Regulation of nuclear envelope permeability in cell death and survival

Christine Strasser, Patricia Grote, Karin Schauble, Magdalena Ganz and Elisa Ferrando-May*

University of Konstanz; Department of Biology; Bioimaging Center; Konstanz, Germany

Keywords: Bcl-2, calcium, NupI53, nuclear pore complex, staurosporine, apoptosis, caspases, calpains, confocal microscopy

Abbreviations: NPC, nuclear pore complex; NE, nuclear envelope; Nup, nuclear pore protein; ER, endoplasmic reticulum; STS, staurosporine; TG, thapsigargin

The nuclear pore complex (NPC) mediates macromolecular exchange between nucleus and cytoplasm. It is a regulated channel whose functional properties are modulated in response to the physiological status of the cell. Identifying the factors responsible for regulating NPC activity is crucial to understand how intracellular signaling cues are integrated at the level of this channel to control nucleocytoplasmic trafficking . For proteins lacking active translocation signals the NPC acts as a molecular sieve limiting passage across the nuclear envelope (NE) to proteins with a MW below -40 kD. Here, we investigate how this permeability barrier is altered in paradigms of cell death and cell survival, i.e., apoptosis induction via staurosporine, and enhanced viability via overexpression of BcI-2. We monitor dynamic changes of the NPC's size-exclusion limit for passive diffusion by confocal time-lapse microscopy of cells undergoing apoptosis, and use different diffusion markers to determine how BcI-2 expression affects steady-state NE permeability. We show that staurosporine triggers an immediate and gradual leakiness of the NE preceding the appearance of apoptotic hallmarks. BcI-2 expression leads to a constitutive increase in NE permeability, and its localization at the NE is sufficient for the effect, evincing a functional role for BcI-2 at the nuclear membrane. In both settings, NPC leakiness correlates with reduced Ca2+ in internal stores, as demonstrated by fluorometric measurements of ER/NE Ca2+ levels. By comparing two cellular models with opposite outcome these data pinpoint ER/NE Ca2+ as a general and physiologically relevant regulator of the permeability barrier function of the NPC.

Introduction

In eukaryotic cells the nuclear envelope (NE) provides a physi­cal barrier that secludes and organizes genomic material within the nucleus. Molecular traffic across the NE occurs exclusively via nuclear pore complexes (NPCs) , multimeric macromolecular channels spanning both lipid layers of the NE.

The NPC acts as diffusion barrier for inert molecules with a MW larger than = 40 kD and facilitates the translocation of much larger proteins up to a flux rate of 1000Is.' This activity is dynamically regulated in response to physiological or pathologi­cal signaling cues.2.3 Functional control of the NPC may occur via alterations in NPC composition4, by post-translational modi­fication of nuclear pore proteins (Nups), 5.G and by modulating the dimensions of the channel itself.? These structural changes impinge on transport capacity and selectivity of the NPC as well as on its size-exclusion limit for passive translocation. Thus, one important layer of regulation of nucleo-cytopl asmie trafficking resides at the NPC itsel f'll.

Active cell proliferation and programmed cell death are examples for cellu lar states known to induce modifications of NPC structure and function. Oncogene-transformed,

'Correspondence to: Elisa Ferrando-May; Emai l: elisa.may@uni-konstanz.de

hyperproliferating cells display larger NPC diameters than their resting counterparts.9,'o In mitotic cells, the level of Nup96 is downregulated to allow efficient GI /5 transition. 1I In apoptotic cells, NPC dismantling and breakdown of the NE permeability barrier are the consequence of caspase-mediated proteolysis of a subset of nUcleoporins (NupS).I2·,G Yet another study has evi­denced that calpains, Ca2'-activated proteases, cleave Nups in neuronal cells undergoing excitotoxic death resulting in nuclear accumulation of the cytoplasmic protein GAPDH.'7 Disruption of NPC components is not the only mechanism of NE per­meabilization, since nuclear leakiness has been observed also in the absence of Nup proteolysis in apoptot ic and virus-infected cells.' 2. 17. IB Also the proapoptotic Bcl-2 family proteins Bax and

Bak were proposed to affect nucleo-cytoplasmic protein parti­tioning: histone HI and nucleophosmi n were shown to redistrib­ute to the cytoplasm as a consequence ofBax/Bak overexpression independently of caspase activity.'9 This result high lights yet another li nk between the apoptosis machinery and nucleo-cyto­plasmic trafficking.

Ca2• functions as second messenger in the cellular response to very diverse endogenous and exogenous signals ranging from noxious environmental insults to growth stimulatory factors.

A

f : i 3

Nuclear 4x Cherry --STS

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time [minJ

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time

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E! 80

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20

0 STS TRAIL

Figure 1. STS, but not TRAIL, induces a pre-apoptotic gradual increase in nuclear perme­ability coincident with dep letion of ER/NE Ca". (A) Time course of nuclear entry of 4xCherry in HeLa cells treated with either STS (open rectangles) or TRAIL (open circles). A region of interest corresponding to the area of the nucleus was obtained by manual segmentation (see Fig. S1 D). The mean intensities measured in the 4xCherry channel are plotted over time on two separate x-axes. On the left x-axis, time points from the start of image acquisition until the onset of nuclear condensation are displayed. On the right x-axis, time points immediately preceding and following nuclear condensation are shown. Here, traces were aligned with respect to the time point of nuclear condensation (time 0, dashed blue line). Red arrows indicate the time points corresponding to the frames shown in (B). Green arrows refer to the time points of ~a" measurement shown in (C) and (D) . .Data points are the aver­age of at least 13 cells. Error bars show the SEM (B) Confoca l images of HeLa ce lls expressing the nuclear permeability marker 4xCherry treated with STS or TRAIL. The time points refer to the time course shown in (A) (red arrows). (C) Characteristic traces of Fluo-4 fluorescence in HeLa ce lls treated to undergo apoptosis. HeLa ce lls were left untreated (black), or treated for 60 min with either STS (light gray) or TRAIL (dark gray), and loaded with the Ca" indica­tor Fluo-4. TG (Sf1m) was added at the indicated time pOint (arrow) to release Ca" from internal stores. (D) Comparison of Ca" content in internal stores after treatment with STS or TRAIL for 60 min. The difference between basal and peak fluorescence was calculated. Bars indicate the amount of CaH mobi lization in treated cells as percentage of control, untreated cells. Experiments were performed in triplicates .•• * p < 0.001.

of the nucleoplasmic basket structure could be distinguished underpinning the role of Ca2• in structural rearrangements of the NPC. 28-31

Intriguingly, Bax and Bak, initially character­

ized as key players in apoptotic cytochrome C

release from mitochondria, were later involved

in maintaining ER [Cah]. Cells lacking both

Bax and Bak were shown to have a drasti­

cally reduced ER [Ca2.] and fail to mobilize Ca2• from the ER to mitochondria.n .33 These

results suggest that Bcl-2 family proteins might impinge on the NPC via modulation of ER/NE Ca2• levels , in line with the abovementioned

findings on Bax/Bak-dependent apoptotic

nuclear protein redistribution.

Prompted by these considerations, we asked

whether changes in ER/NE [Ca2,) could pro­

vide a common denominator for functional

modulation of the NPC under conditions

as diverse as cell death and cell survival. We

addressed this question by investigating first

the NE permeability barrier in cells treated to

undergo apoptosis by staurosporine (STS). This

broad spectrum kinase inhibitor is a well-estab­

lished and efficient apoptosis inducer known to

elicit intracellular [Ca2'] elevation via release from internal storesY·34 We then compared

this death model with overexpression of Bcl-2,

a paradigm for enhanced cell survival and pro­

liferation (reviewed in ref. 35). Bcl-2 is known

to affect ER [Ca2,] through direct interaction with InsP3Rs.3G·37

Our results provide evidence for a major role of ER/NE [Ca2. ] in modulating the NE perme­

ability barrier independently of cellular fate and

identify Bcl-2 as a regulator of the size exclusion

limit of the NE for passive diffusion.

Results

Numerous studies have proposed a role for Ca2• in the regulation

ofNPC structure and function. The underlying mechanisms are

not fully clear yet (reviewed in 20.22). The NE harbours functional

inositol(1 ,4,5)-trisphosphate receptors (InsP3Rs), as well as

ryanodine receptors (RyRs) and nicotinic acid adenine dinucleo­

tide phosphate receptors (NAADPRs) . In response to their cog­

nate agonistic signals these receptors can induce [Ca2,] gradients

in the lumen of the NE - which is continuous to the lumen of

the endoplasm ic reticulum (ER) 23 -, or in the cytosol, or in both

compartments. Depletion of ER/N E Ca2• stores was shown to

inhibit passage of inert molecules and transport cargo in some studies24.25 but not in others. 2G.27 Alterations in NPC topology in

response to Ca2. have been observed by (immuno-)electron and

scanning force microscopy. "Close" vs. "open" conformations

Staurosporine induces a pre-apoptotic, cas­

pase-independent leakiness of the NE. We first

invest igated how STS treatment affects the permeability barrier

function of the NE. To this end, we performed single-cell, con­

foca l time-lapse recordings of HeLa cells expressi ng a reporter

for passive passage across the NE, a tetrameric mCherry fusion

protein (4xCherry) (Fig. lA and B; Fig. SIA and B). Images

were taken approx. every 8 min, and a minimum of 13 cells was

recorded in each exper iment. T he nuclea r inAux of 4xCherry was

quantified over time as described in "Materials and Methods"

and in Figure SID. To monitor apoptosis progression the cel ls

were sta ined with low concentrations of the vital DNA dye

Hoechst 33342. This enabled to visualize nuclear condensation

as a ha llmark of caspase-dependent apoptot ic execution (Fig.

SIC). To account for the asynchronicity of the apoptotic process,

data from each time series were split into two parts and displayed

2

----TRAIL

~ .~ STS on separate x-axes. The left portion of the x-axis

shows traces from the start of the experiment up to time points immediately preceding nuclear conden­sation (pre-apoptotic) . On the right x-axis, traces were aligned with respect to the time of nuclear con­densation (time point 0) . This arrangement properly visualizes changes in nuclear permeability occurring at nuclear execution.

& 1.50 ...... Nup153-GFP :s ..... Nup 153-0349N-GFP

III 1.50 c:

~ .5

~ Q) c:

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~ 0.75 i:.

.~ 0.50

~ 0.25+-.....,.-"'T'"-.----r-l ............... g 9>'0 <pI:. ~ ,.'b !'>'" :-'0 I:. ,,<0

0 ::>

0::

~ 'iij

~ 0.75

0.50

t---1---, STS-treated cells died on average 3.5 h after drug

addition, when 80% displayed apoptotic nuclei (not shown). Traces of nuclear 4xCherry fluores-

time [min]

E 0 c:

0.25 ,,'0

:I- :-~ :-.,," .~ <p'b b'O :I-~'O I:. ,,'0 time [min]

cence displayed a characteristic early-onset and gradual increase in signal intensity indicating that nuclei became partially accessible to the perme­ability marker shortly after drug addition (Fig. lA, left x-axis, open rectangles). At later time points immediately preceding chromatin condensation, a steep increase in nuclear fluorescence was observed, suggesting a rapid collapse of the NE permeability barrier (Fig. lA, right x-axis, open rectangles). To determine whether this behavior is genera lly associ-

Figure 2. Caspase-dependent cleavage of Nup153-GFP coincides with chromatin condensation in TRAIL and STS-induced apoptosis. HeLa cells expressing Nup153-GFP (green line) or its caspase-uncleavable mutant Nup153-0349N-GFP (red line) were treated with STS or TRAIL. The former were additionally preincubated with the pan­caspase inhibitor zVAO (black line). Regions of interest corresponding to the nuclear rim were defined interactively taking care to omit convoluted regions (see also Mate­rial and Methods and Fig. SlO). Normalized Nup153-GFP signal intensities are plotted over time as in Figure 1A. The dashed blue line indicates the time point of nuclear condensation. Data points are the average of at least 15 cells. Error bars show the SEM.

ated with apoptotic cell demise we treated cells with TRAIL, a death receptor ligand triggering cell death via the extrinsic path­way.38 Cells underwent apoptosis quite rapidly as expected ( ~ I.5h) but in contrast to STS treatment nuclei efficiently excluded 4xCherry until chromatin condensation ensued (Fig. lA, open circles, left x-axis). In the final phase, the NE permeability bar­rier collapsed in these cells simi larly to STS-treated cells (Fig. lA, right x-axis). These data suggest that the observed pre-apop­totic increase in nuclear accessibility is a distinctive feature of STS-induced cell death. To address the question about a poten­tial involvement of Ca2 • we determined ER/NE [Ca2 , ] levels at selected time points after treatment with both drugs. We used Fluo-4 as a Ca2 '-specific indicator, in combination with thapsi­gargin (TG), an inhibitor of the ER Ca2·-ATPase.39 TG induces the discharging of ER/NE Ca2• resulting in an abrupt increase of its cytosolic concentration which can be detected with Fluo-4. Thus, TG-induced Ca2

• transients are an established measure of ER/NE Ca2

• content. At 60 min after STS addition we observed a 50% reduction of ER Ca2• levels, contrarily to cells stimu lated for the same time period with the death-receptor ligand TRAIL. The latter had no significant effect on ER/NE [Ca2

. ] (Fig. IC, D). These results suggest that the early increase in the size exclu­sion limit of the NE for passive diffusion observed under STS treatment is associated with the depletion of ER/N E [Ca2 . ] levels induced by this drug.

Early NE leakiness precedes apoptotic cleavage of nucleo­porin Nupl53 and is not dependent on caspase activity. According to previous studies, collapse of the nucleocytoplas­mic permeability barrier in apoptosis is brought about by the caspase-mediated cleavage ofNups .14 To temporally correlate the permeability alterations of the NE observed in STS- and TRAIL­treated cells with NPC proteolysis we monitored a fluorescently labeled fusion of Nup153 over time. Nup153 is a component of the N PC located at the nuclear basket and is effi ciently cleaved by execut ioner caspases.12.16 In control experiments we verified

that the NupI53 -GFP fusion protein is processed upon treatment of HeLa cells with STS or TRAIL in a caspase-dependent fash­ion (Fig. S2). In our confocal time series of apoptotic cells we then quantifid the average intensity of th e NupI53-GFP signal at the nuclear rim by manual segmentation, taking care to avoid regions in which the NE formed convolutions, as seen quite fre­quently in STS-treated cells. Such convolutions appear as out­of-focus regions of the nuclear rim signal (Fig. SID). Both STS and TRAIL treated cells showed a clear and abrupt reduction of NupI53-GFP fluorescence at the nuclear rim at the onset of chromatin condensation. This effect was abrogated in the pres­ence of the pan-caspase inhibitor zVAD and by inactivation of the caspase cleavage site in Nup153 at amino acid position 349 (Fig. 2, right x-axes). In the time window preceding chromatin condensation, NupI53-GFP traces were quite similar for STS and TRAIL treatment and both were not affected by zVAD (Fig. 2, left axes). According to these data, loss of NupI53-GFP sig­nal from the nuclear rim is an indicator of caspase-dependent dismantling of the NPC which occurs concomitantly to chro­matin condensation. We then investigated the effect of zVAD on the kinetics of nuclear 4xCherry influx. Caspase-inhibition prevented the final, abrupt collapse of the permeability barrier in both apoptosis models, but had no effect on the early-onset, moderate increase in nuclear accessibi li ty observed in cells treated with STS (Fig. 3A) . In these cells caspase-3-like activity was not detectable until 2h after stimulation, when partial nuclear entry of 4xCherry was already established. This assay also confirmed that zVAD was highly effective in blocking caspase activation (Fig. 3C). In sum, these results support the existence of an early, caspase-independent leakiness of the NE that precedes the apop­totic dismantling of the NPC triggered by STS.

Early pre-apoptotic permeabilization of the nuclear envelope depends on Ca2., but not on calpain act ivation. The NPC has been shown to be the target of non-caspase proteolytic systems, in particular of the Ca2 '-dependent protease family of ca lpains.

3

A Nuclear 4xCherry B Nuclea r 4xCherry in response to the Ca2• chelator further supports the existence of a caspase­independent effect of STS at the NE which requires mobilization of Ca2 • from imernal scores. At th e final stage of chromatin condensation, the NE permeability barrier collapses, a process dominated by the effect of executioner caspases and evidently not dependent either on Ca2• or on calpains.

~ 'iii 8 ..... STS c:

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... TRAIL + zVAD Ipeplin

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Exogenous expression of Bcl-2 at the nuclear membrane increases NE permeability. Bcl-2 is an antiapoptotic protein with potent pro-survival activity whose expression leads to a reduction in ER/NE Ca2• levels.41 Bcl-2 family pro-o STS + BAPTA-AM

.STS +zVAD

control 1h 2h 4h

----------------

teins Bax and Bak have been suggested to increase nuclear permeabili tyl9 as part of their proapoptotic activity. Their antiapoptotic cousin Bcl-2 has not been investigated so far. Therefore we sought

Figure 3. STS-induced early nuclear leakiness is mediated by Ca2+ but neither by caspases nor cal­pains. (A) Hela cells were treated with STS (open rectangles) and TRAIL (open circles) in the absence (red lines) or presence (black lines) of the pan-caspase inhibitor zVAD. Nuclear 4xCherry intensities were extracted from confocal time series and plotted over time as in Figure 1 A. The dashed blue line indicates the onset of nuclear condensation. (B) Hela cells were treated with STS alone (red open rect­angles), or in presence of the calpain inhibitor calpeptin (black open triangles), or in presence of the Ca" chelator BAPTA-AM (black crosses). Nuclear 4xCherry intensities were extracted from confocal time series and plotted over time as in Figure lA. The dashed blue line indicates the onset of nuclear condensation. (C) Hela cells were treated with STS either in absence (white bars) or presence of the following inhibitors: calpeptin (0.5 fJ.M, dark gray bars), BAPTA-AM (10 fJ.M, light gray bars), and zVAD (20 fJ.M, black bar). The inhibitors were added 30 min prior to STS treatment. Untreated cells served as control. Caspase activity was measured fluorometrically in ceil lysates at the Indicated time points.

to determine whether Bcl-2 expression might impinge on NE permeability and whether this would involve ERI NE [Ca2.]. First, we compared HeLa cells stably expressing wildtype murine Bcl-2 (HeLa-mbcl-2), and SW480 cells expressing the human counterpart (SW480-hbcl-2) (Fig. S4A) with the respective control cell lines using our Nuclear Permeability Assay (NPA).42.43 Cells were partially permeabilized with digitonin, incubated with fluorescendy

In primary cortical neurons, Ca2• overload caused by exposure to excitotoxic glutamate concentrations activates calpains resulting in the degradation of several Nups and the concomitant loss of proper nucleo-cytoplasmic partitioning of reporter molecules.17

To investigate the potential involvement of calpains in STS­mediated nuclear permeabilization in HeLa cells we performed live-cell microscopy experiments in the presence of the cell-per­meable calpain inhibitor calpeptin . The efficacy of the inhibi­tor was confirmed using an in vitro calpain activity assay (Fig. S3). Differendy from neuronal excitotoxicity, calpeptin did not protect from NE permeability changes induced by STS in HeLa cells both on the early and late time scale (Fig. 3B). Early nuclear leakiness, however, was efficien tly prevented in the presence of BAPTA-AM, a cell permeable, high-affinity Ca2• chelator that removes intracellular free [Ca2.] also in internal stores.40 In the presence of BAPTA-AM mobilization of free [Ca2.] from the ER/NE is blocked. This compound did not inhibit the collapse of the NE permeability barrier occurring at nuclear condensation (Fig. 3B). In addition, up to 2h after treatment with STS caspase-3-like activity was not significandy affected eithtr by ca lpeptin or by BAPTA-AM (Fig. 3C) . This lack of correlation between cas­pase activation and the early nuclear redistribution of 4xCherry

4

labeled 70 kD dextran, and imaged by confocal microscopy. Passage of dextran across the NE was quamified by measuring the average fluorescence signal intensity in cell nuclei. In both HeLa and SW480 cells, Bcl-2 expression led to a marked increase in dextran nuclear entry as compared with control (Fig. 4A). This finding was confirmed with tran­sient transfections of HeLa-mBcl-2 cells using the permeability marker 4xCherry. Cells with a homogenously distributed fluo­rescence signal were scored positive for nuclear 4xCherry. Bcl-2 expression resulted in a significan tly higher percemage of these cells as compared with control (Fig. 4B). Finally, we used bead loading co introduce fluoresctnt 70 kD dc::xtran imo Bcl-2 over­expressing HeLa cells, and obtained again similar resuirs (Fig. S4B). Airogether these data demonstrate that overexpression of Bcl-2 leads to an increase in the size exclusion limit of the NE for passive diffusion, a finding quite unexpected from previous stud­ies and from our own data linking nuclear leakiness to apoptotic cell death.

Bcl-2 is known to insert into all intracellular membranes including those of mitochondria, endoplasmic reticulum and nucleus (own unpublished observations and ref. 44). To exam ine whether the effect of Bcl-2 on the nuclear permeabi lity barrier depc::nds on the specific subcd lular loca li zation of this protein

we compared nuclear entry of 4xCherry in cells expressing full length Bcl-2, and Bcl-2 variants targeted either to the endoplas­mic reticulum via fusion to the C-terminus of cytochrome b5 (ER-mBcl245), or to the mitochondria (mito-mBcl246) or to the NE (NE-mBcl2, this study). The latter Bcl-2 variant was gen­erated by replacing the C-terminal transmembrane domain of Bcl-2 with the KASH domain of human Nesprin-2, a protein tethering the actin cytoskeleton to the nuclear membrane47 (Fig. S5A). At high expression levels NE-mBcl2 showed a tendency to mislocalize to the ER (not shown) . We therefore expressed the corresponding gene under the control of a tetracycline-regulat­able promoter in HeLa Tet Off cells. At low expression levels we cou ld achieve an exclusive localization ofNE-Bcl2 at the nuclear rim, as judged by its colocalization with Nesprin-2 in confocal images (Fig. S5B). HeLa cells were transiently transfected with plasmid vectors encoding wildtype, mitochondrial, and ER tar­geted Bcl-2. NE-targeted Bcl-2 was expressed in HeLa Tet Off cells in the presence of doxycyclin. N E-targeted GFP served as control. Nuclear permeability was assessed by contrasfection of 4xCherry and counting cells with equal distribution of the per­meability marker between nucleus and cytoplasm. Bcl-2 local­ization was verified via immunocytochemistry. An increase in N E permeability comparable to that caused by wildtype Bcl-2 was obtained only in cells in which Bcl-2 localized at the ER and the NE but not at the mitochondria. We observed a 2-fold increase with respect to control cells expressing only the per­meability marker (Fig. 5A, B and C). These data indicate that localization of Bcl-2 at the NE is sufficient to mediate its activity on the nuclear permeability barrier.

Bcl-2 effects at the NE are mediated by Ca2 • • The effect of Bcl-2 as regulator ofER Ca2' levels is well documented (reviewed in 48). Based on our observation in STS-treated HeLa cells sug­gest ing that Ca2

• is involved in controlling the size exclusion limit of the NE, and having detected that nuclear membrane­associated Bcl-2 might increase this limit, we inferred a role for Ca2

• as downstream mediator of Bcl-2 at the NE. To address this question, we first determined ER/NE Ca2 • levels in Bcl-2-expressing cells using Fluo-4 and TG. Hela cells stably express­ing wildtype Bcl-2 showed a marked reduction in TG-induced Ca2

• release from internal stores as compared with control cells, in line with previous studies41 (Fig. 6A and B, left panel, left bar). We also recorded capacitat ive Ca2• influx, a secondary Cah

response ensuing in cells with depleted internal stores when Ca2 •

is added back to the cultu re medium.49 In Bcl-2 overexpressing cells, this response was shown to be downregulated as adapta­tion to the long-term reduction of Ca2 • in their internal stores.50

Readdition of Ca2• (1 mM) to HeLa cells previously exposed

to TG evoked a steep increase in fluorophore signal indicative of Ca2

• capacitative influx. This effect was significantly dimin­ished in Bcl-2 overexpressing cells (Fig. 6A and B, right panel, left bar) . T hese data confirm that Bcl-2 overexpression leads to altered Ca2' levels in the ER/N E supporting our assumption of a role of Ca2

• in the Bcl-2-dependent increase in NE permeability in our experimental model. We then sought to mimic the Ca2.­

mediated, Bcl-2 dependent effect on nuclear permeabi li ty by manipulating Ca2• levels in the ER/N E independently of Bcl-2 .

A Nuclear Permeability Assay

~ 1.4 ~ 1.4 'iii HeLa 'iii SW480 co 1.3 j! 1.3 .'!l .S 1.2 .£ 1.2 -g .~ 1.1

-g .~ 1.1

(ij (ij

E 1. E 1. 0 0 co O. c o.

empty mBcl-2 empty hBcl-2 vector vector

B 4xCherry Nuclear-Cytoplasmic Distribution

4xCherry DNA (Hoechst 33342)

Figure 4. 8ci -2 overexpression leads to increased nuciear envelope permeability. (A) Nuclear entry of 70 kD fluorescent dextran w as assessed in HeLa and SW480 cells stably expressing murine and human bci-2, respectively, using the Nuciear Permeability Assay. Cells transfected with the empty vector served as controls. In each experiment, the average nuclear fluorescence intensity was normalized to the respec­tive control measurement. A total of at least 800 nuciei were evaluated semi-automatically .••• p < 0.001. (8) HeLa cells stably expressing murine 8c1 -2 and the corresponding empty vector were transiently transfected with a construct encoding the permeability marker 4xCherry. Left panel : Cell cultures were inspected microscopically for the presence of4xCherry in the nucieus. A representative set of confocal images is shown. Right panel : Quantification of microscopic images. Only cells exhibiting a homogenous distribut ion of the marker protein between nucleus and cytoplasm were scored as positive. The experiment was performed twice counting at least 150 cells per repeat. Scale bar: 10 fLm . ••• p < 0.001.

To this end, we cultured HeLa cells for 18 h in medium at low Ca2 • concentration (0.1 mM), a treatment leading to an adaptive reduction of Ca2 • levels in the ER/NE.50 These cells displayed a marked reduction of TG-induced Ca2

• release as well as a low­ered capacitative Ca2• influx as compared with controls, simi­larly to Bcl-2 overexpressing cells (Fig. 6A and B, both panels, right bars). Analysis of nucleo-cytoplasmic 4xCherry distr ibu­tion after adaptation to low calcium conditions indicated a sig­nificant increase in the number of cells with nuclear 4xCherry, although not to the same extent as following Bcl-2 overexpression (Fig. 6C). Finally, we restored high Ca2

• levels in the ER/NE of Bcl2-expressing Hela cells by coexpressing the ER Ca2>-ATPase SERCA2. Correction of ER/NE [Ca2

. ] by SERCA2 overex­pression has been demonstrated in Bax/Bak double knockout cells, which also display a decreased [Ca2· ] ER.51 In line with our working hypothesis, SERCA2 effectively counteracted the BcI-2 induced increase in nuclear permeability further implicating ERI NE Ca2

• levels in the Bcl-2-dependent regulation of NE perme­ability (Fig. 7A and B).

5

A

conlrol

wt­mBcl-2

mito­mBcl-2

ER­mBcl-2

NE­mBcl-2

NE­GFP

DNA 4xCherry a-mbcl-2 B

50

~ 40 .J:: Ql

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... ...

control wt- mito- ER-

c 50

~ 40 .J::Ql

'j~ 30 ~~

~~ " c:

mBcl-2 mBcl-2 mBcl-2

..

NE- NE-control mBcl-2 GFP

Figure 5. BcI-2localization at the nuclear membrane is sufficient to increase the size excl usion limit of the NE. (A) Confocal images of HeLa cells transiently expressing the permeability marker 4xCherry alone (control) and in combination with either wildtype murine bcl ' 2 (wt-mBcI-2) or bcl-2 variants targeted to the mitochondria (mito-mBcI -2) and the endoplasmic reticulum (ER-mBcI-2). Nuclear envelope targeted BcI-2 (NE-mBcI -2) was expressed in HeLa Tet Off cells in the presence of 1 IJ.g/ml doxycyclin to avoid strong overexpression and mislocalization. Nuclear envelope targeted GFP (NE-GFP) served as control. BcI-2 localization was determined by immunocytochemistry using antibodies specific for murine BcI -2. The NE-Bcl2 protein was not detectable at the nuclear rim under fixation conditions required to preserve 4xCherry localization. NE-BcI-2 expressing cells were nevertheless readily identifiable by a punctuate staining pattern. Proper local ization of NE-Bcl2 was verified in parallel transfection experi­ments using digitonin as a permeabilizing agent (see Supplemental Figure 5 and Material and Methods). Red: 4xCherry; Green: mBcI -2; Blue: DNA (Hoechst 33342). (B) and (C) Quantitation of microscopic images obtained from HeLa cells (B) and from HeLa Tet Off ce ll s (C). Cells expressing mBcI -2 and its variants were inspected for nuclear 4xCherry localization as described in Figure 4. At least 150 cells per experimenta l condition were counted." p < 0.01 . In HeLa Tet off ce lls, expression of NE-targeted mBcI-2 resulted in a 2-fo ld increase in the number of cells displaying nuclear 4xCherry localization with respect to control cells. For wt mBcI-2 this increase was 2A-fo ld .

expression.52.53 The present work focuses on the NPC's activity as molecular sieve and investigates how th is functional parameter is affected by a drug treatment triggering cell death, vs. a condition of increased cell survival and proliferation. In both settings we detect an increase in the size-exclusion limit of the NPC and show data indicating Ca2• as a common mediator of the effect.

In STS-treated cells, we detect an early, pre-apoptotic leakiness of the NE concomitantly to a decrease in NEIER Ca2• levels. The effect is efficiently suppressed by a Ca2• chelator and mimicked by artificial downregulation of [Ca2. ] in internal stores. It is well established that Ca2• release channels, such as IP3Rs and RyRs, carry multiple phosphoryla­tion sites and that phosphorylation status modulates channel activ­ity. To date at least 12 kinases are known to directly phosphorylate the IP3Rs.54 Treatment with STS, a pan-kinase inhibitor with broad sub­strate specifici ty55 may thus directly interfere with Ca2• homeostasis by altering Ca2. channel activity. The associat ion of the IP3R inhibitory protein, IRBIT, to IP3Rs is also regulated by phosphorylation.56

Dephosphorylated IRBIT, which might predominate in kinase-inhib­ited cells, cannot bind to IP3R. As a consequence, the receptor becomes sensitive to IP3 concentrations that exist in cells under resting condi­tions, which might result in a lower threshold for Ca2• release from the NEIER.

Ca2• leakage from the ER of

In sum, these data show that BcI-2 overexpression lowers the size exclusion limit of the nuclear envelope by altering Ca2• homeostasis at the ER/NE, and that localizat ion of the protein at the nuclear membrane is sufficiem for the effect.

apoptotic cells has also been shown to result from caspase-3 dependent cleavage ofIP3Rs, generating a constitutively leaky "channel-only" domain .57 The latter how­ever, represents a late apoptotic event downstream of caspase acti­vation58 which is difficult to reconci le with the ea rly-stage nuclear permeabilization observed here, occurring almost immediately after exposure to STS and prior to the caspase-mediated cleavage of Nups. We can also rule out the involvement of calpain-medi­ated NPC proteolysis in our system, contrarily to what has been reported in neurons challenged with high-doses of glutamate to trigger excitotoxic cell death.

Discussion

The idemification of general mechan isms that govern NPC activ­ity in response to different extra- and intracellular signaling cues is of prime importance for understanding the NPC's recognized involvement in basic nuclear functions, not only nucleo-cyto­plasmic traffickin g but also chrom atin organ ization and gene

6

Our data do not support hyperp hosphorylation of Nup62 as being involved in the STS-dependent early nuclear leakiness.

Nup62, a heavi ly O-glycosylated Nup of the central channel was shown to be excessively phosphorylated in meningo-virus infected cells displaying loss ofNE permeability function. In fact, in that study, STS was used to block virus-mediated permeabilization of the NE .18 A more likely candidate for mediating nuclear leaki­ness in our experimental model is gp21O, a large, N-glycosylated integral nuclear membrane protein associated with the NPC via its short C-terminus exposed toward the cytoplasm. The bulk of the protein extends into the lumen of the NE. gp21O, whose cis­terna l dom ain possesses five EF hand calcium-binding motives, has been proposed to function as Ca2 • sensor within the N~, and to mediate Ca 2. -dependent structural changes of the NPC. 24

Moreover, the C-terminal cytoplasmic tail of gp210 is phos­phorylated in a cell-cycle dependent manner.59 gp210 is required for nuclear envelope breakdown at the onset of mitosis and is possibly involved in early NPC destabilization, functions which depend upon phosphorylation of the C-terminus.60 Thus, gp210 could potentially respond both to alterations in NE Ca2

• levels and to the activity of cytosolic kinases . Whether both signaling cues converge onto gp210 and how this affects NPC structure is not known. Interestingly, stable exogenous expression of Bcl-2 in R6 rat fibroblasts which are devoid of the nucleoporin gp210 does not elicit an increase in basal nuclear permeability (data not shown) further supporting a role of gp210 in transducing Ca2 •

signals to the NPC. So far, the impairment of the permeability barrier function

of the NPC has been mostly associated with stress conditions leading to cell demise. T hus, the finding that exogenous Bcl-2 expression, a condition leading to increased survival and stress resistance also increases basal nuclear permeability was unex­pected. By showing that these cells possess a reduced steady­state luminal ER [Ca2. ], we provide a possible explanation to this seemingly surprising observation . We propose that both, STS treatment as well as Bcl-2 overexpression increase NE per­meability via lowering luminal NEIER [Ca2.]. Bcl-2 has been shown to localize at the nuclear membrane where it could directly interact with IP3Rs.37.61.63 Importantly, we show that targeting Bcl-2 at the nuclear membrane is sufficient for its permeabiliz­ing activity. Direct binding of Bcl-2 to all three IP3R isoforms has been described. 37.64 Recently these interactions have been shown to enha nce the sensitivity of the IP3Rs to IP3 resulting in a decreased steady-state [Ca2 . ] at the ERY We therefore predict that Bcl-2-IP3R interaction at the nuclear membrane will result in a simi lar reduction of [Ca2 . ] in the nuclear cisterna. This cor­relation between ER/NE Ca2 • levels and NE permeability is in agreement with numerous studies on Ca2 '-mediated structural rearra ngements of the NPC.

Our results cannot exclude the existence of fur·ther mecha­nisms of NE permeabi lization unrelated to the NPC. High lev­els of Bcl-2 could destabilize the NE. Alternatively, the marked cytoskeleta l deformations following STS treatment65 might trig­ger limited and controlled disruption of the NE. In parvovirus­infected cells such transient NE disruptions were reported to mediate nuclear delivery of the virus. 66 NE deformations involv­ing changes in the phosphorylat ion status of lamin AIC have been also implicated in SV-40 infection of non-dividing cells.67

A +TG + Ca"

~ 20000

::j 15000 u: ri 10000

5000

0

~ - controt (empty vector)

bcl-2 ... - control (empty vector) In low Ca"

10 20 30 time [min)

B 100 ER [Ca" ] 100 Capacitive Ca" influx

c

80

g60 c: 8 40 '0

* 20

bcl-2 low Ca" bcl-2 low Ca"

4xCherry Nuclear­Cytoplasmic Distribution

£~ 2 .~~ 1 ~2bn B ~ 10 ~o

E control low Ca"

Figure 6, Adaptation to low Ca" conditions reduces ER Ca" levels similarly to BcI-2 expression, and mimics the BcI-2 dependent increase in NE permeability. (A) Typical traces of Fluo-4 fluorescence in HeLa cells stably expressing BcI-2 (light gray), and in control cells transfected with empty vector. The latter were grown either in normal medium (black) or incubated for 1Sh in low Ca2+ medium (0.1 mM, dark gray). Internal stores were depleted by TG addition (5!lm, indicated by the first ar­row). After recovery, extracellular Ca" was added back (1 mM, second arrow) by replacing the medium. A second phase of Ca" mobilization is observed (capacitative Ca2+ influx). Data points are the average of 5 cells. Error bars show the SEM (B) Quantification of ER Ca" levels (left panel) and capacitative Ca" infiux (right panel). The difference between basal and peak fiuorescence values in the first and second phase of Ca" elevation are expressed as percent of the values determined in control (empty vector) cells. Experiments were performed in triplicates. * p < 0.05; *** P < 0.001 . (C) He La control cells (empty vector) were incubated for 6h in low Ca" medium (0.1 mM) or in control medium (1 mM Ca" ) prior to transfection with a construct encoding the permeability marker 4xCherry. After overnight incubation, cell cultures were inspected microscopically for the presence of 4xCherry in the nucleus. Only those cells exhibiting at least a homogenous distribution ofthe marker be­tween nucleus and cytoplasm were scored as positive. A total of at least 150 cells were scored. * p < 0.05.

The observation that in STS-treated cells, early nuclear per­meabilization can be prevented by BAPTA without affecting final apoptotic execution raises the question about the role of early leakiness of the NE in the apoptotic program. In the pres­ence ofBAPTA, STS-treated cells activate caspases (Fig. 3C) and undergo apoptosis. However, we could not identify nuclei with a morphology correspond ing to stage 3 nuclear apoptosis (nuclear

7

mbcl-2

mbcl-2 + SERCA2

100

~ 80 .cQ)

j~ 60 "''<;f"

'iii ffi 40 ~~

2 20

o

-

mbcl-2

.**

mbcl-2 + SERCA2

Figure 7. Replenishment of ERINE Ca" levels via SERCA2 expression restores the NE permeability barrier. (Al Representative confocal images of HeLa cells transfected with a construct encoding 4xCherry alone or in combination with an expression plasmid for SERCA2. (8) Quantification of cells displaying nuclear 4xCherry local ization. At least 100 cells were counted per experiment. Only cells coexpressing SERCA2 and 4xCherry were included in the evaluation. The analysis was performed as described in Figure 4.*** p < 0.001 .

collapse I disassembly)G8 in our confocal movies. In the absence of BAPTA, about 20% of the cells reach this stage within the time of observation (see Fig. 56B). This finding on the one hand cou ld reflect a requirement for Cal . for completing apoptotic chroma­tin fragmentation, as already proposed.G9 On the other hand, it argues against NE dilation being a proapoptotic signal per se. While in the context of apoptosis, early nuclear leakiness might faci litate nuclear disassembly and influence the final apoptotic morphology similarly to what has been reported for the cleav­age of B-type lamins,?o it most likely won't interfere with basic cellular functions in a healthy cell. Our observation that signa l­dependent nuclear transport remains fully functional during the first phase of STS-mediated nuclear pcrmeabilization coft'obo­rates this interpretation (data not shown). Our data showing that in tumor cells such as Bcl-2 expressing HeLa and SW480, more than 60% of the cell population shows equal nucleo-cytoplasmic distribution of an inert permeability marker indicate that large variations in the size-exclusion limit of the NE are well tolerated and compatible with cel l survival and proliferation. Large-scale structural rearrangements of'the NPC are known to occur in connection with the passage of very large cargo, such as mRNP particles7 1 and transient increases in the uptake of transport sub­strates were observed upon stimulation with growth factorsn or

8

during the cell cycle. 'o In bcl-2 overexpressing cells , we observe a constitutive alteration of nuclear membrane permeability which we interpret as an adaptive response to lowered steady­state [Ca2

,] in the lumen of the NE. Whether this persistent enhanced accessibility of the nuclear compartment can be established only in the presence of a general death suppressor such as Bcl-2, or whether high NE permeability may promote oncogenic functions of Bcl-2 associated e.g., with cell cycle regulation or DNA repair73 are challenging questions for the future.

Materials and Methods

Plasmid constructs. p4xCherry was created by consecutively inserting four copies of the cDNA sequence encoding the red fluorescent GFP analog mCherry74 into pcDNA3.1 (Invitrogen) . Removal of the internal start co dons was confirmed by DNA sequence analysis. pRSET-B-mCherry was kindly provided by Roger Tsien (University of California, La Jolla) and used as template for PCR amplification.

Expression constructs encoding murine bcl-2 proteins tar­geted to different subcellular compartments were a kind gift of Prof. Christoph Borner (University of Freiburg): pcDNA3-mbcl2 (full length murine bcl-2) , pcDNA3-mbcl2-cytb5 (ER-targeted murine bcl-2) and pcDNA3-mbcl2-RKIChbcl2X1. (mitochondria-targeted murine bcl-2).

The inducible expression construct for NE-targeted murine bcl-2 (NE-Bcl-2) was designed in analogy to a previously described NE-targeted GFP protein .75 The KASH domain of human Nesprin-2 (aa 6833-6883) was amplified from pEGFP­CI-Tm-Nesprin-2 (kind gift of Dr. Akis Karakesisoglou, University of Durham) by PCR and introduced into the EcoR! and BamHI sites of pTRE MCS (CIon tech) resulting in

pTRE-KASH. pTRE-KASH-mBcl-2 was obtained by inserting a mbcl-2 fragment from pcDNA3-mbcl2 lacking the transmem­brane domain of bcl-2 into pTRE-KASH. The control plasmid pTRE-GFP-KASH was constructed by insertion of the EGFP coding sequence into pTRE-KASH via PCR.

The p5ERCA2 expression construct was kindly provided by Luca Scorrano (University of Padova, Italy). pNup153-GFP was a kind gift of Jan Ellenberg (EMBL Heidelberg). pNup153-D349N-GFP was generated by PCR mutagenesis using the following primers: 153D349NFwd 5'-GTG GGA TAG ATA TCA CAA ATT TTC AGG CCA AAA GAG AAA AG-3' and 153D439NRev 5'-CTT TTC TCT TTT GGC CTG AAA ATT TGT GAT ATC TAT CCC AC-3'.

Cell culture and transfections. HeLa cervix carcinoma cells stably expressing murine bcl-2 (HeLa-mbcl-2) and SW480 colon carcinoma cells stably expressing human Bcl-2 (SW480-hbcl-2) and the respective empty vector control cell lines were a kind gift of Prof. Christoph Borner (University of Freiburg) .

HeLa Tet Off cells expressing the tetracycline controlled transact ivator (Clomech) were kindly provided by Dr. Thomas Meergans (Un iversity of Konstanz).

All cell lines were maintained in DMEM supplemented with 100 U/ml penicillin, 100 f.,lg/ml streptomycin, 2mM L-glutamine

(Invitrogen) and 10% fetal calf serum (Sigma). Cells were cu lti­vated at 37"C with 5% CO2 in a humidi fied atmosphere. For live cell microscopy, cells were seeded in glass-bottom dishes (ibidi) and transfected with Effectene (Quiagen) according to the man­ufacturer's instructions 24 h prior to the experiment.

HeLa Tet Off cells were transfected using the calcium phos­phate co-precipitation method. Cells were used for further exper­iments ~24 h after transfection.

For bead loading experiments, cel ls were seeded in MatTek glass bottom culture dishes with a glass inset of ~I cm2 and grown to a density of ~60% prior to the experiment.

Live cell imaging. Imaging experiments were performed in culture medium devoid of Phenol Red to reduce background fluorescence. After exchanging the medium, 150 fJ..g/ml Hoechst 33342 was added for labeling nuclear DNA, and the cells were incubated at the microscope stage at 37"C and 5% CO

2 for 1-2

h before starting image acquisition. Confocal time-lapse series were recorded at a Zeiss LSM510 Meta confocal microscope equipped with a stage-top incubator and an objective heater using a 63x/1.4 NA Plan-Apochromat objective lens (Carl Zeiss Microimaging). Laser settings were optimized to ensure cell via­bility for the whole duration of the experiments. As an addit ional control for phototoxicity, it was verified that imaging conditions were compatible with cell division (Fig. S6A). Cell nuclei were tracked and focused using an autofocus macro kindly provided by J. Ellenberg.76 For each experiment, 10-13 cells were imaged at a 5x zoom in a time interval of about eight minutes. If neces­sary, a delay between acquis ition rounds was introduced to keep this time frame constant. At each position, a z-stack consisting of three sections at a spacing of 0.6 fJ..m was recorded. After the first imaging cycle, the acquisition routine was paused and the apop­totic inducer STS (Sigma Aldrich, 0.5 fJ..m) or lz-TRAIL (kind gift ofH. Walczak, 300 ng/ml) was added. Inhibitors were added 30 min prior to STS addition at the following concentrations: zVAD-fmk (20 fJ..M), calpeptin (5 fJ..M) and BAPTA-AM (10 fJ..M). For bleaching correction, time courses of untreated cells were recorded under identical condit ions.

Quantification of confocal time series. Image analys is was performed using the open source software Image J. Image series were visually inspected and for each time point one z-section showing the nuclear rim in focus in the Nup153-GFP channel was selected. The images were combined into new stacks for quantification. T his procedure was necessary because cells alter their morphology during the apoptot ic process.

For the same reason, automatic segmentation of the cell nucleus in the Hoechst channel was not applicable. Therefore, all three channels were segmented interactively. Nuclear conden­sation and NE permeabilization were measured in the Hoechst and the 4xCherry channel, respectively. To this end, a ROI cor­responding [Q the inner boundary of the nucleus was first defined in the NupI53-GFP channel and copied into the Hoechst and the 4xCherry images. For quantifying NE integrity, signal intensi­ties were measured in the NupI53-GFP channel in a second ROI including the nuclear rim (Fig. SID). Folded regions of the NE were omitted. Intensity values were background substracted, cor­rected for bleaching, and normalized. The ti~e point of nuclear

condensation in each time series was defined as an increase in Hoechst fluorescence by a factor of at least 1.07 occurrin g within two consecutive frames. Cells go through apoptosis asynchro­nously. To better visualize the abrupt changes in nuclear perme­ability and NE integrity occurring at the final stage of nuclear execution, intensity values for these two channels were plotted on two separate time-axis. In the first part of the time course inten­sity values were displayed from the start of image acquisition up to the time point immediately preceding nuclear condensation. In the second parr, traces were aligned with respect to the t ime point of chromatin condensation as determined in the Hoechst channel.

Assessment of nuclear permeability. Nuclear Permeability Assay (NPA) was performed as described previously.43 Briefly, cells were seeded onto glass coverslips ~20 h before the start of the experiment. Cell membranes were selectively permeabilized with digitonin (24 fJ..g/ml). The semi-permeabilized cells were incubated with the fluorescent permeability marker 70 kDa Texas Red-labeled dextran (Sigma) at a concentration of 20 mg/ ml and then imaged at the confocal microscope (Zeiss LSM510 Meta). Images were quantitated semi-automatically by a custom­made software extracting the average nuclear fluorescence inten­sity. Fluorescence intensity in the nucleus is a measure of nuclear penetration of 70 kDa dextran and thus an indicator of passive permeability of the nuclear envelope.

For the experiment shown in Figure S3B cells were loaded with 70 kDa Texas Red-labeled dextran using glass beads.77.78 Briefly, glass beads with a diameter of ~100 fJ..m (Sigma) were treated overnight with 5 M NaOH, washed with ethanol and dried by evaporation. The culture medium was removed and 20 fJ..I dextran solution was added to the glass inset ofMatTek culture dishes (20 mg/ml 70 kDa Texas Red-labeled dextran (Sigma) in PBS). Subsequently, a monolayer of clean glass beads was added into the dish cavity and the dish was quickly shaken. Beads were then removed by washing with PBS, medium added back and the cel ls incubated for 45 min at 37"C. Confocal images were taken and evaluated as described above.

In cells expressing Bcl-2 and its mutants, nuclear permeabil­ity was assessed by measuring the percentage of cells displaying nuclear loca lization of 4xCherry. Images were taken at fixed time points after transfection of 4xCherry and Bcl-2 expression con­tructs by confocal fluorescence microscopy. Only cells showing at least the same 4xCherry signal in the nucleus as in the cytosol were scored as having a nuclear 4xCherry localization.

Immunocytochemistry. Immunocytochemical detection of mbcl-2 was performed using antibodies specific for murine bcl-2 (mouse monoclonal anti-bcl-2 clone lOC4, Santa Cruz), at a dilu­tion of 1 :500 in 10% NGS/PBS . Cells were shortly washed with 5 mM MgCI/PBS, permeabilised with 0.3% digitonin/PBS for 5 min on ice and rinsed again in 5 mM MgCI/PBS. Fixation was performed with 4% PFA/PBS for 10 min at room tempera­ture followed by washing in PBS. Reactive aldehyde groups were blocked ~ith 50 mM NH4C I for 10 min. Unspecific binding sites were saturated with 1 % BSA/PBS for 30 min . Incubation with the IOC4 anti-bcl-2 antibody was overnight at 4°C. The second­ary antibody conjugated to Alexa488 (Molecular Probes) was

9

diluted 1 :400 in 10% NGS/PBS and incubated for I h. Excessive washing steps between incubations removed unbound antibod­ies. DNA was stained with 200 ng/ml Hoechst 33342 for 10 min.

For co-detection of 4xCherry and Bcl-2, cel ls were fixed over­night at 4°C in 4% PFA/PBS to prevent loss of the permeability marker during permeabilization. After fixation cel ls were perme­abi lized for 5 min with 0.1 % Triton X-I OO/PBS at room tempera­ture, rinsed in PBS and incubated in 50 mM NH4Cl/PBS for 10 min. Subsequently, cells were treated with cold acetone at -20°C for 8 min, rinsed in PBS and incubated in 1% BSA/PBS for 30 min. Antibody incubations and Hoechst staini ng of DNA were performed as described above.

Immunostaining of Bcl-2 at the nuclear membrane required a permeabilization step prior to fixation and was therefore not compatible with simultaneous detection of 4xCherry. In this case, the protocol used for co-detection of 4xCherry and Bcl-2 resulted in a punctuate staining pattern that nevertheless allowed for the identification of cotransfected cells and image quantifi­cation . The correct localization of NE-Bcl-2 was confirmed in parallel transfections using digitonin permeabilization.

For co-detection of 4xCherry and SERCA2, cells were fixed overnight at 4°C in 4% PFA/PBS and processed as decribed above with omission of the acetone treatment. The SERCA2 antibody (lI08, Alexis) was diluted 1 :750 in 1 % BSA/PBS.

Preparation of whole cell extracts and immunoblot analy­sis. For the preparation of cell Iysates, HeLa cell cultures were placed on ice, and protease inhibitors (Complete Mix; Roche Applied Science) and dithiothreitol (lmM) were added directly to the growth media. The cells were then gently scraped off the dish with a rubber policeman, washed in ice-cold PBS, resus­pended in 95°C lysis buffer (50 mM Tris/HCI, pH 8.0, 0.5% SOS, 1 mM dithiothreitol), and heated at 95°C for 10 min. The cell debris was removed by centrifugation at 20,000 x g for 10 min. For the detection of Nup-153, SOS-PAGE was performed according to Thomas and Kornberg,79 while for other proteins it was according to Laemmli.80 Proteins were blotted onto nitro­cellullose using a wet blot chamber (Bio-Rad Trans-Blot Cell) or a Semidry Blotter (Biometra), and filters incubated in TNT buffer (50 mM Tris, pH 8.0, 150 mM NaCI, 0.05% Tween 20) with 5% milk powder at room temperature for 1 h. Incubation with primary antibodies at 4°C was in TNT with milk overnight. Fi lter washings were in TNT alone. Incubations with horseradish peroxidase-coupled secondary antibodies (I :2000, Oako) were in TNT with milk at room temperature for 1 h. The filter strip with the biotinylated molecular weight marker (Bio-Rad) was incubated separately with horseradish peroxidase-coupled avidin for 30 min at room temperature. Immunoblots were visualized with a chem iluminescent image ana lyzer (LAS-lOOO; Fujifi lm) . Murine and human Bcl-2 were detected using the antibody NI9 (Santa Cruz) at a dilution of I :2000. The anti-actin antibody (Chemicon) was used at I :50000 dilution, the anti-Nup-153 I GFP antibody (Progen, clone nup7A8) at I :50 dilution, and the anti-OsRed antibody (Clontech) at 1:500 dilution.

Fluorometric determination of protease activity, and of ER/N E [Cah ]. For measurement of caspase activity, a total of 3 x 105 HeLa cells were seeded in six-well plates 24 h prior to

10

the experiment and treated with STS (0.5 110m). To block cas­pase activation zVAO-fmk (20 110M) was added 30 min prior apoptotic stimulation At the indicated time points, the cells were placed on ice and after the addition of protease inhibi­tors, they were gently scraped off the dish and collected by cen­trifugation. Cells were lysed in 25 mM HEPES, pH 7.5, 5 mM MgCI

2, I mM EGTA, and 0.5% Triton X-lOO, and the cleav­

age of OEVO-7-amino-4-trifluoromethyl coumarin (afc) (40 110M) was monitored fluorometrically in reaction buffer (50 mM HEPES, pH 7.5, 10 mM dithiothreitol, 1% sucrose, 0.1 % CHAPS {3-[(3-cholamidopropyl)-dimethyla'mmonio] -2-hy­droxy-I-propanesulfonate)) over a period of 20 min at 37°C, with a \ , (excitation wavelength) value of 390 nm and a A"n (emission wavelength) value of 505 nm. The activity was ca li­brated by using afc standard solutions. Measurements were run in triplicate.

Calpain activity was measured using a Calpain Activity Assay Kit (Abcam, ab65308) according to the manufacturer's instruc­tions. For apoptosis induction, HeLa cells were treated with 0.5 110M STS for 2h. To block calpain activity, 5 110M calpeptin (Merck) was added 30 min prior to STS treatment. Cell Iysates were prepared from 2 x lOG cells. Protein concentrations were determined using BCA (Pierce Biotechnology). Lysates were spiked with 10 110M 1101 active calpain. After add ition of the cal­pain substrate (Ac-LLY-afc), samples were transferred to a black UV-light permeable 96-well plate and incubated at 37°C for 60 min. Fluorescence emission of afc was detected as above.

For Ca2• measurements, cells were cultured in 96 multiwell

plates for 24 h and washed carefully with Ca2' -buffer (140 mM NaCI, 5 mM KCI, ImM CaCl2, I mM NaHl04, ImM MgS0

4,

5.5 mM Glucose, 20 mM Hepes pH 7.4). Cells were loaded with the [Ca2 . ] indicator Fluo-4 (Invitrogen) by incubation in 150 1101 loading buffer (Ca2 '-buffer conta ining Fluo-4 and pluronic acid at final concentrations of 4 110M and 0.08% respectively) for 60 min at RT in the dark. After two washings with Ca2

• -buffer con­taining I mM probenecid, cells were incubated for 30 min at RT for complete de-esterification of intracellular Fluo-4. Immediately before measurement, the buffer was replaced with fresh Ca2. _

buffer with probenecid. Fluorescence emission was detected with a microplate fluorescence reader (Genios Plus, Tecan) equipped with appropriate filters (\, = 488 nM, \m = 510-570 nM). After measurement of basal fluorescence, 50 1101 of buffer solution were replaced with 50 1101 of thapsigargin (TG) containing Ca2'-buffer (final TG concentration 5 110M) . TG induces release ofCa2 • from internal stores. After Fluo-4 signal intensities had recovered to the basal level (~10 min) the medium was replaced with Ca2._

buffer (1 mM Ca2.) to measure the capacitative Ca2 • influ x. To measure ER/NE Ca2

• and capacitative Ca2• influx in cel ls adapted to low Ca2

• conditions, cells were seeded in 96 multiwell plates two days prior to the experiment. After 24 h the growth medium was exchanged with low-Ca2' -buffer (Cah -buffer con­taining 0.1 mM Ca2 . ). The cells were adapted to low Ca2 • condi­tions for 24 h. Fluorometric measurements were performed as described above using low-Ca2 '-buffer for loading the cells with Fluo-4 and subsequent washing steps. Capacitative Ca2• influx

was measured after readdition of Ca2 •• Control measurements using Ca2'-buffer (1 mM) were conducted in parallel.

Disclosure of Potentia l Conflicts of Interest

No potential confl icts of inten::st were disclosed.

pEGFP-CI-Tm-Nesprin-2, Henn ing Walczak (Imperial College London) for lz-TRAIL, and T homas Meergans for HeLa Tet-Off cells. We are grateful to Fel ix Schonenberger for support in image analysis, and to Anja Holtz for helpfu l discussions and sequence analysis of the 4xCherry expression plasmid.

Acknowledgments

We thank Christoph Borner (Un iversity of Freiburg) for the Bcl-2 expression plasm ids and cell lines, Luca Scorrano (University of Padova) for pSERCA2, Jan Ellenberg (EMBL, Heidelberg) for pNup153-GFP, Akis Karakesisoglou (University of Durham) for

References 1, Ribbcck K. Gorlich D. Kinetic analysis of tra nslo~

carion through nuclear pore complexes. EMBO J 200 I: 20: 1320-30: PMlD: 11250898: http://dx.doi. orgll 0.1 093/emboj/20.6.1320.

2. Walde S. Kehlenbach RH. The Pan and the Whole: functions of nuclcoporins in nucleocyto­plasmic "ansport. Trends Cel l Bioi 20 10: 20:46 1-9: PMlD:20627572: http://dx.doi.org/IO. 1 0 16/j. rcb.20 10.05.00 I.

3. Wente SR, Rout MP. The nuclear pore complex and nuclear transport. Cold Spring Harb Pcrspccl Bioi 20 10: 2:a000562: PMlD:20630994: http://dx.do i. org/l 0.11 0 1/cshperspecl.a000562.

4. Satterly N, Tsai PL, van Dcu rscn J. Nusscll1.vcig DR, Wang Y, Faria PA, ct a1. InAucnza virus targets the mRNA export machinery and the nuclear pore complex. Proc Nad Acad Sci USA 2007: 104: 1853-8: PMlD: 17267598: http://dx.doi .org/ 10.1073/ pnas.0610977 104.

5. Kosako H. Yamaguchi N, Aranarni C, Ushiyama M, Kosc 5, Imamoto N. et al. Phosphoproteomics reveals new ERK MAP kinase targets and links ERK to nucleoporin-mediated nuclear transport. Nat Struct Mol Bioi 2009: 16:1026-35: PMID: 1976775 1: Imp:1I dx.doi .orgl l O. 1038/nsmb.1656.

6. Makhnevych T. Lusk CPo Anderson AM. Ai tchison JD. Wozniak RW: Cell cycle regulated transport con­trolled by alterations in the nuclear pore complex. Cel l 2003: 11 5:8 13-23: PMID: 14697200: http://dx.doi. org/l 0.1 0 16/S0092-8674(03)00986-3.

7. Allen TD. Rutherford SA. Bennion GR. Wiese . C. Riepert S, Kiseleva E, et al. Three-dimensional sur­face structure analysis of the nucleus. Methods Cell Bioi 1998: 53: 125-38: PMID:9348507: http://dx,doi. org/l 0. 1 0 16/S0091 -679X(08)60877 -8.

8. Terry LJ. Shows EB. Wente SR. Crossi ng the nuclear envelope: hierarchical reguladon of nucleo­cytoplasmic transport . Science 2007: 3 18: 1412-6: PMID: 1804868 1: http://dx.doi.org/ 10.1126/sci­ence. 1142204.

9. Feldherr CM. Akin D. T he permeabi lity of the nuclear envelope in dividing and nondivid ing cel l cultures. J Cel l Bioi 1990: II I: 1-8: PMID:236573 I : http:// dx.doi.orgll 0. 1 083/jcb. 1 I 1. 1.1.

10. Fcldherr CM, Akin D. Signal-mediated nuclear trans­pOrL in proliferating and growth-arrested BALB/c 3T3 cells. J Cel l Bioi 199 1: 11 5:933-9: PMID: 1955463: http://dx.doi.org/ 10.1083/jcb. 115.4.933.

II. Chakrabony P, Wang Y. Wei JH. van Deursen J . Yu H. Malureanu L. el al. Nucleoporin levels regulate cell cycle progress ion and phase-specific gene expression. Dev Cell 2008: 15:657-67: PMID: 19000832: http:// dx.doi.orgIlO.IO 16/j.devcel.2008.08.020.

12. Ferrando-May E. Cordes V. Biller I. Godich D. Mirkovic J. NicO[era I~ Caspascs mediate nucleopo­rin cleavage but nor early red istribution of transpOrL factOrs and mRNA in apoptosis. Cell Death Differ 200 I: 8:495-505: PMID: 1142391 0: http://dx.doi. org/ l 0. 1 038/sj.cdd.4400837.

13. Pme M. Tabbert A. Hermann D. Walctak H. Rackwitz HR. Cordes VC, et al. Caspases rarget only two archi­tectu ral components within the core structure of the nuclear pore complex, J Bioi Chem 2006: 28 1: 1296-304: PMID: 16286466: Imp:lldx.doi.orgIl0.1074/jbc. M511 7 17200.

14. Faleiro L. Lazcbnik Y. Caspases disrupt the nucle­ar-cytoplasmic barrier. J Cell Bioi 2000: 151:951 -9: PMID: II 085998: http://dx,do i.orgIl0.1083/ jcb.151.5.951.

15. Ki hlmark M. Imreh G. Hallberg E. Sequential degrada­tion of proteins from the nuclear envelope during apop­tosis. J Cell Sci 200 I: 114:3643-53: PMID: 117075 16.

16. Buendia B. Santa-Maria A, Courvalin Jc. Caspase­dependent proteolysis of integral and peripheral pro­teins of nuclear membranes and nuclear pore complex proteins during apoptosis. J Cel l Sci 1999: 11 2: 1743-53: PMID:103 18766.

17. Bano D. Dinsdale D. Cabrera-Socorro A. Maida S; Lambacher N. McColl B. et al. Alteration of the nuclear pore complex in Ca{2+)-mediated cell death. Cel l Death Differ 2010: 17: 119-33: PMID: 19713973; http://dx.doi.orglI0 .1 038/cdd.2009.11 2.

18. Bardina MV. Lidsky PV. Sheval EV. Fom inykh KY. van Kuppeveld FJ. Polyakov VY. et al. Mengovirus­induced rearrangement of the nuclear pore complex: hijacking cellular phosphorylalion mach inery. J Viro l 2009: 83:3 150-61: PMID:191447 12: http://dx.doi. orgIl0. 1128/JVl.OI4 56-08 .

19. Lindenboim L, Blacher E, BornerC. Stein R. Regulation of strcss-induced nuclear protcin rcdistribmion: a ncw function of Bax and Bak uncoupled from BcI-x(L). Cell Death Differ 20 10: 17:346-59: PMID:198 16507: http://dx,doi.orgIl0. I038/cdd.2009. 145.

20. Bootman MD. Fearnley C. Smyrnias I. MacDonald F. Roderick HL. An update on nuclear calcium signal­ling. J Cell Sci 2009: 122:2337-50: PMID: 1957 111 3: http://dx.doi.orglI0.1242/jcs.028 100.

2t. Sarma A, Yang W. Calcium regulation of nucleocy­toplasmic transport. Protein Cell 20 II : 2:29 1-302: PMID:2 152835 1: hllp:lldx.doi .orgll 0.1 007/s 13238-01 1-1038-x.

22. Gerasimcnko O. Gcrasimcn ko J. New aspects of nudear calcium signa ll ing, J Cell Sci 2004: 11 7:3087-94: PMI D: 15226390: hllp:lldx.doi.orgll 0.12421 jcs,01 295.

23. Wu X, Bers OM . Sarcoplasmic reticulum and nuclear envelope arc one highly interconnectcd Ca2+ store th roughout cardiac myocyte. Cire Res 2006: 99:283-91: PMID: 16794184: htlp:lldx.doi.org/ I0.1161101. RES.0000233386.02708.72.

24. Greber ur, Gerace L. Depletion of calc ium from the lumen of endoplasm ic ret iculum reversibly inhibits passive diffusion and signal-mediated transport into the nudeus. J Cell Bioi 1995: 128:5-14: PMID:7822421: hllp:lldx.doi.orgIiO. 1083/jcb.1 28. 1.5.

25. Stehno-Bittel L. Perez-Tenic C . Clapham DE. Diffusion across the nuclear envelope inhibited by depletion of the nuclcar Ca2+ Store. Science 1995; 270: 1835 -8: pMID:85 25380: 11l tp:lldx.doi. org/ I0. 1126/science.270.5243. 1835,

26. StrUbing C. Clapham DE. Active nuclea r import and export is independent of lumcna l Ca2+ Sl'Ores in inraCl mammalian cel ls. J Gen Physiol 1999: 11 3:239-48: PMID:9925822: http://dx.doi.org/10. 108 5/ jgp.1 13.2.239.

27. Wei X. Henke VG. Strlibing C. Brown EB. Clapham DE. Rea l-time imaging of nuclear permeation by EGFP in single intaer cells. Biophys J 2003: 84: 13 17-27: PMID: 12547812: http://dx.doi.org/IO.1 0 161 S0006-3495(03)74947 -9.

28. Paulillo SM. Powers MA. Ullman KS. Fahrenkrog B. Changes in nucleoporin domain topology in response to chemical efFeerors. J Mol !liol 2006: 363:39-50: PMID:16962 132: http://dx .doi .org/ 10.1016/j . jmb.2006,08.021.

29. Stamer D. Schwart-Herion K. Aebi U, Fahrcnkrog B. Gett ing acl'Oss the nuclear pore comp lex: ncw insights into nucleocytoplasmic transport. Can J Physiol Pharmacol 2006: 84:499-507: PMID: 16902595: Imp:/!dx.doi.orgIl0.11 39/y06-001.

30. StOmer D. Fahrenkrog B. Aebi U. The nuclear pore complex: from molecu lar arch itecmre to functional dynamics. Curr Opin Cell Bioi 1999: 11 :39 1-40 1: PMID: 10395558: hllp:lldx .doi .org/I0.101 6/S0955-0674 (99) 8005 5-6,

31. Perez-Tertic C. Pyle J. Jaconi M. Stehno-Bittel L. Clapham DE. Conformational states of the nuclear pore complex induced by depletion of nuclear Ca2+ Slores. Science 1996: 273: 1875-7: PMID:8791595: http://dx.doi.orglI0.1126/science.273.5283 .1 875.

32. NUll LK. Chandra J. Paraer A. Fang B. Roth JA. Swisher SG. cr al. Bax-mediated Ca2+ mobi lization promores cytochrome c release during apoptosis. J Bioi Chern 2002: 277:2030 1-8: PMID: I 1909872: http:// dx.doi.orgl l 0.1 074/jbc.M20 1604200.

33. Oakes SA. Scorrano L. Opferman JT. Bassik MC. Nishillo M, Pozzan T. et al. Proapoptotic BAX and BAK regulate the type I inosito l trisphosphate recep­[or and calcium leak from the endoplasmic reticu­lum. Proc Nad Acad Sci U SA 2005: 102: 105-10: PMID: 156 13488; http://dx.doi.orgIl0.1073/ pnas.0408352 102.

34. Kruman I. Guo Q. Maltson MP. Calcium and react ive oxygen specics mediale staurosporine-induced mito­chondrial dys function and apoptosis in PC I2 cells. J Neurosci Res 1998: 51 :293-308: PMID:9486765: http://dx.doi.org/ 10. 1002l(SI C I) I 097-4547(19980201)5 1 :3<293::AID-JNR3>3 .0.CO:2-B.

35. Yip KW. Reed Jc. BcI-2 family proteins and can­cer. Oncogene 2008: 27:6398-406: PM1D:18955968: http://dx.doi.org/ 10.1038/0nc.2008.307.

36. Pimon P, Riuuto R. BcI-2 and Ca2+ homeostasis in the endoplasmic ret iculum. Cel l Death Differ 2006: 13: 1409-18: PMID: 16729032: http://dx.doi. orgl l 0. 1 038/sj.cdd,440 1960.

37. Eckenrode EF. Yang J. Velmurugan GV. Foskell JK. White C. Apopto, is protection by Md- I and Bd-2 modul ation of inos ito l 1,4.5-trisphosphate recep­ror-dependcnt Ca2+ signal ing. J Bioi Chern 20 10; 285: 13678-84: PM 10:20 189983: http ://dx.doi. org/ l 0.1 074/jbc.M I 09.096040.

11

38. Walczak H, Degli-EspoSl i MA, Johnson RS, Smolak P) , Waugh )Y, Boiani N, el al. TRAIL-R2: a novel apoplOsis-medialing receptor for TRAIL. EMBO ) 1997; 16:5386-97; PMID:93 11 998; hltp:lldx.doi. orgll 0. 1 093/embojll6. 17.5386.

39. T hastrup 0, Cullen P), Dmbak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter. discharg­es intracellular Ca2+ slOres by specific inhibition of the endoplasmic re ticulum Ca2(+)-ATPasc. Proc Nat! Acad Sci US A 1990; 87:2466-70; PMID:2 138778; Imp:1I dx.doi.orgll 0.1073/pnas.87.7.2466.

40. Presto n SF, Berlin RD. An intracellular calcium store regulates protein synthesis in HcLa ce lls, but it is not lhe hormone-sensitive sto re. Cell Calc ium 1992; 13:303- 12; PMI D: 137798 1; hltp:lldx.doi. org/ l 0. 1 0 16/0 1 43-4 160(92)90065-Z.

4 1. Foyo uzi-Yousscfi It, Arnaudcau 5, Borner C. Kelley WL, Tschopp ) , Lew DP, er al. BcI-2 decreases the free Ca2+ concclllrat ion within the endoplasmic reticulum . Proc Nat! Acad Sci U S A 2000; 97:5723-8; PMID : I 0823933; http://dx.do i.orgIl0. 1073/ pnas.97. 11.5723.

42. Roehrig 5. Tabbert A, Ferrando-May E. In vit ro mea­surement of nuclear permeabili ty changes in apopmsis. Anal Biochem 2003; 3 18:244-53; PMID: 128 14628; hltp:lldx.doi .org/l O. 101 6/50003-2697(03)00242-2.

43. Grote ~ Ferrando-May E. In vitro assay fo r [he quan ~

ti tadon of apopmsis· induced alterations of nuclear envelope permeabili ty. Nal ProlOc 2006; 1:3034-40; PMID: 17406565; hltp:lldx.doi.orgll 0. 1 0381 npro t. 2006,460.

44. Schinzel A, Kaufmann T, Borner C. BcI~2 fa mily mem~

bers: integrators of surviva l and dea th signals in physi~

ology and palhology [co rrecledJ. [corrected] . Biochim Biophys Acta 2004; 1644:95-105; PMID: 14996494; http://dx.doi.org/ 10.101 6/j.bbamcr.2003.Q9.006.

45. Hacki) , Egger L, Monney L. Conus S. Rosse T. Fel lay I, Cl al. Apoplotic crosstalk bctween the endoplas­mic reticulum and mitochondria controlled by BcI-2. O ncogene 2000; 19:2286-95; PMID:10822379.

46. Kaurmann T. 5chlipf 5. San, ) . Neubert K, Stein R, Borner C. CharaC[criza tion of the signal that directs BcI-x(L) , but not BcI-2, to the mi tochon­drial Olller membrane. ) Cell Bioi 2003; 160:53-64; PMID: 125 15824; http://dx. doi.orgIl0.1083/ jcb.2002 10084.

47. Padmakumar Vc. Liborte T, Lu W. Zaim H , Abraham S, Noegel AA, et al. The inner nuclear membrane protein Sun 1 mediates the anchorage of Nesprin-2 to the nuclear envelope. J Cell Sci 2005; 11 8:34 19-30; PM ID: 16079285; http://dx.doi.orgIl0. 1242/ jcs.02471.

48. Rong y, Distelhorst CWo BcI-2 protein fa mily mem­bers: versat ile regulators of ca lcium signaling in cell sur­viva l and apoptosis. Annu Rev Phys iol 2008; 70:73-9 1; PMID: 17680735; l"Ip:lldx.doi.o rgll 0.1 I 46/annurev. physioI.70.02 1507. 105852.

49. Hofer AM, Fasolato C, Pozzan T. Capacitat ive Ca2+ entry is closely linked to the fill ing state of in ternal Ca2+ stores: a study using sim ultaneous measurc­ments of ICRAC and intraluminal [Ca2+] . [Ca2+ J. ) Cell Bioi 1998; 140:325-34; PMID:9442 108; Imp:/! dx.doi.orgll 0. 1 083/jcb.140.2.325.

50. Pin con P, Ferrari D, Magalhaes P. Schulze-Osthoff K, Di Virgi lio F, Po"an T. el al. Reduced loading of intracellular Ca(2+) stores and downregularion of capacitat ive Ca(2+) innux in Bd -2-overexpress ing cell s. J Cell Bioi 2000; 148:857-62; PM ID: 10704437; http://dx.doi.orgIl0. 1083/jcb. 148.5.857.

51. Scorrano L. Oakes 5A. O prerman )T, C heng EH. Sorcinel li MD. Po"an T. el al. BAX and BAK regula­tion of endoplasmic ret iculum Ca2+: a control point for apoplosis. Science 2003; 300: 135-9; PMID: 12624.1 78; hltp:lldx.do i.orgIl 0.1 126/science. 1081208.

S2. Tran E}. Wellle SR. Dynamic nuclear pore com­plexes: lire on Ihe edge. Cell 2006; 125: 104 1-53; PMID: 16777596; htt p://dx.do i.orgIlO. 1 0 16/j. celI.2006.05.027.

12

53. Xylourgidis N, Fornerod M. Acting Ollt of characte r: regulatory roles of nuclear pore complex prote in s. Dev Cell 2009; 17:6 17-25; PMID: 19922867; Imp:1I dx.doi.orgl l 0.1 016/j .devcel .2009. 10.0 15.

54. Vanderheyden V, Devogelaere S, Miss iaen L, De Smedt H. Bultynck G, Parys ) B. Regulat ion orinositol 1.4,S-trisphosphate-induccd Ca2+ release by revers­ib le phosphorylation and dephosphorylation. Biochim Biophys Acta 2009; 1793:959-70; PMID: 19 13330 I; http://dx.doi.orgIlO.1 01 6/j .bbamcr.2008. 12.003.

55. Karaman MW. Hcrrgard S. Treiber DK. Gallant P, Atteridge CE, Campbell BT, el al. A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 2008; 26: 127-32; PMID: 18 183025; http://dx.doi. orgl I0.1 038/nbtl 358.

56. Devogc1aere B, Seullens M, Sammcls E. Deru3 R. Waelkens E. van Lint j, et a!. Protein phosphatase- l is a novel regul ator of the in tcraction between TRBIT and the inos itol 1.4 ,S-rri sphosphate recepLOr. Biochem ) 2007; 407:303- 11 ; PMID: 17635 105; http://dx.do i. orgl I0. 10421B)2007036 1.

57. Nakayama T, Hatto ri M, Uchida K. Nakamura T, Tateishi V, Bannai H, el al. The regulatory domain of the inositol 1 ,4 ,S-trisphosphate receptor is necessary to kecp the channel domain closed: possible phys iologica l significance of specific cleavage by caspasc 3. Biochem ) 2004; 377:299-307; PM ID:1296895 1; Imp:lldx.doi. orgIlO. 1042/B) 20030599.

58. Asscfa Z. Bulrynck G, Szlufcik K. Nadif Kasri N. Vermassel1 E. Goris }, et al. Caspase-3- induced trunca­tion of type I inosi tol trisphosphate rcceptoraccc1erates apoptotic cell death and induces inositol tri sphosphate­independent ca lci um release during apoptosis. J Bioi Chem 2004; 279:43227-36; PMID: 1528424 1; http: // dx.doi.orgll 0.1074/jbc.M403872200.

59. Favreau C. Worman H). Wozniak RW. Frappier T. Courva lin )c. Cell cycle-dependenl phosphoryla­tion of nucleoporins and nuclear pore membrane protein G p2 10. Biochemistry 1996; 35:8035-44; I'MID:8672508; http ://dx.doi.o rgIl0 . 102 1/ bi9600660.

60. Galy V, Antonin W. )aedicke A, Sachse M. Santarella R, Haselmann U, c[ al. A role for gp210 in mito tic nuclear-envelope breakdown.) Cell Sci 2008; 12 1 :3 17-28; PM ID: 182 16332; hup:lldx.doi.orgIl0 . 12421 jcs.022525.

6 1. Hoelelmans R, van Siooten H) , Keij,er R, Erkeland S, va n de Velde Cj, Dierendonck )H . BcI-2 and Bax proteins arc prescnt in interphase nuclei of mammalian ce lls. Cell Death Differ 2000; 7:384-92; PMID: I 0773823 ; http: //dx.doi.org/ 10. 1038/ sj.cdd.4400664.

62. G ivol I. Tsarfa ry I, Resau ) , Rulong S. da Si lva PP, Nasioulas G, et al. Bcl-2 expressed using a rerrov iral vector is localized primarily in the nuclear mem­brane and the endoplasmic reticulum of chicken embryo fib roblasts. Cel l Growth Differ 1994; 5:4 19-29; PMID:80435 16.

63. Monaghan r. RobertSon D, Amos TA, Dyer M) . Mason DY, Greaves ME Ult ras tructural loca liza­tion of bd· 2 protei n. J Histochem Cytochcm 1992; 40: 18 19-25; PMID: 1453000; Imp:lldx.doi. orgll 0.11 77/4 0.12. 1453000.

64. Chen R, Valencia I. Zhong F. McColl KS, Roderick HL, Boolman MD. et al. BcI-2 fun ctionall y inter­acts with inosito l 1,4,5-tri sphosphatc receptors to regulate calciu m release from the ER in response to

inositol I ,4,5-trisphosphate. ) Cel l Bioi 2004; 166: 193-203; I'MID: 152630 17; hllp:lldx.doi.orgll 0. 1 0831 jcb.200309 146.

65. Pelling AE, Vcrailch FS. C hu Cr. Mason C. Horton MA. Mechanica l dynamics of single cells during ea rl y apoptosis. Cel l MOl il Cytoskeleton 2009; 66:409-22; PMID: 19492400; hllp:lldx.do i.orgll 0.1 0021 cm.2039 I.

66. Cohen S. Marr AK. Garcin r. Pante N . Nuclear enve­lope disruption involving host caspas cs plays a role in the parvoviru, replica lion cycle. ) Vira l 20 11 ; 85:4863-74; PMID:2 1367902; http://dx.doi.orgIl 0.1I 28/ j VI.01 999- 10.

67. Butin-Israel i V, Ben-Nun-Shaul O. Kopal. I. Adam SA, Shimi T, Goldman RD. et al. Simian vi rus 40 induces lamin Ale Auctuations and nuclear envelope deformation during ce ll emry. Nucleus 2011 i 2:320-30; PMID:2 194 1 I II; http://dx.doi.orgll0.4 1 6 I I nucl .2.4. 1637 1.

68. Tone S, Sugimoto K, Tanda K, Sud a T. Uehi ra K. Kanouchi H , et al. Threc distinct stages of apop­to tic nuclear condensation revealed by time-lapse imag­ing, biochemical and elec tron microscopy analysis of cell-free apoptos is. Exp Cell Res 2007; 3 13:3635-44; PMID: 17643424 ; http://dx.do i.orgl I0. 101 6/j. yexcr.2007.06.01 8.

69. Oshimi Y, Miya1.aki S. Fas antigen -mediated DNA fragmentation and apoptotic morpho logic changes arc regulated by elevated cytosolic C.2+ level.) Immunol 1995; 154 :599-609; PMID:7529280 .

70. Rao L, Perez D, Wh ite E. Lamin proteolys is faci li­tates nuclear evenrs during apoptos is. } Cell Bioi 1996; 135: 144 1-55; PMID:89788 I 4; hllp:lldx.doi. orgll O. 1083/jcb.135.6. 144 1.

7 1. Kiseleva E, Goldberg MW. Daneho lt B, Allen TD. RN P export is mediated by structu ral rcorganiza tion of Ihe nuclear pore basket.) Mol Bioi 1996; 260:304-II ; PMID :8757794; hup :lldx.do i.orgll 0. 1 0061 jmbi. 1996.040 I.

72. Fcldherr CM, Akin D. Regulation of nuclear trans­POrt in proli ferating and quiescent cells. Exp Cell Res 1993; 205: 179-86; PMID:845399 I ; hup:lldx.doi. orgll 0.1 006/excr. 1993. 1 073.

73. Grcen DR, McKinnon Pl. A survivor hits the breaks. Mol Cell 2008; 29:4 11 -2; PMID:18313378; hllp:1I dx.doi.org/ l 0. 1 0 I 6/j.moiceI.2008.02.003.

74. Shaner NC, Campbell RE, Steinbach PA, G iepmans BN, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. rcd fluorescent protein. Nat Biotcchnol 2004; 22: 1567-72; PMID:15558047; Imp:lldx.doi. orgl 10. 1038/nbti 037.

75. Crisp M, Liu Q, Roux K. Rattner ) B. Shanahan C, Burke B. et al. Coupling of rhe nucleus and cytoplasm: role of the LlNC complex . ) Cell Bioi 2006; 172:4 1-53 ; PMID: 16380439; http://dx.d oi.o rgIl0. 1083/ jcb.2005091 24.

76. Rabul G, Ellenberg ) . Automatic rea l-lime Ihree­dimensional cell tracking by fluorescence microscopy. ) Microsc 2004; 216:1 3 1-7; PMID: 15516224; http:// dx.doi.orgIlO. 1 II IIj .0022-2720.2004.0 1404.x.

77. Grote P, Ferrando-May E. Quantitative measuremelll of nuclear permeability changes in apoptosis. Nat Protoc 2006; 1:3034-4 0; PMID: 17406565 ; hllp:1I dx.doi.orgl I0. 1038/nprot.2006.460.

78. Mcneil PL. Incorporation of macromolecules into living cells. Methods Cell Bioi 1989; 29: 153-73; PMID:2643758; hup:lldx. doi.orgll 0. 1 0 I 6/S009 1-679X(08)60 193-4.

79. Manders EM, Kimura H, Cook PRo Direct im ag­ing of DN A in living cel ls reveals the dynamics of chromosome fo rmalion. ) Cell Bioi 1999; 144:8 13-2 1; PMID: I 0085283; http://dx. doi.org/ 10. 1083/ jcb. 144 .5.8 13.

80. Thomas JOt Kornberg RD. An octamer of hisloncs in chromati n and free in solution. Proc Nat! Acad Sci U S A 1975; 72:2626-30; PMID:24 1077; http://dx.doi. org/ I0. 1073/pnas.72.7.2626.

8 1. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacte riophage T4. Nature 1970; 227:680-5; PM ID:5432063; http://dx.doi. orgl l 0.1 038/227680aO.