Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion
2010; Springer Nature; Volume: 29; Issue: 10 Linguagem: Inglês
10.1038/emboj.2010.54
ISSN1460-2075
AutoresSandra Krull, Julia Dörries, Björn Boysen, Sonja Reidenbach, Lars O. Magnius, Heléne Norder, Johan Thyberg, Volker C. Cordes,
Tópico(s)RNA Research and Splicing
ResumoArticle20 April 2010Open Access Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion Sandra Krull Sandra Krull Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany Search for more papers by this author Julia Dörries Julia Dörries Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany Search for more papers by this author Björn Boysen Björn Boysen Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany Search for more papers by this author Sonja Reidenbach Sonja Reidenbach Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany Search for more papers by this author Lars Magnius Lars Magnius Department of Virology, Swedish Institute for Infectious Disease Control, Solna, Sweden Search for more papers by this author Helene Norder Helene Norder Department of Virology, Swedish Institute for Infectious Disease Control, Solna, Sweden Search for more papers by this author Johan Thyberg Johan Thyberg Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Volker C Cordes Corresponding Author Volker C Cordes Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany Search for more papers by this author Sandra Krull Sandra Krull Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany Search for more papers by this author Julia Dörries Julia Dörries Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany Search for more papers by this author Björn Boysen Björn Boysen Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany Search for more papers by this author Sonja Reidenbach Sonja Reidenbach Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany Search for more papers by this author Lars Magnius Lars Magnius Department of Virology, Swedish Institute for Infectious Disease Control, Solna, Sweden Search for more papers by this author Helene Norder Helene Norder Department of Virology, Swedish Institute for Infectious Disease Control, Solna, Sweden Search for more papers by this author Johan Thyberg Johan Thyberg Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Volker C Cordes Corresponding Author Volker C Cordes Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany Search for more papers by this author Author Information Sandra Krull1,2, Julia Dörries1, Björn Boysen2, Sonja Reidenbach2, Lars Magnius3, Helene Norder3, Johan Thyberg4 and Volker C Cordes 1,2 1Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany 2Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany 3Department of Virology, Swedish Institute for Infectious Disease Control, Solna, Sweden 4Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden *Corresponding author. Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, Göttingen 37077, Germany. Tel.: +49 551 201 2404; Fax: +49 551 201 2407; E-mail: [email protected] The EMBO Journal (2010)29:1659-1673https://doi.org/10.1038/emboj.2010.54 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Amassments of heterochromatin in somatic cells occur in close contact with the nuclear envelope (NE) but are gapped by channel- and cone-like zones that appear largely free of heterochromatin and associated with the nuclear pore complexes (NPCs). To identify proteins involved in forming such heterochromatin exclusion zones (HEZs), we used a cell culture model in which chromatin condensation induced by poliovirus (PV) infection revealed HEZs resembling those in normal tissue cells. HEZ occurrence depended on the NPC-associated protein Tpr and its large coiled coil-forming domain. RNAi-mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin. These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub-compartment and delimiting heterochromatin distribution. Introduction The interphase nucleus of eukaryotic cells is compartmentalized into distinct territories, including areas occupied by transcriptionally active euchromatin and those with highly condensed, transcriptionally more inert heterochromatin. Whereas only small amounts of heterochromatin occur in proliferating cultures of permanent cell lines, it can occupy much of the nucleus in terminally differentiated cells (e.g., Francastel et al, 2000). In transmission electron microscopy (TEM) such heterochromatin appears as dense patches deep within the nucleus and aligned along the inner surface of the nuclear envelope (NE). However, even conspicuous amassments of condensed nuclear-peripheral chromatin are known since long to be gapped by electron-lucent zones free of heterochromatin (e.g., Swift, 1959; Watson, 1959). Such heterochromatin exclusion zones (HEZs) can differ in length and expansion, sometimes appear to segment large areas of the nuclear interior (e.g., Davies et al, 1974), and are often found associated with the nuclear pore complexes (NPCs) that serve as gateways of nucleo-cytoplasmic transport. However, even though different models exist of how nuclear compartmentalization might be accomplished (e.g., Jackson, 2003; Misteli, 2005, 2007; Cremer et al, 2006; Branco and Pombo, 2007; Hancock, 2007; Lanctôt et al, 2007; Rippe, 2007; Schneider and Grosschedl, 2007; Richter et al, 2008), the cellular factors or mechanisms that establish the perinuclear HEZs have remained unknown. While these zones are sometimes also referred to as euchromatic, chromatin-free, or interchromatin channels, or as part of a reticular inter-chromosomal compartment traversing the nucleus, the designation HEZ used in this study refers to the NPC-proximal parts of these zones. The NPC is a macromolecular structure of eightfold rotational symmetry that perforates the NE. Fibrillar appendices have been found attached to the NPC's nuclear side that vary significantly in length in different cell types, sometimes projecting deep into the nuclear interior as in vertebrate oocytes (e.g., Franke and Scheer, 1974; Scheer et al, 1988; Ris and Malecki, 1993; Goldberg et al, 1997). There, as well as in insect salivary gland cells and in the protozoan Dictyostelium, eight NPC-attached fibrils interdigitate with each other laterally, forming a structure called the nuclear basket (NB) (Ris, 1989; Jarnik and Aebi, 1991; Kiseleva et al, 1996; Beck et al, 2004). NB-reminiscent structures have also been described in yeast (Kiseleva et al, 2004) and proposed to exist in mammalian cells (e.g., Iborra et al, 2000; Frosst et al, 2002; Krull et al, 2004). In fact, an NPC-attached 'fish trap'-like assembly of nuclear fibrils in monkey cells was reported as early as 1976 (Maul, 1976). However, due to the delicate nature of these fibrils and difficulties in visualizing them by conventional TEM, it has remained uncertain whether these findings reflect the occurrence of an NB common to all NPCs. Moreover, the protein composition of fibrils and NBs has not yet been unambiguously determined, and different proteins are considered as their potential components (e.g., Krull et al, 2004; Prunuske et al, 2006; Brown and Silver, 2007; Köhler and Hurt, 2007). Two of the candidate proteins formerly proposed as fibrillar appendices of the NPC are called Nup153 and Nup98 (e.g., Sukegawa and Blobel, 1993; Powers et al, 1995; Radu et al, 1995; Fahrenkrog and Aebi, 2002; Frosst et al, 2002). Both belong to a subfamily of NPC proteins (nucleoporins) that contain large, natively unfolded domains with FG-tandem repeats (Denning et al, 2003). In human HeLa cells though, both the FG-repeat and the NPC-anchor domains of Nup98 were found at the NPC proper (Griffis et al, 2003; Krull et al, 2004), indicating that they are not very likely components of fibrillar material that may be peripherally attached to the NPCs in this particular cell type. The NPC-targeting domain of Nup153 too was found located at the NPC in HeLa and in other cell types (e.g., Walther et al, 2001; Fahrenkrog et al, 2002; Krull et al, 2004), but the location of its large FG-repeat domain remains enigmatic. Found within the NPC proper in fission yeast (Balasundaram et al, 1999), the FG-repeat domain of the vertebrate orthologue might form extended and highly flexible fibrils capable of projecting into the nuclear interior (e.g., Fahrenkrog et al, 2002, 2004). Another constituent of fibrillar NPC appendices is a 267-kDa protein called Tpr (e.g., Mitchell and Cooper, 1992; Cordes et al, 1997; Frosst et al, 2002). It forms long coiled-coil homodimers of rod-like shape via its N-terminal domain of 190 kDa, which also harbours a short segment required for anchorage to the NPC (Bangs et al, 1998; Cordes et al, 1998; Hase et al, 2001). By contrast, its C-terminal 'tail' domain does not homodimerize but appears largely unfolded and flexible (Hase et al, 2001). In different mammalian cell types Tpr's NPC-binding segment was indeed found positioned at the NPC, whereas other parts of the rod domain and the C-terminal tail were located deeper within the nucleus (Cordes et al, 1997; Frosst et al, 2002; Krull et al, 2004; Hüve et al, 2008). To investigate whether such a protein plays a role in HEZ establishment, one could attempt eliminating it in a cellular background in which HEZs are prevalent. However, in tissue cells with clearly contoured HEZs, target protein elimination is generally difficult to achieve. On the other hand, target protein levels can be easily knocked down by RNA interference (RNAi) in permanent cell lines (Elbashir et al, 2001). In such cells, however, condensed chromatin is usually scarce, and HEZs are imperceptible at most NPCs. Therefore, a suitable model system would need to concurrently allow rapid chromatin condensation and RNAi-mediated protein knockdown to possibly visualise HEZs and then study their fate after RNAi. Hyper-condensation of chromatin can be induced by infecting cultured cells with picornaviruses (Bienz et al, 1973). Within a well-defined timeframe, such condensation rapidly spreads throughout the nucleus of the infected cell, whereas HEZs are not trespassed even late after infection (Belov et al, 2004; Lidsky et al, 2006). This results in the contouring of HEZs at essentially all NPCs, thereby revealing shapes similar to those in terminally differentiated somatic cells. Here, we have exploited this possibility of visualizing HEZs by infecting HeLa cells with poliovirus (PV). Using this in combination with RNAi, we found Tpr to be an element essential for HEZ establishment and for delimiting perinuclear heterochromatin distribution. Results NPC-associated HEZs in PV-infected cells In cell lines such as HeLa, only small amounts of heterochromatin are aligned along the NE between neighbouring NPCs (Figure 1A). Local amassments of such heterochromatin occur only sporadically (Supplementary Figure S1A). Furthermore, the frequency of NPCs in these contact regions between heterochromatin clusters and the NE can be notably lower than that in the neighbouring areas (see also Maul, 1977; Garcia-Segura et al, 1989). Therefore, ultrathin sections of such cells only infrequently include perpendicular sections through NPCs juxtaposed to heterochromatin amassments. The few present, however, generally reveal an NPC-associated heterochromatin-free zone, often with seemingly hyperbolic contours or shaped like an isosceles triangle or trapezium with its basis next to the NPC. Similar clearance zones are also seen when the granular, pre-ribosomal material of the nucleolus is positioned directly in front of an NPC (Supplementary Figure S1A). Again though, NPCs are comparatively rare in such contact regions (Maul, 1977). Figure 1.NPC-associated HEZs of distinct size and shape withstand the expansion of condensed chromatin after PV infection. (A1) TEM of a perpendicular NE cross-section of a non-infected HeLa cell in mid-interphase, with a thin layer of NE-associated heterochromatin between the NPCs. (B1) A PV-infected HeLa cell (assembled from two micrographs) at 12 h post-infection, illustrating the characteristic nuclear distortion and emergence of NPC-associated HEZs (arrows). (A2, B2) Higher magnification views of NE cross-sections of non-infected HeLa cells (A2) and of cells 12 h after PV infection (B2), illustrating the contouring of NPC-associated HEZs by condensed chromatin (cc). Nucleolar materials (no) were excluded from these zones too. Chromatin hyper-condensation was also accompanied by gradual loss of the electron-dense NPC midplanes seen in non-infected cells (white arrowhead), and often by dilation of the NE lumen (arrow) late in the infection process. Bars: 200 nm; same magnification for panels A1 and B1, and panels A2 and B2. Download figure Download PowerPoint However, PV infection of HeLa causes amassments of condensed chromatin, first at the NE and later also deep in the nucleus, without trespassing ostensive boundaries in front of most, if not all, NPCs. This happens within a few hours post-infection and results in the contouring of HEZs of different lengths and shapes, with a seemingly temporal order of predominant appearance. Longer HEZ section areas of rectangular shape or with contours reminiscent of parabolic or hyperbolic curves as from sections of elongate cones, were more often observed in the earlier stages of chromatin condensation. Although notably varying in length, the majority of measured section heights (hy) were between 100 and 150 nm, some also reaching 150–250 nm (Supplementary Figure S1B). Very rarely, exceptionally long rectangular HEZ sections up to 460 nm in length were observed (Supplementary Figure S1B3), reminiscent of channel-like HEZs seen in other cell types (e.g., Visser et al, 2000; Rego et al, 2008). Analysis of consecutive cell sections showed that some of the long HEZs were longitudinally surrounded by condensed chromatin, indicative that they can be of cylindrical shape. Later in the infection process, the elongate types of HEZ sections were less frequently observed and shorter HEZ sections of triangular and trapezoid-like appearance or with hyperbola-like contours were prevalent. Although also observed in the earlier stages of chromatin condensation, these shorter HEZ section shapes were strikingly predominant in cells late in PV infection, with highly distorted nucleus and amassments of condensed chromatin. Their mean size did not appear to diminish further, suggestive of a rather steady residual HEZ 'core' region (Figure 1B and Supplementary Figure S1B). To estimate the size of such minimal HEZs, we chose a stereometric approach, on the basis of the simplifying assumption that the underlying HEZ shape can be approximatively described as a cone-like space. To this end, we measured the 2D-shape parameters of longitudinally sectioned HEZs next to perpendicularly sectioned NPCs and normalized for non-diametric section planes. These values were used for trigonometric calculations and approximation of HEZ dimensions and shape (Figure 2 and Supplementary Figure S2). Figure 2.Stereometric size approximation of the NPC-associated minimal HEZ. (A) A perpendicularly but non-diametrically sectioned NPC-associated HEZ enclosed by condensed chromatin, from the late stage of PV infection. Bar: 50 nm. (B1) Schematic depiction of a non-diametrically sectioned HEZ and measuring tracks (double-headed arrows) of intra-membrane NPC channel diameter (2rn), of HEZ-section diameter at the base (2rb) and parallel to the base at 40 nm distance (2r40), and of HEZ-section height (hy). A total of 121 perpendicularly cross-sectioned HEZs from the late stages of PV infection were measured and normalized for non-diametric section planes as described in Supplementary Figure S2. (B2) Schematic depiction of the mean normalized diametric HEZ section, with a basis diameter 2Rb of 119 nm and heights hyfr and hapex of 74 and 96 nm, respectively. (C) The 3D shape of the corresponding mean HEZ, showing a cone with flattened top. Schemes are drawn in scale to an NPC with an intra-membrane channel diameter set to 84 nm; for possibly smaller channel diameters in PV-infected cells and the effect on calculated HEZ dimensions, see Supplementary Figure S2. Download figure Download PowerPoint Even though the resulting model was likely to be slightly smaller than the authentic size (Supplementary Figure S2), it provided an estimate of the minimal dimensions of the HEZ 'core' region, showing a cone with flattened top, with a diameter (2Rb) of 119 nm at its basis and an approximated height (hapex) of 96 nm. This was slightly smaller than the calculated means of the rare HEZs found in non-infected HeLa cells, yielding a truncated cone with a 2Rb value of 129 nm and a hapex of 107 nm (Supplementary Figure S2D). Noteworthy, the shapes and dimensions of the trapezoid-like HEZ sections from PV- and non-infected HeLa cells closely resembled those from terminally differentiated cells (e.g., Tokuyasu et al, 1968; Maul, 1977). Maintenance of NPC-associated HEZs after PV infection correlates with the proteolytic insensitivity of Tpr's coiled-coil domain PV infection causes silencing of host cell transcription and specific degradation of a small number of nuclear proteins (e.g., Crawford et al, 1981; Davies et al, 1991; Rubinstein et al, 1992; Shen et al, 1996; Yalamanchili et al, 1997). Two nucleoporins, Nup153 and Nup62, were found degraded in PV-infected HeLa cells too (Gustin and Sarnow, 2001). Whereas Nup62 is an FG-repeat protein of the NPC core, the structurally unfolded C-terminal half of Nup153 possibly projects into the nucleus. Its degradation, though, would indicate that it is dispensable for the HEZs seen in such cells. Moreover, NPC anchorage of the coiled-coil protein Tpr is mediated by a short segment of Nup153 located within its N-terminal half (Hase and Cordes, 2003). Complete Nup153 degradation might therefore prevent or destabilize NPC binding of Tpr and thus also exclude a role for Tpr in HEZ establishment. However, Tpr's actual fate during PV infection, and that of other FG-repeat nucleoporins, such as Nup98, was unknown at the beginning of this work. We hence studied these and other proteins by immunoblotting of total HeLa proteins, collected at different time points after PV infection. SDS–PAGE showed that the bulk of cellular proteins remained unaffected by PV-induced proteolysis (Figure 3A). Furthermore, immunoblotting showed that lamins (Figure 3B) and most NPC-core proteins were left unharmed (Supplementary Figure S3). This included those of the Nup160 subcomplex, which represent the fundament for direct or indirect NPC anchorage of other NPC-associated proteins such as Nup98, Nup153, and Tpr (e.g., Vasu et al, 2001). By contrast, most FG-repeat nucleoporins (Figure 3C and D, and Supplementary Figure S3) had their FG-repeat domains removed. Tpr was also partially degraded (Figure 3C and E). Figure 3.Nup153 and Nup98 appear largely degraded upon PV infection whereas the coiled-coil rod domain of protein Tpr remains intact. (A) SDS–PAGE and Coomassie staining of whole-protein extracts from HeLa cells before and at 2–12 h post-infection, showing the bulk of cellular proteins unaffected by PV-induced proteolysis. The extracts were from the same HeLa cell cultures analysed by TEM in Figure 1. (B) Immunoblotting of extracts used in panel A, showing that lamin A and lamin C, like lamin B (not shown), remain unaffected. This indicates that rearrangements within the nuclear lamina or its NE attachments, but not lamin proteolysis, are required for the NE upfolding observed. (C) Epitope sites of Nup153, Nup98, and Tpr antibodies indicated by arrowheads. Different antibodies targeting the same protein are numbered as in panels D and E. (D, E) Immunoblotting of Nup153, Nup98, and Tpr, using cell extracts shown in panel A; (see also Supplementary Figure S3). Target regions are given in parentheses; Δ indicates mAbs for which actual epitopes within defined protein segments are unknown. (D) At 8–12 h post-infection, when HEZs are visible at almost all NPCs, Nup153 (full-length proteins marked by arrows) appears largely degraded, except for a small segment (double-asterisk) comprising at least part of the Tpr-binding region. Additionally, only minor amounts of a 120-kDa degradation product (asterisk) and some unspecific cross-reactions (u) are seen with some Nup153 antibodies. Nup98 is degraded more rapidly, with only its C-terminal domain (asterisk) resisting proteolysis slightly longer. (E) Whereas Tpr's C-terminal domain is being degraded around 8 h post-infection, its entire rod domain (double-asterisk) withstands proteolysis. The membrane marked 1622–1640 was first incubated with rb-anti-Tpr-4 (2063–2084), then stripped and re-incubated with gp-anti-Tpr-3 (1622–1640). Download figure Download PowerPoint Proteolysis of the FG-repeat proteins, including Nup153 and Nup98, first affected their unfolded FG-repeat domains. The one from Nup98 was eliminated earliest, in line with another recent study of PV-mediated Nup98 degradation (Park et al, 2008). The NPC-binding domains of these nucleoporins withstood degradation longer but when the HEZs became visible, the anchor domains appeared largely degraded too, except for those of Nup358, an FG-repeat nucleoporin located at the NPC's cytoplasmic side (Supplementary Figure S3), and Nup153 (Figure 3D). Whereas most of Nup153 appeared degraded at 12 h post-infection, a small segment of ∼26 kDa withstood degradation. This segment likely harbours the sequences essential for NPC anchorage and Tpr binding, both residing within a region comprising aa 228–439 (Enarson et al, 1998; Vasu et al, 2001; Hase and Cordes, 2003; Griffis et al, 2004). Tpr degradation, in contrast, was restricted to its unfolded C-terminal tail domain whereas its large coiled-coil domain (aa 1–1630), including Tpr's NPC- and Nup153-binding region, remained unharmed. Moreover, immunofluorescence microscopy (IFM) showed that these Tpr rods remained bound to the NE even late after PV infection. Similarly, the NPC-anchor segment of Nup153 remained attached to the NE, as did residual copies of Nup98's NPC-anchor domain. By contrast, already earlier after infection, neither other parts of Nup153, nor the FG-repeat domains of Nup98 and other nucleoporins, were detectable at the NE any longer (Figure 4 and Supplementary Figure S4). Figure 4.The coiled-coil rod domain of Tpr remains anchored to the NPC even late after PV infection. IFM of HeLa cells at 10 h post-infection, showing that antibodies against Tpr's coiled-coil domain (anti-Tpr-2 (636–655), -3 (1622–1640), -5 (1370–1626Δ)) and the Tpr-binding domain of Nup153 (anti-Nup153-2 (391–404)) still label the NE. At this time point, and earlier ones (not shown), the other parts of Nup153, and the FG-repeat domain of Nup98, are no longer detectable whereas the NPC anchor of Nup98 and Tpr's C-terminal tail are still present at some NEs. Nucleoporins of the Nup107 subcomplex (also Supplementary Figure S3), representing direct and indirect anchor sites for Tpr, Nup153, and Nup98, remain bound to the NPC. DNA staining and differential interference contrast (DIC) micrographs show nuclear-peripheral chromatin accumulation and cell rounding, characteristic for later stages of PV infection. Bar: 10 μm; same magnification for all the micrographs. Download figure Download PowerPoint NPC-associated HEZs can no longer be established after in vivo depletion of Tpr If fibrillar NPC appendices mark the periphery of the NPC-associated HEZ, a largely degraded Nup153, and Tpr's tail domain could not be regarded as prime candidates for maintaining the HEZs still visible late in PV infection. By contrast, Tpr's rod domain, distinguished by its integrity and persisting attachment to the NPC, remained a potential candidate. To test this further, we attempted to PV-infect cells after having depleted them of Tpr. In principle, elimination of Tpr was known to be achievable in HeLa cells by RNAi. Such Tpr-depleted cells are still capable of cell-cycle progression and nucleo-cytoplasmic transport of proteins and mRNAs (e.g., Hase, 2003; Hase and Cordes, 2003; Lee et al, 2008; and data not shown; see also Shibata et al, 2002). Furthermore, chromosome positioning as monitored by telomere and centromere detection, as well as the patterns of several epigenetic histone modifications, appeared unaffected in most Tpr-deficient cells (Supplementary Figure S5). Furthermore, it had been shown that activation of the RNAi machinery does not necessarily trigger antiviral responses, and siRNA-transfected cells can remain susceptible for subsequent PV infection (Gitlin et al, 2002). Nonetheless, to avoid innate cellular immune responses that could impair PV infection (e.g., Ida-Hosonuma et al, 2005), we first screened different Tpr siRNAs for high knockdown efficiencies without off-target effects unrelated to Tpr deficiency (data not shown). Several pre-selected siRNAs, complementary to non-overlapping ORF segments of the Tpr mRNA, caused a clear knockdown of Tpr protein levels at day 4 after transfection (Figure 5A). The mean intensities of residual Tpr staining in the transfected cells could be as low as 4–6% as determined by IFM, whereas residual Tpr levels in immunoblots of total cell extracts commonly ranged between 10% and less than 20% (Figure 5B). TEM of such cell populations allowed sporadic detection of perpendicularly sectioned NPCs that were juxtaposed to heterochromatic or nucleolar material but now mostly lacked distinct exclusion zones. This indicated that Tpr might at least play a role in the establishment of the few HEZs seen in non-infected HeLa cells. By contrast, even though correspondingly positioned NPCs were similarly sparse in simultaneous controls, they were still characterized by distinct HEZs (Figure 5C; see also Supplementary Text to Figure 5, and Supplementary Figure S6). Figure 5.RNAi-mediated Tpr knockdown in HeLa cells. (A) Confocal IFM of Tpr at day 4 after transfection with different Tpr siRNAs (Ib3, IV2, IV4) or target-less control siRNAs. Only traces of Tpr staining are seen in most cells after Tpr RNAi; bright nuclear rim staining shown as reference is visible only in cells that remained untransfected. For occasionally observed dots of residual Tpr staining at otherwise largely Tpr-deficient NEs, see Supplementary Figure S6. Bar: 20 μm. (B1) SDS–PAGE and Coomassie staining of serial dilutions of whole-protein extracts from non-transfected cells (Ctrl 2), and cells treated with Tpr siRNAs (Ib3, III4, III5, IV2, IV4) or transfection reagent alone (Ctrl 1), showing that the bulk of cellular proteins remains unaffected by siRNA treatment. (B2) Immunoblotting of identical loadings as in panel B1. Incubations with anti-Tpr and anti-Nup98 (asterisks: unrelated cross-reactions) were on different halves of the same membrane. Efficient Tpr knockdown was achieved with all Tpr siRNAs without eliciting distinct effects on other NPC proteins, including Nup93, Nup107, Nup133, and gp210 (not shown). Cells transfected with III4 and III5, however, were later found to not allow for a normal PV-infection process, so that these siRNAs were not used further. Of the infection-compatible siRNAs, Ib3 and IV4 were used for all subsequent PV-infection experiments in parallel. (C) TEM of non-transfected cells, and of cell populations at day 4 after treatment with transfection reagent alone, and transfection with Tpr siRNAs Ib3, or non-target control siRNAs (Ctrl 3). NPCs juxtaposed to heterochromatic or nucleolar material (arrows) in control cells remained characterized by HEZs but mostly lacked such exclusion zones after Tpr RNAi. Bars: 200 nm; same magnification for all the images. Download figure Download PowerPoint Tpr-deficient cell populations were then tested for susceptibility to PV infection, paralleled by infecting control cells that had been transfected with a target-less siRNA, treated with transfection reagent only, or had been left untreated. For several Tpr siRNAs, PV infection of transfected cells was found to appear normal. Characteristic changes in morphology, such as cell rounding, and their time points of occurrence were similar to that in the controls. At the molecular level, the characteristic NPC-protein degradation patterns were basically the same, except that the time points of complete degradation were slightly delayed in the siRNA-transfected cells (Figure 6). Figure 6.Post-transcriptional tpr gene silencing by RNAi does not impair subsequent PV infection and degradation of nucleoporins. Four days after transfection with Tpr siRNAs or mock transfection with non-target control siRNAs (Ctrl 1), or after incubation with transfection reagent alone (Ctrl 2), cells were either infected with PV or not, and harvested 10 h later. Total cell proteins were analysed by immunoblotting. Regardless of whether Tpr had already been eliminated by RNAi before PV infe
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