Disposiciones Generales

Monitoring phosphorylation levels by microinjection of Fab fragments has two pitfalls: 1. antibody binding kinetics are rate-limiting, and 2. binding of antibodies to the receptor may stearically inhibit receptor interactions resulting in attenuated receptor transphosphorylation. These pitfalls prevent accurate determination of the spatial extent of receptor phosphorylation at early time points. Therefore, in order to more accurately determ ine the extent o f phosphorylation, experim ents w ere perform ed on

paraformaldehyde fixed cells (see section 2.3.5). Firstly, the precise spatial distribution of receptor phosphorylation at early time points after bead stimulation was analysed. Liquid handling during fixation, antibody incubation and washing of cells on coverslips is liable to remove cell-bound beads. However, by avoiding excessive turbulence during liquid handling, cell-bound beads could be retained in their original position on cells due to the strong interaction between EGF and the ErbBl receptor. This enabled the spatial distribution of receptor phosphorylation to be correlated with EGF-bead position in fixed samples (Fig. 3.4). At stimulation times shorter than 1 minute after bead application, cells could be found where receptor phosphorylation was present in spatially confined ‘patches’ or polarised across one side of a cell. Under these conditions, bead position could be correlated with the polarised phosphorylation (10s and 30s, Fig. 3.4 A). In contrast, after 1 minute of bead stimulation all cells exhibited a distributed phosphorylation pattern around the entire rim of the cell that was no longer polarised (60s, Fig. 3.4 A). Elevated phosphorylation levels in a rim around the edge of cells were also observed with soluble EGF stimulus.

The apparent presence of higher phosphorylation levels around the rim of cells can be explained as follows. In general, most transfected cells contain two spatially distinct populations of ErbBl-G FP receptors: a major portion of the receptors are localised to the plasma membrane, but some are present in membranes of the secretory pathway or in endosomes. The FLIM images are acquired in a wide field microscope and consequentially the fluorescence measured at each pixel is contributed by GFP both on the surface of the cells and intracellular GFP. Intracellularly-retained ErbB l-G FP is inaccessible to ligand and remains nonphosphorylated, therefore where the cells are thickest (in the centre of the cell) there is a greater contribution of fluorescence from internal, nonphosphorylated receptors. This gives rise to lower apparent levels of phosphorylated receptors measured in the centre of the cells. In contrast, cells are thinner at the edge, so there is less contribution from intracellular ErbBl-GFP. Therefore, around the edge of cells, the signal from surface phosphorylated receptors predominates and phosphorylated receptor populations appear higher at the edge than in the centre.

The data from fixed cells confirmed that, after focal stimulus of ErbBl receptors, phosphorylation is propagated to ligand-unoccupied receptors across the entire plasma membrane. However, the presence of free EGF or PTP inhibiting compounds in the bead preparation, or leakage of non-covalently absorbed EGF from the beads, might also

____________________________________________________________________Chapter 3 produce the observed widespread phosphorylation pattern. Some observations were made which rule out these possibilities. In Fig 3.4 B two cells imaged at 60 seconds after bead stimulus are shown. The bottom cell exhibiting raised phosphorylation levels is in contact with a bead whereas the top cell that is not directly contacted, but lies proximal to several beads, is quiescent. This demonstrates that contact of an EGF-coated bead with a cell is required to induce E rbB l-G FP phosphorylation and that possible leaching of non- covalently bound EGF from the beads did not occur. As a further precaution in several experiments beads were also washed again prior to use, but this had no significant effect on the results indicating that the preparation was free from contaminants.

In order to quantitatively compare the levels of phosphorylated receptor generated after both homogeneous and focal stimulation, large sets of cells were processed and imaged after stimulation for different times (5 seconds - 30 minutes). The average percentage of phosphorylated receptors was quantified at different time points after both soluble and focal stimulation (Fig. 3.5 C). Examples of cells used to calculate each data point are presented in Fig. 3.5 A and B. Both types of stimuli induced phosphorylated receptor levels exceeding those in non-stimulated cells as early as 5 seconds after stimulation and this phosphorylated population increased over time reaching a maximum at 1 minute after stimulation. Cells treated with soluble EGF exhibited a homogeneous pattern of phosphorylated receptor at the pre-1 minute time points (Fig. 3.5 A) whereas focal stimulation often resulted in the appearance of polarised receptor phosphorylation, as observed previously (Fig. 3.5 B). In accordance with this, the average phosphorylated receptor levels observed in cells at early time points after focal stimulus are lower than those detected after a soluble stimulus (Fig. 3.5 C).

The levels of phosphorylated receptors detected after 1 minute with both forms of stimulus were equivalent (EGF = 52 ± 3%; EGF-beads = 47 ± 7%). Additionally, the population images of these cells showed that, regardless of the means of stimulus, a similar pattern of phosphorylation was apparent after 1 minute: cells showed a homogeneous distribution of phosphorylated receptors in pixels around the entire periphery of the cell. In these pixels, the populations of phosphorylated receptors approached 100%. The populations of phosphorylated receptors fell after 1 minute (Fig. 3.5 C), in keeping with previously published data. This is due to the shielding of epitopes by phosphotyrosine binding proteins that associate with the receptor after internalisation (Wouters & Bastiaens, 1999).

3.4 A morphological study of cells provides independent evidence of receptor