ANÁLISIS DE LA POSICIÓN DEL TRIBUNAL CONSTITUCIONAL PERUANO COMO

From the analysis of the distribution profiles of RyR subtypes and αIgp120

labelling, perhaps the most striking observation is that dense clusters of both αIgp120

and RyR3 labelling were found within the perinuclear region of PASMCs. The similar distribution profiles of both αIgp120 and RyR3 labelling would suggest that

lysosomes may couple with a specific subtype of RyRs, namely RyR3. With this in mind, the co-localisation between each RyR subtype and lysosomes within isolated PASMCs was investigated. This was achieved by analysing the total density, and the regional distribution of co-localisation between labelling for αIgp120 and each RyR

subtype.

3.3.3.1 Lysosomes preferentially co-localise with ryanodine receptor subtype 3 in isolated pulmonary arterial smooth muscle cells.

RyR3 showed significantly greater co-localisation with αIgp120 labelling than

αIgp120 and RyR3, which are both predominantly localised in the perinuclear region.

Example cells co-labelled for αIgp120 and either RyR3, RyR2 or RyR1 are shown in

Fig. 3.8A, B and C respectively. In each example panel a shows a transmitted light image of an exemplar cell and panel b shows the distribution of labelling for αIgp120

(green) relative to the DAPI labelled nucleus (blue) in a 3D reconstruction of a series of deconvolved Z-sections (focal depth 0.28 µm, Z step 0.2 µm). Panel c shows

another 3D reconstruction of deconvolved Z-section in the same cell representing the distribution of labelling for the given RyR subtype (red) relative to the DAPI labelled nucleus. To visualise areas of co-localisation Z-sections were superimposed resulting in a deconvolved Z-section showing the distribution of RyR subtype (red), αIgp120

(green) and areas of co-localisation visualised in yellow, shown in panel d. Panel e

shows a 3D reconstruction of the superimposed deconvolved Z-sections showing the distribution of labelling for the given RyR subtype (red), αIgp120 (green) and co-

localisation between the two labels (yellow) relative to the DAPI labelled nucleus. In order to allow for visualisation of the position of co-localisation between αIgp120 and

a given RyR subtype, panel f shows the distribution of each individual element of co- localisation (yellow) in a 3D reconstruction of deconvolved Z-sections with non-co- localised elements of αIgp120 and RyR-labelling removed.

Visual comparison of the cells presented in Fig. 3.8 would suggest that although

αIgp120 co-localises with all RyR subtypes, there is a greater degree of co-

localisation with RyR3 than with either RyR2 or RyR1. This is further illustrated in Fig. 3.9A which shows the mean (± S.E.M.) density of co-localisation between

αIgp120 and each RyR subtype across the entire cell volume of the PASMCs

examined (i.e. no regional subdivision). RyR3 showed a significantly greater density of co-localisation with αIgp120 (0.006 ± 0.0006 µm3 per µm3, n = 11; Appendix 1,

Table 3.27) when compared to either RyR2 (0.0034 ± 0.0007 µm3 per µm3; n = 14; P < 0.05; Appendix 1, Table 3.23) or RyR1 (0.0031 ± 0.0007 µm3 per µm3, n = 10; P <

0.05; Appendix 1, Table 3.19). This 2-fold greater density of co-localisation of RyR3 with αIgp120 provides further confirmation that lysosomes preferentially co-localise

with RyR3 in PASMCs. This is also evident from the fact that within the whole cell, RyR3 was found to co-localise with 26 ± 3 % (n = 11; Appendix 1, Table 3.31) of the total volume of lysosome labelling, whereas labelling for RyR2 and RyR1 co-

localised with only 16 ± 2 % (n = 10; Appendix 1, Table 3.31) and 15 ± 3 % (n = 14; Appendix 1, Table 3.31) of the total volume of lysosomal labelling respectively.

Fig. 3.8. Lysosomes preferentially co-localise with ryanodine receptor subtype 3 within isolated pulmonary arterial smooth muscle cells

Example cells showing the degree and spatial distribution of co-localisation between αIgp120 labelling and labelling of RyR3 (A), RyR2 (B), and RyR1 (C). In each case the panels show the following: Panel a shows a transmitted light image of the isolated PASMC imaged; Panel b shows a 3D reconstruction of a series of deconvolved Z-sections (Z step 0.2 µm) taken through the cell shown in

panel a showing the spatial distribution of αIgp120-labelled lysosomes (green) in relation to the plasma membrane (dotted line) and nucleus (dark blue); Panel c shows a 3D reconstruction of deconvolved Z-sections showing the distribution of RyR labelling (red) in relation to the plasma membrane and nucleus as indicated in panel b; Panel d shows a deconvolved Z-section (focal depth 0.28 µm) taken through the PASMC showing the distribution of labelling for αIgp120, the given RyR subtype and the co-localisation between the two in relation to the nucleus of the cell (dark blue) and the plasma membrane, with areas of co-localisation shown in yellow; Panel e shows a 3D representation of RyR subtype and αIgp120 labelling (red and green respectively); Panel f is a 3D reconstruction showing individual volumes of co-localisation between αIgp120 and a given RyR subtype.

The preferential co-localisation of lysosomes with RyR3 is further demonstrated in Fig. 3.9B, which shows the Pearson product-moment correlation coefficient values for the co-localisation between αIgp120 and each RyR subtype. This coefficient

value (ranging from -1 to 1) is an indication of the strength of association between two continuous variables. Fig. 3.9B shows that the co-localisation between αIgp120

and RyR3 had a significantly greater correlation (0.26 ± 0.01, n = 11; Appendix 1,

Table 3.27) than observed for either RyR2 and αIgp120 labelling (0.18 ± 0.01, n =

14; P < 0.05; Appendix 1, Table 3.23) or RyR1 and αIgp120 labelling (0.16 ± 0.01, n

= 10; P < 0.05; Appendix 1, Table 3.19). It is important to note, that the Pearson

product-moment correlation coefficient value is a measure of the correlation in the whole cell and not focussed on any particular region. Therefore, despite the greater degree of correlation between αIgp120 and RyR3 than for either RyR2 or RyR1, this

is likely to be an underestimate of the degree of correlation between αIgp120 and

RyR3 in the perinuclear region. The greater Pearson coefficient values, together with the greater density of co-localisation across the entire cell volume does, however, provide further evidence in support of the proposal that lysosomes preferentially co- localise with RyR3 in PASMCs.

Fig. 3.9. Co-localisation is greater in cells co-labelled for RyR3 than those co-labelled for RyR2 and RyR1

A Bar chart showing the density of co-localisation (µm3 per µm3; mean ± S.E.M.) between αIgp120 labelling and each RyR subtype within the whole cell volume. B The Pearson product-moment correlation coefficient for each RyR subtype indicating linear dependence (mean ± S.E.M.). *

indicates a statistically significant difference ( P< 0.05) when compared to the RyR3/αIgp120 co- localisation density or Pearson correlation coefficient (RyR1, n = 10; RyR2, n = 14; RyR3, n = 11).

3.3.4 Examination of the regional distribution of co-localisation between lysosomes