The finding that NAADP mediates Ca2+bursts by mobilizing Ca2+from non-SR stores (Boittin, et al., 2002) which are Bafilomycin A1-sensitive provides evidence in support of the view that acidic, lysosome-related Ca2+ stores represent an NAADP-sensitive Ca2+ store in pulmonary artery smooth muscle cells (Kinnear, et al., 2004). In order to further examine this proposal I investigated whether there was any evidence to suggest that the distribution of lysosomes within these cells may underlie the generation of the Ca2+ bursts
evoked by NAADP and the spatially restricted Ca2+ release events evoked by Bafilomycin A1. In order to do this I used the fluorescent indicator dye LysoTracker Red. As mentioned previously, LysoTracker Red is a weak base and accumulates within acidic organelles, such as lysosomes (see Chapter 2, Section 2.3.2). This allowed me to determine the spatial organisation of these sub-cellular organelles.
In deconvolved Z-sections (focal depth 0.28 m) taken through isolated pulmonary artery smooth muscle cells loaded with LysoTracker Red (0.5 – 2 nM, 30 min) the distribution of LysoTracker Red labelling revealed that the acid organelles formed clusters that were either located in a ring around the perimeter of the cell, or as spatially restricted clusters located close to the centre of the cell. However, it was noted that smaller clusters/individual lysosomes were also observed in other parts of the cell. Two typical examples of the cellular distribution of LysoTracker Red labelling are shown in Fig. 3.10. It was important to determine whether or not the organelles that were being labelled with LysoTracker Red were indeed lysosomes and, in order to
Fig. 3.10: LysoTracker Red fluorescent labelling of acidic organelles in isolated pulmonary artery smooth muscle cells.Panel A, left-hand image shows a brightfield image of the cell under investigation. The right-hand panel shows the corresponding LysoTracker Red (excitation 577 nm, emission 590 nm) fluorescence image from a deconvolved Z section (depth 0.28 m) taken through the centre of the cell. Note, the largest area of clustering located centrally within the cell as indicated by the arrow; Panel B, left-hand image shows a brightfield image of the cell under investigation. The right-hand panel shows the corresponding LysoTracker Red fluorescence image from a deconvolved Z section taken through the centre of the cell. Note, the cell shows areas of lysosomal clustering associated at the perimeter of the
do this, I carried out investigations using the compound glycl-L-phenylalanine 2-napthylamide (GPN). GPN is a substrate of cathepsin C, which leads to rupturing of lysosomes via osmotic lysis. Cathepsin C is a lysosomal cysteine protease and it can sequentially remove dipeptides from the N- termini of various peptides and proteins (Coffey and de Duve, 1968). It breaks GPN down into its component amino acids which, due to their polarity, are unable to exit the lysosome, leading to increased osmotic stress until eventually the lysosome ruptures (Jadot, et al., 1984; Berg, et al., 1994). Cathepsin C has previously been shown to only be expressed in end stage lysosomes (Jadot, et al., 1984; Berg, et al., 1994; Jadot and Wattiaux, 1995). Therefore, addition of GPN to cells will only lead to rupture of end stage lysosomes and subsequent loss of fluorescence from these organelles.
That LysoTracker Red labelled organelles lost fluorescence in a time- dependent manner following addition of GPN to the experimental chamber confirmed that these LysoTracker Red labelled organelles were indeed lysosomes. The osmotic lysis of lysosomes caused by the application of GPN may induce contraction of the cells due to the release of lysosomal Ca2+stores or as a result of altering cellular pH, which can alter Ca2+ homeostasis within the cell. In order to prevent any misinterpretation of results which may arise due to movement of cells as a result of contraction, cells were incubated for fifteen minutes with the compound ML-9, an inhibitor of myosin light chain kinase, which has previously been shown to prevent contraction of smooth muscle cells (Saitoh, et al., 1986). GPN has previously been shown to trigger a loss of LysoTracker Red fluorescence from acidic vesicles in a time-dependent manner, and subsequently abolish NAADP-mediated Ca2+ release from these vesicles in sea urchin eggs (Churchill, et al., 2002). Consistent with the findings by Churchill et al. in sea urchin eggs, it was noted that GPN (200
M), eliminated LysoTracker Red fluorescence within 15 minutes of application to pulmonary artery smooth muscle cells (Fig. 3.11; n = 6), which is consistent with the labelled organelles being lysosomes. The spatial distribution of Ca2+ burst events have been shown to be manifested in two primary forms (Boittin, et al., 2002). Firstly, Ca2+bursts were seen to generate
a ring of Ca2+ release around perimeter of the cell, proximal to the plasma membrane, or were seen as spatially restricted focal Ca2+bursts covering an
Fig. 3.11: GPN depletes LysoTracker Red labelling of lysosomes in a time dependent manner:(i)Shows a brightfield image of an isolated pulmonary artery smooth muscle cell,(ii) corresponding LysoTracker Red fluorescence in a deconvolved Z section (depth 0.28 m) taken through the centre of the cell in (i).(iii – iv)LysoTracker Red fluorescence 5(iii), 10(iv) and 15(v)minutes after application of glycylphenylalanine 2- napthylamide (GPN; 200M).
area of between 2 – 10 m across the cell. Consistent with the spatial distribution of the Ca2+ bursts generated in response to NAADP (Fig 3.12; A (ii) and B(ii), experiments carried out by Dr. F.X. Boittin), the large areas of LysoTracker Red labelling observed within isolated pulmonary artery smooth muscle cells appeared in either one or two distinct forms. Firstly, the LysoTracker Red staining was seen to form a “ring” proximal to the plasma membrane of the cell (Fig 3.12A(i)). This ring was seen to be approximately 2
m in diameter in the cells examined (n = 5). Secondly, LysoTracker Red fluorescence was seen to form spatially restricted units that were primarily located in the centre of the cell, close to a large area devoid of labelling which may indicate the position of the nucleus within the cell (Fig. 3.12B(i)). These tight lysosomal clusters were seen to be approximately 2 – 6m across (n = 6). Thus, we may conclude that the spatial organisation of lysosomes in pulmonary arterial smooth muscle cells approximates that of the primary forms of Ca2+ bursts induced by NAADP.
Fig 3.12:Distribution of lysosomal clusters closely mirrors NAADP-mediated Ca2+bursts in isolated pulmonary artery smooth muscle cellsPanel A:(i)Shows a bright field image of an isolated pulmonary artery smooth muscle cell and(ii) the corresponding LysoTracker Red fluorescent image in a deconvolved Z section (depth 0.28 m) taken through the cell. The arrow indicates LysoTracker staining running proximal to the plasma membrane.(iii)shows a pseudocolour representation of a Ca2+ burst (indicated by the arrow) arising proximal to the plasma membrane in response to intracellular dialysis of NAADP (10 nM) in a different pulmonary artery smooth muscle cell.Panel B: (i)Shows a bright field image of a different isolated pulmonary artery smooth muscle cell and,(ii); the corresponding LysoTracker Red fluorescent image in a deconvolved Z section (depth 0.28 m) taken through the cell. The arrow indicates a spatially restricted lysosomal cluster located in the centre of the cell. (iii) Shows a pseudocolour representation of a spatially restricted Ca2+burst elicited in response to NAADP (10 nM) in a different isolated pulmonary artery smooth muscle cell as indicated by the arrow.
Following 3D reconstruction of deconvolved Z stacks (depth 0.28 m, Z-step 0.2 m) using the Softworx software (Fig. 3.13(iii)) I was able to examine the volume occupied by the spatially restricted primary lysosomal clusters within cells (Fig. 3.13(iv) and Appendix 1, Table 3.15). 3- dimensional
reconstructions of cells were rotated around the X- or Y-axis through 0o, 90o, 180o and 270o. At each point of rotation, measurements were taken of length and height of the largest discernable lysosome clusters (Fig. 3.12(v – viii). The volumes of the lysosomal clusters were then determined. In order to limit the amount of dead space in the volume measurements, clusters which appeared cylindrical upon rotation volumes were determined using the equation for the volume of a cylinder:
Volume = h xπx r2 Equation 3.1
Where h is the height of the cluster measured in m,π is taken to be equal to 3.14, and r2 is the square of the radius. The radius is equal to half of the diameter, and the diameter is taken to be the length across the cluster, measured in m, when viewed face on. Lysosomal clusters which were not seen to be cylindrical in appearance had there volume determined by applying the equation used to determine the volume of a box:
Volume = l x b x h Equation 3.2
Where l is the length of the cluster in m, b is the width of the cluster in m and h is the depth of the cluster in the Z-plane measured in m. These measurements allowed for calculation of the approximate volume occupied by the lysosomal clusters. From the measurements gathered from these cells, the volume occupied by the largest separable lysosomal clusters identified in each cell studied, was seen to range from 7.84 m3to 42.83m3. This represented a mean volume of 27.42 ± 10m3(n = 3, Appendix 1, Table 3.15).
Fig. 3.13: Measurement of the largest individual cluster of lysosomes in an isolated pulmonary artery smooth muscle cell: (i) shows a bright field image of an isolated pulmonary artery smooth muscle cell, (ii) the corresponding deconvolved LysoTracker Red fluorescence image from a Z section (depth 0.28m) through the middle of the cell, (iii)the corresponding 3- dimensional reconstruction of deconvolved stack of Z sections (40 Z’s, depth 0.28m, Z-step 0.2m); note lysosomes form a spatially restricted cluster ~ 6m across as indicated by the white rectangle;(iv)shows a schematic diagram of the volume occupied by the largest lysosomal cluster in the cell shown in (iii), as indicated by the white rectangle;(v – viii)three dimensional reconstruction of the largest lysosomal cluster in the cell shown in panel (iii) as indicated by the white rectangle , measured for volume through 0o(v), 90o(vi), 180o (vii)and 270o (viii); white lines indicate the distance measured for volume measurements in micrometers (m).
These data show that lysosome-related organelles in pulmonary artery smooth muscle cells form dense clusters that may provide an NAADP-sensitive Ca2+ store with a high degree of spatial organisation. Thus, the spatial organisation of these acidic organelles within isolated pulmonary artery smooth muscle cells is quite different to the granular pattern of discrete vesicles observed in sea urchin eggs (Churchill, et al., 2002).