**12. Subtle changes make a big difference**

In signalling systems there are many components all vying for attention, and to initiate a response. However, even though it is individual components which are studied in many cases, the overall response of the cell will be dictated by the sum of the signalling which is taking place, an idea modelled by Rachmilewitz & Lanzavecchia (2002). Even so, within this holistic approach it needs to be realised that subtle changes in the levels of some signalling components can give a large effect even if there is no change in other proteins and molecules. Certainly some signals have been described as dominant over others. An example here would be a paper by Reya and Clevers (2005) who write "Current evidence indicates that the Wnt cascade is the single most dominant force in controlling cell fate along the crypt–villus axis". Therefore, a small change in a dominant pathway would initiate a significant response regardless of the activity of less dominant pathways.

Music is often like this, with the single notes in amongst many others having a profound effect. If a pianist plays a major chord, perhaps a C, E and G, to produce a chord of C major, the effect is recognisable, and being major will sound cheerful. However, keeping the C and G the same, but lowering the tone of the E by a semitone to E flat and the chord becomes minor. This is a significant effect. One note amongst the three has changed by a relatively small amount, but the chord is now recognisably different, and the resultant sound has gone from a happy major to a rather sad minor. A small musical change, with a large result. Beethoven uses the alteration of one note in amongst many to great effect in the first movement of the "*Moonlight Sonata*" (Piano Sonata no. 14 in C sharp minor - Op. 27 no 2) for example.

### **13. Background, volume and thresholds**

60 Biomedical Science, Engineering and Technology

Fig. 7. Cytochrome *c* has more than one function. Cytochrome *c* is normally found in the mitochondria. It resides associated with the inner mitochondrial membrane, where it acts to shuttle electrons from Complex III to Complex IV. However, there is a signalling pathway which leads to cell suicide, or apoptosis, in which cytochrome *c* leaves the mitochondria and moves into the cytoplasm. Here it interacts with the caspase system which leads to ultimately

to cell death. Therefore cytochrome *c* has two very distinct and disparate functions.

role they play at any moment in time.

**12. Subtle changes make a big difference** 

Musicians and their instruments are like such moonlighting proteins. They may play a violin, but in fact such instruments can be used in multiple music genres. During the day the violinist may be in an orchestra, but some evenings may be playing jazz in the local bar. As with the proteins, their roles are not fixed, and translocation from one venue to another will allow them to partake in a new role. Furthermore, many if not most musicians are experts at more than one instruments. Therefore if an orchestra is short of a viola player, then perhaps a violinist can take their place. Or the change of instrument can be more dramatic, with a flutist can take over on the kettle drums. Just like many proteins, temporal and spatial location of the player, and the interactions in which they may partake, may dictate the exact

In signalling systems there are many components all vying for attention, and to initiate a response. However, even though it is individual components which are studied in many cases, the overall response of the cell will be dictated by the sum of the signalling which is taking place, an idea modelled by Rachmilewitz & Lanzavecchia (2002). Even so, within this holistic approach it needs to be realised that subtle changes in the levels of some signalling components can give a large effect even if there is no change in other proteins and molecules. Certainly some signals have been described as dominant over others. An example here would be a paper by Reya and Clevers (2005) who write "Current evidence indicates that the Wnt cascade is the single most dominant force in controlling cell fate along the crypt–villus axis". Therefore, a small change in a dominant pathway would initiate a

Music is often like this, with the single notes in amongst many others having a profound effect. If a pianist plays a major chord, perhaps a C, E and G, to produce a chord of C major, the effect is recognisable, and being major will sound cheerful. However, keeping the C and G the same,

significant response regardless of the activity of less dominant pathways.

Signalling in cells needs to take place in the presence a background level of "signalling noise". Cells are bombarded by extracellular signals all the time, whether from the environment of the organism or from other cells of the same organism. The demands on a cell will be constant and varied in many cases. Therefore, if a major response is needed, the signalling that is invoked needs to be "heard" above the noise of the rest of the activity of the cell.

Most signalling systems will in fact be in a state of equilibrium. Often levels of signalling components are measured, perhaps before and after a treatment. However, rarely do the levels of activity, levels of signalling molecules or levels of phosphorylation go from a base level of zero to a higher level. In the vast majority of cases the levels rise from a low level to a transiently higher level. Therefore researchers define threshold levels for signals, (for example Pereyra *et al*., 2000).

How individual signals get heard may be liken to phrases in the orchestra when one instrument temporally is dominate and can be clearly heard, especially an instrument such as a kettle drum. In music there are often many instruments play all at the same time, and often it is hard to discern the exact contribution of any one instrument. The holistic effect may be pleasing but the parts played by individuals are assumed to be part of the whole. However, one instrument can dominate over the others and be heard above the rest. Perhaps a trumpet is playing a strident part. It will be heard above the other hundred instruments which make up the orchestra. And the audience will follow the trumpet, the tune from which will carry the music and the mood. Cells will have a similar system. Many signals are all contributing, but the arrival of a new hormone may need to dominate. A pathway may be activated, and the activity of the players in that pathway will reach a threshold allowing them to have their effect above that of the other signalling components, which will after all be carrying on doing what they were doing before. However, transiently, the pathway with the "volume" which is dominant will be able to invoke the cellular response needed.

Some instruments such as a bagpipe rely on a background tone, or drone (Nordquist & Ayers, 2009). The highland bagpipe has a tonic note (that is the base note of the scale) of A. Therefore, the other notes are played over this, but the tonic gives the constant tone to the music. It is the other notes which will dominate to give the tune and harmonies. In cells there are various parameters upon which activities and functions of proteins and signalling components will need to contend. In cellular compartments pH is crucial, but as well as this there is the redox state of the environment. In the cytoplasm for example, there is a high concentration of reduced glutathione which will endeavour to maintain the redox state relatively constant (Schafer & Buettner, 2001). This is important because proteins contain reactive side groups which may be affected by the redox of the medium in which they function. Such groups include the thiol groups of cysteine residues for instance. Here, two cysteines may react together in an oxidation reaction to create a disulphide bridge which may stabilise the protein. Alternatively they may react with signalling molecules such as nitric oxide (to be S-nitrosylated) or hydrogen peroxide (to be oxidised). Disruption of the redox state of the cell towards the oxidised state is referred to as oxidative stress, a condition of cells with is extremely important not only to control cell function but also to regulate processes such as apoptosis. Oxidative stress has been implicated in numerous diseases, including degenerative disease (Kadenbach *et al.*, 2009). Therefore it is extremely important for this basal redox state to be maintained, much like a basal note of the bagpipe. It needs to be there, allowing continuity of the harmony of the cell. However, there does need to be the involvement of signals such as hydrogen peroxide and nitric oxide. It may be that the basal redox state in some cases maintains the thiol groups in a state to enable compounds such as nitric oxide to react and have its effect. This would be like the pitched notes on the bagpipe being strident above the background tonal level. On the other hand, if the background is disturbed, perhaps during oxidative stress, such thiols would have already reacted with for example hydrogen peroxide and be no longer available for a reaction it would normally partake in. A disruption of the basal background harmony has altered the effect of the other signals, and the overall effect is quite different. For normal signalling to resume, the background "tonal" redox state would need to be restored.
