MARCO SITUACIONAL

Figure 3.3.

Primary Gradients Secondary Gradients

B 60-1 5 0 - 1 4 0 - 0) % 3 0 - o 2 2 0 - 1 0 -

I

I

I

I

1

iC È l

il

A P-hexosaminidase, stimulated 25mins □ pH]-serotonin, resting

A pH]-serotonin, stimulated 25mins ■ HRP-P-selectin, resting

♦ HRP-P-selectin, stimulated 5mins • HRP-P-selectin, stimulated lOmins

HRP-P-selectin, stimulated 25mins

X 7) 73 fil !■ 85

contrast, when cells are stimulated to exocytose for 25 minutes, 47% of the

secretory lysosome markers are released. This 47% release is in good agreement with the 45% fall in p-hexosaminidase activity from the subcellular profile of the secretory lysosome in Figure 3.3.A.

3.5 Effects of mutations in the cytoplasmic tail of HRP-P-selectin on secretorv lysosomal targeting

I compared the targeting of wt HRP-P-selectin to secretory lysosomes with that of tetra-alanine mutant chimeras of the cytoplasmic sequences KCPL, YGVF and DPSP and of the deletion mutant HRP-P-selectin, which lacks all three motifs (Table 3.1). These three motifs have been implicated in lysosomal, secretory granule and SLMV targeting (Figure 1.4 of Chapter One).

HRP-P-selectin cvtoolasmic tail

wt HRP-P-selectin RKRFRQKDDGKCPLNPHSHLGTYGVFTNAAFDPSP KUHL HRP-P-selectin _{RKRFRQKDDGAAAANPHSHLGTYGVFTNAAFDPSP} Yüvh HRP-P-selectin RKRFRQKDDGKCPLNPHSHLGTAAAATNAAFDPSP uKsp HRP-P-selectin _{RKRFRQKDDGKCPLNPHSHLGTYGVFTNAAFAAAA}

/Ba HRP-P-selectin RKRFRQKD

Table 3.1 Schematic il ustration of the cytoplasmic tail of HRP-P-selectin chimeras. showing the name of each chimera to the top left, which represent sequences that have been replaced by alanine (underlined and in bold), or deleted.

Rbl cells were transiently transfected with wt, and HRP-P- selectin and cultured for three days. Fractionation was then carried out and the subcellular profile across the primary sucrose gradient for each chimera is shown in Figure 3.4.A. The secretory lysosome fractions are highlighted in grey (14-20). The majority of wt, and HRP-P-selectin activity is found in the secretory lysosome peak, all three having very similar profiles. However, much lower levels of HRP activity within the secretory lysosome peak are found with the mutants and HRP-P-selectin, which coincide with a significant accumulation of HRP activity elsewhere on the gradient. The profiles for and HRP-P-selectin

differ in that there is a significant shoulder to the major peak for HRP-P-selectin such that it covers fractions 6-15, while the peak for HRP-P-selectin is sharper (fractions 10-15). Mutating the sequence KCPL, which is implicated in lysosomal targeting to tetra-alanine has a similar effect on targeting to the secretory lysosome as the complete removal of the last 27 residues of the cytoplasmic domain.

Expression of HRP-P-selectin in H.Ep.2 cells results in its localisation to transferrin-positive compartments, and HRP-P-selectin is localised at the plasma membrane (Blagoveshchenskaya et al. 1998b, Blagoveshchenskaya et al. 1998a). Figure 3.4.B illustrates the distribution of [^^^l]-transferrin following loading of transfected cells for 1 hour, and internal stores of [^^®l]-transferrin after stripping transferrin bound at the plasma membrane (reproduced from Figure 3.2.A). Internal transferrin peaks at fractions 11-12, while plasma membrane-bound transferrin peaks at fractions 5-6. A substantial proportion of HRP activity for the mutants and HRP-P-selectin cod istri butes with the internal endosomal load of [^^^1]- transferrin (fractions 11-12). The shoulder of HRP activity for HRP-P-selectin (fractions 6-15) codistributes in part with the plasma membrane and mainly with endosomes. All the HRP chimeras in Figure 3.4.A show a peak of activity at fraction 4 that does not codistribute with any marker assayed, and may represent newly synthesised material within the ER and Golgi.

To investigate the targeting of each mutant to the two peaks of the secretory lysosome identified on secondary gradients, I pooled fractions 14-20 (highlighted) and ran them on a secondary sucrose gradient as in Figures 3.2.B, 3.3.B and 3.3.D. It is clear from Figure 3.4.C, that and HRP-P-selectin show a similar distribution as the wt chimera, localising to the two peaks that also contain P-hexosaminidase and [^H]-serotonin. Hence there is no differential targeting of the mutant chimeras between the two peaks. The HRP activity that appeared to be within the secretory lysosome peak for '^^^'■and HRP-P-selectin in Figure 3.4.A is now seen to partially codistribute with the f ^^l]-transferrin positive peak (fraction 8), rather than with the secretory lysosome markers.

Figure 3.4

Subcellular distribution of HRP-P-selectin chimeras

Rbl-2H3 cells were transiently transfected with wt, and HRP-P- selectin three days prior to analyses. Cells were homogenised and the PNS run on primary (A) and secondary gradients (C), and analysed for HRP activity, as

described in Figure 3.3. Individual traces for each mutant are shown as a

percentage of total HRP activity on the gradient, and are plotted on the same scale axis. The subcellular markers assayed in Figure 3.2 are shown again in B and D,

to highlight areas of codistribution. Shaded areas of A and B represent fractions 14-20 that were pooled and fractionated on secondary gradients (C and D).

Figure 3.4 Primary Gradients Y G V F K C P L 3H]-serotonin 6-hexosamin 25|]-tfn + strip Secondary Gradients 0 5 0 Fraction number 10 15 20 25 Fraction number n 89

3.6 Immunofluorescence microscopy of HRP chimeras and serotonin

To confirm the subcellular localisation of the HRP chimeras I visualised HRP using Tyramide Signal Amplification (ISA), which uses HRP activity to catalyse the deposition of cyanine 3-tyramide immediately adjacent to the immobilised HRP-P- selectin chimeras. Cells were also stained with an antibody to the secretory

lysosome marker serotonin and Figure 3.5 shows the fluorescence pattern of HRP and serotonin in cells transfected with wt, and HRP-P-selectin. The majority of wt HRP-P-selectin is found to co-localise with serotonin-positive structures, as highlighted by the magnification of one of the processes of the cell. A few

structures stain only for serotonin and not HRP, these most likely reflect secretory lysosomes formed before or after the transient expression of wt HRP-P-selectin. Also present are a small number of HRP-positive structures that appear devoid of serotonin, which may represent the biosynthetic traffic of HRP-P-selectin en route to the secretory lysosome. In contrast, HRP-P-selectin and HRP-P-selectin localise to very distinct structures from serotonin. HRP-P-selectin label is especially enriched at the ends of the processes of the cells. The localisation of 763HRp-p-seiectin is more diffuse, mainly at the plasma membrane, and also within internal structures that do not colocalise with serotonin.

3.7 Intracellular proteolvsis (TX114 assav)

Once HRP-P-selectin is delivered to and accumulates within protease-rich

environments, the chimera becomes subject to proteolytic attack such that soluble HRP is released from the P-selectin membrane anchor. This method has

previously been used as a monitor of lysosome targeting (Blagoveshchenskaya and Cutler 2000a, Blagoveshchenskaya et al. 1999a, Blagoveshchenskaya et al. 1998b, Blagoveshchenskaya et al. 1998a). I made use of this phenomenon as an independent measure of targeting to the secretory lysosome. Partitioning of HRP activity between two phases of TX114 reveals the extent of proteolysis for each of the HRP chimeras (Figure 3.6), with 48.6% of wt HRP-P-selectin HRP in its soluble form and 24.2% proteolysis for HRP-P-selectin. HRP-P-selectin has a very similar value to that of the -deletion mutant (27.4%), but the extent of