Dismantling of the Components of the Nuclear Pore Complex
during Apoptosis
DISSERTATION
zur Erlangung
des akademischen Grades
des Doktors der Naturwissenschaften des Fachbereichs für Biologie
der Universität Konstanz
vorgelegt von Monika Patre
Tag der mündlichen Prüfung: 12 Juli 2004
Referent: Prof. Alexander Bürkle
Referent: Prof. Werner Hofer
Part of this work was submitted for publication:
M. Patre, A. Tabbert, H. Walczak, V. Cordes and E. Ferrando-May (2004) Caspases target essential architectural elements of the nuclear pore complex
Acknowledgements
I would like to thank first and foremost, my supervisor Dr Elisa May, for providing interesting research and her continuous support and perseverance with this work.
I would like to extend my gratitude to Prof. Alexander Bürkle for being involved in the supervision of this research, for stimulating discussions and his constant support and motivation.
Furthermore, I would like to thank Prof. Werner Hofer for reviewing my thesis and Prof. Dr.
Hartung for agreeing to be on the thesis committee.
I am grateful to Prof. Pierluigi Nicotera for suggesting carrying out a PhD in Germany …it was a character building experience!
I am particularly indebted to Dr Volker Cordes for his continuous supply of anti-nucleoporin antibodies and Dr Henning Walczak, for providing TRAIL. Also Dr Ralph Kehlenbach, Dr J.
Koeser and Dr Dirk Gorlich for their kind gifts of antibodies.
I would like to thank former and current members of the lab for their invaluable help and suggestions. In particular, Tina Wunsch and Petra Schildknect for their technical expertise, Momo Arsic for his encouragement, Tina Baur for suffering the thymidine block with me, Sebastian Rohrig and Sascha Beneke for their friendly advice and Thilo Sindlinger for dealing with my laptop issues.
Furthermore, I would like to thank the following for their contribution to this work, Daniela Hermann for her assistance with western blots, Marco Kuster for performing the mutagenesis and Anja Tabbert for carrying out the cell death ELISA assays.
I would like to thank Dr Rupert Marshall for his friendship and proof reading the thesis.
A very special thanks goes to Ronald, for taking an interest in my research, his help with the graphics and his endless support and encouragement over the past three year
INTRODUCTION ...1
1.1 Overview of apoptosis ...1
1.2 Signalling in apoptosis...2
1.2.1 Caspases: Initiators and executors...2
1.2.1.1 Activation of caspases...3
1.2.1.2 Receptor-mediated apoptosis by TNF-related apoptosis inducing ligand (TRAIL) ...7
1.2.1.3 Etoposide-induced DNA damage ...9
1.2.1.4 Subcellular localisation...11
1.2.1.5 The execution of apoptosis by caspases...12
1.3 Nuclear Pore Complex ...14
1.3.1 The structure of the nuclear pore complex ...14
1.3.2 Molecular constituents of the nuclear pore complex: the nucleoporins ...17
1.3.2.1 Localisation of Nucleoporins ...17
1.3.2.2 Distinct Nucleoporin Sub-complexes of the NPC...21
1.3.3 Nuclear Pore Disassembly and Assembly ...23
1.3.4 Nuclear Pore and Apoptosis...24
2 AIMS ...26
3 MATERIALS AND METHODS...27
3.1 Materials...27
3.1.1 Machines and technical devices ...27
3.1.2 Chemicals...28
3.1.3 Kits...29
3.1.4 Antibodies ...29
3.1.5 Cells ...30
3.1.6 Cell culture material ...30
3.1.7 Plasmid...30
3.2 Methods ...31
3.2.1 Culturing of cells ...31
3.2.2 Cell synchronisation via double thymidine block. ...31
3.2.3 Flow Assisted Cell Sorting (FACS) analysis...32
3.2.4 Pre-treatment protocol for protease inhibitors ...32
3.2.5 Cell viability assays ...33
3.2.5.1 SYTOX/Hoechst assay ...33
3.2.5.2 DNA-fragmentation ELISA ...33
3.2.6 Measurement of caspase activity...34
3.2.7 Immunostaining...34
3.2.7.1 Double staining for cytochrome c and Bax...34
3.2.7.2 Double staining for caspase-3 and cytochrome c ...35
3.2.8 Preparation of whole cell extracts ...36
3.2.9 Determination of protein content ...36
3.2.10 SDS-polyacrylamide-gel electrophoresis and western blotting ...37
3.2.11 Isolation of Plasmid DNA...41
3.2.12 DNA digestion with restriction endonucleases ...42
3.2.13 Agarose gel electrophoresis ...42
3.2.14 DNA sequencing ...42
3.2.15 Primers ...43
3.2.16 Digitonin permeabilisation...44
CONTENTS
4 RESULTS...45
4.1 Apoptotic models ...45
4.1.1 TNF related apoptosis inducing ligand (TRAIL) induced apoptosis ...45
4.1.2 Dose dependent toxicity of etoposide in HeLa cells ...48
4.1.3 Cell Synchronisation...49
4.1.4 Dose dependent toxicity of etoposide in synchronised HeLa cells ...51
4.1.5 Cell synchronisation does not induce spontaneous apoptosis...53
4.1.6 Synchronisation potentiates etoposide toxicity in HeLa cells ...54
4.1.7 Etoposide induces cytochrome c release ...58
4.1.8 Etoposide induces Bax translocation...62
4.2 Fate of nuclear pore proteins during apoptosis...67
4.2.1 Proteolysis of NPC during TRAIL-induced apoptosis ...67
4.2.2 Proteolysis of nuclear pore proteins during etoposide-induced apoptosis...70
4.2.3 Lamin B and PARP are cleaved during TRAIL- and etoposide-induced... apoptosis...72
4.2.4 Search for putative caspase cleavage sites...74
4.2.5 TRAIL-induced nucleoporin cleavage is prevented by caspase inhibitors..77
4.2.6 Etoposide-induced nucleoporin cleavage is prevented by caspase inhibitors ...80
4.2.7 Cleavage of PARP during etoposide-induced apoptosis is not inhibited by ... z-VAD-fmk ...83
4.2.8 Construction of a reporter system to investigate the degradation of the cytoplasmic side of the nuclear pore ...84
4.2.8.1 Transfection of full length Ran GAP-GFP and caspase mutants ...85
4.2.8.2 Cellular localisation of RanGAP-GFP and caspase mutants ...86
4.2.8.3 Digitonin permeabilisation of RanGAP-GFP ...87
4.2.8.4 Cleavage of RanGAP-caspase-2-GFP and RanGAP-caspase-3-GFP ...88
5 DISCUSSION...91
5.1 TRAIL induces apoptotic cell death in HeLa 229 cells ...91
5.2 Etoposide induces apoptotic cell death in HeLa 229 cells ...92
5.2.1 Cell synchronisation potentates etoposide toxicity ...93
5.2.2 Etoposide induces cytochrome c release in synchronised HeLa cells...94
5.2.3 Bax translocation triggers cytochrome c release...96
5.3 The proteolysis of nucleoporins is caspase-dependent ...97
5.4 The nuclear pore undergoes sequential proteolysis during apoptosis...98
5.5 Construction of a reporter system to investigate the degradation of the cytoplasmic side of the nuclear pore...102
5.6 Conclusion ...103
6 SUMMARY...104
7 REFERENCES ...107
INTRODUCTION
1.1 Overview of apoptosis
The observation that cell death is part of normal development was made over 150 years ago (Vogt, 1842). The term programmed cell death (PCD) was proposed to describe an observation that some cells were destined to die during insect and tadpole metamorphosis, as if driven by a cell intrinsic program (Lockshin and Williams, 1965). The term
“apoptosis” was introduced over 30 years ago to describe cell death associated with a set of common morphological features (Kerr et al., 1972). In more recent years, numerous biochemical characteristics have been observed exclusively within this mode of cell death and now contribute to the definition of apoptosis (Hengartner, 2000). These distinguishing features include cell shrinkage or the loss of cell volume, chromatin condensation, internucleosomal DNA fragmentation, membrane blebbing, phosphatidylserine exposure and the formation of apoptotic bodies that are subsequently cleared by phagocytosis without evoking an inflammatory response to the intracellular components, which occurs in necrosis (Kerr, 2002). Depolarisation of the mitochondrial membrane potential (Waterhouse et al., 2001), release of cytochrome c (Adrain and Martin, 2001) and activation of specific proteases have also been suggested to play critical roles in the apoptotic process (Degterev et al., 2003).
The studies of C.elegans cell lineage revealed that 131 of 1090 C.elegans somatic cells invariably die during normal development (Horvitz and Sulston, 1980). This finding led the way for genetic characterisation of the critical components of the apoptotic molecular machinery (Degterev et al., 2003). The central components of the PCD machinery in C.elegans are three ced genes, namely ced-3, ced-4 and ced-9 (Hengartner, 1999; Yuan and Horvitz, 1992). Homologues of these genes have been shown to regulate apoptosis in higher eukaryotes, although via more complex mechanisms (Yuan et al., 1993). The mammalian homologue of ced-3 is a family of cysteine proteases, which are responsible for the execution of apoptosis. Apaf-1 is a mammalian ced-4 homologue which is involved in the regulation of cytochrome c release from the mitochondria into the cytosol (Zou et al., 1997). The Bcl-2 family members are mammalian homologues of ced-9 and
INTRODUCTION
are important regulators of mitochondria-dependent apoptosis (Hengartner and Horvitz, 1994).
Apoptosis plays a vital role in the elimination of unwanted cells during the development and homeostasis of multicellular organisms (Raff, 1998). A balance between cell death and cell proliferation is essential to maintain a homeostatic cellular state and deviation from this cellular balance may result in human disease. Excessive apoptosis can play a role in neurodegenerative disease, osteoporosis, AIDS and ischaemic damage. In contrast, cell accumulation via from insufficient apoptosis can contribute to cancer and autoimmune diseases (Cryns and Yuan, 1998).
1.2 Signalling in apoptosis
1.2.1 Caspases: Initiators and executors
Apoptotic signalling pathways converge on a common machinery of cell destruction that is activated by a family of cysteine proteases, known as caspases, which cleave their substrates adjacent to aspartate residues (Alnemri et al., 1995; Martin and Green, 1995).
These proteins were first linked to apoptosis, when it was found that the mammalian gene encoding interleukine-1b-converting enzyme (ICE, caspase-1) shows high homology to the C. elegans ced-3 gene, and that both gene products are similarly able to induce apoptosis (Miura et al., 1993; Yuan et al., 1993). To date, 14 mammalian caspases, termed caspase-1 to -14, 10 of which are human, have been identified. Caspases are typically constitutively present within cells as inactive zymogens that contain an N-terminal pro-domain followed by the region that contains a small and a large subunit with the catalytic domain. The proforms of caspases require proteolytic cleavage at specific aspartate residues to achieve their active configuration (Nicholson and Thornberry, 1997). An initial cleavage event separates the C-terminal small subunit from the rest of the molecule, allowing assembly of an active caspase that autocatalytically cleaves off its pro-domain. A mature active caspase is composed of a heterotetrameric complex of two large subunits and two small subunits (Lamkanfi et al., 2002). Despite their common requirement for cleavage after the aspartate residues at the substrate P1 site, caspases are highly specific in their substrate preferences. The substrate specificity is mainly determined by the sequence of four smino
small subunit, being the most critical determinant of specificity (Nunez et al., 1998).
Based on specificity studies, caspases can be classified into three groups (Lien et al., 1999;
Rano et al., 1997). The optimal tetrapeptide recognition motif for group I caspases (caspase-1, -4 and -5) is (W/L)EHD whereas group II caspases (caspase-2, -3, and –7) prefer DEXD. Group III caspases (caspase-6, -9, -8 and –10) prefer the sequence (L/V)EXD (Tozser et al., 2003).
Caspases can be divided into initiators and executors, based on their structure and order of activation during apoptosis signalling (Cohen, 1997). Initiator caspases possess long prodomains containing one of the two characteristic protein-protein interaction motifs: the death effector domain (DED) (caspase-8 and –10) and the caspase activation and recruitment domain (CARD) (caspase-1, -2, -4, -5, -9, -11 and –12), providing the basis for interaction with upstream adaptor molecules (Degterev et al., 2003). The caspases that perform the downstream execution steps of apoptosis by cleaving multiple cellular substrates are usually processed and activated by upstream caspases. Caspase-3, -6 and -7 are members of the executioner caspases and are characterised by a short or absent pro- domain (Cohen, 1997). Their preferred cleavage motif appears in many proteins that are cleaved during cell death (Slee et al., 1999).
1.2.1.1 Activation of caspases
There are at least three distinct pathways for caspase activation in mammalian cells; (1) recruitment-activation, (2) trans-activation and (3) auto-activation (Nicholson, 1999). Two examples of caspase activation following recruitment of multiple homologous proenzymes to a common site are recruitment of pro-caspase-8 to an oligomeric activation complex following ligation of a death receptor such as CD95/Fas/Apo-1 or TRAIL/Apo-2 (Krammer, 1999) and oligomerization of pro-caspase-9 forming part of the mitochondrial apoptotic pathway (Li et al., 1997) (Fig.1). Ligand induced receptor multimerization results in the formation of the death-inducing signalling complex (DISC) containing multiple adaptor molecules including FADD (Fas-associated death domain), TRADD (TNF associated death domain), DAXX, RIP, (Receptor interacting protein kinase) RAIDD (RIP associated protein with death domain), FLIP (Flice-like inhibitory protein) (Peter and Krammer, 2003).
INTRODUCTION
Figure 1: Two principle caspase signalling pathways in apoptosis Adapted from Los et al., 1999
FADD is recruited to the DISC via is terminal C-terminal death domain (DD) and interacts through its N-terminal DED with the DED of caspase-8 (Walczak and Sprick, 2001). The recruitment and oligomerization of caspase-8 in the DISC results in its autocatalytic activation and is essential for initiation of cell death. Caspase-2 and -10 may be recruited to the DISC, however their role in receptor mediated cell death remains unclear (Kischkel et al., 2001; Sprick et al., 2002; Wang et al., 2001b). The activation of caspase-8 leads to the processing and activation of the executioners, caspase-3 and -7. These latter caspases cleave a variety of cellular substrates leading to the branched cascades of caspase activation, which amplify apoptotic signalling (Boyce et al., 2004).
Apoptotic signalling via the death receptor may also depend on a mitochondrial amplification step (Fig.1) (van Loo et al., 2002). This may occur when there is insufficient active caspase-8 or downstream caspases (Degterev et al., 2003). Caspase-8 cleaves the cytosolic BH3-only proapoptotic Bcl-2 family member Bid. The processing of Bid results in its translocation from the cytosol to the mitochondria, where it causes the release of
cytochrome c through the oligomerisation of the proapoptotic Bcl-2 family members Bax and Bak (Lim et al., 2002a).
Apaf-1 is a protein that consists of three distinct domains: an N-terminal Caspase recruitment domain (CARD) that recruits procaspase-9, a CED-4-homologous region that promotes oligomerization, and a regulatory C-terminus with multiple tryptophan/aspartic acid repeats (WD40 repeats) that appears function as a cytochrome c-responsive domain (Creagh and Martin, 2001). Release of cytochrome c into the cytoplasm abrogates the inhibitory action of the WD40 repeat region on Apaf-1. In the presence of cytochrome c and dATP, Apaf-1 and caspase-9 assemble into a complex that known as the mitochondrial apoptosome (Cecconi, 1999). This complex is a heptamer consisting of seven Apaf-1 adaptor molecules, each bound to one molecule of cytochrome c and a dimer of the initiator caspase-9 (Salvesen and Renatus, 2002). Caspase-9 is activated through an apoptosome-induced conformational change and processes the executioner caspase-3 and - 7 to initiate the execution of apoptosis in a similar manner to caspase-8 (Creagh and Martin, 2001). This mitochondrial pathway is regulated at several steps, including the release of cytochrome c from the mitochondria, the binding and hydrolysis of dATP/ATP by Apaf-1, and the inhibition of caspase activation by the proteins that belong to the inhibitors of apoptosis (IAP) (Shi, 2001). Another component of the apoptotic machinery, known as SMAC (second mitochondrial-derived activator of caspase) is localised in mitochondria and is released into the cytosol during apoptosis (Bratton and Cohen, 2003).
It appears to lower the threshold for entry into apoptosis by binding to IAPs, thereby removing their inhibitory activity on caspases (Verhagen and Vaux, 2002).
INTRODUCTION
Figure 2: Mitochondrial cytochrome c release promotes assembly of the apoptosome and a cascade of caspase activation events
It is important to note that caspase-2 has been found to act upstream of the mitochondria to promote cytochrome c release during etoposide-induced apoptosis (Robertson et al., 2002). Adapted from Creagh and Martin, 2001.
Trans-activation of one caspase by another is a second well established mechanism for proenzyme maturation and activation. The upstream initiator caspases cleave and activate downstream executioner caspases via proteolysis of the Asp-X site between the large and small subunits (Nicholson, 1999). Trans-activation is assisted by Hsp60 activation, which suggests that the vulnerability of executioner caspases to activation by initiator caspases is
1999). Autocatalytic activation is a third mechanism of caspase activation, which has been has been identified in studies whereby RGD peptides directly stimulate the autoactivation of procaspase-3(Buckley et al., 1999).
1.2.1.2 Receptor-mediated apoptosis by TNF-related apoptosis inducing ligand (TRAIL)
Tumour Necrosis Factor (TNF)-related apoptosis-inducing ligand (TRAIL) was first identified based upon sequence homology to other members of the TNF superfamily (Wiley et al., 1995). TRAIL, alternatively known as Apo-2L (Pitti et al., 1996) is a characteristic type II transmembrane protein and exhibits highest homology to the CD95 ligand, with 28% amino acid similarity in the extracellular binding motifs. Early investigations also identified two other unique characteristics of TRAIL. Firstly, TRAIL can selectively induce apoptosis in transformed or tumourigenic cells but not in normal cells. Secondly, TRAIL mRNA is expressed constitutively in a wide variety of tissues and cell types, unlike other members of the TNF family whose expression are more often transient on activated cells (Wiley et al., 1995).
TRAIL induces apoptosis through receptors, TRAIL-R1 (DR4) (Pan et al., 1997) and TRAIL-R2 (DR5/TRICK2/KILLER) (MacFarlane et al., 1997; Walczak et al., 1997), both of which contain a cytoplasmic death domain motif that exhibits homology to the death domains found in CD95 and TNF receptor 1 (TNF-R1). Two additional receptors, TRAIL- R3 (DR3/DcR1/ TRID/LIT) (Degli-Esposti et al., 1997; Mongkolsapaya et al., 1998;
Sheridan et al., 1997) and TRAIL-R4 (TR4/DcR2/TRUNDD) (Degli-Esposti et al., 1997;
Marsters et al., 1997; Pan et al., 1998), are unable to signal for cell death and are known as
“decoy” receptors (Pan et al., 1997; Sheridan et al., 1997). TRAIL-R3 lacks an intracellular domain and is a glycosylphosphatidylinositol-linked cell surface protein, whereas TRAIL-R4 contains a truncated intracellular domain and, thus, an incomplete death domain lacking residues critical for engaging apoptosis. In addition to these four membrane associated receptors, the secreted TNFR-homologue (osteoprotegerin) OPG can also bind TRAIL, however with lower affinity (Emery et al., 1998).
The engagement of TRAIL-R1 or TRAIL-R2 by TRAIL results in the recruitment and activation of caspase-8 as well as cleavage of Bid and cytochrome c release from the
INTRODUCTION
mitochondria, events that subsequently lead to the activation of the caspase cascade (LeBlanc and Ashkenazi, 2003). It has been suggested that TRAIL-R1 or TRAIL-R2 mediate apoptosis through the Fas-associated death domain protein (FADD). However, there are conflicting views regarding the ability of the death receptors to recruit or bind these adapter proteins. FADD deficient mice cells are resistant to apoptosis induced by CD95, TNFR1 AND DR3 but are fully responsive to DR4. This implies that TRAIL is linked to caspases via a FADD independent pathway (Barkett and Gilmore, 1999). Other studies have demonstrated that apoptosis mediated by DR5 requires the recruitment of FADD and caspase-8 (Sprick et al., 2000). However, proof of an essential role for FADD was obtained when the native TRAIL DISC was finally isolated (Bodmer et al., 2000).
Analysis of the DISC revealed that FADD was present together with caspase-8 and that in a cell line lacking either FADD or caspase-8, TRAIL-induced apoptosis was completely abrogated (Bodmer et al., 2000; Kischkel et al., 2000; Sprick et al., 2000). Experiments involving co-transfection or co-immunoprecipitation have also revealed that TRADD may be recruited by both TRAIL-R1 and -R2 (Chaudhary et al., 1997). This observation raised the possibility that TRAIL, similar to TNF, may signal for NF-kB and JNK. There is evidence to suggest that RIP and TRAF2 are important effectors of TRAIL-induced NF-B and JNK activation, but that neither are required for TRAIL-induced apoptosis (Li et al., 2000). Recent studies suggest that DAP3, a GTP-Biding protein, may play a role in linking TRAIL death receptors to FADD and the downstream apoptotic machinery, thus acting as an adapter molecule required for TRAIL-induced apoptosis (Miyazaki and Reed, 2001) Cell death induced by TRAIL death receptors can be regulated at several levels.
Intracellular anti-apoptotic molecules can block the apoptotic signalling pathway or re- direct it into different responses. Examples of such molecules include cellular FLICE-like inhibitory protein (c-FLIP), which competes with caspase-8 for binding to FADD (Irmler et al., 1997), or XIAP, cIAP-1 and cIAP-2, that can act to inhibit active caspases. Another mechanism of cellular resistance to members of the TNF family is through activation of the transcription factor NF-B and up-regulation of NF-B-regulated anti-apoptotic genes.
Other studies have reported that RIP can be cleaved by caspase-8 to produce a dominant negative fragment, which inhibits TNF-induced NF-B activation (Lin et al., 1999).
Further investigations have demonstrated the recruitment of the NF-kB-activating kinase RIP to the native TRAIL DISC. It has been suggested that caspase-mediated cleavage of
RIP could significantly inhibit the capacity of TRAIL-sensitive cells to activate NF-kB (Harper et al., 2001).
1.2.1.3 Etoposide-induced DNA damage
Etoposide is a topoisomerase II inhibitor and is widely used as an antineoplastic drug (Clapp and Hande, 2002). DNA topoisomerases are nuclear enzymes, which induce transient breaks in the DNA allowing DNA strands or double helices to pass through each other (Toonen and Hande, 2001). By this action topoisomerases solve topological problems of DNA in replication, transcription, recombination and chromosome condensation as well as de-condensation. DNA topoisomerases can be classified as type I enzymes that induce single stranded cuts in DNA or type II enzymes that cut and pass double stranded DNA.(Champoux, 2001).
In the absence of topoisomerase II, cells are unable to segregate daughter chromosomes and subsequently die of mitotic failure. Eukaryotic topoisomerase II is homodimeric in nature (Warburton and Earnshaw, 1997). Each protomer contains an active site tyrosine (Tyr-805) that is responsible for cleaving one strand of the double helix. In order to maintain the integrity of the genetic material during the double stranded DNA passage reaction of topoisomerase II, each active site residue forms a covalent phosphotyrosyl bond with one newly generated 5’terminus (Warburton and Earnshaw, 1997). This covalent topoisomerase II-cleaved DNA complex is known as the cleavable complex (Wilstermann and Osheroff, 2003).
Despite the physiological importance of topoisomerase II, the cleavage complex is potentially toxic. Usually, cleavage complexes are present at very low levels and are not toxic to the cell. However, conditions that increase the cellular concentration of topoisomerase II-associated nucleic acid breaks lead to DNA recombination and mutagenesis (Baguley and Ferguson, 1998). When these breaks overwhelm the cell, they trigger programmed death pathways (Kaufmann, 1998). Etoposide induces apoptosis by increasing the concentration of topoisomerase II-DNA cleavage complexes (Wang et al., 2001a) This action converts topoisomerase II to a potent cellular toxin that fragments the genome. It has been known for more than a decade that etoposide stabilizes topoisomerase II-associated double-stranded DNA breaks by inhibiting the ability of the enzyme to ligate
INTRODUCTION
cleaved nucleic acid molecules (Osheroff, 1989; Robinson and Osheroff, 1991). These breaks are recognized by the multiprotein complex DNA dependent protein kinase (DNA- PK), in particular the heterodimer of Ku (Ku70/60) subunits that bind to DNA. On binding to DNA, Ku recruits and activates the catalytic subunits. The activation of DNA-PK is essential to the apoptotic cascade as it provides the link between the recognition of DNA damage and downstream signaling events (Fig.1). Among the substrates of DNA-PK is the tumour suppressor protein p53. Phosphorylation of p53 results in its stabilization and activation of its transactivation factors leading to regulation of proteins implicated in cell cycle control and apoptosis(Burns and El-Deiry, 1999).
The interaction of p53 with the pro-apoptotic Bcl-2 family protein, Bak causes oligomerization of Bak and release of cytochrome c from mitochondria (Leu et al., 2004).
Recent evidence has revealed an unexpected role for the linker histone H1.2 in DNA damage-induced apoptosis (Konishi et al., 2003). It was observed that DNA double strand breaks induced translocation of nuclear H1.2 to the cytoplasm, where it promoted the release of cytochrome c from mitochondria by activating the Bcl-2 family protein, Bak (Gillespie and Vousden, 2003). Bax is another pro-apoptotic Bcl-2 family protein that is transcriptionally regulated by p53. The pro-apoptotic action of Bax is thought to be mediated by its interaction with the mitochondria, in particular, its insertion into the outer mitochondrial membrane (Goping et al., 1998). The overexpression of Bax leads to mitochondrial permeabilisation and cell death (Pastorino et al., 1998). However, it has been suggested that other mechanisms in addition to an increase in the content of the protein are necessary for Bax to translocate from the cytosol to the mitochondria (Wolter et al., 1997). It has been proposed that a conformational change in Bax results in the exposure of its N-terminal domain, an event that may free the hydrophobic C-terminal membrane- anchoring domain (Roucou and Martinou, 2001). Several mechanisms have been proposed to account for such a Bax conformational change, including an alteration in intracellular pH, an alkalinisation of the cytosol or an interaction with the pro-apoptotic protein Bid.
Recent studies have identified that Ku70 suppresses the apoptotic translocation of Bax to mitochondria (Sawada et al., 2003). On translocation to the mitochondrion, Bax can cause the release of cytochrome c (Lim et al., 2002b). The mechanism by which Bax releases cytochrome c is currently a matter of debate (Eskes et al., 1998; Gogvadze et al., 2004;
Martinou et al., 1999; Ott et al., 2002). Interestingly, Robertson et al., have shown that
etoposide-induced apoptosis involves the early activation of caspase-2, which acts upstream of the mitochondria to regulate cytochrome c release (Robertson et al., 2002).
1.2.1.4 Subcellular localisation
The majority of pro-caspases have been localised in the cytoplasm where both receptor and apoptosome-mediated caspase activation occurs. However, caspase activation may also occur in subcellular compartments besides the cytosol and the translocation of these proteins during apoptosis may contribute to caspase activation (Ritter et al., 2000;
Zhivotovsky et al., 1999)
Caspase localisation may vary depending on the cell type and pro-caspase-3 has been shown to be localised in both the cytoplasm and the intermembranous space of the mitochondria in HeLa cells (Shi, 2002). Apoptotic stimuli result in the release of caspase- 3 from the mitochondria. It has been demonstrated that pro-caspase-3 in the mitochondria forms a complex with heat shock proteins Hsp60 and Hsp10 and that activated caspase-3 is dissociated from Hsp and released from the mitochondria (Samali et al., 1999;
Xanthoudakis et al., 1999). It has been shown that pro-caspase-2 and -9 are localised in the mitochondria and active forms released during apoptosis (Susin et al., 1999). Cohen et al., have demonstrated that both pro-caspase-3 and caspase-3 are localised in the cytosol, using bio-fractionation of mouse hepatocytes (Cohen, 1997). In contrast pro-caspase-7 was localised in the cytosol and microsomal fragments, whereas active caspase-7 was redistributed to the mitochondria and microsomal fractions (Chandler et al., 1998).
It has been demonstrated that the prodomain of pro-caspase-2 has a nuclear localisation signal (NLS) and that both the precursor and active caspase-2 localise to the cytoplasm and nucleus (Paroni et al., 2002). Similarly, TNF-induced translocation of pro-caspase-1 to the nucleus is mediated by a NLS in the pro-domain (Mao et al., 1998). It has been suggested that caspase-3 is translocated to the nucleus through active transport. Immunostaining has revealed the presence of pro-caspase-3 in the nuclei of some cells (Ramuz et al., 2003) and affinity labelling has shown active caspases in the nuclei of apoptotic HL-60 myeloid cells (Martins et al., 1997). Interestingly, Eguchi et al., have found that ATP is required after caspase-3 activation for completion of apoptosis in Fas-stimulated cells, probably for nuclear transport (Eguchi et al., 1999).
INTRODUCTION
1.2.1.5 The execution of apoptosis by caspases
Execution or effector caspases are thought to be responsible for the actual demolition of the cell during apoptosis and the characteristic hallmarks of apoptosis. A number of studies have suggested caspase-3 to be the major executioner caspase whereas caspase-6 and -7 play minor roles (Creagh and Martin, 2001)
Active caspases promote cellular destruction by targeting key structural and regulatory proteins within the cell for degradation by activating other destructive enzymes such as DNases or by promoting cytochrome c release from the mitochondria via proteins such as Bid. Structural proteins cleaved during apoptosis include nuclear lamins, fodrin, vimentin, and actin (Lazebnik et al., 1995; Martin et al., 1995; Mashima et al., 1995; Neamati et al., 1995; Oberhammer et al., 1994; Prasad et al., 1998). Their degradation may be involved in the rounding up of the cell, condensation of the chromatin, and the packaging of cellular constituents into small and easily clearable apoptotic bodies. Ruchaud et al., have recently revealed that caspase-6 activity is essential for lamin A cleavage and that when lamin A is present it must be cleaved in order for the chromosomal DNA to undergo complete condensation during apoptotic execution (Ruchaud et al., 2002).
One of the nucleases primarily responsible for effecting internucleosomal DNA fragmentation during apoptosis is called DNA Fragmentation Factor 40 (DFF40) or Caspase-activated DNase (CAD) (Liu et al., 1999). DFF40/CAD is activated by caspase-3 that cleaves the nuclease's inhibitor DNA Fragmentation Factor 45/Inhibitor of Caspase- activated DNase (DFF45/ICAD). The nuclease preferentially attacks chromatin in the internucleosomal linker DNA. DFF40/CAD-mediated DNA fragmentation triggers chromatin condensation that is another hallmark of apoptosis (Widlak, 2000).
Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme, which is responsible for synthesis of poly(ADP-ribose) in response to DNA damage caused by numerous agents. Cellular repair enzymes such as PARP, are inactivated by caspases presumably to avoid squandering energy resources of the dying cell (Los et al., 2002) Caspase-3 is primarily responsible for the cleavage of PARP during cell death. The sequence at which caspase-3 cleaves PARP (DEVD) is very well conservedin the PARP protein from very distant species, indicating thepotential importance of PARP cleavage in
ROCK-1 (Rho-associated kinase 1) has been identified as a caspase substrate and is thought to be responsible for apoptotic cell contraction and membrane blebbing (Coleman et al., 2001; Sebbagh et al., 2001). The Rho GTPases and ROCK proteins are intracellular signalling molecules that regulate the actin cytoskeleton. ROCK-1 is cleaved by caspase-3 during apoptosis resulting in a truncated molecule with increased kinase activity. This increased ROCK activity and consequent membrane blebbing are necessary for redistribution of fragmented DNA from the nucleus into membrane blebs and apoptotic bodies (Coleman et al., 2001; Sebbagh et al., 2001). The cleavage of inhibitory molecules such as the X-linked apoptosis inhibitor protein, (XIAP) further enhances caspase activity (Deveraux et al., 1999).
Following proteolytic cleavage by caspase-8, active Bid mediates the death signal to the mitochondria and induces cytochrome c release (Luo et al., 1998). The negative regulator of apoptosis, Bcl-2, is a further caspase substrate. Its cleavage does not only result in the loss of its anti-apoptotic function, but also produces a fragment that promotes apoptosis, therefore accelerating the whole cell death process (Cheng et al., 1997). The translocation of phosphatidylserine to the cell surface as recognition molecule for phagocytes also appears to be caspase-dependent (Martin et al., 1996).
INTRODUCTION
1.3 Nuclear Pore Complex
1.3.1 The structure of the nuclear pore complex
The nuclear pore complex (NPC) is one of the largest supramolecular assemblies in the eukaryotic cell. It was originally described from thin-section transmission electron micrographs as an electron dense structure within a pore formed where the inner and outer membranes of the nuclear envelope (NE) join (Aaronson and Blobel, 1974). The NPC has a molecular mass of approximately 125 MDa in vertebrates (Reichelt et al., 1990) and comprises 30 proteins termed nucleoporins (Cronshaw et al., 2002). Yeast pore complexes are also composed of a similar number of nucleoporins and have a molecular mass of 66 MDa (Rout and Blobel, 1993). A vertebrate cell nucleus contains approximately 2000 NPCs, whereas the smaller yeast nucleus contains in the order of 200.
The structure of the NPC has been extensively studied by transmission and scanning electron microscopic techniques and a consensus on its basic architectural framework has emerged (Akey and Radermacher, 1993; Jarnik and Aebi, 1991; Reichelt et al., 1990;
Unwin and Milligan, 1982). It is cylindrically shaped, 125 nm in width and 150-200 nm in height (including peripheral structures). Accordingly, the vertebrate NPC exhibits a tripartite architecture composed of a spoke complex encompassing a central channel complex, which is sandwiched between a cytoplasmic and nuclear ring. When viewed perpendicular to the plane of the nuclear envelope (NE), the intact NPC, spoke complex and both rings exhibit an 8-fold rotational symmetry (Pante and Aebi, 1995). Its 55 MDa central framework is a ring-like assembly built of eight multi-domain spokes consisting of two roughly identical halves. Hence the entire spoke complex yields 8-2-2 symmetry (Reichelt et al., 1990), so that its asymmetric unit (i.e.one half-spoke) represents 3.3MDa, one 16th of its mass or roughly the size of a ribosome. This central framework is sandwiched between a 32MDa cytoplasmic ring and a 21MDa nuclear ring. Surface imaging techniques such as field emission in lens scanning electron microscopy (FEISEM) (Goldberg and Allen, 1993) and metal shadowing in TEM (Jarnik and Aebi, 1991) have identified the peripheral structures of the NPCs. From the cytoplasmic ring eight short, kinky fibrils emanate, whereas the nuclear ring anchors a “basket” or “fish trap”,
diameter ring. The asymmetry of the peripheral structures of the NPC might be related to its functional asymmetry, since both nuclear import and export involve vectorial cargo movement (Suntharalingam and Wente, 2003). The cytoplasmic fibrils may act as initial docking sites for nuclear import cargoes, whereas the nuclear basket, and in particular its distal ring, may serve as initial docking site for nuclear export cargoes (Marelli et al., 2001;
Rout and Aitchison, 2001). The nuclear basket appears to anchor distinct intranuclear filaments (Cordes et al., 1997), whereas the cytoplasmic fibrils might connect nucleoplasmic cytoskeletal elements to the NPC (Davis, 1995). FEISEM studies have identified other structures including a star ring underlying the cytoplasmic and nucleoplasmic rings. (Goldberg et al., 1997). A filamentous structure of 8-10nm has also been observed termed the nuclear lattice. This appears to be different from the nuclear lamina and may connect the distal rings of the nucleoplasmic basket to each other (Goldberg and Allen, 1992). The NPCs attach to the nuclear lamina via the spoke ring complex and adjacent NPCs appear to be interconnected via small radial arms within the NE lumen (Goldberg and Allen, 1996).
The ring-like, 8-2-2--symmetric central framework embraces the central pore of the NPC, which acts as a gated channel (Adam, 2001). The central pore is often plugged with a distinct particle, called central plug or transporter, of highly variable appearance whose molecular architecture and functional significance remain to be established (Stoffler et al., 1999). Despite the transporter element being a labile structure and often difficult to preserve for detailed EM studies (Feldherr and Akin, 1997), a 3-D structural model has been proposed based on cryo-electron adsorption and desorption of the substrate/receptor complex to nucleoporins that line the transporter element (Akey and Radermacher, 1993).
According to this model, the transporter is approximately 625Å in length, and is made up of two hollow globular domains, each having a diameter of about 420Å at the widest point.
The diameter at the waist of the transporter, where the globular domains join, is approximately 320Å. The variable presence and inconsistent shape of the central plug suggests the possibility that it is not a true structure but instead consists of material caught in transit through the central channel (Stoffler et al., 2003).
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Figure 3: Schematic diagram of the Nuclear Pore Complex
The structure has an apparent eight rotational symmetry perpendicular to the plane of the nuclear membrane.
However, certain facing portions have been removed to reveal the architecture and central regions.
Structures found in vertebrate NPCs are indicated, with the cytoplasmic face on top. Adapted from model in Rout and Wente 1994.
The yeast NPC was originally described as having a much simpler structural architecture compared to the vertebrate NPC (Rout and Blobel, 1993). The basic organisation is similar to vertebrates but it lacks the cytoplasmic and nuclear rings and the lumenal ring of the central spoke domain (Yang et al., 1998). Recent studies using field emission scanning electron microscopy to probe NPC structure found that yeast, like higher eukaryotic NPCs contain similar peripheral components (Kiseleva et al., 2004). In this study, cytoplasmic and nucleoplasmic rings in yeasts were shown to be present in yeast. A filamentous basket was present on the nucleoplasmic face and evidence for cytoplasmic filaments was apparent. A central structure was observed, possibly the transporter, which may be linked to the cytoplasmic ring by internal filaments (Kiseleva et al., 2004).
1.3.2 Molecular constituents of the nuclear pore complex: the nucleoporins
Major progress has been made in the analysis of NPC structure and nucleoporins as a consequence of the convergence of two lines of research. Firstly, the completion of the yeast genome has facilitated the identification of possible NPC proteins by sequence homology to known nucleoporins (Bassett et al., 1996). Secondly, the purification of the yeast NPC (Allen et al., 2001) as enabled the potential identification of all pore-complex proteins by proteomic analysis (Blobel and Wozniak, 2000; Miller and Forbes, 2000).
On the basis of mass calculations from structural studies, the initial estimates of the total number of proteins required to form the NPC varied from 50 in yeasts to over 100 in vertebrates (Davis, 1995; Doye and Hurt, 1997; Pante and Aebi, 1995). These proteins known as nucleoporins were initially identified via biochemical, genetic and immunological studies. Recent advances in proteomics pinpoint the nucleoporins in highly enriched, purified fractions from yeast and rat liver nuclei (Cronshaw et al., 2002; Rout et al., 2000). A surprising finding is that despite a mass twice that of the yeast structure, the vertebrate nuclear pore is also comprised of 30 distinct nucleoporins. If one considers the ribosome, with a mass of 4MDa containing approximately 75 different proteins, it is remarkable that so few proteins are required for the much larger NPC (Suntharalingam and Wente, 2003). However, this can be explained by the higher than average molecular mass of many Nups, averaging approximately 100kDa in yeast and the high copy number of Nups in a given NPC with a minimum of 8 based on rotational symmetry (Cronshaw et al., 2002).
1.3.2.1 Localisation of Nucleoporins
A considerable effort in the field has been made to determine the location of specific proteins within the NPC structure and with which nucleoporin it interacts. Of the 30 vertebrate nucleoporins, 22 have homologues or orthologues in yeast, and two others have possible functional equivalents (Cronshaw et al., 2002). A complete map of nucleoporins is not yet available for the vertebrate NPC, since genetic approaches are more difficult in vertebrate systems. Consequently, the determination of localisation of nucleoporins in the vertebrate NPC has required the generation of antibodies to the particular nucleoporin of
INTRODUCTION
interest. However, not all antibodies have proven equally useful for electron microscopy and immunofluorescence studies, and thus a number of vertebrate nucleoporins have not been precisely localized or have conflicting localisations reported by different groups.
Recent studies have used a FRET assay to investigate structural organization of the yeast NPC. This study defines spatial relationships for 13 pairs of nucleoporins and have applied the data to generate a refined molecular model of the yeast NPC (Damelin and Silver, 2002).
Nucleoporins located at the nuclear side of the vertebrate NPC comprise Nup153(Sukegawa and Blobel, 1993), Nup50 (Guan et al., 2000; Smitherman et al., 2000), components of the Nup160-Nup133-Nup96-Nup107 (Belgareh et al., 2001; Vasu and Forbes, 2001b)and the Nup93-Nup188-Nup205(Grandi et al., 1997; Miller et al., 2000) sub-complexes and a 267kDa protein known as Tpr(Byrd et al., 1994; Cordes et al., 1997) Immunofluorescence and immunoelectron microscopy studies have recently revealed that Nup98 is found on both sides of the pore complex (Griffis et al., 2003). The p62-Nup58- Nup54-Nup45 sub-complex maps in near the central transporter region (Davis and Blobel, 1987). The cytoplasmic filaments are known to contain Nup214/CAN, Nup88, Nup358/RanBP2, RanGAP and RanBP1. A specific ubiquitin-like SUMO-1 modification targets RanGAP to Nup358/RanBP2 on the cytoplasmic filaments (Swaminathan et al., 2004). The integral membrane proteins gp210, with a large lumenal domain and short cytoplasmic domain and POM121, with a small lumenal domain and a large FG cytoplasmic domain are thought to anchor the NPC to the membrane (Soderqvist and Hallberg, 1994).
Nup153 at the distal ring of the basket is thought to be involved in the initial assembly of nuclear membranes and lamina assembly, before pore formation (Ullman et al., 1999a;
Walther et al., 2001). In the pore, it has an important role in the terminal steps of import and RNA export (Ullman et al., 1999b). Nup98 is involved in RNA export (Powers et al., 1997). It has a unique binding site for the transport factor Rae1/Gle and binds to transport factors such as Crm and Tap on its amino-terminal GLFG repeats (Blevins et al., 2003).
The carboxy terminus of Nup98 is the binding site for the Nup160 complex. Nup50 is a FG repeat nucleoporin and has a direct role in export and probably serves as a binding site on the nuclear side of the pore complex for export receptor-cargo complexes (Guan et al.,
However, it has been suggested that this nucleoporin may be functionally redundant since null Nup50 fibroblasts do not exhibit any major cellular defects (Smitherman et al., 2000).
Tpr is localised to the inranuclear filaments of the nuclear basket. A number of functions of Tpr have been proposedincluding roles in intranuclear and nucleocytoplasmic transport and as a scaffolding element of the nuclear interior and theNPC (Fontoura et al., 2001;
Frosst et al., 2002; Shibata et al., 2002). RanBP2 has been identified as having a role in nuclear mRNA export (Singh et al., 1999). Recent sudies have demonstrated that RanBP2 provides a major Biding site for NXF1-p15 heterodimers which promote the nuclear export of bulk mRNA across the NPC (Forler et al., 2004).
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Figure 4: Localisation of the nuclear pore proteins
Schematic representation of the approximate localisation of nucleoporins and major sub-complexes in relation to sub-structures of the vertebrate nuclear pore complex. FG-repeats containing nucleoporins are indicated in red type. Established physical interactions between complexes are shown with solid lines,
1.3.2.2 Distinct Nucleoporin Sub-complexes of the NPC
When assembled in the NPC, a number of nucleoporins mutually interact to form distinct sub-complexes. These sub-complexes have been identified in vertebrate NPC either by separation of the sub-complexes formed during NPC disassembly at mitosis or via chemical extraction of purified nuclear envelopes (Vasu and Forbes, 2001b). Since it is not possible to substitute a tagged form of a nucleoporin gene into the genome, the introduction of modified nucleoporins into the NPC has been limited to a nuclear reassembly method in Xenopus egg extracts. Despite the fact that tagged nucleoporins can be reconstituted into the extracts and assemble into NPCs, the NPC in a reassembled nucleus is attached to the lamina and thus contaminated with chromatin and other nuclear components.
Miller et al., have overcome this problem by designing a two-step organelle trap assay. In this assay, soluble proteins from a Xenopus egg extracts are applied to an affinity column containing a ligand of interest (Miller et al., 2000). The bound proteins are eluted biotin tagged and reconstituted into NPCs of annulate lamellae assembled in vitro. A novel vertebrate nucleoporin was indentified using this approach, Nup188 and was found to be complexed to Nup93 and Nup205 (Miller et al., 2000). These known nucleoporins have yeast homologues which also form a complex with yeast Nup188. Despite yeast Nup188 and vertebrate Nup188 have limited sequence similarity the presence of the two proteins in similar complex suggests that they and their associated proteins form a conserved sub- complex within the NPC (Miller and Forbes, 2000).
Two-hybrid screens and immunoprecipitation experiments have revealed a direct and evolutionarily conserved interaction between Nup133 and Nup107 and indicated that hNup133 and hNup107 are part of a NPC sub-complex that contains two other nucleoporins, the previously characterized hNup96 and a novel nucleoporin designated as hNup160 (Vasu et al., 2001). These data support the previously reported interaction between Nup107 and Nup96, which was based on their co-sedimentation as a unique band on a sucrose gradient from solubilised NPCs (Fontoura et al., 1999). Furthermore the members of the vertebrate Nup107-160 complex have homology to the previously identified yeast Nup84 complex (Nup84p, Nup85p, Nup120p, Nup145Cp, sec13, and seh1) (Belgareh et al., 2001). The reconstitution of nuclei lacking the Nup107-160 complex
INTRODUCTION
results in the most severe defect in NPC assembly seen so far, with a complete loss of the nuclear pore complex (Belgareh et al., 2001). It would appear that the Nup107-160 complex is an essential core element of the NPC and is used very early in the decision to form a nuclear pore and may provide a point for regulation of nuclear pore complex assembly (Harel et al., 2003). It has been observed that Nup98 interacts with the Nup160 complex as well as the Nup88 complex through direct binding to Nup96 (Griffis et al., 2003). Interestingly, the same site within Nup98 is involved in binding to both Nup88 and Nup96 (Griffis et al., 2003). It has been shown that vertebrate Nup133 and Nup107 are localised on both faces of the NPC to which they are stably associated at interphase, and remain associated as part of a NPC sub-complex during mitosis (Walther et al., 2003).
During mitosis, a fraction of Nup133 and Nup107 localises to the kinetochores, thereby revealing a surprising connection between structural NPCs constituents and kinetochores (Belgareh et al., 2001).
Biochemical studies of solubilised NPC have identified p62 as being stably associated with three other O-linked glycoproteins p58, p54 and p45 to form a “p62” complex (Guan et al., 1995). The p62 complex has sequence homology and similar domain organisation to a complex of NPC proteins characterized in Saccharomyces cerevisiae consisting of Nsplp, Nic96p, Nup57p, and Nup49p (Grandi et al., 1995). However, it is not known whether the yeast Nspl complex is functionally homologous to the mammalian p62 complex. The localisation of the p62 complex relative to the eight-fold symmetry axis of the NPC is similar to where nuclear import ligands accumulate near the gated channel. The complex has a direct role in nuclear protein import as a consequence of its interaction with at least one cytosolic transport factor, NTF2 (Paschal and Gerace, 1995). Furthermore when Xenopus egg extracts are immunodepleted of the central transporter components, Nup62/58/54/45, the resulting nuclei are defective for import (Finlay et al., 1991). The presence of the p62 complex on both the nucleoplasmic and cytoplasmic faces of the NPC may also indicate a role in nuclear protein export (Dargemont et al., 1995).
The vertebrate Nup88 is found to be associated in a dynamic sub-complex with the oncogenic nucleoporin CAN/Nup214, which appears to have a number of essential roles.
Depletion of the Nup88/Nup214 sub-complex may result in defective import-export processes and eventually cell cycle arrest (Fornerod et al., 1997). In overexpressing cells it
1997). Recent studies have shown that Nup88 and Nup214 mediate the attachment of Nup358 to the NPC and that localisation of the export receptor CRM1 at the cytoplasmic face of the NE is Nup358 dependent (Bernad et al., 2004).
1.3.3 Nuclear Pore Disassembly and Assembly
Animals from Drosophila to man undergo open mitosis (Vasu and Forbes, 2001a). The nuclear envelope (NE) undergoes disassembly, which involves dismantling of the NPCs and the lamina and removal of nuclear membranes followed by its rapid reassembly as the cell leaves mitosis. The 120 MDa NPC is dismantled into sub-complexes of up to a million Dalton. A major open question is the manner in which the nuclear pore complex assembles and disassembles with each cell cycle. It has been suggested that pore disassembly is initiated via direct phosphorylation of a subset of nucleoporins by mitotic cdc2/cyclin B kinase (Collas, 1998; Ganeshan and Parnaik, 2000; Macaulay et al., 1995a;
Stoffler et al., 1999).
A monoclonal antibody that recognises nuclear pore antigens has identified nuclear pore sub-complexes in mitotic extracts of human somatic cells (Matsuoka et al., 1999). This study revealed that the NPC disassembles into at least three sub-complexes, termed sub- complexes A, B and C. The direct partial amino acid sequencing of the components of these sub-complexes indicates that the A sub-complex contains CAN/Nup214/p250 and p62 and the B sub-complex also contains p62, indicating that p62 is contained in two different sub-complexes. Sub-complex C was shown to consist of Nup98 and human RAE1, a human homolog of yeast Rae1p/Gle2p. It has been suggested that some of the mitotic sub-complexes may both be formed by functionally related proteins since Nup98 and Rae1p/Gle2p have been implicated in mRNA export (Pritchard et al., 1999).
The steps of NPC assembly following mitosis are not fully understood. Ultrastructural observations of post-mitotic nuclear envelope assembly indicate that the association of nuclear membranes with the chromosome surface occurs prior to the formation of NPCs (Goldberg et al., 1997; Macaulay and Forbes, 1996). Using confocal immunofluorescence microscopy, a time course of post-mitotic assembly for a group of NPC components relative to the integral nuclear membrane protein LAP2 and the NPC membrane glycoprotein gp210 has been defined (Bodoor et al., 1999). Nup153 associates with
INTRODUCTION
chromatin towards the end of anaphase, in parallel with the inner nuclear membrane protein, LAP2. However, the initial Nup153 chromatin association is thought to be membrane-independent. Recent studies have shown Nup153 to play a vital role in directing COPI to the nuclear membrane at mitosis and thereby providing feedback to other aspects of nuclear disassembly (Liu et al., 2003) Assembly of the remaining proteins follows that of the nuclear membranes and occurs in the sequence POM121, p62, CAN/Nup214 and gp210 and Tpr. Since p62 remains as a complex with three other nucleoporins (p58, p54, p45) (Guan et al., 1995) during mitosis and CAN/Nup214 maintains a similar interaction with its partner, Nup88 (Bastos et al., 1997), the relative timing of assembly of these additional four proteins may also be inferred. These findings imply that there is a sequential association of NPC proteins with chromosomes during nuclear envelope reassembly and the recruitment of at least eight of these precedes that of gp210 (Liu et al., 2003).
1.3.4 Nuclear Pore and Apoptosis
The dismantling of the nuclear pore during apoptosis is a relatively novel field of research and a clear sequence of events have yet to emerge. The fate of the nuclear envelope (NE) in different human cells committed to apoptosis by actinomycin D or etoposide has been investigated (Buendia et al., 1999). In this study, antibodies against marker proteins of the three domains of the NE were used, namely lamin B (LB) for the lamina, transmembrane proteins LBR and LAP2 for the inner nuclear membrane, and nucleoporins p62, Nup153 and gp210 for the NPCs. Since LB, LAP2 and Nup153 are exposed at the inner face of the nuclear envelope and all interact with chromatin (Vlcek et al., 2001), it has been suggested their cleavage enables the detachment of NE from chromatin and the clustering of NPCs in the plane of the membrane, two conserved morphological features of apoptosis observed in this investigation (Buendia et al., 1999).
Further studies have shown that the degradation of nuclear envelope marker proteins occur in a specific order (Kihlmark et al., 2001a). POM121 degradation occurred early and was initiated prior to nucleosomal DNA degradation and was completed before clustering of the nuclear pores. The proteolysis of Nup153 and lamin B coincided with the onset of
p62 was degraded much later. It appears that POM121 degradation may be an important early step in propagation of nuclear apoptosis. Interestingly this is also the first pore protein to be recruited in the reassembly of the NPC following mitosis (Kihlmark et al., 2001a).
Caspases contribute to the disassembly of the cell by disrupting the nuclear-cytoplasmic barrier (Faleiro and Lazebnik, 2000) and a recent study proposes that caspase-dependent disassembly of nuclear pores and disruption of the nucleocytoplasmic barrier paves the way for nuclear entry of caspases and subsequent activation of CAD-mediated DNA fragmentation (Kihlmark et al., 2004). Investigations in our laboratory have demonstrated that whereas nucleoporins are substrates for caspase-3 activity during apoptosis, nuclear transport factors such as Ran, importin- and importin- are not proteolytically processed, but redistribute across the nuclear envelope independently and prior to caspase activation (Ferrando-May et al., 2001).
AIMS
2 AIMS
Caspases are the major members of the apoptotic proteolytic machinery. Two main pathways have been elucidated, which lead to their activation, one triggered by death receptors at the plasma membrane (extrinsic pathway), the other involving the mitochondria (intrinsic pathway). The relationship between dismantling of nuclear pores, disruption of the nucleocytoplasmic barrier, and nuclear entry of caspases is unclear. On one hand, caspases may translocate from the cytosol into the nucleus as active species.
Alternatively they may be directly activated from within the nucleus, which would imply the existence of an autonomous nuclear caspase activation pathway. The sole communication channel between the nucleus and the cytosol is the nuclear pore, which may act as a sensor for caspase activation.
The present study was designed to:
• investigate 2 models of apoptosis, one initiated at the plasma membrane by engagement of the TRAIL death receptor and the other triggered at the nucleus by the DNA-damaging
• investigate the proteolysis of an extensive number of nuclear pore proteins as an indicator of caspase activation on the nucleoplasmic and cytoplasmic side of the nuclear membrane
• compare the time course of proteolysis of nucleoporins within sub-complexes and those restricted to either the nuclear interior or the cytosol in the two different models of apoptosis
3 MATERIALS AND METHODS
3.1 Materials
3.1.1 Machines and technical devices
Cameras: MP-4 Land (Polaroid, Hertfordshire, UK); Contax 167 MT (YASHICA Kyocera GmbH, Hamburg, Germany). Centrifuges: Biofuge fresco and Megafuge 1.0 R (Hereus Instruments, Hanau, Germany). Confocal microscope system: Zeiss LSM 510 Meta. Electrophoresis chambers: Mini-PROTEAN 3 Electrophoresis Cell and power supply Power Pac 300 (BioRad Laboratories GmbH, Munich, Germany). Electrophoretic transfer cell: Mini Trans-Blot® Electrophoretic Transfer Cell, Tran-Blot® SD Wet Transfer Cell and power supply Power Pac 200 (BioRad Laboratories GmbH, Munich, Germany). ELISA-Reader: SLT Spektra (SLT Labinstruments, Crailsheim, Germany).
FACS: BD LSR (Becton Dickinson, Franklin Lakes, USA). Film material: Fuji Medical X-ray film (Fuji Photo Film, Dusseldorf, Germany. Fluorimeter: Microplate Fluorescence Reader FL 600 (Deelux Labortechnik, Godensorf, Germany). Image reader: Gel Jet Imager (Intas GmbH, Germany), Luminescent Image Analyser LAS-1000 CH, acquisition software Image Reader LA-1000 (Fuji Photo Film Co., Ltd., Tokyo, Japan), and Advanced Image Data Analyser (AIDA) software (Raytest GmbH, Staubenhardt, Germany). Imaging camera: Dage-72 CCD camera (Dage-MTI, Michigan City, USA) and image analysis system MCID (Imaging Research Inc., St. Catherine, Ontario, Canada). Incubator: Model BB 6220 (Heraeus Instruments, Hanau, Germany).
Laminar Flow: LaminAir® HB 2448 and LaminAir® HB48 (Heraeus Instruments, Fellbach, Germany); Microflow Laminar Flow Workstation (Nunc GmbH, Wiesbaden, Germany). Membrane: Nitrocellulose Hybond™ ECL (Amersham-Buchler GmbH &
Co. KG, Braunschweig, Germany). Microscopes: Fluorescent microscope Axiovert 25 (Zeiss, Oberkochen, Germany), Leitz DM IRB and Leitz DM IL (Leica Microscopy and Systems GmbH, Wetzlar, Germany). Pipettes: Eppendorf (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany) and Gilson (Abimed, Langenfeld, Germany). Thermomixer:
Eppendorf Thermomixer (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany).
MATERIALS AND METHODS
3.1.2 Chemicals
Bachem Biochemica GmbH, Heidelberg, Germany: z-VAD-fmk (z-Val-Ala-Dl-Asp- fluormethylketone).
Bender & Hobein GmbH, Heidelberg: Pierce BCA protein assay reagent.
Biomol, Hamburg, Germany: Asp-Glu-Val-Asp-aminotrifluoromethylcoumarine (DEVD-afc), N-Val-Asp-Val-Ala-Asp-aminofluoro-methylcoumarine (VDVAC-afc).
BioRad Laboratories GmbH, Munich, Germany: Biotinylated SDS-Page Standards.
Calbiochem-Novabiochem, Bad Soden, Germany: caspase-2 inhibitor I /z-VDVAD-fmk (Z-Val-Asp-Val-Ala-Asp-FMK), caspase-3 inhibitor II /z-DEVD-fmk (Z-Asp-Glu-Val- Asp-FMK) and caspase-6 inhibitor I /z-VEID-fmk (Z-Val-Glu-Ile-Asp-FMK).
FMC BioProducts, Rockland, ME, USA: agarose SEA Kem GTG, low melting point agarose SEA plaque GTG.
Eastman Kodak Company, Rochester, USA: Kodak GBX Fixer and replenisher, Kodak GBX developer and replenisher.
MBI Fermentas, St. Leon-Rot, Germany: Prestained Protein Molecular Weight Marker.
Merck, Darmstadt, Germany: _-mercaptoethanol, sucrose.
Molecular Probes Europe BV, Leiden, Netherlands: Hoechst 33342, SYTOX green nucleic acid stain.
Peptide Institute, Osaka, Japan: cathepsin B inhibitor (CA-074 Me).
PIERCE, Rockford, USA: Super Signal West Pico Chemiluminescent Substrate.
Polyscience Inc, Warrington, USA: Aquapolymount.
Qbiogene Inc., Heidelberg, Germany: jetPEI™.
Roche, Germany: complete protease inhibitor.
Roth GmBH & Co., Karlsruhe, Germany: ethanol, glycine, HEPES, ponceau S, Rotiphorese Gel A, Rotiphorese Gel B, Rotiphorese Gel 30, sodium chloride, Tris.
Serva, Heidelberg, Germany: ammonium persulfate, Coomassie® Brilliant Blue G250, paraformaldehyde, TEMED.
Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany: 7-Amino-4-trifluoromethyl- coumarin (AFC), bovine serum albumin, 3-[(3-cloamidopropyl)-dimethylammonio]- propane sulphate, digitonin, dimethylsulphoxide, dithiothretiol (DTT), ethylenediamine tetra-acetic acid (EDTA), ethyleneglycol-bis- (-aminoethylether) tetra-acetic acid (EGTA), ethidium bromide, etoposide, glycine, glycerol, goat anti-rabbit HRP, normal
goat serum, sodium dodecylsulfate, staurosporine, thymidine, Tris-base, Triton X-100, trypan blue 0.4%, Tween 20.
TRAIL was a kind gift from Dr. H. Walczak (German Cancer Research Centre, Heidelberg, Germany).
3.1.3 Kits
Cell Death Detection ELISA: Boehringer Mannheim, Mannheim, Germany.
QIAprep Spin Miniprep Kit : Qiagen, Hilden, Germany.
EndoFree Plasmid Maxi Kit : Qiagen, Hilden, Germany.
3.1.4 Antibodies
Monoclonal anti-actin antibody: Chemicon, Temecula, CA, USA.
Alexa™ 488-conjugated anti-mouse IgG antibody, Alexa™ 596-conjugated anti-rabbit IgG antibody: Molecular Probes Europe BV, Leiden, Netherlands.
Bax rabbit polyclonal antibody directed against the Bax N-terminus: Upstate Biotechnology, Lake Placid, New York, USA.
Cleaved caspase-3 (Asp 175) antibody: Cell Signaling Technology, Beverly, MA, USA.
Monoclonal anti-cytochrome c antibody for immunostaining (clone 6H2.B4): Pharmingen, San Diego, USA.
Monoclonal antibody to human lamin A/C (clone JOL-3) : Abcam, Cambridge, UK.
Lamin B affinity-purified goat polyclonal antibody: Santa Cruz, California, USA.
Mouse monoclonal antibody to poly(ADP-ribose) polymerase (clone C2-10): Biomol, Hamburg, Germany.
-IgG mouse HRP polyclonal antibody, Peroxidase-conjugated affinity-isolated goat anti- rabbit serum: DAKO AIS, Glastrup, Denmark.
Polyclonal anti-mouse HRP and HRP Peroxidase conjugated goat anti-mouse immunglobulin specific polyclonal antibody: Pharmingen, Hamburg, Germany.
The following antibodies were generously provided by Dr. V. Cordes (Karolinska Institute, Stockholm, Sweden): Anti-Nup50 affinity purified rabbit antibody, Anti-Nup93-3 affinity purified guinea pig antibody, Anti-Nup96-1 affinity purified guinea pig antibody, Anti- Nup98-2 affinity purified guinea pig antibody, Anti-Nup107-1 affinity purified guniea pig antibody, Anti-Nup205-2 affinity purified guinea pig antibody, Anti-Nup153 mouse
MATERIALS AND METHODS
monoclonal antibody, Anti-RanBP2 affinity purified guinea pig antibody and Anti-Tpr mouse monoclonal antibody.
Anti-Can/Nup214 monoclonal rabbit antibody was a kind gift from Dr. R. Kehlenbach (Hygiene Institute, Department of Virology, Heidelberg, Germany). Anti-Nup160 and anti-Nup188 antibodies were generously provided by Dr. J. Koeser (Basel University, Switzerland). Anti-RanGAP was a kind gift from Dr. D.Gorlich (ZMBH, Heidelberg, Germany).
3.1.5 Cells
HeLa 229 human cervix carcinoma cells were obtained from the American tissue culture collection (ATTC No.CCL-2.1, Rockville, MD, USA).
3.1.6 Cell culture material
Cell culture flasks and plates: Costar GmbH, Bodenheim, Germany and Greiner GmbH Frickenhausen, Germany. Dulbecco´s modified Eagle medium: Invitrogen GmbH, Karlsruhe, Germany. Fetal calf serum: Biochrom AG/Seromed®, Berlin, Germany.
Penicillin: Invitrogen Corporation, Karlsruhe, Germany. Glutamate: Invitrogen Corporation, Karlsruhe, Germany.
3.1.7 Plasmid
VLP35 was kindly provided by Dr. Melissa Rolls, University of Oregon, USA (Rolls et al., 1999).
3.2 Methods
3.2.1 Culturing of cells
Cell culture was performed aseptically in a micro-flow laminar workstation. HeLa 229 cells were cultured in either a 75cm2 or 175cm2 flask using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated foetal calf serum, 5% L- glutamine, 100units/ml penicillin and 100g/ml streptomycin in a humidified atmosphere of 5% CO2 in air at 37°C. They were passaged routinely every two to three days in a ratio of 1:5 or 1:10 respectively, thus enabling logarithmic growth to be maintained.
Cells were washed with 5ml 1x PBS (10x PBS: 137mM NaCl, 10mM NaH2PO4, 3mM KH2PO4, pH 7.4), treated with 5ml trypsin-EDTA (trypsin-ethylenediamine tetra-acetic acid) and then incubated at 37°C for 1 minute to facilitate detachment of adhered cells.
The action of trypsin was terminated via the addition of 10ml of media. The cell suspension was centrifuged at 1000rpm for 5 min and the resulting cell pellet was resuspended in 10ml of media. For counting, 10µl of trypan blue was added to 50µl of the cell suspension. The cells were counted using a haemocytometer and plated at the required density. Cells used in experiments were from passage numbers 1-30.
3.2.2 Cell synchronisation via double thymidine block.
Synchronised cells are achieved by imposing metabolic blocks such as thymidine, resulting in a synchronous population of cells at the beginning of S phase (Collins, 1978). The first thymidine block on exponentially growing cells is for a period equivalent to G2+M+G1.
High concentrations of thymidine inhibit DNA synthesis in S phase cells by depleting the nucleotide precursor pools of deoxycytidine triphosphate (dCTP). Cells in G2, M and G1 are not affected and continue to traverse the cell cycle until reaching G1-S phase boundary when the onset of DNA synthesis is inhibited. On completion of the first block approximately half of the cell population is uniformally distributed throughout S phase and half at the beginning of S phase. The first block is released for a period equivalent to S phase which allows the cells accumulated at the G1-S phase and those blocked throughout the S phase to pass through S phase. Now with half the cell population in early G2 and other cells distributed between G2, M and G1, a second thymidine block is imposed for a
MATERIALS AND METHODS
period equivalent to G2+M+G1. At completion all the cells are at the beginning of S phase. On release of the second thymidine block the cells synchronously traverse S, G2, mitosis and G1 (Collins, 1978).
Cells were plated at a density of 5x104cells/ml and allowed to grow for 24h. The cells were then treated with a final concentration of 2.2mM thymidine (stock solution: 13.2mM in medium, sterile filtered) for 12h. The cells were released from the first thymidine block by removing the medium, washing twice with 1x HBS (10x HBS: 122mM NaCl, 2.67mM KCl, 9.4mM glucose, 14mM NaH2PO4, 20mM Hepes, pH 7.4) and then adding fresh medium. The second thymidine block was imposed 9 h later. The cells were once more treated with 2.2mM thymidine for 12h. The cells were released from the second thymidine block for 4h prior to induction of apoptosis.
3.2.3 Flow Assisted Cell Sorting (FACS) analysis
HeLa 229 cells were seeded at 5x104cells/ml in 10cm petri dishes and allowed to grow for 24h. After experimental treatment, the cells were gently scraped on an ice pack in 2ml of cold PBS, transferred to a Falcon tube and centrifuged at 1500rpm at 4°C. The pellet was resuspended in 200ml of PBS and the cells were fixed in 800ml EtOH (100%) at 4°C for 30 min and stored overnight at 4°C. The samples were centrifuged for 5 min at 1500rpm and the pellet resuspended in 800ml of PBS, 100ml of RNase A-solution (final concentration 100l/ml) and 100ml of propidium iodide (final concentration 40g/ml). A mininum of 10 000 cells were analysed.
3.2.4 Pre-treatment protocol for protease inhibitors
For protease inhibitor assays, cells were pre-incubated with the inhibitor for 30 min prior to the addition of the apoptosis-inducing agent and remained in the medium during the incubation. Inhibitors tested for their protective potency were caspase inhibitors: z-VAD- fmk (N-benzyloxycarbonyl-Val-Ala-aspartyl-fluoromethylketone), cathepsin B inhibitor (CA-074-Me), caspase-3 inhibitor/z-DEVD-fmk (N-benzyloxycarbonyl-Asp-Glu-Val-Asp- fmk, caspase-6 inhibitor/z-VEID-fmk (N-benzyloxycarbonyl-Val-Glu-Ile-Asp-fmk) and caspase-2 inhibitor/z-VDVAD-fmk). All inhibitors were added to a final concentration of 20µM except cathepsin B at 100µM.
3.2.5 Cell viability assays
3.2.5.1 SYTOX/Hoechst assay
To analyse living cells for apoptosis and necrosis, cells were stained with a combination of the fluorescent chromatin dyes Hoechst 33342 (500ng/ml; membrane permeant, stains all nuclei) and SYTOX (500M, membrane impermeant, stains nuclei of lysed cells) by adding the dye solution (100x in DMSO) 10 min prior to investigations. Using a Leica DM-IRB fluorescence microscope and lenses providing 400 x final magnification, cells with condensed or fragmented nuclei were scored as apoptotic; lysed cells with non- condensed nuclei were scored as necrotic. For each data point at least 200 cells were scored in 3 different microscopic fields.
3.2.5.2 DNA-fragmentation ELISA
Histone-containing oligonucleosomal DNA-fragments were quantitated using the Cell Death Detection ELISA. The basis of this assay is a sandwich-enzyme-immunoassay principle using monoclonal antibodies against DNA and histones respectively. This allows the specific determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates.
Cells (5 x 104) were lysed in 500l incubation buffer (included in the assay kit) for 30 min at 4°C. Then, organelles and other high molecular weight structures (e.g. non-fragmented DNA) were separated by centrifugation (1500rpm, 10min, 4°C) from the cytosolic fraction, in which oligonucleosomal DNA fragments also remained. The supernatant was diluted 1:20, and either stored at -20°C or assayed immediately. Following the manufacturer’s guidelines, lysate obtained from about 500 cells was sufficient to perform the test. Colour reaction was detected at = 405 nm. The values were averaged and the background value substracted.
