Resultados del análisis de brechas de pertinencia

membrane proteinlgp120

4.2.2.1 Regionalisation of isolated pulmonary artery smooth muscle cells in order to determine the sub-cellular distribution of fluorescent labelling

In order to determine the sub-cellular distribution of fluorescent labelling within isolated pulmonary artery smooth muscle cells, each cell was divided into four regions. These regions were termed the ‘nuclear region’ ‘perinuclear region’, the ‘extra-perinuclear region’ and the ‘sub-plasmalemmal region’, as depicted in the schematic diagram of a cell shown in Fig. 4.3. The processing applied to pulmonary artery smooth muscle cells in order to define these regions and quantify the fluorescent labelling within each is outlined below.

Fig. 4.3 Designation of regions within a model isolated pulmonary artery smooth muscle to allow for examination of the spatial distribution of fluorescent labelling.

Volumetric analysis of cells was carried out using Volocity software (Improvision, UK). Measurements of cellular volume were achieved by drawing a region of interest (ROI) around the perimeter of the cell and measuring its volume. Pulmonary artery smooth muscle cells were seen to occupy a volume of 1754.1 ± 258.4m3(n = 24; Appendix 2, Table 4.2).

The volume of the nucleus of the cell was defined by initially measuring the labelling of the fluorescent probe DAPI (excitation 360 nm, emission 457 nm). DAPI binds to the major groove of the double helix of DNA, once bound it fluoresces in response to ultraviolet light (Barcellona, et al., 1990); note, accurate measurements of the volume of DAPI, and all other fluorescent probes, was achieved through the application of threshold filters to exclude any labelling with fluorescence intensities lower than the background levels (Chapter 2, section 2.4.2). Following this, an ROI was drawn around DAPI labelling to define the boundary of the nucleus. The volume of this ROI was measured and the nucleus was seen to occupy 105.7 ± 13.4 m3 of the volume of cells (n = 24, Appendix 2, Table 4.2). The volume of the nucleus, and any fluorescent labelling contained within, were excluded from all subsequent analyses (see below).

Once the nuclear boundary had been established, an ROI was drawn in the cytoplasm within 1.5 m of the nucleus and termed the perinuclear ROI. The volume of the perinuclear region was then determined by subtracting the volume of the nucleus from the volume of the perinuclear ROI. The perinuclear region of cells occupied a volume 580.4 ± 103.9 m3 (n = 24; Appendix 2, Table 4.2).

A second ROI was then drawn within the cytoplasm of the cell and was termed the non sub-plasmalemmal ROI. This ROI included both the perinuclear and nuclear regions of the cell, but excluded the sub-plasmalemmal region. Subtraction of the volumes of the perinuclear and nuclear regions of cells, from the measures obtained from the non sub-plasmalemmal ROI provided a measure of the volume of the extra-perinuclear region of cells. The extra-perinuclear region occupied a volume of 418 ± 75m3(n = 24; Appendix 2, Table 4.2).

The sub-plasmalemmal region of the cell was determined as being the area of cytoplasm located within 1.5 m of the plasma membrane of the cell. Subtraction of the volume of the non sub-plasmalemmal ROI from the ROI encompassing the whole cell gave a measure of the volume of the sub- plasmalemmal region of cells. The sub-plasmalemmal region of cells occupied a volume of 673.2 ± 135.2m3of cells (n = 24; Appendix 2, Table 4.2).

In areas of cells where the plasma membrane encroached within 1.5m of the nucleus, the distance that the perinuclear region extended from the nucleus was altered in order to prevent misleading calculations of the distribution of labelling. In order to do this, the distance from the edge of the nucleus to the plasma membrane of the cell was measured. The boundary of the perinuclear region was then readjusted to extend half the distance from the nucleus to the plasma membrane of the cell at that point. Therefore, if the nucleus was positioned 1m from the plasma membrane at a given point, the perinuclear region was readjusted to extend 0.5 m from the nucleus at that point. Similarly, the boundary of the sub-plasmalemmal region was altered to extend half the distance from the nucleus to the plasma membrane within these regions. Therefore, within these regions the sub-plasmalemmal and perinuclear regions bordered one another, with exclusion of the extra-perinuclear region. In some cells, the distance between the nucleus and the plasma membrane was smaller than the accurate resolution limit of the imaging system. Therefore, any fluorescent labelling located within these areas of cells was excluded from analysis as it could not be confidently assigned to a given region of the cell.

As illustrated in Table 4.2 (Appendix 2), the volume of cells was not distributed evenly between the three regions. To allow comparison of measurements of fluorescent labelling between regions, a process of normalisation was applied to the labelling within each region of the cell. To achieve this, the volume of fluorescent labelling within a given region was divided by the volume occupied by that region in order to provide the volume of fluorescent labelling per m3of a given region of the cell. Thus, the density of fluorescent labelling in a given region was measured, thereby allowing direct comparison of the distribution of labelling between different regions and between cells.

4.2.2.2 Determination of lysosomal distribution in methanol-fixed isolated pulmonary artery smooth muscle cells

The distribution of Lysosomes within immunocytochemical

investigations was visualised through the use of an antibody raised in mouse against the integral lysosomal membrane glycoprotein, lgp120 (GM10; Grimaldi, et al., 1987). The glycoprotein lgp120 has previously been shown to be expressed predominantly on the membrane of lysosomes, with little expression detected on endosomes, the plasma membrane, or within the golgi cisternae (Lewis, et al., 1985). Binding of this primary antibody was visualised via the binding of goat anti-mouse secondary antibody conjugated to the fluorescent probe FITC (excitation 490 nm, emission 528 nm).

A typical example of the distribution of lgp120-labelling within isolated pulmonary artery smooth muscle cells is shown in Fig. 4.4. Fig. 4.4(i) shows a transmitted light image of an isolated pulmonary artery smooth muscle cell, while Fig. 4.4(ii) shows a deconvolved Z-section (focal depth 0.28 m) through the cell, corrected to remove background fluorescence, as determined from matched control slides (Chapter 2, Section 2.4.2). Lysosomal distribution and the position of the nucleus are visualised in green and blue, respectively. Consistent with the other cells examined, intense areas of lysosomal labelling are located in close proximity to the nucleus of the cell shown in Fig. 4.4(ii) and an example of one of these areas of labelling is indicated by arrow 1. Lysosomal labelling located further from the nucleus of the cell is evident; however, these areas are fewer in number and appear to be more diffuse than elements located close to the nucleus (Fig. 4.4(ii) arrows 2 and 3). Fig. 4.4(iii) shows a 3-dimensional (3D) reconstruction of a series of deconvolved Z- sections (focal depth 0.28m, Z step 0.2 m) obtained through the cell. It can be seen that the densest areas of lysosomal labelling appear to be located close to the nucleus of the cell. Analysis of cells was then carried out to determine the volume of labelling present within cells. Under carefully controlled ‘optimal’ experimental conditions, the DeltaVision imaging system is able to accurately resolve elements of labelling smaller than 0.2m in size in each of

the X-, Y- and Z-planes. However, the precisely controlled environment under which these ‘optimal’ measurements are obtained cannot be recreated under the experimental conditions in my experiments. Therefore, in these and all subsequent analyses, I set a more conservative value on the limit of resolution in my experiments. To this end, I included only those volumes of labelling measuring≥0.5m in the X-, Y- and Z-planes (volume≥0.125m3) and any element of labelling smaller than these limits were excluded from consideration. Fig. 4.4(iv) shows a 3D representation of the positioning of each individual volume ≥ 0.125 m3, in purple, of lysosomal labelling measured from the cell. Thus, examination of cells showed thatlgp120- labelling

Fig. 4.4 Visualisation of lgp120 labelling within an isolated pulmonary artery smooth muscle cell:(i)transmitted light image of an isolated pulmonary artery smooth muscle cell.(ii) deconvolved Z-section (focal depth 0.28 m) taken through the cell shown in(i)showing the distribution of lgp120 labelling, indicative of the distribution of lysosomes in green. The nucleus of the cell is shown inblue. Arrows 1 – 3 indicate areas of lysosomal labelling.(iii)3D reconstruction of a series of Z-sections (focal depth 0.28m, Z step 0.2m) obtained through the cell shown in(i)showing the distribution oflgp120 labelling ingreen. The nucleus of the cell is shown inblue.(iv)3D representation of the distribution of individual volumes≥ 0.125 m3(inpurple) oflgp120 labelling measured in cells. Individual volumes of labelling were determined using Volocity software (Improvision, UK). (v)shows the individual volumes of lgp120 labelling as in(iv)coloured to indicate the region of the cell in which they are located; volumes within the perinuclear, extra-perinuclear and sub-plasmalemmal regions are shown in orange, pinkandblue, respectively.

occupied a volume of 29.84 ± 3.47 m3 in pulmonary artery smooth muscle cells (n = 24; Appendix 2, Table 4.3). This equated to a density of labelling

within cells of 0.019 ± 0.003m3 of labelling per m3of cell volume (n = 24; Appendix 2, Table 4.3). The spatial distribution of these volumes of labelling was then examined to provide a measure of the fluorescent labelling within each of the three different regions of the cell. Fig. 4.4(v) shows a 3D visual representation of the regionalised distribution of each individual volume of labelling within the cell shown in Fig. 4.4(iv). Each volume of labelling in Fig. 4.4(v) is coloured to indicate the region of the cell in which it is located. Thus, volumes of lysosomal labelling located in the perinuclear, extra-perinuclear and sub-plasmalemmal regions are visualised in orange, pink and light blue, respectively. Volumetric analysis of the lysosomal labelling located within the three different regions of cells indicated that both the volume and the density of labelling were greater in the perinuclear region of cells than either the extra- perinuclear or sub-plasmalemmal regions. Lysosomal labelling occupied a volume of 16.25 ± 3.18 m3 in the perinuclear region of cells (n = 24; Appendix 2, Table 4.4). This equated to a density of 0.032 ± 0.006 m3 of labelling per m3 of the perinuclear region (Fig. 4.5; Appendix 2, Table 4.4). In contrast, lysosomal labelling in the extra-perinuclear region occupied a volume of 6.72 ± 1.47m3(n = 24; Appendix 2, Table 4.5), accounting for a density of 0.018 ± 0.006 m3 of labelling per m3 of the region (Fig. 4.5; Appendix 2, Table 4.5). The sub-plasmalemmal region of cells contained both the lowest volume and the lowest density of lysosomal labelling measured in cells. In the sub-plasmalemmal region, lysosomal labelling occupied volume of 5.93 ± 1.68m3(n = 24; Appendix 2, Table 4.5) and a density of 0.01 ± 0.003

m3of labelling perm3of the region (Fig. 4.5; Appendix 2, Table 4.6).

A direct comparison between the densities of lysosomal labelling within the three regions of the cells examined is indicated in the bar chart in Fig. 4.5. Statistical comparison of the densities of labelling confirmed that a significantly greater density of labelling was present in the perinuclear region than either the extra-perinuclear (P = < 0.05; Appendix 2, Table 4.7) or the sub-plasmalemmal regions of cells (P = < 0.05; Appendix 2, Table 4.7). Furthermore, the density of labelling within the sub-plasmalemmal region of cells was significantly lower than that detected in the extra-perinuclear region (P = < 0.05; Appendix 2, Table 4.7). Therefore, these data suggest not only that

lysosomal labelling predominates within the perinuclear region but also that the density of labelling declines progressively as one moves from the nucleus to the plasma membrane of cells.

Fig. 4.5 Comparison of the density (m3 of labelling per m3 of region) of lysosomal labelling within the 3 regions of isolated pulmonary artery smooth muscle cells. * indicates statistical difference (P = ≤ 0.05) when compared to the perinuclear region. indicates statistical difference (P =≤0.05) when compared to the extra-perinuclear region (n = 24).

A comparison was then carried out to examine whether there was a difference in the mean volumes of individual elements of lysosomal labelling within the different regions of cells. Separate elements of fluorescent labelling which are closer together than can be resolved with the imaging system used to acquire data are recorded as single elements of labelling. Thus, the larger the individual volumes of lgp120 labelling detected, the larger the degree of lysosomal clustering. The difference between the mean volumes of labelling within the regions of the cell is indicated in the bar chart in Fig. 4.6. The mean volume of labelling within the perinuclear region of cells was 0.76 ± 0.14 m3 (n = 24; Fig. 4.6; Appendix 2, Table 4.4). The mean volumes of labelling were seen to decrease out with the perinuclear region of cells. Thus, the mean volume of labelling within the extra-perinuclear region of cells was 0.59 ± 0.13

elements of lysosomal labelling within the sub-plasmalemmal region of cells was 0.32 ± 0.06m3(n = 24; Fig. 4.6; Appendix 2, Table 4.6).

Although Fig. 4.6 shows a clear trend for a decrease in the mean volume of elements of labelling between the perinuclear and extra-perinuclear regions of cells, this decrease was not statistically significant (P = > 0.05, Appendix 2, Table 4.8). However, the mean volume of individual elements of labelling within the sub-plasmalemmal region of cells was significantly smaller than those observed in either the perinuclear (P = < 0.05; Appendix 2, Table 4.8) or extra-perinuclear regions (P = < 0.05; Appendix 2, Table 4.8). Therefore, the mean volume of individual elements of lysosomal labelling appeared to decrease across the cell from the perinuclear region to the sub- plasmalemmal region (Fig. 4.6).

Fig. 4.6Comparison of the mean volume (m3) of lysosomal labelling within the 3 regions of isolated pulmonary artery smooth muscle cells. * indicates statistical difference (P =≤ 0.05) when compared to the perinuclear region. indicates statistical difference (P = ≤ 0.05) when compared to the extra-perinuclear region (n = 24).

I can conclude, therefore, that the density of lysosomes was greatest in the perinuclear region of pulmonary artery smooth muscle cells, where a high degree of lysosomal clustering was also evident. Given these data, coupled with the finding that lysosomal clusters colocalised with a subpopulation of RyRs in pulmonary artery smooth muscle cells (Section 4.3.1), I proceeded to

examine whether areas of colocalisation were formed between lysosomes and a specific subtype of RyRs.

4.2.3 Spatial distribution of ryanodine receptor subtypes within pulmonary