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First published online April 1, 2009
doi: 10.1242/10.1242/jcs.040840
Research Article |
1 Department of Biomedicine, University of Bergen, Bergen, Norway
2 Institute of Clinical Biochemistry, University of Oslo, Rikshospitalet Medical Centre, Oslo, Norway
Author for correspondence (e-mail: stig.ove.boe{at}rr-research.no)
Accepted 2 December 2008
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Summary |
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and can be readily detected within the APL cell line NB4. These results provide insight into a dynamic pool of cytoplasmic nucleoporins that form a complex with the tumor suppressor protein PML during the G1 phase of the cell cycle.
Key words: AML, CBP, Cytoplasm, Nuclear pore complex, Nucleoporins, PML
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; Borden, 2008; Everett and Chelbi-Alix, 2007; Salomoni et al., 2008). In addition, this protein is linked to the pathogenesis of a subtype of acute myelogenous leukemia (AML) termed acute promyelocytic leukemia (APL), because PML is almost invariably expressed as an oncogenic fusion protein (PML-RAR) in APL blasts as a result of the t(15;17) translocation (de The et al., 1990; Kakizuka et al., 1991).
PML is the defining unit of the subnuclear compartments termed PML nuclear bodies (PML-NBs). The ability of PML to assemble into these structures is, in part, stimulated by intermolecular interactions between SUMOylated residues and a SUMO-interaction domain present on the PML protein (Shen et al., 2006). PML also recruits a multitude of other proteins (with diverse functions) to PML-NBs, many of which contain SUMOylated residues or a SUMO-interacting motif (Lin et al., 2006). In APL blasts, the PML-NBs become disrupted as a result of expression of the PML-RAR fusion protein (Dyck et al., 1994; Koken et al., 1994; Weis et al., 1994).
In addition to the PML-NBs that are present within interphase nuclei, PML can also be detected in subcellular mitotic structures, termed MAPPs (mitotic accumulations of PML proteins) (Dellaire et al., 2006; Everett et al., 1999) and in cytoplasmic subdomains that appear during the G1 phase (Dellaire et al., 2006; Everett et al., 1999) or in response to virus infection (Borden et al., 1998; McNally et al., 2008; Turelli et al., 2001). Whereas the nuclear PML-containing bodies have been intensely studied over the past 15 years, comparatively little information is available regarding the biochemical composition or function of MAPPs and the cytoplasmic PML-containing particles.
Nuclear pore complexes, the protein assemblies that form channels through the nuclear envelope, might also contribute to the pathogenesis of certain types of AML. This is suggested by the identification of recurrent chromosomal translocations in AML blasts that involve the genes encoding the nucleoporins Nup98 or Nup214/CAN (Borrow et al., 1996a; Dash and Gilliland, 2001; Graux et al., 2004; Kraemer et al., 1994; Nakamura et al., 1996). In addition, the transcriptional co-activator CREB-binding protein (CBP), which also represents a recurrent target for chromosomal translocation in AML blasts (Borrow et al., 1996b; Dash and Gilliland, 2001), has been shown to functionally interact with nucleoporin-specific FG-repeat sequences (Kasper et al., 1999). Nuclear pore complexes have long been known to have a major role in bidirectional transport across the nuclear membrane (Tran and Wente, 2006; Weis, 2003), but have in recent years also been linked to other cellular functions, such as gene expression regulation (Brown et al., 2008; Faria et al., 2006; Kurshakova et al., 2007; Mendjan et al., 2006). The precise function of nucleoporins in AML pathogenesis, however, has not yet been determined.
In addition to their presence in the nuclear pore complexes of the nuclear membrane, nucleoporins can also be detected in the cytoplasm. In infected cells, as well as in oocytes and embryos of various organisms, fully assembled nuclear pore complexes can be detected at the electron microscopic level as membranous cisternae termed annulate lamellae (Kessel, 1992). In addition, a large excess of soluble, membrane-free nucleoporins in the cytoplasm has also been reported (Onischenko et al., 2004). Although the existence of nucleoporins and nuclear pore complexes in the cytoplasm has been known for several decades, their functions remain largely unknown.
In the present study, we identified a cytoplasmic compartment that contains high concentrations of nucleoporins, as well as the tumor-suppressor proteins PML and CBP. These structures, which we refer to as CyPNs (cytoplasmic accumulations of PML and nucleoporins), form immediately following exit from mitosis, move on microtubule filaments and have the ability to dock at the nuclear membrane. We also find that the cytoplasmic distribution of nucleoporins becomes affected by siRNA-mediated depletion of PML and by expression of the PML-RAR fusion protein, suggesting a functional interaction between PML and nucleoporins that might contribute to APL pathogenesis.
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). CyPNs were detected in 8-10% of the cells in an asynchronously growing population of HaCaT cells, and the number of CyPNs that could be detected within a single cell generally ranged from 1 to 20. Notably, colocalization between PML and nucleoporins was detected exclusively in the cytoplasm and not in the nuclear membrane or within the PML-NBs. We also noted that CyPNs frequently contacted the nuclear membrane, indicating an affinity of these structures for the nucleus (Fig. 1A). The formation of CyPNs was not specific for HaCaT cells, because these structures could also be detected in HeLa, MCF7, U2OS and primary fibroblasts in approximately 4%, 5%, 3% and 2% of the cells, respectively. Additionally, in several cells that we examined, the cytoplasmic accumulations of PML were found to be restricted to a subset of nucleoporin-positive structures, suggesting differences in the biochemical composition between different cytoplasmic domains enriched in nucleoporins (Fig. 1B). We were not able to convincingly detect cytoplasmic annulate lamellae in cells that contained CyPNs (e.g. HaCaT cells) by electron microscopy and could therefore not determine whether these two types of structure are related.
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; Everett et al., 1999). This is consistent with the results described above, which show that only a fraction of asynchronously growing cells contain CyPNs. To verify that CyPN formation is cell-cycle dependent, we analyzed HaCaT cells that had been arrested by culturing in serum-free medium for 3 days and subsequently released into the cell cycle by serum stimulation. Analysis by laser-scanning cytometry revealed that these cells synchronously entered S-phase at approximately 13 hours after serum stimulation, and, following progression through G2 and mitosis, reached a second G1 phase approximately 30 hours after cell-cycle release (Fig. 2A). To determine the level of CyPNs at different cell-cycle stages we scored cytoplasmic foci that stained positive for PML and the nucleoporin Nup153 at different time points after cell-cycle release. By estimating the percentage of CyPN-containing cells, as well as the average number of CyPNs per cell, we found that the level of CyPNs was consistently low during the first 24 hours after cell-cycle release. At 24-30 hours, however, concomitant with exit from mitosis and entry into G1, we observed a considerable increase in the number of these cytoplasmic compartments (Fig. 2A).
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A similar result was obtained by analyzing HaCaT cells that had been synchronized by nocodazole-induced mitotic arrest and subsequent release from the cell-cycle block by drug removal. In this case too, we observed an increase of CyPNs as the cells traversed from mitosis to G1 (Fig. 2B). Together, these results show that CyPNs primarily form in G1 cells, and that their assembly is stimulated by a preceding mitosis.
That formation of CyPNs primarily occurs during the mitosis-to-G1 transition was also supported by high-resolution confocal microscopy of HaCaT cells fluorescently labeled with anti-PML and the nucleoporin-specific antibody Mab414. During all stages of mitosis, PML was mainly found to accumulate within MAPPs, whereas the nucleoporins exhibited a dispersed distribution in prophase, metaphase and telophase, and tethered to condensed chromatin during cytokinesis. Immediately after mitosis, concomitant with the formation of two relatively small closely paired daughter nuclei with decondensed chromatin, colocalization between nucleoporins and PML within CyPNs was almost invariably detected (Fig. 2C). These results show that complex formation between nucleoporins and PML is prevented during mitosis but occurs immediately after its completion.
CyPNs move on the microtubular network
We next studied the dynamics of CyPNs using live-cell imaging. For this purpose we monitored the movements of YFP-tagged PML-III transiently expressed in U2OS cells. As shown in Fig. 3A, YFP-PML-III colocalizes with nucleoporins in the cytoplasm and exhibits a cytoplasmic distribution that is consistent with that of endogenous CyPNs. By using a spinning disc confocal microscope, we collected a Z-stack of images every second for a total imaging time of 2 minutes. This experiment revealed that some YFP-PML-III-containing particles move rapidly from one cytoplasmic location to another in short bursts, whereas others remained stationary during the recording period (supplementary material Movie 1). By increasing the intervals between Z-stack acquisitions to 30 seconds and by using a total recording period of 40 minutes, we found that the majority of YFP-labeled CyPNs were capable of moving extensively within the cytoplasm (supplementary material Movie 2). In these longer movies, we also observed several examples of fusions between YFP-labeled cytoplasmic bodies (supplementary material Movie 2). Interestingly, most CyPNs appeared to be highly motile as long as they did not come into contact with the nuclear surface, but as soon as contact with the nuclear envelope had been made, they remained stably connected (Fig. 3B; supplementary material Movie 3). This demonstrates an affinity of CyPNs for the nucleus and might explain why these structures are frequently detected at or within the nuclear envelope (see Fig. 1A).
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To confirm the association between cytoplasmic PML and microtubule filaments, we coexpressed CFP-PML-III and YFP--tubulin in U2OS cells. By using a capture rate of 1 frame per second and a total imaging period of 2 minutes, we were able to visualize CyPNs that moved on microtubule filaments. One example is shown in supplementary material Movie 5 and as still images in Fig. 3D. In this particular example, an YFP-PML-III-containing particle is observed to move in two consecutive short bursts along two microtubule filaments running in different directions.
The nuclear localization sequence of PML is required for CyPN targeting
Several different isoforms of PML are expressed in human cells as a result of alternative splicing (Jensen et al., 2001). The different splice variants contain a tripartite TRIM domain (comprising a RING-finger, two B-boxes and a coiled-coil) in their N-terminus and a variable C-terminus that might contribute to isoform-specific functions (Fig. 4A). To assess the ability of different PML isoforms to target CyPNs, we studied the subcellular localization of PML-I, PML-II, PML-III, PML-IV, PML-V and PML-VII that were transiently expressed in U2OS cells. All isoforms, except the cytoplasmic PML-VII splice variant, were found to target CyPNs (Fig. 4B). Notably, the transiently expressed PML-III isoform appeared to be able to target CyPNs more effectively than the other nuclear isoforms (Fig. 4B).
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To further examine a link between nuclear import and the ability of PML to form complexes with CyPNs, we mutated Arg486 and the Lys487, two amino acids known to be crucial for nuclear import (Duprez et al., 1999), to Ala (a mutant that we termed PML-IIInls) (Fig. 4A). This import-defective mutant was detected exclusively in the cytoplasm following transfection into U2OS cells, but no colocalization between PML-IIInls and cytoplasmic nucleoporins was detected in any of the cells examined (Fig. 4C). This suggested that the capacity of PML to target CyPNs is linked to its nuclear-import capabilities.
We also mutated the RING-finger domain of PML, because this motif is thought to be crucially important for several PML functions. To achieve this, we substituted two of the zinc-coordinating cysteines (Cys57 and Cys60) with Ser (Fig. 4A) (Shen et al., 2006). This mutant (that we termed PML-IIIringS) retained its capacity to target CyPNs, but induced an altered morphology and distribution of these structures (Fig. 4C,D). Most notably the PML-IIIringS mutant induced CyPNs that were generally larger in size, fewer in number and were commonly detected in cytoplasmic regions more distal to the nuclear envelope compared with the wild-type protein. Furthermore, live-cell analysis of YFP-tagged PML-IIIringS revealed that this mutant was completely impaired in its ability to form stable interactions with the nuclear membrane (Fig. 4D). Since a RING-finger domain containing Cys-to-Ser mutations might retain some zinc-binding activity, we also constructed a less conserved ring finger mutant (PML-IIIringA) where Cys57 and Cys60 were replaced by Ala (Fig. 4A). The subcellular distribution of PML-IIIringA was found to be indistinguishable from that of PML-IIIringS (Fig. 4C).
Finally, we analyzed the cytoplasmic distribution of a SUMOylation-defective PML-III protein that contained Lys-to-Arg substitutions at Lys65, Lys160 and Lys490 (a mutant that we termed PML-IIIsumo) (Fig. 4A). This mutant colocalized with the cytoplasmic CyPNs in a manner that was comparable with that seen for wild-type PML-III (Fig. 4C). Thus, PML SUMOylation appears to be dispensable for CyPN formation.
Loss of PML causes increased levels of nucleoporins in the cytoplasm
We next assessed the effect of PML loss on nucleoporin distribution. For this purpose, we transfected U2OS cells with two different siRNAs specific for PML and a siRNA designed to target the firefly luciferase gene as a control. The two PML-specific siRNAs, but not the control siRNA, caused downregulation of PML expression and a concomitant increase of nucleoporins in the cytoplasm (Fig. 5). This result suggests a functional role of PML in regulating the cytoplasmic levels of nucleoporins. The increase of cytoplasmic nucleoporins was not due to alteration in cell-cycle distribution, because analysis of PML-depleted and control cells by laser-scanning cytometry revealed similar cell-cycle profiles (Fig. 5, right panels). Notably, the phenotype of PML-depleted U2OS cells reported here is reminiscent of that previously seen for Nup98-negative cells (Wu et al., 2001).
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and MLL. In agreement with previous reports (Dellaire et al., 2006; Everett et al., 1999), the PML-NB-resident portions Daxx and SUMO1 were readily detected in PML-NBs but were not observed to colocalize with MAPPs or with PML-containing cytoplasmic particles (data not shown). The majority of the other antibodies that we tested did not reveal any affinity for CyPNs, suggesting that the ability to target CyPNs is restricted to a small group of proteins and is not a general characteristic of proteins containing a nuclear-targeting signal. The only protein in addition to PML and nucleoporins that we were able to detect within CyPNs by using the antibodies mentioned above was the transcription coactivator CBP. Interestingly, this protein was also readily detected within MAPPs (Fig. 6). A cytoplasmic association between PML and CBP was also noted in a previous study in which CBP and an exogenously expressed nuclear-import-defective mutant of PML were found to colocalize within cytoplasmic subdomains (Bellodi et al., 2006).
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CyPNs are targeted by the PML-RAR fusion protein
Previous studies have revealed a more dispersed distribution of PML and other PML-NB constituents in APL cells compared with normal cells, an effect that was attributed to expression of the oncogenic PML-RAR fusion protein (Dyck et al., 1994; Koken et al., 1994; Weis et al., 1994). To determine whether this protein chimera also has the capacity to alter the morphology of CyPNs, we overexpressed PML-RAR in U2OS cells and subsequently analyzed the distribution of nucleoporins and the expressed fusion protein by immunofluorescence. Using antibodies targeted to both the PML and the RAR moiety of PML-RAR, we detected cytoplasmic clusters of nucleoporins and the expressed fusion protein in 15-20% of the transfected cells. Notably, the number of CyPNs was frequently observed to be markedly higher in PML-RAR-transfected cells compared with untransfected cells (Fig. 7A).
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, and cells regain prominent PML-NBs in response to differentiation-induced treatment with all-trans retinoic acid (ATRA) (Dyck et al., 1994; Koken et al., 1994; Weis et al., 1994). In untreated NB4 cells, we detected prominent CyPNs in more than 30% of the cell population. Moreover, by culturing these cells in the presence of ATRA, which is known to cause degradation of the PML-RAR fusion protein, we observed a gradual decrease in the number of cells containing detectable CyPNs. In addition, CyPNs in ATRA-treated cells appeared smaller than those detected in unstimulated cells (Fig. 7B). Thus, in contrast to the nuclear PML bodies, the morphology of CyPNs is not disrupted by PML-RAR expression.
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The formation of CyPNs might be transitionally linked to MAPPs, which represent a cellular compartment specific for the mitotic phase of the cell cycle (Dellaire et al., 2006; Everett et al., 1999). This is suggested by the finding that CBP, the only non-nucleoporin protein in addition to PML that we so far have been able to detect within CyPNs, also accumulates within MAPPs during mitosis. In addition, the formation of CyPNs during early G1 of the cell cycle was greatly stimulated by a preceding mitosis, suggesting the involvement of mitotic proteins and/or structures for their formation. Thus, PML might form a complex with CBP (and possibly other proteins) within MAPPs during the mitotic phase of the cell cycle, and subsequently, following completion of mitosis, might become stably attached to nucleoporins. A contributing role of MAPPs to the formation of cytoplasmic PML particles has also been proposed in previous reports (Chen et al., 2008; Dellaire et al., 2006).
The ability of PML to become linked to cytoplasmic nucleoporins in early G1 cells appears to be critically dependent on its nuclear-targeting domain, because both the cytoplasmic PML-VII isoform, as well as the nuclear import defective PML-IIInls mutant, failed to accumulate within CyPNs. This argues that PML is capable of associating stably with cytoplasmic nucleoporins by interacting directly with components of the nuclear pore complexes that mediate its nuclear import. The PML protein could therefore potentially become targeted to these cytoplasmic compartments by interacting with import adaptor proteins, such as importin- or importin-β. Consistent with this notion, previous studies show that both of these import receptors are present in nuclear pore complexes of cytoplasmic annulate lamellae in Xenopus egg extracts (Cordes et al., 1997; Miller and Forbes, 2000). However, since PML contains an unconventional nuclear localization signal that does not match the consensus of any of the nuclear localization motifs that currently are known to bind import factors (Duprez et al., 1999), it might be equally possible that the nuclear-targeting sequence of PML contacts other components of the nuclear pore complex. In this respect it is of interest to note that CBP, the other CyPN constituent discovered in this study, has been reported to interact directly with nucleoporin-specific FG repeats (Kasper et al., 1999). Further studies should determine the precise protein interactions that take place between the nuclear-targeting domain of PML and components of the nuclear pore complexes.
The dependency of the PML nuclear-targeting sequence for association with cytoplasmic nucleoporins might also indicate that CyPNs represent nonfunctional complexes that form because of fortuitous interactions between PML and nuclear-import receptors present on fully assembled cytoplasmic nuclear pore complexes. However, several findings made in the present study suggest that cotargeting of PML and nucleoporins to common cytoplasmic compartments reflects a functional interaction between PML and constituents of the nuclear pore complexes. First, the PML-IIIring mutant, which contains a disrupted RING-finger domain, induced the formation of morphologically deformed CyPNs, which, in contrast to CyPNs containing wild-type PML-III, were impaired in their ability to dock at the nuclear membrane. Second, siRNA-mediated depletion of PML caused a considerable increase in the levels of cytoplasmic nucleoporins, suggesting a functional role of PML in regulating the balance between cytoplasmic versus membrane-bound nucleoporins. Last, during the course of this study, more than 15 different proteins were examined for their ability to target CyPNs, and only two of these (PML and CBP) could be detected within these compartments. Thus, although it is expected that other proteins in addition to those identified in the present study are present within CyPNs, these structures appear to be highly selective for a relatively small group of nuclear proteins and do not generally attract proteins that contain a nuclear localization signal.
Since both PML and CBP are known to be involved in various aspects of gene expression, this might indicate a role of CyPNs in transcriptional regulation. This idea is consistent with several studies demonstrating a role of nuclear pore complexes in the regulation of gene expression. Most notably several studies in yeast have demonstrated activation of gene expression following the recruitment of genetic elements from the interior of the nucleus to nuclear pore proximal sites at the nuclear periphery (Casolari et al., 2004; Menon et al., 2005; Schmid et al., 2006; Taddei et al., 2006). In a recent study performed on human cells, several binding sites for the nucleoporin Nup93 were mapped to regions of transcriptional regulation on chromosomes 5, 7 and 16, indicating a similar role of nuclear pore complexes in mammalian gene expression (Brown et al., 2008). An intriguing possibility is that some protein complexes that participate in transcriptional regulation might be routed to the nucleus by a mechanism that involves preassembly of nucleoporins and transcription complexes in the cytoplasm and subsequent exposure of these transcription factors to the nuclear environment following insertion of new nuclear pore channels into the nuclear envelope.
It is also possible that the observed colocalization between PML and cytoplasmic nucleoporins reflects a role of these proteins in nucleocytoplasmic transport. In agreement with this, PML has been implicated in the export of selected mRNA species involved in cell-cycle regulation through an interaction with the translation initiation factor eIF4E (Cohen et al., 2001; Culjkovic et al., 2005; Culjkovic et al., 2006; Lai and Borden, 2000).
A third possibility could be that CyPNs represent sites of protein degradation and that the presence of nucleoporins within these structures in early G1 phase reflects an excess of nuclear pore complex constituents that were not incorporated into the progeny nuclei following completion of mitosis. This theory is consistent with previous studies supporting a role of PML in protein degradation (Chai et al., 1999; Janer et al., 2006). Furthermore, a role of PML in degrading excessive nucleoporins is in agreement with the observed increase of cytoplasmic nucleoporins in PML-depleted cells.
Although it has long been known that the cytoplasm contains PML and nucleoporins that are readily visualized by fluorescence microcopy, very little functional information exists regarding the cytoplasmic life of these proteins. The data presented here reveal a dynamic interplay between nucleoporins, the tumor suppressor proteins PML and CBP, and the nuclear membrane. Furthermore, several of the CyPN-resident proteins, including Nup98, Nup214, PML and CBP are encoded by genes that are at the break points of chromosomal translocations that recurrently are identified in AML blasts. Further research into the composition and function of CyPNs might therefore provide new insight into the molecular mechanisms that determine the development of this type of cancer.
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Antibodies and immunofluorescence
The antibodies used were mouse anti-PML (PG-M3), rabbit anti-PML (H238) and rabbit anti-CBP (A-22) from Santa Cruz, mouse anti-Nup153 (QE5), Mab414, mouse anti-His (HIS.H8) rat anti-Nup98 (2H10) and rabbit anti-Nup214 (Ab40797) from Abcam and mouse anti-HA (12CA5) from Roche. Fixation and immunofluorescence labeling of cells was performed as previously described (Jul-Larsen et al., 2004).
Plasmids, siRNAs and transfection
His-tagged PML isoforms I to V cloned into pcDNA3 were kindly provided by Kun-Sang Chang at the University of Texas (Xu et al., 2005). His-tagged PML-VII was derived from pcDNA-PML-V by first introducing a ClaI site at nucleotide positions 1345-1350 by silent mutagenesis and subsequently placing a synthetic ClaI-XbaI linker containing the PML-VII specific C-terminal sequence into the ClaI-XbaI site. Plasmids expressing YFP-PML-III or CFP-PML-III were kindly provided by Karien Wiesmeijer and Roeland Dirks at Leiden University Medical Centre in the Netherlands. The plasmid expressing YFP-tagged -tubulin was kindly provided by Michaël Marie at the University of Bergen, Norway. The pCNA-PML-RAR was a kind gift from Pier G. Pelicci at the European Institute of Oncology, Milan, Italy. The mutants PML-IIInls (R486A; K487A), PML-IIIringS (C57S; C60S), PML-IIIringA (C57A;C60A) and PML-IIIsumo (K65R; K160R; K490R) were generated by using the QuikChange kit from Stratagene.
Plasmid transfections were performed using the FuGENE6 transfection reagent from Roche. In all experiments, cells were analyzed or fixed at 12-24 hours following transfection. Transfections with siRNAs were performed using Oligofectamine (Invitrogen, CA) according to the manufacturer's instructions. Cells were fixed and processed for immunofluorescence at day 3 after transfection. siRNA oligos used were: PML siRNA1, 5'-GUUUCUGCGCUGCCAGCAAtt-3' (Eurogentec, Belgium); PML siRNA2, 5'-AGAUGCAGCUGUAUCCAAGtt-3' (Ambion, TX); control siRNA (against fire fly luciferase), 5'-UCGAAGUACUCAGCGUAAGtt-3' (Eurogentec).
Laser-scanning cytometry (LSC) and quantification of CyPNs
To determine the cell-cycle profile of asynchronous and synchronously gowning cells on coverslips DNA was stained with TO-PRO-3 (Invitrogen), and subjected to laser-scanning cytometry (CompuCyte, Cambridge, MA). CyPN quantification was performed by immunofluorescence labeling using antibodies against Nup153 and PML. For each sample 6-8 randomly selected microscopic fields were captured by using a Leica DMRXA fluorescent microscope equipped with a x63 oil-immersion objective and a DC500 camera. Quantification was performed on merged Z-stacks of 8-12 images comprising the entire cell, and CyPNs were scored positive if the localization of Nup153 and PML labeled particles overlapped. Both the percentage of cells containing CyPNs as well as the average number of CyPNs per cell was calculated. Quantification of CyPNs in transfected cells was performed by analyzing a total of 100 randomly selected transfected cells (identified by His-tag-positive staining) that had been labeled with anti-His and anti-Nup153 antibodies. Cells were scored positive if His- and Nup153-positive cytoplasmic particles colocalized.
Confocal microscopy and live-cell imaging
Confocal microscopy images were obtained using a Zeiss LSM 510 Meta laser confocal microscope (Jena, Germany) equipped with a x63 oil-immersion lens (Zeiss). Live cell imaging was performed using an Ultraview spinning disc confocal microscope from Perkin Elmer (Wellesly, MA) attached to a x63 oil-immersion lens. Cells were grown in 6 cm glass bottom dishes from MatTek (Ashland, MA) and transfected 1 day before imaging. During imaging, cells were maintained at 37°C and 5% CO2 using an environmental chamber. For short sequences, Z-stacks of 1-3 images were collected every second for 2 minutes. For longer periods of imaging (30 to 40 minutes total imaging time) Z-stacks of 8-12 images were collected at intervals of 30 seconds. Exposure times for each of the images varied from 30 mseconds to 60 mseconds. Velocity diagrams were obtained by determining the shortest distance between a YFP-labeled particle to the nuclear membrane using the measuring tool in Adobe Photoshop.
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Footnotes |
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This work was supported by the Cancer Gene Therapy Program funded by the Norwegian Health Department and Helse-Vest. Support from the Norwegian Cancer Society and the Norwegian Research Council is also acknowledged. Live-cell imaging was performed at the National Technology Platform Molecular Imaging Center, supported by the functional genomics program (FUGE) in the Research Council of Norway.
* These authors contributed equally to this work 
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