Aplicación de los Principios:

FRET between ErbBl-GFP and a Cy3-labelled anti-phosphotyrosine antibody can be used to detect receptor phosphorylation in cells using FLIM (Wouters & Bastiaens, 1999). Here, a global analysis of the FLIM data was employed (Verveer et a i, 2000a), based on the assumption that each ErbBl-GFP molecule should be associated with one of two spatially-invariant lifetimes corresponding to the presence (phosphorylated receptor) or absence (nonphosphorylated receptor) of FRET. This has allowed the percentage of phosphorylated receptors present in single cells to be quantified.

Focal stimulus of cells with beads covalently-coupled to EGF results in rapid and extensive propagation of ErbBl receptor phosphorylation across the surface of the entire cell (Fig. 3.3, 3.4 and 3.5). The level and spatial distribution of phosphorylation obtained after one minute of stimulus is equivalent whether growth factor is applied as a saturating soluble EGF dose or using EGF-beads (Fig. 3.5). EGF-beads can initiate the same changes in cell morphology and ErbBl localisation as soluble EGF (Fig. 3.6). The spread of phosphorylation is not due to leaching of EGF from the bead because direct contact between beads and a cell is required for activation of ErbBl receptors (Fig. 3.4 B).

___________________________________________________________________Chapter 3 Instead, a bead-position correlated, polarised distribution of phosphorylation captured at pre-1 minute time points suggests that the phosphorylation signal spreads from the point source of ligand (Fig. 3.4 A). A lag in the levels of phosphorylated receptor detected after bead stimulus, as compared to soluble stimulus, supports the presence of an intermediate phase of signal spreading that occurs prior to the peak levels detected at 1 minute (Fig. 3.5 C). It is concluded that a focal stimulus with EGF results in lateral propagation of ErbB 1 receptor phosphorylation in the plane of the plasma membrane.

There are several conceivable interpretations as to the mechanism via which the spreading of phosphorylation might occur. It is convenient to split these hypothetical models into two broad categories: models that are dependent on long-range diffusion of protein tyrosine kinases and those that are based on a wavefront propagation of phosphorylation.

A. Diffusion-based models

(1). A cytosolic or membrane-associated protein tyrosine kinase (PTK), other than ErbBl, is activated by bead-bound ErbBl receptors and can then diffuse across the cell phosphorylating ligand-unoccupied ErbBl receptors (Fig. 3.7 A). This could be tested by measuring lateral propagation of phosphorylation under conditions which inhibit downstream signalling to PTKs.

(2). Rapid diffusion of ErbBl receptors through the area under the bead leads to phosphorylation of the receptors as they pass through (Fig 3.7 B). This could occur via one of two mechanisms. For example, passing receptors could be transphosphorylated by a stable pool of active, bead-bound receptors. Alternatively, receptors could become phosphorylated as a consequence of transiently binding EGF presented on the beads. In the latter case, a rapid exchange of bead-bound receptors with the free receptor pool would be necessary to elicit such an effect. In both cases, after activation at the bead, ErbB 1 must diffuse away from the bead and into the periphery of the cell to account for the widespread distribution of phosphorylated receptors observed after bead stimulus. The relevance of this model can be tested by characterising the diffusion of ErbBl-GFP in living cells.

B. Wavefront propagation models

(1). ErbB l receptors bound to the EGF-beads are able to transphosphorylate adjacent ligand-unoccupied ErbBl molecules, thereby activating the kinase activity of these receptors. The signal is then propagated outside the bead contact area via consecutive transphosphorylation events between adjacent ligand-unoccupied receptors (Fig. 3.7 C). The phosphorylation signal therefore proceeds via a ‘domino effect’ that spreads in a wavefront across the plasma membrane.

(2). A localised increase in ErbBl kinase activity underneath the bead inhibits the activity of protein tyrosine phosphatases (PTPs). In this model, ErbBl tyrosine kinase activity drives PTP inhibition, which in turn permits ligand-unoccupied receptors outside the bead contact point to become phosphorylated. Activation of these receptors invokes more PTP inhibition, which in turn leads to phosphorylation of more receptors i.e. a positive feedback mechanism is invoked. This is expected to also spread the phosphorylation signal in the form of a wavefront (Fig. 3.7 D). Significantly, this allows ligand- unoccupied ErbB 1 receptors outside the bead contact point to become phosphorylated without any requirement to be directly phosphorylated by bead-coupled receptors. This is in contrast to wavefront propagation model 1.

In wavefront propagation model 1, spreading of the phosphorylation signal is dependent on an enhanced ErbBl tyrosine kinase activity that is induced by tyrosine phosphorylation of the receptors, whilst in model 2, ErbBl tyrosine kinase activity is independent of the tyrosine phosphorylation status of the receptor, but sustained PTP inhibition is required. In order for either model 1 or 2 to be true, it must be the case that transphosphorylation can occur in transient, ligand-independent receptor dimers. In order for model 2 to be correct, pharmacological agents that inhibit PTPs must be able to induce ligand-independent phosphorylation of ErbBl to a similar extent as EGF-beads.

In the next Chapter the mechanisms underlying the observed signal spreading are further investigated. Experiments are performed to test predicted features of the above models in order to decipher between them.

Chap te rs Figures Figure 3.1 Global analysis of FLIM data from an ErbBl-GFP phosphorylation assay

A. Tyrosine phosphorylation of GFP-tagged ErbBl can be detected by measuring the fluorescence lifetime of the donor GFP when a Cy3-labelled antiphosphotyrosine antibody (Cy3-PY72) is bound. For the purpose of global analysis it is assumed that in all experiments: 1. all nonphosphorylated ErbBl-G FP molecules within the cell possess a unique, spatially invariant fluorescence lifetime, Tj, and 2 . all receptors

bound to antibody are associated with a unique, spatially invariant fluorescence lifetime, B. At every resolvable volume element of a cell (pixel) there will exist phosphorylated and nonphosphorylated ErbBl-G FP receptors (white box). In the given example, it is assumed that nonphosphorylated receptors are all associated with a lifetime of 2 . 2 ns ( r j and all phosphorylated receptors with a shorter lifetime of 0 . 8

ns (T2) due to FRET to the acceptor on the bound antibody. In the volume element

(pixel) shown, 40% of the receptors have a lifetime of 0.8 ns and 60% have a lifetime of 2.2 ns. Conventional analysis of frequency-domain FLIM data returns two measures of the fluorescence lifetime at each pixel (see Fig. 1.9). These estimates, calculated from the phase shift and demodulation values at each pixel, are frequency- weighted averages of the fluorescence lifetime which are dependent on the lifetimes of each individual species and the relative abundance of each species. For the given example, a conventional analysis of FLIM data acquired at a single modulation frequency of 80 MHz would yield a phase lifetime estimate of 1.62 ns for the pixel. In contrast, a global analysis o f the FLIM data would yield the lifetimes of each individual species (i.e. 2.2 and 0.8 ns) and the fraction of each species present (i.e. 0.4 and 0.6) in the pixel. C . The global analysis of ErbBl-GFP FLIM data makes use of the assumptions described in A and fits the data from each pixel to a two lifetime component model. This yields the true lifetime of each species (Tj and ; blue box), and the relative populations of each species as a molar fraction, on a pixel-by-pixel basis ( a map; yellow box). Colour bar indicates populations o f the Tj species (phosphorylated receptors) as a decimal fraction at each pixel.

Fig. 3.1