The fact that not all nucleoporins were proteolysed indicates that the nuclear pore complex does not simply undergo simultaneous destruction upon induction of apoptosis. Our investigation has revealed that the nuclear pore is dismantled in a stepwise manner and is consistent with previous findings (Kihlmark et al., 2001a). However, the mechanism of nuclear pore breakdown during apoptosis has not yet been fully elucidated. Nucleoporin cleavage during apoptosis may i) be analogous with disassembly of the NPC during mitosis, ii) reflect the accessibility of effector caspases to the NPC, iii) depend upon the origin of caspase activity in the nuclear compartment or iv) identify a linchpin nucleoporin whose proteolysis facilitates the disassembly of the subset of nucleoporins.
A dramatic event during mitosis in higher cells is the disassembly of the nuclear envelope, which involves the dismantling of the NPC lamina and the removal of nuclear membranes (Cotter et al., 1998). The mechanism of nuclear pore disassembly during mitosis may shed some light on the sequence of events that occur during apoptosis. The disassembly and assembly is a complex, orchestrated fundamental process whose understanding remains elusive (Kiseleva et al., 2001). Disassembly appears to be more synchronous compared to assembly. This would be expected because the triggering of mitosis and NE breakdown is a rapid, almost sudden, switch regulated by a positive feedback loop leading to the phosphorylation of nucleoporins (Macaulay et al., 1995b). The order of disassembly of particular substructures probably reflects their accessibility to mitotic kinases, with the internal structures only becoming accessible when structures that are more peripheral have been solubilised.
If nucleoporins are degraded by a common set of downstream effector caspases, it is feasible to expect the order of degradation also to be restricted by accessibility. It has been proposed that apoptosis proceeds in a centripetal direction as a gradient of activated caspases commencing in the cytoplasm and working its way into the nucleus (Kihlmark et al., 2001a). If this were the case then proteins accessible from the cytoplasmic face of the nuclear pore complex would be degraded first and those accessible from the nuclear side degraded later since activated caspases would first have to enter the nucleus. Our studies revealed that degradation of two nucleoporins exclusively found on the cytoplasmic face of the NPC, CAN/Nup214 localised near the cytoplasmic coaxial ring and RanBP2/Nup358 localised at the cytoplasmic filaments, did not precede degradation of nucleoporins located on the nuclear side of the NPC namely Nup153 and Tpr. This pattern of proteolysis appears to contradict a study by Kihlmark et al., who reported degradation of RanBP2 and POM121, an integral membrane protein to precede Nup153, a peripheral protein located in the nucleoplasmic basket (Kihlmark et al., 2001b). However, this study does not show the proteolytic fragments of nucleoporins cleaved during apoptosis. Moreover, the investigators used staurosporine to induce apoptosis, which is not targeted to a specific compartment but inhibits protein kinases in both the nucleus and the cytoplasm (Swannie and Kaye, 2002).
However, this model does not explain the resistance of proteolysis by Nup107, Nup205, Nup98 and p62 observed in both receptor-mediated apoptosis and DNA damage. An alternative interpretation of the sequence of nucleoporin disassembly as suggested by Kihlmark et al., is that initially only a particular group of key targets are attacked by upstream proteases. This primary attack may consequently lead to further destruction at a later time point, facilitated by loss of pore function and increased permeability through the pore. The contribution to NPC structure by Nup93 and Nup96 is essential for establishment of normal NPC function and for cell viability. Therefore, an initial degradation of these proteins, as observed in both apoptotic models may destabilise the NPC and allow entry of nucleases and effector caspases into the nucleus. This model is supported by a study by Faleiro and Lazebnik who have shown that caspase-9 contributes to cell disassembly by disrupting the nuclear-cytoplasmic barrier (Faleiro and Lazebnik, 2000). In terms of the proteolytic cascade, caspase-9 is activated earlier than caspase-3 (Creagh and Martin, 2001) and it has been revealed that active caspase-9 is required for caspase-3 translocation from its predominantly cytoplasmic localisation to the nucleus
DISCUSSION
(Faleiro and Lazebnik, 2000; Kihlmark et al., 2004). Furthermore, activation of caspase-9 has been identified as responsible for alterations in nuclear transport (Wilkinson et al., 2003). Since activation of caspase-9 has been implicated in both TRAIL- AND etoposide-induced apoptosis, this model may be particularly applicable to the nuclear pore disassembly observed in these two models.
Active import does appear to be necessary to translocate some pro-apoptotic factors into the nucleus. Caspases themselves and caspase-activated molecules such as DEDD, CAD and Acinus are thought to exert their effects following uptake into the nucleus (Wilkinson et al., 2003). The caspase-dependence of the exchange between the nucleus and other cytoplasmic compartments during apoptosis is unclear. Furthermore, an open question remains regarding the origin of caspase activity in the nuclear compartment. On one the hand caspases may be translocated into the nucleus as active species, on the other caspases may be activated within the nucleus, which may indicate an autonomous nuclear caspase activation pathway. One might expect this to be reflected in the disassembly of nuclear pore proteins when the apoptotic stimulus acts primarily on the nucleus.
Our findings indicate that early cleavage of Nup93 and-96 is conserved in the two different apoptotic models whereas that the sequence of cleavage of the peripheral nucleoporins appears to be dependent upon the apopotic trigger.. However there are a number of common features between the TRAIL and etoposide apoptotic model: 1) Nup93 and Nup96 are both cleaved early, 2) Tpr and Nup153 are cleaved concomitantly and 3) CAN/Nup214 is cleaved late in both apoptotic models. Nup93 and Nup96 exist in sub-complexes that are located on both the cytoplasmic and nuclear face of the NPC and during etoposide-induced apoptosis are cleaved concomitantly with Nup153 and Tpr, which are both exclusively localised to the nuclear face. Nup153 and Tpr act as binding partners whereby direct interaction with Nup153 mediates binding of Tpr to the periphery of the NPC (Hase and Cordes, 2003), hence supporting our observation that these two nucleoporins are cleaved simultaneously. Our findings may indicate a pool of active caspase within the nucleus as well as in the cytosol.
The nuclear pore complex is a protein assembly, which comprises a number of distinct evolutionary conserved sub-complexes (Boehmer et al., 2003; Lutzmann et al., 2002;
Powers and Dasso, 2004). Another possible model for dismantling of the nuclear pore
nucleoporin whose proteolysis facilitates the disassembly of the subset of nucleoporins.
This dissociation will ultimately have consequences for the binding dynamics of structural nucleoporins and may lead to an alteration in the permeability barrier.
Nup107, Nup160, Nup133, Nup96 and Nup85 form a hetero-oligomeric complex referred to as the Nup107-160 sub-complex (Boehmer et al., 2003). This sub-complex is an essential core element of the nuclear pore complex and Forbes et al have found that reconstitution of nuclei lacking the Nup107-160 sub-complex results in the most severe effect in NPC assembly reported i.e. a total loss of the nuclear pore complex (Boehmer et al., 2003). Our studies have revealed that Nup96 is the only nucleoporin to be cleaved within this sub-complex, suggesting that this protein plays an important role in maintaining the structural integrity of this sub-complex. Our findings appear to contradict other studies, which have implicated Nup107 as the keystone nucleoporin that is required for the assembly of a subset of nucleoporins into the nuclear pore complex. However, in the present study Nup107 was found not to undergo proteolysis in either TRAIL- or etoposide-induced apoptosis.
A further vertebrate nuclear sub-complex, Nup93-Nup188-Nup205 has been identified as a structural building block of the nuclear pore (Miller et al., 2000). C.elegans homologues of vertebrate Nup93 and Nup205 have been found to be required for normal nuclear pore complex formation in the nuclear envelope. The depletion of Nup93 and Nup205 has been shown to cause a failure in nuclear exclusion of non-nuclear macromolecules of 70kDa without preventing active nuclear import or assembly of the nuclear envelope (Galy et al., 2003). Interestingly an increase in pore size correlated with partial NPC disassembly. Our investigation so far has implicated Nup93 to be cleaved within this sub-complex.
The p62 complex, an assembly of 62, 58, 54 and 45kD O-linked glycoproteins localised near the central gated channel of the nuclear pore complex has been directly implicated in the nuclear protein import (Emig et al., 1995). Our investigations so far have not identified a target nucleoporin within this sub-complex (not shown).
DISCUSSION
5.5 Construction of a reporter system to investigate the degradation of the cytoplasmic side of the nuclear pore
Until recently, it was widely believed that specific nucleoporins were assigned to the cytoplasmic or nucleoplasmic face of the nuclear pore. Recent literature has revealed that many nucleoporins are located symmetrically within sub-complexes (Belgareh et al., 2001;
Griffis et al., 2003; Harel et al., 2003; Matsuoka et al., 1999). A reporter system was constructed to study the degradation of the cytoplasmic side of the nuclear pore because of limited antibodies directed against the nucleoporins on the cytoplasmic side of the pore.
This reporter consisted of a fusion protein comprising full length Ran GAP linked to GFP via a linker region containing a caspase cleavage site. Our data indicated that we achieved efficient transfection of RanGAP-GFP and caspase mutants that were correctly localised at the nuclear rim. Immunostaining with HA antibody was negative and sequencing revealed a 7bp deletion at the C-terminus in the region of the HA tag (data not shown). One would have expected RanGAP-GFP-caspase-3 to be cleaved during staurosporine-induced apoptosis since the ability of this protein kinase inhibitor to activate caspase-3 is well documented in the literature (Johnson et al., 2002; Kovacs et al., 1999; Thuret et al., 2003).
One would have expected the cleavage fragment to increase the intensity of the band representing endogenous RanGAP, since they should have a similar molecular weight.
RanGAP was the first identified target for SUMO modification and it has been demonstrated that the localisation of RANGAP to the nuclear pore is SUMO1-dependent (Matunis et al., 1998; Seewald et al., 2003). The association of RANGAP with RanBP2 depends on the modification of RanGAP with a single molecule SUMO1 molecule (Seewald et al., 2003). Our results (4.2.8.4) indicated the presence of endogenous and transfected-sumoylated RanGAP whose MWs are in agreement with previous studies (Swaminathan et al., 2004) . The western blot also revealed a 65kDa band, which may represent transfected, non-sumoylated RanGAP. This may be explained by the saturation of the sumoylation process as a consequence of the cells exhibiting high expression of RanGAP, which was observed by immunostaining (Fig.33). This experiment was performed in parallel to that of digitonin permeabilisation (4.2.8.3) and the next step would be to repeat this experiment following digitonin permeabilisation of transfected cells.
Further work may include the repair of the RanGAP sequence with regards to the HA tag.
Additionally, since the sequence also contains a myc tag (Fig.1) and GFP sequence, reprobing the western blots with an anti-myc-tag or anti-GFP antibody may be able to