Characterisation of Ras1 signalling in Sz pombe
A Vector pREP3x-Ras
4.4. CONSTITUTIVE EXPRESSION OF RAS1 103
0 10 20 30 40 Vector Ras1 JY1351
pREP3x JY544 (ras1+)
JY1351 pREP3x-Ras1
JY1351
pREP3x pREP3x-Ras1JY1351 JY544 (ras1+)
Figure 4.18. Expression of Ras1 from pREP3x does not restore mating in Δras1 cells
The mating of Δras1 (JY1351) cells transformed with vector alone and pREP3x- Ras1 was assessed using the quantitative mating assay detailed in section 4.2.1. The ras1+ strain JY544 expressing Sxa2 from pREP41x is included as a control. No significant mating was seen in JY1351 transformed with either vector alone or pREP3x-Ras1. Data shown representative of three independent determinants + SEM.
alone control. This was reflected in the lack of iodine staining observed in this population, indicating a lack of mating and spore formation. By contrast,
JY544 (ras1+) transformed with pREP41x-Sxa2 exhibited a cfu recovery of 32
+ 2 % upon incubation with cells of the opposite mating type (Figure 4.18).
4.4.2. Constitutive Ras1 expression alters cell morphology and the localisation of the cell tip marker Tea1.
Images of cells expressing Ras1 from pREP3x indicate an enlarged and more rounded morphology than that expected for wild-type cells (Figure 4.19A). This observation could indicate a change in the regulation of polar growth
when Ras1 expression is not regulated by theras1 promoter. Thus, the locali-
transformed with pREP3x alone and pREP3x-Ras1, in order to establish the ability of Ras1 expressed from pREP3x to maintain polar cell organisation.
Images of these cells were obtained using a Nikon E800 epifluorescence microscope fitted with an Andor EM-CCD camera. In addition, images of
Δras1 cells (JY1279) expressing Ras1 from pREP3x, and expressing GFP
from pREP4x, were obtained using a Leica SP5 scanning confocal microscope. Cell circularity was then determined for 30 individual cells in these populations using Quimp software (Figure 4.19).
As is seen in Δras1 cells and the vector alone control, Tea1-mCherry in
cells containing pREP3x-Ras1 showed some preferential localisation to oppo- site sides of the cortex, but Tea1-mCherry could be seen over the extent of the cell periphery (Figure 4.19C). Additionally, cells expressing Ras1 from pREP3x displayed a rounded morphology similar to the vector alone control
( 85.9 + 1.0 % compared to 87.2+ 1.8 %) (Figure 4.19A and B). In contrast,
cells expressing Ras1 from the ras1 locus (JY544) exhibit the discrete locali-
sation of Tea1-mCherry to opposite poles of the cell, and a significantly lower
percentage cell circularity (73.5+ 1.2%, P = 0.001). These data suggest that
the regulation of Ras1 expression is also key to correct Ras1 function through Scd1-Cdc42.
4.4.3. Constitutive Ras1 expression causes the formation of mul- tiple shmoo tips.
A key aspect of mating in fission yeast is the polar growth of a cell towards a cell of the opposite mating type. As a consequence, the loss of polarity ob- served in cells expressing Ras1 from pREP3x (Figure 4.19) could account for some of the loss of mating seen in these cells (Figure 4.18). Examination of JY1279 expressing Ras1 from pREP3x indicates that a number of cells form multiple projection tips (indicated by black arrows) upon stimulation with 10
A
Vector Ras1 JY544 (ras1+)
B
C
JY1279
pREP3x JY544 (ras1+)
JY1279 pREP3x-Ras1
JY1569
pREP3x JY544 (ras1+)
JY1569 pREP3x-Ras1 JY1279
pREP3x pREP3x-Ras1JY1279 JY544 (ras1+) ***
Figure 4.19. Expression of Ras1 from pREP3x disrupts polar cell morphology
Images of Δras1 cells (JY1279) transformed with vector alone and pREP3x-Ras1, expressing GFP from pREP4x, were obtained using a Leica SP5 scanning confocal microscope. Representative DIC images are displayed in panel A. Cell circularity was determined in these populations using Quimp software (B). Data representative of 30 individual cells + SEM. Cells containing pREP3x-Ras1 displayed a rounded morphology. Tea1-mCherry localisation was determined in aΔras1 strain (JY1569) transformed with vector alone and pREP3x-Ras1 using a Nikon E800 epifluorescence microscope fitted with an Andor EM-CCD camera (C). Tea1 was distributed around most of the cortex in cells containing pREP3x-Ras1. The scale bar represents 10 μm. JY544 (ras1+) is also displayed to represent a wild-type phenotype.
regulation of Ras1 expression is important in directional sensing during mating and shmoo formation, and may explain the lack of mating observed in Figure 4.18.
Ras1 OE gives messed up shmoos
Figure 4.20. Expression of Ras1 from pREP3x in Δras1 cells causes the formation of multiple shmoo tips
The morphology of cells expressing Ras1 from pREP3x was examined following incubation in the presence of 10μM pheromone for 16 h. A number of cells within the population displayed multiple shmoo tips. DIC images were taken using a Leica SP5 scanning confocal microscope. The scale bar represents 10μm.
4.5. The localisation of Ras1 4.5.1. Constructing a GFP-Ras1 fusion.
Many previous studies have demonstrated the use of N-terminal GFP-ras fu- sions as a means of assessing ras localisation, while maintaining ras function
(Choy et al. 1999; Chiu et al. 2002; Matallanas et al. 2006; Onken et al.
2006). A direct in-frame N-terminal gfp-ras1 fusion was created at the ras1
locus, to allow analysis of Ras1 localisation. Integration of gfp-ras1 in the
strain JY1247 (ras1::ura4+, sxa2>lacZ) was achieved using homologous re-
combination between flanking regions of the ras1 locus and homologous re-
gions flanking the gfp-ras1 fusion in a linearised vector (Figure 4.21A). FOA
selection was used to select for the loss of the ura4+ cassette and integration
of the fusion, generating the strain JY1390. Immunoblotting using an anti-ras RAS10 antibody gave a chemiluminescent signal consistent with the predicted
size of the GFP-Ras1 fusion (∼52kDa) in whole cell extracts generated from
JY1390. Little breakdown of the fusion was also observed (Figure 4.21B).
4.5.2. Ras1 is localised to the plasma membrane.
To determine the cellular localisation of Ras1, fluorescence images of cells
from the strain JY1390 (gfp-ras1), grown in DMM, were obtained using a
Nikon E800 epifluorescence microscope fitted with an Andor EM-CCD camera (Figure 4.22). The fluorescence level observed from these cells was very low, indicating that the GFP-Ras1 fusion was expressed at low levels. Despite this, the localisation of GFP-Ras1 could be observed at the plasma membrane, and was particularly apparent at the septa of dividing cells. Some localisation to peri-nuclear endomembrane structures may also have been present. The lo- calisation of GFP-Ras1 has previously been described in one study, in which
GFP-Ras1 was expressed from integrated plasmids containing the ras1 pro-
4.22 is the first localisation of GFP-Ras1 from the endogenous ras1 locus de- scribed. The pattern of localisation seen in Figure 4.22 closely matches that previously observed.
X
X
5’UTR 5’UTR 5’UTR 3’UTR 3’UTR 3’UTR gfp-ras1 EcoRI BglII ura4 cassette A JY1247 ura4 ORF gfp-ras1 JY1390 GFP-Ras1 integration 225 150 102 76 52 38 31 26 17 225 150 102 76 52 38 31 26 17 kDa Ras1 GFP-Ras1 BFigure 4.21. Integration of GFP-Ras1
Integration of GFP-Ras1 in theras1::ura4+,sxa2>lacZ strain JY1247 was achieved by homologous recombination between flanking regions of theras1 ORF and homol- ogous regions on a linearised vector (A). FOA selection was used to select for the loss ofura4. Expression was confirmed via immunoblotting using a RAS10 antibody (B). Expression of GFP-Ras1 (JY1390) was demonstrated, and a chemiluminescent signal was observed at a position consistent with the size of the fusion (∼ 52kDa). Coomassie stains of whole protein are included above the immunoblot as loading controls.