This chapter can be divided into three main sections. The first being to confirm the previous findings of Koffer et al (1990) and Norman et a! (1994/1996) regarding the cytoskeletal and secretory responses of intact and permeabiiised mast cells. This was undertaken as these systems were to be used in the following chapters, to investigate the link between the actin cytoskeleton and secretion in RPMC. The second part of this chapter was to provide evidence to confirm or dispute the existence of a monomer pool regulated by GTPyS. This was investigated by studying how this nucleotide alters actin leakage from permeabiiised mast cells. Finally a method needed to be developed to visualise G-actin, as one of the aims of this thesis was to visualise and study a theorised pool of G-actin regulated by GTPyS. A few points regarding this method conclude the discussion.
3.6.1 The secretory and cytoskeletal responses of intact RPMC
The secretory responses of intact mast cells agreed with previous observations Koffer et al (1990) and Norman et al (1994/1996). However a few notable differences were observed regarding the effects of these triggers on the actin cytoskeleton. With regard to intact cells, Norman et al (1994) had reported that compound 48/80 induced the formation of F-actin within the cell interior, forming a similar cytoskeletal structure induced by GTPyS. In this study compound 48/80 induced both an increase in cortical F-actin and cell spreading: inducing no relocalisation/polymerisation of actin within the cell interior (Fig 3.1A). The reason for this apparent difference was discovered to be due to differences in the duration of incubation with compound 48/80. In this study the cells were exposed to compound 48/80 for 20mins, in comparison to 2min used by Norman et al. A study undertaken by Dr M Holt showed that 48/80 initially induced the formation of F-actin within the cell interior which relocalised to the cell cortex and adhesion plane over a period of -10-20min (personal communication). It is possible that, in permeabiiised cells, GTPyS is inducing this first stage of actin polymerisation but is incapable of relocalising actin to the cell cortex: possibly due to the loss of cell components during permeabilisation. This relocalisation of F-actin from the cell interior to the cortex and adhesion plane may be mediated through an actomyosin contractile mechanism.
3.6.2 The secretory and cytoskeletal responses of permeabiiised RPMC
The secretory responses of permeabiiised mast cells observed in this study are in agreement with those previously reported by Koffer et al (1990) and Norman et al (1994/1996). The cytoskeletal responses were also similar to those previously observed by Norman et al (1994). This study confirmed that GTPyS does indeed increase the amount of F-actin within the cell interior of washed permeabiiised cells. Quantification of the F-actin content of both basal and G/E/A triggered cells (refer to Fig 4.8B) showed that G/E/A increases the relative F-actin content of RPMC by 410 % .
Furthermore confocal images in chapter 6 (Fig 6.4) confirmed the earlier finding that ATP
increases the proportion of cells that respond to GTPyS i.e. exocytose and exhibit and increase in F-actin (Norman et al. 1994). Norman et al did not investigate this role of ATP. Chapter 6
presents data to show how ATP is thought to amplify actin polymerisation mediated GTPyS.
Actin polymerisation induced by GTPyS/ATP (G/E/A) was altered by calcium (G/C/A). Quantification showed that calcium reduced this increase in relative F-actin content by -30%. As calcium alone (C/A) reduced the relative F-actin content by -30%, this finding suggests that calcium/ATP is depleting cortical F-actin via a signalling pathway that is G-protein independent. These two triggers certainly induce different effects on the actin cytoskeleton (Fig 4.7), which correlates with theory that they are acting via different signalling pathways. The findings of Sullivan et al (awaiting publication) are partially in agreement with these findings. Their work showed that calcium depletes cortical F-actin via calmodulin (CaM) signalling to MLCK, in a Rho- independent manner. However they also found that Rho induces a small enhancement C/A mediated cortical F-actin depletion.
3.6.3 GTPyS: The existence of a monomeric actin pooi reguiated by GTPyS
The ability of GTPyS to induce de novo actin polymerisation in cells containing negligible amounts of soluble actin suggests that permeabiiised mast cells contain a pool of actin monomer. One of the aims in this thesis was to visualise and study the regulation of this pool (chapter 6). This study found that GTPyS (G/E/A) significantly reduced actin leakage from permeabiiised cells (-30%) (Fig 3.2A). This is unlikely to be due to retention of cortical F-actin as the cortical RP
staining in GTPyS (G/E/A) treated cells is no brighter than seen in control (E/A) cells (Fig 3.1 B). Thus the source of actin retained by GTPyS is most likely to be unpolymerised at the time of triggering. This monomeric pool must be associated with internal structures as permeabilisation and washing results in the loss of soluble actin (-70-80%). However the possibility that this actin represents stabilisation of the F-actin cortex cannot be ruled out. The results presented in chapter 6 however favour the first hypothesis.
3.6.4 The localisation of G-actin in RPMC
The finding that mast cell actin is predominantly if not solely g-actin is not unexpected, as this is the prevailing isoform in non-muscle cells: erythrocytes containing only g-actin. The observation that phalloidin partially masks the a-g-actin epitope was surprising as their binding sites on actin do not overlap. Phalloidin binding to G158, D179, R177 which lie close to the nucleotide binding cleft (Belmont et al. 1999), while a-g-actin binds to the N-terminal (refer to chapter 2). Thus prevention of binding must arise from some steric effect, although as phalloidin is a small molecule this is slightly unexpected.