1Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, University of Uppsala, 2Department of Microbiology, Swedish University of Agricultural Sciences
Bjerling, P., Olsson, I., Meng, X. n. Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast. J. Vis. Exp. (59), e3454, doi:10.3791/3454 (2012).
Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.
1. Fission yeast culture
2. Sample preparation
4. Quantitative measurement of subcellular distances
5. Representative Results
Strain PJ1185:(h+ his7+::dis1placR-GFP Chr1[::ura4+ hphMX6 lacO] sid4-mRFP::kanMX6 cut11-mCherry::natMX6 ura4-D18 leu1-32 ade6-DN/N ) was grown in EMM. A sample was withdrawn and mounted in a small growth chamber and pictures were taken (Fig. 1A +N). Subsequently the growth media was replaced with EMM w/o ammoniumchloride (EMM-N), and cells were grown for 15 minutes while shaking. The nitrogen starved cells were then mounted in a growth chamber with EMM-N and pictures were taken (Fig. 1A -N). The measuring tool was used to measure the distance between the locus (GFP) and the SPB (Fig. 1B and Table 1). In addition, the distance between the locus (GFP) and the NM was measured (Fig. 1B and Table 2). The median subcellular distances before and after nitrogen depletion were compared using the SigmaStat-3.5 software (Table 3). There was a statistically significant shift in the localisation of the gene cluster moving away from the NM towards the SBP. The data measuring the distance between the GFP and the SBP had a normal distribution and hence the mean distances (1.777 μm +N and 1,587 μm -N) could be compared using a t-test (Table 1 and 3). There was a significant difference between the two mean values (P = 0.008, t-test). The data measuring the distance between the GFP and NM did not have a normal distribution and hence the median distances (0 μm +N and 0.390 μm -N) were compared using a Mann-Whitney Rank Sum test (Table 2 and 3). There was a significant difference between the two median values (P < 0.001, Mann-Whitney Rank Sum test).
Figure 1. The localisation of a cluster of genes named Chr1, marked by GFP, changed after the nitrogen starvation. (A) Left column, +N, a representative cell nucleus from a cell grown in EMM right column, -N, a representative cell nucleus from a cell grown in EMM-N. Green is the GFP signal labelling the Chr1 cluster, red is the mRFP and mCherry labelling the SPB and NM, respectively. (B) Same cell nucleus as (A) but now with measured subnuclear distances; yellow: the diameter of the cell nucleus, blue: the distance between SPB and GFP signal and pink: the distance between the GFP signal and the nuclear membrane.
Table 1. The measured subnuclear distances in μm of the PJ1185 strain grown in EMM. First row, diameter of the cell (d), second row, distance between the GFP and the SPB, third row, distance between the GFP and the NM.
Table 2. The measured subnuclear distances in μm of the PJ1185 strain grown in EMM-N. First row, diameter of the cell (d), second row, distance between the GFP and the SPB, third row, distance between the GFP and the NM.
Table 3. Descriptive statistics of the observed subnuclear distances in strain PJ1185 before (+N) and after (-N) nitrogen starvation. First row: number of cell measured, second row: mean diameter (d), third row: median diameter (d), forth row: mean distance between the GFP and SPB, fifth row: median distances between the GFP and NM.
During the last decade the use of live cell imaging to monitor cellular events has become increasingly popular. It started with the use of Green Fluorescence Protein from jellyfish Aequorea victoria and now many different fluorochromes are available emitting fluorescence through a wide spectra from cyan (475 nm) to far red (648 nm) 11. One of the major advantages of live cell imaging over immunofluorescence is that the cells are not fixed by formaldehyde or ethanol/acetone treatment before microscopy, hence avoiding possible artefacts from the fixation process. In addition, live cell imaging offers the possibility to follow individual cells and take pictures at frequent intervals during hours or days of incubation, making it possible to get movies of cellular events 12. Mammalian cells have the advantage of having a larger size, with diameters of around 100 μm, as compared to the smaller yeast cell with a diameter of about 3-4 μm. On the other hand, the advantage with yeast is the easily manipulated genome. Homologous recombination occurs very efficiently in yeast and is used to fuse proteins of interest to different fluorochromes 3. Moreover, using targeted integration of lacO sequences and subsequent expression of LacR-GFP protein allows the detection of a specific part of the genome within the cell nucleus 2. Using S. pombe cells in microscopy has additional advantages since they are natural unicellular organisms that grow with a fast generation time in low-cost cell culture conditions. Moreover, S. pombe is an excellent eukaryotic model organism since it has metazoan homologous genes.
One of the major limitations of this technique is autofluorescence in the yeast cell disturbing the detection of the true signal. This problem can be overcome by using minimal growth media with filter sterilised glucose instead of autoclaved. In addition the yeast cells should be grown for 2 days in log phase before mounting them. The protocol presented here offers a relatively simple, but yet quantitative method to determine the subcellular localisation of proteins within the yeast cell. Moreover by taking pictures at different time points we can follow cellular events.
We have nothing to disclose.
We thank Professor Hiraoka for sending us strains. We acknowledge support from the Goran Gustafssons Foundation and the Swedish Cancer Society (2008/939).
|Silicon grease||Dow Corning|
Laser scanning microscopeLSM 700
|Carl Zeiss, Inc.||LSM 700||Other confocal microscope could be used.|
|SigmaStat3.5||Statcon||SigmaStat3.5||Any software for statistical analysis could be used.|