Componentes del Sistema Primario de Distribución

The nuclear actin identified in this study was found to be absent in resting intact mast cells. A number of lines of evidence, presented in this chapter indicate that nuclear actin forms, not as an artefact of permeabilisation, but as a result of ATP depletion. Addition of exogenous ATP (E/A) was found to reduce nuclear actin present in permeabiiised mast cells (Fig 6.3B). Further investigation revealed that actin translocates into the nucleus during permeabilisation (EGTA + SL-0), and that this translocation is prevented by ATP (Fig 6.7). This is supported by the findings of Koffer (1993) who showed that permeabilisation (by SL-0) of mast cells for 1.5min at 30®C, reduced the total cellular ATP content to -18% of that present in intact cells. As stated translocation of actin into nuclei does not appear to arise as an artefact of permeabilisation. This

is indicated by the observation that ATP depletion in intact cells (via metabolic inhibition) also induces actin to translocate into the nucleus (Fig 6.8). Koffer et al (1993) reported that metabolically inhibiting (Ml) intact mast cells for lOmin reduces the cellular ATP concentration to -5% of its original value, and to an undetectable level after 20min. However the level of phosphoprotein was found to have decreased by only 30% after 20 minuets of Ml. This decreased by a further 30% when the cells were Ml for an additional 40 minuets. The level of phospholipids did not appreciably change during a GOmin period of Ml. The confocal image? in Fig 6.8 show that nuclear actin increases progressively over an 80 min period of Ml. This suggests that dephosphorylation of protein(s) maybe is responsible for formation of nuclear actin (Fig 6.8). This is discussed in the following section.

Actin does not contain a nuclear localisation sequence (NLS) and therefore it needs to associate with an actin-binding protein, which contains such a sequence, to be actively transported into the nucleus. This study has shown that ATP depletion is the signal, which induces actin to translocate into the nucleus, or prevents its export out of nuclei.

A number of groups have shown that dephosphorylation of the actin-binding protein cofilin, induces its translocation into the nucleus (refer to section 5.8.3). However, the progressive increase in nuclear actin induced by metabolic inhibition was not associated with an increase in a-cofilin staining within the nucleus (Fig 6.9). Cofilin may carry actin into the nucleus, where it then dissociates and exits the nucleus. Alternatively, cofilin may not be required to transport actin into the nucleus under these conditions. Nuclei of basal (EGTA) permeabiiised cells are intensely stained by a-cofilin. The intensity of this staining was substantially reduced when the cells were exposed to ATP: paralleling the effects of ATP on nuclear actin. This simplest explanation for this effect of ATP is that it promotes phosphorylation of cofilin, most probably on residue Ser 3. Phosphorylation of this residue results in the dissociation of any bound actin (Sun et al. 1995), which is presumably responsible for the loss of both actin and cofilin from the nucleus. This hypothesis corroborated by the observation made by NebI et al (1996) who found that expression of a non-phosphorylatable form of cofilin results in its accumulation in the nucleus. A good candidate for the kinase activated by ATP is LIM-K which, as discussed in section 5.8.5, has recently been identified as a kinase, which phosphorylates cofilin (Aber et al. 1998). This group

found that expression of LIM-K prevented the formation of cofilin-actin rods induced by overexpressing a-cardiac actin, most probably through phosphorylation and thereby inactivation of cofilin. A representation of these hypothesised signalling events is summarised in figure 6.11. Another explanation is that cofilin carries actin into the nucleus, and that export of cofilin out of the nucleus may not require ATP, while that of actin does.

The effect of GTPyS on the localisation of cofilin, in both the absence and presence of ATP and/or calcium, was also investigated in this study. This showed that GTPyS alone does not appear to have any effect on the localisation of nuclear cofilin. However it should be noted that in this set of experiments GTPyS did not induce a decrease in the nuclear actin or the associated increase in F-actin within the cell interior. The localisation of nuclear cofilin therefore needs to be investigated in those cells which respond to GTPyS alone (G/E). This could not investigated here, as no more a-cofilin was available. The findings obtained from cells triggered with GTPyS in the presence of ATP however indicate that GTPyS may export cofilin from the nucleus. In those cells exposed to ATP (E/A) alone only a proportion of the nuclear actin was exported from the nucleus. Exposing cells additionally to GTPyS induced most if not all of both cofilin and actin to be exported from the nucleus.

The proposed role of ATP in protein phosphorylation can be easily tested by addition of serine kinase inhibitors. The use of compound Y-27632 (Yoshitomi Pharmaceutical Industries) which inhibits ROCK, and 03 which ADP-ribosylates Rho and N17Rac (a dominant negative Rac) may provide insight into whether GTPyS is inducing export of actin and cofilin from the nucleus via the Rho - ROCK and Rac signalling pathway.

The effects of GTPyS on secretion correlate well with its ability to export and polymerise this nuclear actin within the cell interior. The findings in chapter 4 and those of Norman et al (1996) suggests that polymerisation of actin is not involved in secretion. It may however play a role in the endocytocic signalling pathway of RPMC. This could be tested by investigating how depletion of this nuclear actin (using E/A) modulates the endocytotic pathway, using such methods as fluorescently labelling sphingomyelin which becomes internalised during the endocytosis.