There are a large number of proteins which regulate actin polymerisation., these include Wiskott-Aldrich Syndrome Protein (WASP) interacting protein (WIP). With rapid replication, similarity to mammalian cells in both signalling pathways and cytoskeletal organization, and ability to be manipulated, the species Saccharomyces cerevisiae presents as a model organism in studying the role that WIP has in the organisation of actin in the mammalian cell. Verprolin protein [Vrp1p], as the homologue of the WIP present in the S. cerevisiae, contains two highly conserved proteins at the monomeric actin-binding site (2). Because of this, in high amounts, the expression of WIP can effectively rescue a Vrp1p-knockout.
Vrp1p is expected to be highly functioning in actin-polymerisation, primarily as a multivalent chaperone, conveying long-term conformational changes through transient binding of associated proteins (Figure 1). This conformational change leads to the termination of the inhibitory effects of the Hof1p SH3 domains on actin-polymerase. Additionally Vrp1p converts Las17p to an actin-branching form. Strains of S. cerevisiae where the Vrp1p has been knocked out has been studied (4), and has been shown to have definite morphological alterations due to defective production of actin filament. Furthermore, the knockouts showed difficulty in movement due to the important role the WASP proteins play in regulation of cell motility. At 24 degrees celciusthe Vrp1p is important, however at 37 degrees Celsius it is essential.
The results from our electrophoresis were error prone beyond any recognition, with the agarose gel failing to set, and negligible results being taken. Due to this, results were borrowed from Joseph Burke, Bench F of tutor Angela Johnstone.
Methods
After being prepared and observed on a microscope slide under a phase-contrast objective of 100x, the aliquots of VRP1-knockout mutant and wild type [WT] S. cerevisiae cells were each streaked onto two YPD plates. Cells were then put into incubation for 48 hours, one plate at room temperature and one plate at 37°C (figure 2).
figure 2: different temperatures of incubation of YPD plates each containing wild type and VRP1 mutant S. cerivisiae, BIOL2200 Lab Manual
The DNA of each strain of yeast was isolated using a technique known as the "smash and grab" technique. As well as the DNA, primers, essential chemicals and Taq polymerases were added to PCR tubes in preparation for polymerase chain reaction (PCR). A tube with no DNA acted as a PCR control. Tubes were then moved to a thermal cycler so as to amplify the DNA sequence that contains the VRP1 mutant. These were then run on agarose gel.
A VRP1 mutant sample was used for transformation with lithium acetate, which leads to pore formation in the cells, salmon sperm, which acts as a vehicle for plasmid uptake, and PEG3350, which is altered extracellular DNA for uptake. Transformation occurred with the mutant DNA either with a plasmid containing the intact VRP1 gene of a control of purified water. Once transformed, cells were spead on plates and incubated at 37°C for 48 hours, at which point they were observed.
Results
When observed, it was evident that the wild type cells were smaller than mutant cells, in both length and width with the length ratio of WT:Mutant equating to 4.3µm:5.2µm, and the width ration of WT:Mutant equating to 4.3µm:5µm. Significance was weak in both width and length differences with p-values of 0.0568 and 0.1178 respectively. Under phase-contrast microscopy, it was observed that mutant cells grew in patches, and also appeared to be more circular, as opposed to WT, which grew with even distribution and with a less circular structure.
The mutant VRP1-knockouts showed a reduction in growth in the YPD plates incubated at RT, but a complete cell death at 37°C, where as the WT streaked plates showed only an intermediate decrease between colonies incubated and RT and 37°C. (ASK JOEYYYYYY)
There was no differentiation seen in the running of DNA through electrophoresis between WT, mutant and the negative control containing no DNA. As well as this, the WT, mutant and negative control all ran through the gel faster than the all types of DNA in the ladder compound.
Discussion
The major finding of the experiment was that in VRP knockout yeast cells, the morphology and tolerance to temperature increase was significantly different to that of the WT cells. The mutants demonstrated proliferation difficulties at normal temperatures, and complete inhibition while incubating at 37°C. This outcome has been highly published and replicated (4,6). The mutant phenotype is thought to be the cause of loss of function of the signalling molecule, verprolin, in the actin polymerisation pathway. It is thought that in WT replication actin is organised via cables directed toward the budding cell in concentrations of cortical patches inside the but in the VRP1-knockout mutants, during replication, actin is presented with no form of organisation which leads to slow and irregular replication. The formation and contraction of the S. cerevisiae is thought to be controlled to a certain degree by actin. As an important structure in cytokinesis, deficiencies in the formation and contraction of this ring may be a contributing factor to decreased mutant cell proliferation. The regulation of actin polymerisation is also a contributing factor in abnormal morphologies(2). The morphological differences can cause unusual cell structures and loose cytoskeletal arrangements allowing an increase in cell size.
The decreased temperature tolerance of the mutant strands was shown when the plates were incubated at 37°C. The increase rate of cell death, attributed to lysis, lead the cells to die within an hour of 37°C incubation.
In the experimental results acquired from Bench F, results of the PCR products contained errors that can be attributed to floors in the experimental protocol. The errors can be attributed to different experimental mistakes, for example, when using the "smash and grab" technique, removal of small amounts of the wrong layer could leave unwanted chloroform in the sample. With ideal results, the WT DNA would have moved further through the agarose gel, and hence more accurate results would lead to significant differentiation of DNA types in distance moved through gel due to size of fragments. As the rescued mutants were still susceptible to decreased growth at 37°C, it was suggested that some cells transformed the plasmid incompletely. This incomplete transformation allows the actin polymerisation to return to mutant levels. Contamination is shown by growth on the control plates, and could pose as an issue to result viability. Due to this contamination, improvements in experimental protocol in terms of infection control could ensure a reduction in possible result corruption. To increase completion in transformation of plasmids, the use of plasmids with a gene encoding an antibiotic resistance could insert selective pressure.
