoter CTT1. The induction of the Msn2/4 genes during osmotic stress requires Hog1 and these transcription factors recruits Hog1 to the promoter CTT1 and HSP12 (O’Rourke et al., 2002). The HSP12 plays a role in the yeast stress response by stabilising the plasma membrane against heat shock and other stresses. The protein folds into a helical structure and binds to specific lipids structures, this interaction changes the organisation of the membrane by increasing its stability and integrity (Welker et al, 2010).
These studies established that genes and pathways that were involved in one type of stress response also play a role in other stresses. In S.cerevisiae, the HOG pathway has been seen to be active during both osmotic stress and heat stress and the Sho1, a membrane bound sensor, mediates heat stress by the activation of Hog1 (Winkler et al, 2002). It was also evident that a PKC pathway is triggered due to increased temperature and that this pathway is linked to the HOG mediated signalling. Under stressful conditions, the cells adapt by accumulating osmolytes and polypols such as glycerol and fatty acids in the cell membrane (Nasser and El-Moghaz, 2010), where high levels of salt ions increases the viscosity of the cells membrane by the increase in unsaturated fatty acid levels (Nasser and El-Moghaz, 2010). These changes in the cell structure lead to the activation of the HOG pathway (Alonso-Monge et al, 2001) and the synthesis of glycerol. Glycerol is an integral compound for phospholipids that is involved in signalling (Siderius et al, 2000). This leads to the suggestion that the glycerol levels and its interaction with the lipid composition may play a role in the transient nature of HS response since, the HOG pathway is activated by heat resulting in glycerol synthesis.
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These molecular findings helped to understand the results obtained during osmotic shock via KCl, which was that KCl increased the temperature of HS response by changing the membrane structure. The increase in molarity increased the external concentration of the cell, resulting in a strong response at 0.5M and then a much stronger response at 1.0M. The increase in the internal accumulation of glycerol may explain the shift in the maximal temperature for HS response at 42oC for 0.5M and 45oC for 1M, resulting in an increased tolerance to temperature (Figures 10 and 11). Furthermore, this provides evidence, that the cell membrane and the lipid composition seems to influence the response the cells have to HS and explains how the compaction of the membrane provides heat tolerance.
4.1.2 OLE1 gene
Homologous recombination was used to knock out the OLE1 gene, which encodes for monounsaturated fatty acids (see section 1.3 and 1.6). It was conducted to understand the influence of fatty acids on the transient nature of HS response. However, sufficient results could not be obtained to support or disapprove the theory due to time constraints and experimental difficulties. Preliminary steps towards obtaining results were completed. The HIS5 gene was correctly amplified where a thick band could be visualised in Figures 16, 18 and 19. The fragment size of 1.26kb was calculated using the Log graph (Figure 20), which roughly correlated to the estimated size of 1.238kb.
Initial analysis of the PCR tube content, using gel electrophoresis and UV did not show any banding as seen in the results (Figures 12, 13, 14, 15 & 17). It was originally suggested that the correct annealing temperature should be established, to allow specific binding of the hybrid primers. At high annealing temperatures, the primers were more likely to bind to a specific sequence. However, it could also cause the hydrogen bonds to become weaker, and subsequently break or result in primers not binding. At low annealing temperatures, the hydrogen bonding was stronger and the primers could bind easily to both specific and non-specific binding sites. Therefore, it was necessary to find the ideal annealing temperature. The first analysis did not show any bands at an annealing temperature of 52oC suggesting, it was either too high or too low (Figure 13). As a result, PCR was repeated at 50oC and 54oC respectively, however, these temperatures did not produce any banding (Figure 14 and 15). It was then suggested that the primers may have been stored in the freezer for too long, consequently affecting the ability to anneal properly. PCR was then run using a new hybrid primer and the annealing temperature of 52o; analysis of the result showed correct banding size similar to the expected amplified HIS5 gene size (Figure 16).
4.2 Limitations and Further experiments
Few limitations were encountered throughout the project. The most difficult and most time-consuming issue was the fact that the yeast cells were not growing to the right OD, in order to carry out the rest of the experiment. This was seen even after using frozen glycerol stocks; however, inoculating from the glycerol stock obtained roughly similar ODs making the experiment results more accurate. Reasons behind the inconsistency of the growth in each flask that was inoculated at the same time are still not very clear, since the media, the amount and the stock used were the same. Another major limitation was the difficulty in obtaining the β-galactosidase enzyme hydrolysis (Table 3); this may have been due to breakage of pellets not being conducted properly and/or the addition of excessive glass beads. This was confronted by adding the equal amount of glass beads for breakage and increasing the amount of spins conducted to 15x30sec instead of 10x30sec. The OD also seems to affect the β-galactosidase activity observed and it was discovered that the yeast grown to an OD around 0.1-0.5 instead of 0.05-0.1 seem to provide a good ONPG hydrolysis. This may have been because of the large amount of yeast cells that produces the HS response, which results in an increased expression of enzymes. Therefore, a higher and faster ONPG assay reading was obtained if heat shock had occurred. Manually adjusting the water baths for different temperatures also proved difficult, as they seemed to fluctuate and increased the chances of human error; however, it was carefully controlled using hot water and cold water to improve accuracy of the results. Furthermore, to increase the accuracy during PCR and Gel analysis, a larger amount of PCR product could be pipetted in the wells resulting in a larger amplified DNA band that can be highlighted much more intensely via UV light.
Regarding further experiments, a few approaches could take place in order to confirm the findings found in this project and to conduct the experiment that could not be completed. Repeats should be carried out for the heat shock profile for both 0.5M and 1.0M KCl to confirm the trend shown in Figures 10 & 11. The yeast cells could be exposed to a range of KCl concentrations, to find out if the trend can be seen throughout, the higher concentrations should be exposed to temperatures above 47oC, and finally, the yeast cells could be exposed to hyposmotic stress in order to compare responses.
The amplified HIS5 gene could be used to knockout the OLE1 gene in yeast cell to produce the mutant-DBY747-HSE1lacZ strain (see section 1.6). In order to test for the homologous recombination, the yeast should be grown on media containing L-leucine and L-tryptophan only, as the mutant strain contains the histidine gene therefore, is able to grow on media without L-histidine. This mutant strain should then be exposed to various temperatures and the effect of HS response should be recorded. The predicted behaviour of the OLE1 mutant when exposed to HS is that it would be able to produce HSPs however, could not be able to switch off this response after a prolonged period of time compared to an OLE1 wildtype. This is because the membrane cannot be altered, as unsaturated fatty acids cannot be synthesised and it is known that increased fatty acid levels produce tolerance to heat. Therefore, a transient nature should not be seen (Section 1.3).
The amplified HIS5 gene is available for the knockout of OLE1 gene, creating a mutant-DBY747-HSE1lacZ strain (see section 1.6). This can be used to test whether the OLE1 gene that encodes for unsaturated fatty acids affects the transient nature of the HS response. It is also seen that both heat stress and osmotic stress cause changes in the membrane and can be detected by Sho1 and Sln1 sensors, which activate HOG1. This leads to the synthesis of glycerol and results in the restoration of the cell. It was identified that structural changes in the lipid composition of the membrane via HSP12 and glycerol appear to play an important role in the dynamics of the HS response. These confirmed the findings that suggested the HS response is lipid mediated (Chatterjee et al, 1997). However, further studies are needed in order to confirm the findings seen in this project and to understand the relationship between the cells’ membrane and HS response.
4.4 Significance and Implications
The technique used to alter the membrane via OLE1 knockout may show a relationship between the membrane, the HS response and increased tolerance. Therefore, transferring this knowledge may help other organisms such as the plant species, where it could play a role in the increased tolerance to high temperatures during growth or could help to address tolerance towards drought and poor soil conditions. Furthermore, HSPs have been found to be very useful in increasing the efficacy of cancer vaccines and treatments; understanding more about the function could play a huge role in therapeutic medicine.