Resultados del análisis de brechas de cantidad

Given that NAADP has been shown to mediate Ca2+ bursts from a lysosome-related Ca2+ store that are then amplified into global Ca2+ waves through CICR via RyRs on the SR, I used the fluorescent probes LysoTracker Red (0.5 – 2 nM; excitation 577 nm, emission 590 nm) and BODIPY-FL ryanodine (1 M; excitation 480 nm, emission 510 nm) to examine whether there was an underlying structural basis for this mechanism of Ca2+ signalling within freshly isolated pulmonary artery smooth muscle cells. To this end I examined whether or not lysosomes colocalised with RyRs expressed on the SR.

Fig. 4.1 shows 2 typical representations of the distribution of lysosomes and RyRs in isolated pulmonary artery smooth muscle cells. Fig. 4.1A(i) shows a transmitted light image of an isolated pulmonary artery smooth muscle cell. Fig. 4.1A(ii) shows the corresponding LysoTracker Red fluorescence, visualised in red, in a single deconvolved Z-section (focal depth 0.28 m) acquired through the centre of the cell shown in Fig. 4.1A(i). In this cell, there is clear evidence of a number of small lysosomal clusters diffusely distributed throughout the cytoplasm, with the largest area of lysosomal clustering positioned in the centre of the cell, as highlighted by arrow 1. Fig. 4.1A(iii) shows BODIPY-FL ryanodine fluorescence, visualised in green, acquired in a deconvolved Z-section (focal depth 0.28m), acquired at the same Z-depth as A(ii). BODIPY-FL ryanodine labelling was seen throughout the cytoplasm in all cells studied, as would be expected given that RyRs have previously been shown to be located upon both peripheral and central sarcoplasmic reticulum (SR) in vascular smooth muscle cells (Lesh, et al., 1998; Gordienko, et al., 2001; Yang, et al., 2005). In isolated pulmonary artery smooth muscle cells BODIPY-FL ryanodine labelling was seen to form ‘ribbons’ weaving through the cytoplasm, an example of which is highlighted by arrow 2 in Fig. 4.1A(iii).

Fig. 4.1 Lysosomes and RyRs colocalise in arterial smooth muscle cells: Panel A(i) transmitted light image of an isolated pulmonary artery smooth muscle cell.; Panel A(ii) deconvolved Z-section (focal depth 0.28 m) through the same cell as in A(i) shows lysosomes labelled with LysoTracker Red (0.5 nM, 45 min. incubation). A large area of lysosomal clustering is indicated with arrow 1;A(iii)deconvolved Z-section through the same cell as inA(i)shows the distribution of ryanodine receptors (RyRs) labelled with BODIPY-FL ryanodine (1 M, 60 min incubation). Arrow 2 indicates a ribbon of BODIPY-FL ryanodine labelling;A(iv) composite image ofA(ii) andA(iii) shows close association, as indicated by arrow 3, 4 and 5, of lysosomes and a subpopulation of RyRs. Panel B(i) transmitted light image of a different isolated pulmonary artery smooth muscle cell.B(ii)deconvolved Z-section (focal depth 0.28 m) through the same cell as in B(i) shows lysosomes labelled with LysoTracker Red (1 nM, 45 min. incubation).B(iii)deconvolved Z-section through the same cell as inB(i)shows the distribution of RyRs labelled with BODIPY-FL ryanodine (1M, 60 min. incubation).B(iv) composite image ofB(ii) andB(iii)shows close association between lysosomes and RyRs, arrow 1 indicates an area of close association between lysosomes and a subpopulation of RyRs.

These ribbons of BODIPY-FL ryanodine labelling are consistent with the distribution of the SR previously described within vascular smooth muscle cells (Devine, et al., 1972). Images of LysoTracker Red labelling and BODIPY-FL ryanodine were then superimposed upon one another. From this it was clear that there was an extremely close association, within certain areas of the cells, between lysosomes and a subset of RyRs. Areas of colocalisation are visualised in yellow (Fig. 4.1A(iv)). A large, dense area of colocalisation can be seen between the largest cluster of lysosomal labelling and RyRs in the centre of the cell indicated by arrow 3 in Fig. 4.1A(iv). The largest area of colocalisation within the cells examined was seen to be located next to an area largely devoid of labelling. The lack of labelling of RyR or lysosomes within this region may be indicative of the position of the nucleus. It is also worthy of note that while dense areas of colocalisation were observed within cells, a number of smaller areas of colocalisation were observed throughout the cytoplasm of the cell, as shown in the areas surrounding arrows 4 and 5 in Fig. 4.1A(iv). A second example showing the close association between LysoTracker Red- and BODIPY-FL ryanodine-labelling in a different isolated pulmonary artery smooth muscle cell can be seen in Fig. 4.1B(i). Once again, the colocalisation between dense lysosomal labelling and RyRs is evident within this cell as indicated by arrow 1 in Fig. 4.1B (iv). As with the example shown in Fig. 4.1A, the largest area of colocalisation in Fig. 4.1B(iv) is located close to an area devoid of labelling which may indicate the positioning of the nucleus of the cell.

Given that lysosomes represent a discrete, sub-cellular organelle, and that RyRs are expressed on the SR, the distance between these two sites of Ca2+release could have important functional implications. I therefore sought to determine the maximal distance between lysosomes and RyRs that existed within the same focal plane. To achieve this I examined 3-dimensional reconstructions of a series of deconvolved Z-sections acquired from isolated pulmonary artery smooth muscle cells (focal depth 0.28 m, Z-step 0.2 m) labelled with LysoTracker Red and BODIPY-FL ryanodine. An example of a 3-dimensional reconstruction of deconvolved Z-sections is shown in Fig. 4.2(i). In this cell a dense central plaque of lysosome and RyR colocalisation is

clearly evident, while a number of smaller areas of colocalisation can be seen throughout the cytoplasm of the cell. Following the generation of the 3- dimensional (3D) reconstruction, each area of colocalisation was examined and the cell was rotated in 1osteps through 360oaround the X- and Y-axis in order to determine whether, at any point, the two fluorescent labels could be separated from one another in the same focal plane (Fig. 4.2(ii)). When the two fluorescent labels could be separated, the cell was rotated to determine the point at which they lay within the same plane (Fig. 4.2(iii)a). In order to achieve this aim the cell was rotated until the lysosomal labelling was seen to

Fig. 4.2.Formation of trigger zone between closely associated lysosomes and RyRs:(i)3D reconstruction of deconvolved Z-sections (depth 0.28 m, Z-step 0.2 m) taken through an isolated pulmonary artery smooth muscle cell shows close association of lysosomes labelled with LysoTracker Red (0.5 nM, 45 min. incubation) and a subpopulation of ryanodine receptors (RyRs) labelled with BODIPY-FL ryanodine (1M, 60 min. incubation);(ii)shows the cell in(i)rotated 284° around theyaxis;(iii) two sequential enlargements (aandb) of the section of the cell in(ii)indicated with awhite rectangle:(iii) bselected area shows distance (d =≤0.4m) measured between a LysoTracker Red-labelled organelle and RyRs labelled with BODIPY-FL ryanodine colocalised in the same focal plane.

be located directly above the RyR labelling, and a note of the extent of rotation was taken. After this, the cell was again rotated until the RyR labelling was positioned directly above the lysosomal labelling; once more the degree of rotation was noted. From these measurements the point at which the fluorescent labels were positioned side by side at the same focal depth was determined and the cell was rotated to this point. A measurement of the distance between the BODIPY-FL ryanodine and the LysoTracker Red

Appendix 2, Table 4.1). From the measurements taken, it was seen that RyRs and lysosomes that were closely associated within the same focal plane were separated by a distance of≤0.4m (Fig. 4.2(iii)b; Appendix 2, Table 4.1; n = 12 areas from 9 cells). However, the majority of areas of colocalisation between RyRs and lysosomes were much smaller than could be resolved using the techniques at my disposal. Thus, it would appear that the junction or cleft between lysosomes and RyRs represents a very tight association between these elements. This close association between lysosomes and RyRs is well within the estimated maximum distance that Ca2+may diffuse within the cytoplasm of cells (≤ 5 m; Allbritton, et al., 1992). Therefore, this close association is ideally suited to a role in RyR-dependent amplification of lysosomal Ca2+ signals.

It is clear from these experiments that a large proportion of BODIPY- FL ryanodine labelled RyRs are not associated with LysoTracker Red labelled organelles. There are three known subtypes of RyRs found in mammalian cells, namely; RyR1 (Inui, et al., 1987b; Lai, et al., 1988; Takeshima, et al., 1995), RyR2 (Inui, et al., 1987a; Takeshima, et al., 1998) and RyR3 (Hakamata, et al., 1992; Sorrentino, et al., 1993; Takeshima, et al., 1996). All three of these RyR subtypes have previously been shown to be expressed in several types of

vascular smooth muscle, including pulmonary artery smooth muscle

(Herrmann-Frank, et al., 1991; Neylon, et al., 1995; Jeyakumar, et al., 1998; Coussin, et al., 2000; Mironneau, et al., 2001; Yang, et al., 2005). Thus, a particular subpopulation of RyRs may colocalise with lysosomes to form a trigger zone for Ca2+ signalling by NAADP. Therefore, I sought to determine whether or not a specific subtype of RyR colocalised with lysosomes in order to comprise the proposed trigger zone for NAADP-mediated Ca2+signalling.

4.2.2 Examination of lysosomal distribution in methanol-fixed pulmonary