Nuclear actin is partially associated with Cajal bodies in human cells in culture and relocates to the nuclear periphery after infection of cells by adenovirus 5

Experimental Cell Research 303 (2005) 229 – 239 www.elsevier.com/locate/yexcr

Nuclear actin is partially associated with Cajal bodies in human cells in culture and relocates to the nuclear periphery after infection of cells by adenovirus 5 L.J.E. Gedgea, E.E. Morrisonb, G.E. Blaira, J.H. Walkera,* a

School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK b CRUK Clinical Centre at Leeds, St. James’ University Hospital, Leeds, LS9 7TF, UK Received 1 March 2004, revised version received 11 June 2004

Abstract Cajal bodies are intra-nuclear structures enriched in proteins involved in transcription and mRNA processing. In this study, immunofluorescence microscopy experiments using a highly specific antibody to actin revealed nuclear actin spots that colocalized in part with p80 coilin-positive Cajal bodies. Actin remained associated with Cajal bodies in cells extracted to reveal the nuclear matrix. Adenovirus infection, which is known to disassemble Cajal bodies, resulted in loss of actin from these structures late in infection. In infected cells, nuclear actin was observed to relocate to structures at the periphery of the nucleus, inside the nuclear envelope. Based on these findings, it is suggested that actin may play an important role in the organization or function of the Cajal body. D 2004 Elsevier Inc. All rights reserved. Keywords: p80 coilin; Adenovirus; Nuclear matrix; Actin

Introduction Despite great progress in our understanding of replication, transcription, and RNA processing, much less is understood about the internal organization of the cell nucleus. Increasing evidence suggests that the nucleus is compartmentalized and that there may be an underlying matrix responsible for its spatial and functional organization. Actin-based structures are of major importance in the cytoplasm where they are involved in cell movement, organelle transport and structural support. There has recently been a resurgence of interest in the role of actin in the nucleus [1–3]. Nuclear actin has been detected in diverse cell types ranging from cultured mammalian cells to yeast. Currently, the best-established role for nuclear actin is

* Corresponding author. School of Biochemistry and Molecular Biology, University of Leeds, LS2 9JT, UK. E-mail address: [email protected] (J.H. Walker). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.06.034

in chromatin remodeling [4]. Nuclear actin has also been implicated in transcription and RNA processing, however, the mechanisms involved have yet to be elucidated [5–9]. Cajal bodies (coiled bodies) were first described by Ramon y Cajal in 1903 and have been found in the nuclei of both plant and animal cells [7,8]. Cajal bodies are characterized by the presence of a marker protein, p80 coilin [9]. p80 coilin is concentrated in Cajal bodies and is also associated with speckles distributed throughout the nucleoplasm. Cajal bodies contain a number of nuclear proteins involved in transcription and processing of nuclear RNA [10]. However, despite the presence of snRNPs involved in splicing, they lack polyadenylated RNA and the essential splicing factors, U2AF and SC35, and are therefore not believed to be involved directly in splicing. Instead, Cajal bodies may be involved in snRNP biogenesis. When GFP-sm proteins were expressed in cells, they were first detected in Cajal bodies and nucleoli prior to moving to nuclear speckles associated with splicing, suggesting that Cajal bodies were involved in snRNP modification [11]. Furthermore, evidence has been

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presented showing that both RNA polymerase II and TATA box-binding protein are present in Cajal bodies [12] indicating a role for Cajal bodies in transcription. Cajal bodies have also been shown to be dynamic and associated with chromatin in an ATP-dependent manner [13]. Actin has not been identified as a constituent of Cajal bodies although the actin-binding protein profilin I has been shown to colocalize with these structures [6]. In this study, we have investigated the distribution of actin in the nuclei of mammalian cells in culture. We have found that a pool of actin is associated with Cajal bodies and that adenovirusinduced disassembly of coiled bodies correlates with the loss of actin from these structures.

Materials and methods Materials The monoclonal antibody to actin (clone C4) was purchased from ICN (Hampshire, UK) or from Chemicon International (Harrow, UK). Rabbit a-skeletal actin standard (AK99) was obtained from Cytoskeleton (Denver, CO, USA). Chemicals were purchased from Sigma (Poole, Dorset, UK) or BDH (Poole, Dorset, UK) unless indicated otherwise. Methods Cell culture HeLa and A549 cells were grown in Dulbecco’s minimal essential medium supplemented with 20 mM lglutamine, 100 U/ml penicillin, 100 Ag/ml streptomycin, and 0.3% (w/v) sodium bicarbonate. All cell culture reagents were purchased from Invitrogen (Paisley, UK). Cells were grown in NUNC k (Nalgene Inc.) flasks maintained at 378C, in a humidified atmosphere containing 5% CO2. Immunocytochemistry HeLa or A549 cells were seeded onto cover slips in 6well plates and grown to sub-confluence. The immunofluorescence method was adapted as described [14]. Cells were washed twice with PBS (9 mM Na2HPO4d 12H2O, 1 mM Na2HPO4d 2H2O, 0.15 M NaCl pH 7.4) pre-warmed to 378C, fixed for 5 min in 10% neutral buffered formalin (Sigma HT50), washed three times with PBS and permeabilized with 0.1% (v/v) Triton X-100 (from a 10% w/v stock) in PBS for 5 min, then refixed for 5 min in 10% neutral buffered formalin. After further PBS washes, cells were incubated in 1 mg/ml NaBH4 in PBS (3  5 min) to eliminate autofluorescence. Cells were blocked with 5% (v/v) goat serum in PBS containing 2 mM NaN3 for 3 h. Primary antibodies were incubated overnight in PBS containing 5% (v/v) goat serum and 2 mM NaN3. After

further PBS washes, cells were incubated with secondary antibodies for 3 h. All antibody incubations were carried out at room temperature. In some cases, cells were counterstained by incubating in 1 Ag/ml DAPI (4V, 6-diamidino2-phenylindole) for 2 min and washed twice in PBS prior to mounting in Citifluor (Agar Scientific, Hertfordshire, UK). Conventional fluorescence microscopy was performed using a Nikon Optophot microscope or with a Zeiss Axioplan 2 microscope coupled to an imaging system with IP lab software (Digital Pixel Ltd., Brighton, UK). For confocal microscopy, a Leica TCS SP spectral confocal imaging system coupled to a Leica DM IRBE inverted microscope was used. To ensure optimal resolution, averages of four scans were made of each confocal section. Individual confocal sections were 0.365 Am thick. Double and triple labeled cells were scanned sequentially to prevent signal bleed through between channels. Densitometric analysis of cell staining Densitometric scans of single confocal images were carried out using NIH Image 1.62 software. Adenovirus infection Sub-confluent HeLa cells were infected with replication competent Ad5 luc3, in which the viral E3 gene is replaced by a luciferase reporter gene [15] at a multiplicity of infection of approximately 10 fluorescence-focus forming units (FFU) per cell as previously described [16]. Cells were washed twice in PBS at 378C and incubated in 200 Al of virus in serum-free medium for 1 h and were agitated four to five times during this period. Serum-free medium was added in the presence or absence of 25 Ag/ml cytosine arabinoside to observe the effects of early and late viral infection, respectively. Cells were incubated at 378C for 23 h prior to processing for immunofluorescence. In situ nuclear matrix extractions In situ, the following extractions were performed essentially according to Ref. [17] with minor modifications. In brief, cells were washed twice with PBS and lysed with CSK buffer (9 mM PIPES-NaOH pH 6.8, 90 mM NaCl, 290 mM sucrose, 2.7 mM MgCl2, 0.9 mM EGTA, 0.45% (v/v) Triton X-100, 0.9 mM PMSF and 20 mM vanadyl ribonucleoside complex) for 2 min at RT and then digested with 40 units of RNAse-free DNAse I per coverslip (Roche, East Sussex, UK) for 30 min at room temperature. Cells were then extracted with 0.25 M (NH4)2 SO4 added dropwise from a 2-M stock. This was removed and cells were covered with CSK and an equal volume of 4 M NaCl was added drop-wise to a final concentration of 2 M. Cells were then fixed and processed for immunofluorescence microscopy as described above. Preparation of whole cell lysate Whole cell extracts of HeLa cells for SDS PAGE analysis were harvested by washing cells with PBS followed by

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Results Specificity of the anti-actin antibody

Fig. 1. Specificity of the anti-actin antibody determined by Western blotting. A–C shows a 10% SDS PAGE gel stained with Coomassie Brilliant Blue. (A) Mr markers-Biorad broad range; (B) HeLa cell lysate (20 Ag protein); (C) a-skeletal muscle actin standard (1.5 Ag). (D) Western blot analysis of HeLa cell lysate (20 Ag protein) incubated with anti-actin. (E) Western blot analysis of purified actin incubated with anti-actin. HRPconjugated goat anti-mouse IgG and ECL were used for detection of bound antibody.

The monoclonal anti-actin antibody, clone C4, has been extensively characterized previously and binds to an epitope mapped to residues 23–39 of actin (Prof. J. Lessard, personal communication). The antibody recognizes all isoforms of actin [20] and has been used previously in studies on nuclear actin [21,22]. The specificity of this antibody was confirmed on Western blots of total cell proteins derived from the epithelial cell line HeLa (Fig. 1D). Highly purified actin was also recognized by the antibody (Fig. 1E). Fig. 1 shows that the antibody was completely specific for an immunoreactive component of 42 kDa as expected for actin.

incubation for 5 min on ice with 1 ml lysis buffer (2% SDS, 1 mM EDTA, 1 mM EGTA, 2 Ag/ml Aprotinin, 2 Ag/ml Leupeptin, 2 Ag/ml Pepstatin A, 1 mM PMSF). Cell material was collected using a cell scraper, boiled for 1–2 min and sheared by passage through a 25-gauge needle. Determination of protein concentration Protein concentration was determined by the BCA assay according to manufacturer’s instructions (Pierce, Cheshire, UK), using BSA as a standard. Protein electrophoresis Proteins were separated by SDS PAGE using 1 mm thick 10% (w/v) polyacrylamide gels and a discontinuous buffer system [18] with the dMighty smallT SE200 series vertical slab gel apparatus (Hoeffer Scientific Instrument, Staffs, UK). Electrophoresis was performed at 20 mA. Proteins were stained with Coomassie blue (0.25% (w/v) in 40% methanol 7% acetic acid) for 1 h and destained in 10% ethanol/10% acetic acid overnight. Western blotting Proteins were electrophoretically transferred onto nitrocellulose membranes [19]. Transfer was carried out for 3 h at 400 mA or overnight at 80 mA using a Biorad Trans-Blot cell. Transfer was confirmed by staining the membranes with 0.1% (w/v) Ponceau S, 3% (v/v) acetic acid. After washing with PBS containing 0.1% (v/v) Triton X-100 (PBST), blots were blocked with PBST supplemented with 5% (w/v) low fat milk (Safeway plc, UK), (PBSTM) and incubated with primary antibodies in PBSTM for 3 h or overnight. After extensively washing blots with PBST, bound antibody was detected by incubating with secondary antibody conjugated to HRP for 1 h followed by ECL (Pierce) according to the manufacturer’s instructions.

Fig. 2. Conventional immunofluorescence microscopy locating actin in HeLa cells. Cells were fixed with 10% neutral buffered formalin and permeabilized with 0.1% Triton X-100 in PBS followed by incubation with a monoclonal antibody against actin. Bound antibody was detected with FITC-conjugated goat anti-mouse IgG. In A, the microscope was focused on the cell surface in contact with the coverslip. In B, the microscope was focused on the cell nuclei. Cells incubated with FITC-conjugated goat antimouse IgG alone were unstained (not shown). The scale bar represents 15 Am.

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Actin in the nuclei of human cells Initially, the location of nuclear actin was investigated in HeLa, A549, CHO, 3Y1, and EA.hy.926 cells by conventional indirect immunofluorescence microscopy. Several different cell lines were investigated to ensure that the staining patterns observed were not exclusive to one cell type. In all cell types, stress fibers were evident especially when the microscope was focused on the cell surface attached to the cover slip. When the focus was adjusted to focus on nuclei, all cells were found to contain intranuclear actin immunoreactivity. This is illustrated in Fig. 2 for HeLa cells, a cell line used extensively for studies on intranuclear structure. In Fig. 2A, with the microscope focused on the cell surface in contact with the cover slip, stress fibers and cell edges are clearly labeled. Diffuse staining of the cytoplasm is also present. When the microscope was focused on the nuclei, staining of intranuclear structures seemed to be evident (Fig. 2B). Laser-scanning confocal microscopy was used to determine more rigorously whether actin was present within nuclei. A projected image is shown in Fig. 3D of all the confocal sections through a HeLa cell. This shows stained stress fibers and closely resembles the results in Fig. 2. Single confocal sections (0.365 Am thick) through central

regions of the nuclei of HeLa and A549 cells are shown in Figs. 3A and B. Actin immunoreactivity is present in nuclei as bright spots and fine speckles of staining throughout the nucleoplasm. The bright spots were evident in all cells examined. In an asynchronous population of Hela cells, these bright spots were 0.57 Am (F0.21 Am, SEM) in diameter and on average 3–8 per nucleus. Fig. 3C shows densitometric analysis through a single confocal section through the nucleus. The staining of the bright intranuclear spots in a confocal section through the nucleus were brighter than cytoplasmic actin staining. The smaller peaks in Fig. 3C correspond to the fine speckles in the nucleoplasm whereas the three large peaks correspond to the bright spots. In all cases, controls in which primary antibody was omitted were unstained (data not shown). Actin associates with Cajal bodies and the association persists despite extraction to reveal the nuclear matrix The observation that monoclonal anti-actin antibody stained discrete spots within the nucleus of similar size and number to Cajal bodies prompted experiments to compare the distributions of nuclear actin and p80 coilin. p80 coilin is a protein highly concentrated in Cajal bodies and also distributed throughout the nucleoplasm in speckles.

Fig. 3. Confocal sections comparing the location of nuclear actin in human cell lines. Cells were fixed with 10% neutral buffered formalin and permeabilized with 0.1% Triton X-100 in PBS followed by incubation with a monoclonal antibody against actin. Bound antibody was detected with goat anti-mouse IgG conjugated to Alexa 488. (A) HeLa cells; (B) A549 cells; (C) surface plot showing the distribution of actin immunoreactivity in HeLa cells; (D) Projected image of HeLa cells. Data was generated by using NIH Image 1.62 to process a single confocal section through the centre of the nucleus. Confocal sections 0.365 Am in thickness are shown. Scale bar = 20 Am.

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Fig. 4. Cajal body-associated actin in HeLa cells. Cells were fixed with 10% neutral buffered formalin and permeabilized with 0.1% Triton X-100 in PBS prior to labeling with mouse anti-actin and rabbit anti-p80 coilin. Bound antibodies were detected with goat anti-mouse IgG conjugated to Alexa 488 and goat antirabbit IgG conjugated to Alexa 594. Cells were counter-stained with DAPI. Ai, monoclonal anti-actin; Aii, polyclonal rabbit p80 coilin; Aiii, merged image of i and ii; Aiv is a merged image of i, ii, and DAPI. Scale bar = 10 Am. In B and C enlarged images of Cajal bodies compare the distribution of actin to p80 coilin. Bi, Ci, monoclonal anti-actin; Bii, Cii, polyclonal rabbit p80 coilin. Biii is a merged image of i and ii. Ciii is a merged image of Ci and Cii. Scale bar = 1 Am. Confocal sections 0.365 Am in thickness are shown.

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Fig. 5. Cajal body associated actin and p80 coilin colocalize in cells extracted to reveal the nuclear matrix. HeLa cells were sequentially extracted as described in the materials and methods. Panels A to C show conventional epifluorescence micrographs of control cells labeled with antibodies against p80 coilin (panel A, detected with goat anti-rabbit IgG conjugated to Alexa 594) and actin (panel B, detected with goat anti-mouse IgG conjugated to Alexa 488). The cells were counter-stained with DAPI (panel C). Panels D to F show a cell treated to remove DNA and reveal components of the nuclear matrix and subsequently labeled with antibodies against p80 coilin (panel D), actin (panel E) and counter-stained with DAPI (panel F). Nuclear focus is shown.

Asynchronous HeLa cells were incubated with the monoclonal anti-actin antibody, and simultaneously with a polyclonal rabbit anti-serum raised against p80 coilin. Cells were subsequently incubated with fluorescently conjugated secondary antibodies and with DAPI. The results showed that the large actin spots colocalized with Cajal bodies (Fig. 4). The smaller, nucleoplasmic speckles of actin and p80 coilin did not colocalize. Fig. 4A shows a representative 0.365 Am thick confocal section, showing the location of actin, p80 coilin, and DNA. Figs. 4B and C show two individual Cajal bodies enlarged to show the distribution of p80 coilin and actin within them. p80 coilin had a homogenous distribution throughout the Cajal body. Actin immunoreactivity was more structured within the Cajal body and was often observed in a central location, surrounded by a halo of p80 coilin (Figs. 4Biii and Ciii). To investigate whether actin remains associated with Cajal bodies in the nuclear matrix, HeLa cells were sequentially extracted with Triton X-100, DNAse I and 2 M NaCl to extract soluble nuclear proteins (Figs. 5D and E). The absence of DAPI staining after DNAse I and salt treatment confirmed the complete digestion and removal of DNA (Fig. 5F). Nuclear actin remained associated with Cajal body-associated p80 coilin. The effect of adenovirus infection on Cajal body-associated actin Ad2 and Ad5 are almost genetically identical and follow an infectious program of approximately 30 h in HeLa cells at a high multiplicity of infection [23]. Cajal bodies have previously been shown to disassemble into micro-foci in the late phase of Ad2 infection [24] and form both clusters of dots and homogenous masses in cells infected with Ad5 [25]. Here, Ad5 infection was used as a tool to perturb Cajal

bodies and to assess its effect on the colocalisation of nuclear actin and p80 coilin. HeLa cells were infected with Ad5 (10 FFU/per cell). Initially, the virus was titrated to determine an appropriate FFU/per cell required to observe both infected and uninfected cells in the same microscopic field (data not shown). Cells were incubated in the presence of 25 Ag/ml cytosine arabinoside to investigate the effect of early Ad5 infection on p80 coilin and nuclear actin. Cytosine arabinoside inhibits viral DNA replication preventing late gene expression. Cells were fixed and processed for immunofluorescence microscopy at 24 h post-infection. E1A is an immediate early gene product of Ad5 and was used as a marker for early Ad5 infection as it is expressed prior to viral DNA replication [26]. Early Ad5 infection had no effect on the p80 coilin (data not shown) in agreement with previous work on Ad5 [25]. There was also no effect on the distribution of actin (data not shown). To permit Ad5 to progress to the late stage of the infectious cycle, cells were infected with Ad5 in the absence of cytosine arabinoside and fixed 24 h post-infection. The late phase of infection was characterized by the expression of the penton base, a product of late Ad5 gene expression. Cells were labeled with a rabbit polyclonal anti-serum raised to the penton base and a mouse monoclonal antibody raised against p80 coilin. The penton base was expressed in cells to different levels. In cells with a low level of expression, the penton base was distributed as fine speckles throughout the nucleus (faintly visible in Fig. 6Bi, cell a). In cells with higher expression, the penton base exhibited large aggregates of staining concentrated at the nuclear periphery together with internal nuclear aggregates (Fig. 6Bi, cell b). Cells with the faint pattern of speckles in the nucleus (Fig. 6Bi, cell a) showed a breakdown of p80 coilin-positive

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Fig. 6. Localisation of p80 coilin and penton base in single confocal optical sections. HeLa cells were infected with approximately 10 FFU/per cell of recombinant Ad5 (i), or mock-infected (ii) and fixed 24 h post-infection with 10% neutral-buffered formalin. Cells were then permeabilized with 0.1% Triton X-100 in PBS and incubated with the following antibodies: (A) monoclonal anti-p80 coilin followed by goat anti-mouse IgG conjugated to Alexa 488; (B) polyclonal rabbit anti-serum raised to recognize the penton base, followed by goat anti-rabbit IgG conjugated to Alexa 594. In B(i) cell a has a low level of expression of penton base compared to cell b. Confocal sections 0.365 Am thick are shown. Scale bars = 10 Am.

Cajal bodies. p80 coilin was distributed in these cells as bright compact structures or micro-foci that seemed to be clustered into discrete territories (Fig. 6Ai, upper cell). In cells with a high level of expression of penton base (Fig. 6Bi, cell b), p80 coilin-positive structures were distributed more homogeneously through the nucleus (Fig. 6Ai, lower cell). They were also less compact than the structures seen in cells with lower levels of penton base. Once the staining pattern of p80 coilin in cells at the late stage of Ad5 infection had been established in relation

to penton base expression, the relationship between actin and p80 coilin in Ad5-infected cells could be investigated. Fig. 7 shows that the Cajal body-associated actin is lost as a result of Ad5 infection. Both infected and uninfected cells can be seen in the same field (Fig. 7A). In the lower uninfected cell, distinct coiled bodies are visible and are p80 coilin (Fig. 7B) and actin (Fig. 7A) positive. In the infected cell, p80 coilin immunoreactivity is distributed into micro-foci in regions throughout the nucleoplasm (Fig. 7B, upper cell), whereas nuclear actin redistributes

Fig. 7. Colocalisation of coilin and actin is lost late in Ad5 infection. HeLa cells were infected with approximately 10 FFU/per cell recombinant Ad5 and fixed 24 h post infection. Cells were then permeabilized with 0.1% Triton X100 in PBS and incubated with the following antibodies: (A) monoclonal anti-actin followed by goat anti-mouse IgG conjugated to Alexa 488; (B) polyclonal rabbit anti-serum raised to p80 coilin followed by goat anti-rabbit IgG conjugated to Alexa 633. Sections, 0.365 Am. Scale bars = 5 Am.

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mainly to patches at the nuclear periphery (Fig. 7A, upper cell). Actin staining was frequently found adjacent to the penton base aggregates around the nuclear periphery Fig. 7 shows that the Cajal body-associated actin previously observed in the nuclei was redistributed as a result of Ad5 infection. Cells exhibiting a breakdown of p80 coilin into micro-foci in the nucleoplasm often had strong staining of actin around the nuclear periphery. This observation was further characterized using the viral penton base as a marker for late gene expression. The actin staining was frequently found adjacent to the penton base aggregates around the nuclear periphery (Figs. 8Ci and D). Speckles of actin were also evident within the penton base aggregates. The actin and penton base aggregates resided inside the nuclear envelope (Fig. 8D). In infected cells, the distribution of DNA labeled with DAPI became less diffuse and strong peripheral staining was observed (Figs. 8Ci and 8D). Mockinfected cells showed the normal distribution of nuclear actin consisting of Cajal body-associated actin and nucleoplasmic speckles (Fig. 8Aii). No penton base immunoreactivity was observed in mock-infected cells (Fig. 8ii).

Discussion Actin is associated with Cajal bodies We have demonstrated that actin is associated with Cajal bodies. The clone C4 monoclonal antibodies against actin have been used to localize nuclear actin previously in a mammalian cell lines [21,22]. Actin has also been previously shown to co-localize with snRNP aggregates [27]. In this study, a specific marker for Cajal bodies has confirmed the colocalization. The anti-actin antibodies labeled structures 0.57 Am in diameter that were also positive for p80 coilin, the specific marker for Cajal bodies (Fig. 4). Actin and p80 coilin are also associated with two independent populations of nucleoplasmic speckles that are smaller than Cajal bodies. This suggests that actin and p80 coilin are not associated except in Cajal bodies. Actin was frequently found to occupy a central location within the Cajal bodies. Recently, profilin I, a key player in the regulation of actin dynamics, has also been shown to be concentrated at the centers of Cajal bodies [6], suggesting that G-actin and profilin may be associated at the core of this structure. Profilin has also been recently demonstrated to act as a cofactor of actin export [28]. It is of interest that other anti-actin antibodies tested did not reveal actin aggregates that correspond to Cajal bodies (data not shown). Many nuclear actin-binding proteins have been reported [1] that may mask the epitopes to which the other actin antibodies have been raised. Consistent with this hypothesis, other anti-actin antibodies surveyed were either

raised to the C-terminus or the epitope to which they have been raised is unknown. In contrast, the monoclonal antiactin antibody (clone C4) used here has been recently mapped to residues 23–39 (Prof. J. Lessard, personal communication). Epitope masking has previously been proposed to account for the reactivity of a monoclonal antibody that recognizes SDS-denatured actin from many species but exhibits a selective pattern of staining by immunofluorescence microscopy including labeling of unidentified intranuclear structures [29]. Epitope masking has also been observed for the intranuclear structural proteins, lamins A and B [30]. Cajal bodies have been shown previously to resist various extraction conditions and be retained in the nuclear matrix [31]. We have shown that the actin remains associated with Cajal bodies despite extraction to reveal the nuclear matrix. This argues for a tight association of actin within Cajal bodies that could be consistent with an association between actin and other proteins. Adenovirus infection results in loss of actin from Cajal bodies Previous studies looked at the result of Ad2 and Ad5 infection on the structure of p80 coilin-positive Cajal bodies. Late in Ad2 infection, p80 coilin was shown to be present as microfoci throughout the nucleoplasm [24]. Ad5 infection also resulted in the redistribution of p80 coilin into clusters of tiny dots [25]. In agreement with these data, we also found that, late in infection with the Ad5, p80 coilin localized to microfoci. Interestingly, cells expressing high levels of a marker for late infection (penton base) showed microfoci throughout nuclei, whereas, in cells stained weakly for the penton base, p80 coilin disassembled into microfoci that were clustered together in domains that may reflect an early stage of Cajal body breakdown. Early in Ad5 infection, actin remains associated with Cajal bodies and with smaller intranuclear speckles. Late in infection by Ad5, actin was not associated with the microfoci containing p80 coilin indicating that it must have dissociated from the Cajal bodies as they disassembled. However, our results do not indicate whether Ad5 infection results in a loss of actin from Cajal bodies resulting in their disassembly, or vice versa. Late in Ad5 infection, actin was found frequently to be concentrated in patches around the nuclear periphery, often adjacent to the penton base. This may simply be fortuitous or could argue for a role for actin in the later stages of adenovirus synthesis and assembly. Possible roles for Cajal body actin Cajal body-associated actin may be responsible for maintaining a pool of G-actin in complex with its binding proteins in order to maintain a local concentration of the protein.

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Fig. 8. Localisation of actin, penton base and DNA in Ad5 infected cells. HeLa cells were infected with approximately 10 FFU/per cell recombinant Ad5 (i), or were mock-infected (ii). Cells were then fixed with 10% neutral buffered formalin 24 h post-infection, permeabilized with 0.1% Triton X-100 in PBS and incubated with the following antibodies: (A) anti-actin followed by goat anti-mouse IgG conjugated to Alexa 488; (B) polyclonal anti-serum raised against the penton base followed by goat anti-rabbit conjugated to Alexa 633 (a, high penton base expression; b, low penton base expression); (C) a merged image of A, B and DAPI; (D) enlarged nucleus showing the location of actin, penton base and DNA. Confocal sections 0.365 Am thick. Scale bars = 10 Am.

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G-actin is implicated in transcription. It sequesters the transcription factor MAL in the cytoplasm prior to its translocation to the nucleus [32]. It is possible that G-actin may also sequester transcription factors within the nucleus. G-actin has also been suggested to be a transcription initiation factor for RNA polymerase II [33] and has been demonstrated to directly stimulate transcription with its action enhanced by profilin [5]. Furthermore, an actinribonucleoprotein interaction has been shown to be involved in transcription by RNA polymerase II [34]. Cajal bodies contain a large number of factors involved in transcription and have been suggested to provide sites for the assembly of transcription complexes [35]. G-actin at the core of the Cajal body could play a role in such assembly processes. Actin could also play a role in RNA processing. One intriguing possibility is that actin could act as a factor that carries nascent RNA into the Cajal body for modification and remains associated until the mRNA is released into the cytoplasm to facilitate nuclear export. Actin binds to the heterogeneous RNP36 (hrp36) and is associated with the RNA transcript via hrp36 during transfer of RNA all the way from the gene to the polysome [36]. Furthermore, nuclear actin was demonstrated to associate with a specific subset of hnRNP A/Btype proteins from pre-mRNP complexes and it has been suggested that actin may bind pre-mRNA and mediate its packaging into an RNP particle and facilitate its export [37]. Cajal bodies have also been shown to be involved in RNA processing. snRNPs accumulate in Cajal bodies prior to accumulation in speckles and the nucleolus [11]. Cajal bodies have also been implicated in histone premRNA processing and associate with histone genes [38,39]. Additionally, dguideT RNAs have been identified in Cajal bodies that may direct modification of snRNAs [40]. In conclusion, our data argue for an important role for actin in Cajal body function. Further work will be required to distinguish the precise role for actin which may have an involvement in nuclear processes such as RNA processing.

Acknowledgments We are grateful to Dr. Torkjel Matzow for providing Ad5, Dr. Claire Thomas for the anti-penton base antibody, Avril Trejdosiewicz for carrying out the FFU assay, Prof. Angus Lamond, University of Dundee, Dundee, for monoclonal and polyclonal antibodies against p80 coilin and Prof. James L. Lessard, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH for sharing unpublished data concerning the epitope recognized by the anti-actin antibody. This work was funded by a BBSRC quota studentship, Cancer Research UK and Yorkshire Cancer Research.

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