VIII.1. Caspase-independent regulation of the nucleocytoplasmic barrier in apoptosis
A serine protease that can be inhibited by Pefablock has been found to have an impact on the nucleocytoplasmic barrier in apoptosis.
This serine protease is not identical with the mitochondrial serine protease OMI because OMI can not be inhibited by Pefablock. Nevertheless, OMI was found to alter nuclear permeability when added to S-20 extracts from control Jurkat cells using NPA.
Additionally, a degradation fragment was detected when OMI was incubated with purified nuclei.
Determination of the origin of this degradation fragment would contribute to the understanding of how OMI influences NPC properties. This could be achieved by immunoprecipitation of the fragment and subsequent protein sequencing.
Furthermore, it would be interesting to test if OMI activates another serine protease already present in control extracts or in the HeLa cell nuclei used in the NPA. Addition of OMI and Pefablock to the reaction would answer this question.
To investigate the role of OMI on nuclear permeability in vivo, an siRNA based approach could be used. Knock-down of OMI by siRNA has been shown to confer resistance to various apoptotic stimuli in a number of different cell lines [174, 267, 268]. If OMI would directly or indirectly mediate the increase in nuclear permeability e.g. after apoptosis-induction with STS, this increase would be abolished in OMI-depleted cells.
VIII.2. Alteration of the nucleocytoplasmic barrier by Bcl-2
A basal increase in passive nuclear permeability due to overexpression of Bcl-2 in HeLa cells was observed in this study. To gain further insight into the mechanism of alteration of NPC properties by Bcl-2 further investigations are necessary.
To clarify if Bcl-2 could mediate changes in NPC properties through direct interaction with nucleoporins we are currently investigating whether Bcl-2 is located at NPCs by electron microscopy. Additionally, a Bcl-2 mutant located solely to the NE but not to the ER is constructed by replacing its transmembrane domain by the KASH domain of Nesprin-2 (in analogy to an NE-targeted GFP protein constructed by Crisp et al. [269]).
With the help of this construct it will be investigated whether NE localization of Bcl-2 is sufficient to alter nuclear permeability or if additionally, its localization at the ER is necessary.
A further question to be addressed concerns the possibility that Bcl-2 alters NPC properties via alterations of ER/NE-calcium-contents. To this end Bcl-2 overexpressing HeLa cells should be stably transfected with SERCA2, which has been found to reverse the increase in nuclear permeability. Calcium measurements should help to find out, if the calcium concentration is indeed decreased in Bcl-2 overexpressing cells and if this decrease can be reversed by SERCA2. If this is the case, AFM studies should be performed in these cell lines to visualize NPC structures and link them to their different functional states.
Furthermore it would then be interesting to see if calcium is modulated by Bcl-2 through interaction with and/or alteration of IP3R- or SERCA-function. To this end immunoprecipitation studies and activity measurements should be performed. These analyses are of special importance in view of the controversial data concerning the subject of Bcl-2 and calcium regulation.
VIII.3. Apoptosis-induced alterations of the nucleocytoplasmic barrier: future perspectives
A confocal microscopy time lapse procedure to visualize functional and structural changes of the NPC during the time course of apoptosis was established in the present work. The method allows to directly correlate apoptosis-induced alterations in nuclear permeability, degradation of nucleoporins, and condensation of DNA in single living cells. Special image analysis tools for the representation and evaluation of the data were developed.
Results obtained so far revealed that alterations of NPC permeability occur early in the apoptotic time course after apoptosis-induction with the general kinase inhibitor STS and late (concomitant with DNA condensation) after activation of the extrinsic apoptosis pathway by TRAIL. Caspase-dependent degradation of nucleoporins occurred at a late stage in both apoptosis models.
The imaging method established here opens the door to a comprehensive characterization of the regulation of nucleocytoplasmic transport in cell death.
Based on the results obtained in this study the following questions should be addressed in the future:
1) Is an early or late increase in nuclear permeability a characteristic feature of intrinsic or extrinsic apoptosis pathways, respectively?
The data obtained from cells stimulated with STS or TRAIL should be validated by examining further apoptosis models of intrinsic and extrinsic pathways.
2) Is an increase in nuclear permeability during apoptosis correlated with an impairment of active nuclear transport?
The imaging of different fluorescently labeled transport receptors and/or specific fluorescent reporters for active transport will show, if and at what time point specific transport pathways are altered during apoptosis.
3) Is an increase in nuclear permeability during apoptosis associated with the loss of specific nucleoporins from the NPC?
Imaging of a variety of fluorescently tagged nucleoporins would answer this question. A loss of specific nucleoporins from the NPC during apoptosis would be in analogy to the observed nucleoporin loss and increased permeability during mitosis in A. nidulans [98]. In this context, a detailed analysis of apoptosis-induced posttranslational modifications of nucleoporins would be of value, as these are known to occur during mitosis and to influence specific transport pathways [96, 97].
4) What is the impact of Bcl-2 on the regulation of the nucleocytoplasmic barrier during apoptosis?
A comparison of results obtained from the above mentioned experiments with corresponding data from Bcl-2 overexpressing Hela cells would show if and which transport pathways are affected by Bcl-2. Apoptosis-induced translocation of several proteins was found to be inhibited in Bcl-2 overexpressing cells [199, 201].
Additionally, they were found to display an increased nuclear permeability (this study). These experiments could lead to a further insight into the cytoprotective mechanisms of Bcl-2.
5) Are alterations of the nucleocytoplasmic barrier in apoptosis downstream or upstream of the translocation of apoptosis-related proteins?
The experimental system could be exploited to further investigate the translocation of apoptosis-related proteins as for example AIF or p53 (Table 2), and to correlate them with increased nuclear permeability, nucleoporin degradation, and DNA condensation.
6) Do variations in calcium-levels play a role in apoptosis-induced alterations of the nuclear permeability barrier?
Concomitant visualization of alterations in nuclear permeability and variations in calcium levels during apoptosis would help to further characterize a possible link between calcium, calcium regulated proteins and nuclear transport. Again results from these experiments could be compared to results obtained from Bcl-2 expressing HeLa cells, which have altered basal ER/NE calcium levels ([126], see also chapter VII.2.1.2.2).