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Images of RP stained of cells, treated with LB (5 |ig/ml and 40 pig/ml) both before and after permeabilisation “L B ^ SL-0 -> Trigger + LB”, are shown in the bottom two panels of Fig 4.7. The extent of F-actin depletion in cells exposed to 5 |ig/ml LB was heterogeneous: some cells possessing minimal F-actin while others retained significant amounts of cortical and or, nuclear F- actin. These different actin morphologies account for three cell populations revealed by the flow cytometry data (Fig 4.6). At the higher concentration of LB (40 ng/ml) both basal (E/A) and triggered cells contained minimal cortical actin. Quantification of the relative F-actin content of these cells confirmed that LB treatment induces a dose dependent loss of F-actin - basal cells exposed to LB (40 iig/ml) containing only - 20% of the F-actin present in control cells (Fig 4.8A and C). Fig 4.8C confirms the observation that LB (20 ^ig/ml), present both before and after

permeabilisation, completely abolishes the ability of GTPyS (G/E/A and G/C/A) to increase F- actin (Fig 4.8B): this was observed at even the lowest [LB] used (5 ^ig/ml).

These findings agree with the predicted effect of this protocol on the F-actin cytoskeleton (see Table 4.1) i.e. depletion of the F-actin cortex and inhibition of F-actin polymerisation.

Depletion of cortical F-actin together with the prevention of actin polymerisation, (protocol “ LB (40 |ig/ml) -> SL-0 -> Trigger + LB”), inhibited G/E/A and G/C/A induced secretion by only -30%. F-actin depletion alone using the protocol “LB (40 |ig/ml) SL-0 Trigger” inhibited G/E/A and G/C/A to a similar extent. Thus F-actin polymerisation appears to play only a minor role in GTPyS/EGTA/ATP and GTPyS/calcium /ATP mediated secretion.

Calcium/ATP induced secretion however, was inhibited by -65 % when LB was present both before and after permeabilisation “ LB SL-0 Trigger + LB”, (as compared to only -35% when LB was present only prior to permeabilisation “ LB SL-0 -> Trigger” (Fig 4.5). This suggests that the actin cytoskeleton plays a signficant modulatory role in C/A mediated secretion.

4.7.3 LB present only after permeabilisation • “SL-0 -> Trigger + LB”

Cells treated using this protocol exhibited: (1) no increase in F-actin upon cell activation, (2) cortical F-actin depletion and (3) no nuclear F-actin (not shown - refer to chapter 5 Fig 5.2). This protocol was expected to only prevent actin polymerisation induced by cell activation (refer to table 4,1). The depletion of cortical F-actin may have arisen as a consequence of permeabilisation: the released monomers are able to leak out from the cells, shifting the equilibrium towards depolymerisation. Moreover permeabiiised cells were exposed to LB for lOmin prior to triggering which may have been sufficient time to deplete cortical F-actin. This depletion of cortical F-actin has thus to be considered when interpreting the effects of this protocol.

Secretion and the actin cytoskeleton

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Latruncuiin concentration (|ag/ml)

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E/A C/A G/E/A G/C/A

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Figure 4.8: Effect of LB on the total F-actin content of (A) resting and (C) activated attached cells

Confocal micrographs of equatorial slices from attached cells, treated using the protocol “LB ->

SL-0 -> Trigger + LB”, were analysed for relative F-actin content using the same method as used in Fig 4.3. (A) Effect of LB (0-40 pg/ml) on the relative F-actin content basal cells. (B) The effects of calcium/ATP (C/A), GTPyS/EGTA/ ATP (G/E/A) and GTPyS/calcium/ATP (G/C/A) on the relative F-actin content of mast cells. (C) The effect of LB (20 |ug/ml) on the cytoskeletal responses induced by these triggers. F-actin content of basal control cells (E/A) was taken as 100 %. Error bars represent SE, n=50.

4.8 Summary

Intact cells

• LB -> Trigger (attached cells): This protocol as predicted depleted cortical F-actin without preventing compound 48/80 mediated actin polymerisation. Cortical F-actin depletion maximally inhibited compound 48/80 mediated secretion by only -30%. (Fig 4.1 B)

• Trigger + LB (attached cells): This protocol as predicted prevented compound 48/80 mediated actin polymerisation while maintaining cortical actin during exocytosis. Prevention of actin polymerisation had no effect on the secretory response to compound 48/80.

• LB -> Trigger + LB (attached and suspended cells): This protocol as predicted depleted cortical F-actin and prevented compound 48/80 mediated actin polymerisation. The virtual loss of F-actin had profound inhibitory effects (-80% inhibition) on secretion

from both attached and suspended cells. Suspended cells were more sensitive to the inhibitory effects of LB.

• All the LB protocols prevented compound 48/80 induced cell spreading

• The protocols LB SL-0 -> Trigger and LB SL-0 -> Trigger + LB induced the formation of substantial amounts of RP stained nuclear F-actin. This effect of LB had not been previously reported. (Fig 4.2) RP stained nuclear F-actin did not however form in response to the protocol “SL-0 ^ Trigger + LB”.

Permeabiiised ceils

• LB SL-0 -> Trigger (suspended and attached cells): This protocol depleted cortical actin, but only at high concentrations, without preventing GTPyS mediated actin polymerisation (Fig 4.7). A small reduction in RP staining was also observed in flow cytometry (Fig 4.6). This apparent lack of cortical F-actin depletion however is misleading, as it represents the amount of F-actin following cell stimulation - the cells partially recovering from LB treatment during stimulation, not the amount of F-actin present at the point of triggering. Secretion in these cells was maximally inhibited by 25% to 35% in attached and suspended cells. However at lower

concentrations, LB promoted G/E/A and G/C/A mediated secretion in suspended cells and to a lesser extent in attached cells.

• SL-0 Trigger + LB (suspended cells): This protocol as predicted inhibited actin

polymerisation in response to triggering. However, cortical F-actin was also depleted, which may partly result from the 10min pre-incubation with LB. Secretion was more potently inhibited in these cells as compared to those treated using the protocol “LB SL-0 -> Trigger”, in which only the F-actin cortex had been depleted. This greater potency was especially apparent at low concentrations of the toxin. (Fig 4.4)

• LB ^ SL-0 -> Trigger + LB (attached cells): This protocol as predicted depleted cortical F- actin and prevented GTPyS mediated actin polymerisation (Fig 4.7 and 4.8A and 80).

• This virtual loss of F-actin maximally inhibited G/E/A and G/C/A mediated secretion, by approximately the same extent as that induced by cortical F-actin depletion alone (Fig 4.5). Secretion induced by C/A was inhibited to a much greater extent (-65% inhibition) when both the F-cortex is depleted and actin polymerisation is prevented.

• C/A stimulated secretion was the most sensitive to the inhibitory effects of LB, irrespective of the protocol used.

• The protocols LB ^ SL-0 Trigger and LB -> SL-0 -> Trigger + LB induced the formation of substantial amounts of nuclear F-actin. In comparison only marginal amounts of RP stained nuclear actin formed using the protocol “SL-0 -> Trigger + LB”. Moreover this small amount of nuclear actin was present in only a small proportion of the cell population. This phenomenon is investigated in chapter 5 (refer to Fig 5.2).

4.9 Discussion

The aim of this study was to clarify the role actin played in the exocytotic signalling pathways of primary mast cells. It also aimed to ascertain whether cell attachment significantly alters the cell behaviour. This investigation studied used intact and permeabiiised cells to study the role of the actin cytoskeleton at both the early/mid and late stages of the exocytotic signalling pathway. The discussion of the results is thus split up into two main sections: “Intact cells” and “Permeabiiised cells”