Correspondence to: Michael J. Hendzel, Department of Oncology, Cross Cancer Institute, University of Alberta, 11560 University Ave., Edmonton, Alberta, Canada T6G 1Z2. Tel:(780) 432-8439 Fax:(780) 432-8892 E-mail:michaelh{at}cancerboard.ab.ca.
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Abstract |
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Compartmentalization of the nucleus is now recognized as an important level of regulation influencing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concentration to sites of active transcription, but splicing factors are also thought to exist in a freely diffusible state. In this study, we examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASFGFP) using time-lapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We find that ASFGFP moves at rates up to 100 times slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of an RNA polymerase II transcription inhibitor and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites.
Key Words: ASF/SF2, IGCs, FRAP, cell nucleus, nuclear matrix
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Introduction |
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It is well established that the cell nucleus is spatially organized through compartmentalization of functionally related biomolecules (
Although a subject of widespread investigation, the acceptance of such a framework has suffered from the inability of years of ultrastructural research to reveal this nuclear component without prior elution of chromatin (
The potential implications of a structural basis to nuclear compartmentalization have arisen in recent studies. The observation that small portions of speckles appear to break away from the larger domain and move to sites of newly transcribing genes (
We set out to determine whether the movement of splicing factors throughout the nucleus occurs mainly due to a directed transport of macromolecular assemblies, or whether significant quantities are able to freely diffuse. In this study, we used three-dimensional (3-D) time-lapse (4-D) fluorescence deconvolution microscopy to confirm the movement of ASF-enriched structures described by
After the determination of structure mobility, we then evaluated molecular mobility. We used the GFP protein to define the mobility of a protein capable of moving relatively freely throughout the nuclear space. Surprisingly, the mobility of ASF into and out of speckles is virtually identical to the mobility of ASF in the nucleoplasm. Importantly, we find the mobility of ASF to be many times slower than free diffusion. The redistribution of ASF into photobleached areas is not dependent upon the reported translocation of ASF-enriched domains to and from nuclear speckles. We found that under conditions where the translocation of microdomains enriched in ASF has ceased, the ASFGFP recovers with similar kinetics. A slight increase in mobility in both the nucleoplasm and in nuclear speckles was observed under conditions of RNA polymerase II transcriptional inhibition.
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Materials and Methods |
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Cell Culture and Transfections
Mouse 10T1/2 cells, human intestinal smooth muscle cells, human lung cancer cells, human neuroblastoma cells, or Indian muntjac fibroblasts were plated onto glass coverslips and cultured 12 d in growth medium until 50% confluent. At this point, complete medium was removed and cells were transfected using lipofectamine (GIBCO BRL) and an ASFGFP fusion protein construct described previously (24 h before imaging. In some cases, staurosporine (Sigma-Aldrich) or 5,6-dichlorobenzimidazole riboside (DRB; Sigma-Aldrich) were added at 100 µg/ml and 75 µg/ml, respectively, between 2 and 4 h before imaging.
Live Cell Imaging
Coverslips were placed on glass slides containing several drops of media surrounded by vacuum grease. The vacuum grease allows an airtight seal to form. Cells are capable of growing in these conditions for least 24 h at 22°C. For 4-D imaging, a Zeiss AxioPlan II microscope, a 100x 1.4 NA lens, and a 12-bit cooled CCD (Cooke SensiCam) were used to collect images. Images were further processed by digital deconvolution using AutoQuant Autodeblur. For FRAP, the laser scanning microscope (Zeiss LSM 510) was set to laser scanning mode and the initial imaging conditions were determined. A 25x 0.8 NA lens was used for these experiments and pixel sampling was set between 90 and 120 nm/pixel. The argon laser spectral line at a wavelength of 488 nm was set to an intensity of no greater than 1.25% of its total power (15 mW) for image collection. A mask, which typically covered half of the cell nucleus, was then photobleached using 100 iterations at 3.75 mW laser power. 12-bit images were collected before, immediately after, and at defined intervals after bleaching. Our total experiment time was set to 500 s, which was sufficient for complete recovery from photobleaching.
For quantitative imaging, a 3-µm wide strip of the cell was photobleached and then scanned during recovery. Photobleaching was completed in 2 s. Similar proportions of GFP (
25%) and ASFGFP (
30%) total nuclear fluorescence were lost during photobleaching. Because the GFP protein recovered very rapidly, it was necessary to only scan the photobleached region in order to adequately sample GFP recovery. For simplicity in the graphical presentation of the data, the initial post-bleach value of the photobleached region was normalized to zero by subtraction and the maximal value obtained during recovery was normalized to 100. The t1/2 of recovery can then be directly read off of the graph as the 50% value. Identical results were obtained when 2-µm spots, rather than strips, were bleached. To determine the immobile population of ASFGFP, the maximal recovered value was divided by the starting value of the photobleached region after correction for total nuclear fluorescence lost during photobleaching. This correction factor was determined by measuring the total nuclear fluorescence immediately after photobleaching and dividing this value by the total nuclear fluorescence immediately before bleaching. The laser intensity during recovery was set so that no measurable loss of fluorescence was observed during the monitoring period.
To track the mobility of HDAC4GFP domains and small foci enriched in ASFGFP we used the track points function in Universal Imaging's MetaMorph 4.0 imaging software. The image sequences were viewed on screen at 200% magnification and the center of each focus was identified with a mouse. For HDAC4 domains, the structures sometimes moved out of the plane of focus. In this instance, the foci could still be followed by identifying slightly elevated regions of signal intensity. Although difficult to identify in single frames, these could be easily identified by rapidly moving between the several frames where the domains move into and out of focus. This occurred for two of the five foci tracked. These foci, however, remained in focus for most of the time course.
Photobleaching and Cell Viability
Photobleaching experiments have an undeserved reputation of being photodestructive to living cells. Excessive photodamage, if it occurred, would complicate collection and interpretation of FRAP-based experimental data. However, bleaching GFP using the monochromatic laser at low intensities needed for bleaching and image acquisition, does not noticeably perturb live cells (reviewed
Online Supplemental Material
Video 1 further depicts Fig 1. A cell expressing ASFGFP is shown in 3-D red/green stereo time-lapse. Individual frames were captured at 2-min intervals.
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Videos 2 and 3 further depict Fig 2. A cell expressing an HDAC4GFP fusion protein (video 2) or ASFGFP (video 3) was captured as a 2-D time-lapse experiment at 1-s intervals for 60 s. These videos are available at http://www.jcb.org/cgi/content/full/150/1/41/DC1.
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Results |
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4-D Dynamics of the ASF Splicing Factor in Living Cells
Previous observations using the ASFGFP fusion protein clearly indicate that nuclear speckles are structurally dynamic subnuclear domains (
Large Nuclear Domains Are Capable of Random Motion within the Cell Nucleus
The movement of nuclear structures as large as those that originate and migrate away from nuclear speckles could be defined by macromolecular crowding in the nucleoplasm. In particular, chromosomal territories have been demonstrated to occupy, collectively, a considerable portion of the nuclear volume (
A complete characterization of the relationship between the size of the structure and its mobility characteristics will be presented elsewhere. At this point, we wished to simply address the issue as to whether structures in the size range of the buds derived from nuclear speckles are capable of rapid and random motion in the cell. To address this, cells expressing nuclear HDAC4GFP complexes of 500 nm diameter were imaged by time-lapse microscopy. Exposures were taken every second for 60 s. The results are shown in Fig 2 and are also presented in supplemental materials online as video 2 (available at http://www.jcb.org/cgi/content/full/150/1/41/DC1). Fig 2 shows an individual frame that demonstrates the presence of small spherical domains in a cell transiently expressing HDAC4GFP (top left panel). The top right panel shows the tracks followed by five individual domains over a 60-s time course. The middle panel illustrates the individual domains tracked. This is also illustrated in video 2. The largest distance traveled by HDAC4 in this cell was
1.0 µm in 1 s. This occurred in the right uppermost nuclear focus between frames 3 and 4 (video 2). Some foci move in and out of the plane of focus during the time course. We suspect that the observed movement reflects the probability of a collision, although the establishment of large compartments with physical barriers to movement of large structures cannot be ruled out. For comparison, an identical time course and process was carried out with a cell expressing ASFGFP (Fig 2, video 3). The motion observed for individual ASF foci is considerably more restricted over this very short time course than is observed for HDAC4 domains of similar size. This indicates that the slow movement of ASF foci in the longer time course shown in Fig 1 and video 1 does not represent spatial averaging of undersampled more rapid movement. In addition, the movement of ASF subdomains is distinct from the rapid motion potential realized by HDAC4 domains of similar size.
FRAP Analysis of ASFGFP Movement
We have confirmed the dynamic properties of nuclear speckles and splicing factorenriched structures that fuse or bleb from these domains. By using an HDAC4 construct with novel expression properties, we have clearly shown that the movement of the speckle domain buds is distinct from random motion. Although structures as large as speckles are physically limited in their abilities to migrate through the nucleoplasm (Hendzel, M.J., and D. MacDonald, manuscript in preparation), structures in the size range of the smaller splicing factorenriched domains (e.g., HDAC4 foci) are capable of rapid and random motion. The mobility of these structures, when compared with ASF, indicates that the smaller ASF-enriched domains are not moving freely through nuclear space and slowed only by their high molecular mass. Since it has been demonstrated that these splicing factor subdomains can be recruited to sites of transcription, and, hence, may function in the efficient processing of pre-mRNAs, it is important to understand the mobility properties of the pools of molecules associated with these structures.
To do this, we characterized the movement of the ASF protein, which is present both within and outside of nuclear speckles, using FRAP-based experiments. As a control, we characterized the fluorescence recovery rate of GFP in the absence of the ASF protein. The top two panels of Fig 3 show a photobleaching experiment performed on an Indian muntjac fibroblast cell expressing GFP. The first image was taken before photobleaching and the second image was taken within 1 s of the completion of photobleaching. Under conditions in which a photobleached region is easily generated in the ASFGFP expressing cell (bottom panels), the 3-µm photobleached strip through the cell was not evident in the GFP expressing cell. This is because the image scan time, 1 s, is large relative to the rapid mobility of the freely diffusing GFP protein. The partial equilibration of the GFP protein during scanning results in the observed decrease in fluorescence intensity throughout the entire cell. This is particularly evident in the nucleus and is consistent with the free mobility of the bulk of the pool of GFP within the cell. The bottom panels show the photobleaching of a 3-µm strip through an ASFGFP expressing cell. The photobleached region remains quite evident even at the 10-s time point. Complete recovery is not observed until at least 30 s. The immobile fraction of ASFGFP was calculated for both human intestinal smooth muscle and mouse 10T1/2 fibroblasts. There were no differences between cell types. The mean immobile population was calculated to be 6.7 ± 4.7%. Control experiments, in which the entire nuclear fluorescence was bleached, demonstrated that de novo synthesis, import of a cytoplasmic pool, or refolding of the GFP molecule was not a significant contributor to fluorescence recovery (data not shown).
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To quantitatively assess how much slower the ASF protein migrated through the cell nucleus compared with the freely mobile GFP protein, we performed experiments in which a 3-µm strip extending through the cell was scanned at rapid intervals. This enabled us to collect images every 50 ms. This was necessary because under conditions in which a clear bleach zone is observed with ASFGFP, the GFP protein infiltrates the photobleached region sufficiently fast that only a gradient of intensity is observed and recovery is nearly complete during the time required to acquire a single frame (1-s scan time). A plot of the recovery of the GFP protein is shown in Fig 4. The estimated t1/2 of recovery is <300 ms. Fig 4 also shows the same experiment performed on an ASFGFP expressing cell. The estimated t1/2 is
20 s. The mean recovery time of several cells from four different cell lines is summarized in Table 1. Differences were observed between cell lines in the mobilities of both GFP and ASFGFP and there was a significant amount of variability in the recovery rates between individual cells. This is reflected in the high standard deviation and may reflect variability in expression levels. Cells expressing very low levels of ASFGFP are difficult to image and so the data set was biased towards cells expressing intermediate or higher levels of ASFGFP. However, only cells expressing the normal speckled ASF distribution were imaged and, although there was a tendency for the highest expressing cells to recover faster than the lower expressing cells, this relationship was not absolute. Additionally, the level of expression could not be used to predict the amount of free ASFGFP. If we use the amount of fluorescence lost during the photobleach period from nucleoplasmic regions distant from the bleaching site as an estimate of the amount of free ASFGFP, fluorescence loss was measured at between 0 and 25.5% with a mean of 10.9% and a standard deviation of 8.4%. This value did not correlate with expression level of the transiently expressed protein. Out of the entire data set, only one cell showed a recovery time <10-fold slower than GFP. It is clear from these results that the majority of the ASFGFP population does not behave as a freely mobile molecule.
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Movement of Splicing Factors During Transcription or Kinase Inhibition
Transcriptional inhibition causes speckles to reorganize, becoming both larger and more spherical (5 min after bleaching, which was similar to results obtained with cells treated with staurosporine (Fig 5 D). We found that the ASFGFP moves into and out of speckles and between individual speckles with kinetics that are similar to untreated control cells expressing ASFGFP (Fig 5 B), but is considerably slower than GFP itself (Fig 5 A). These drugs do, however, affect the mobility of some nuclear proteins. Histone H1 mobility, for example, is dramatically reduced by treatment with either drug (Lever, M.A, J.P.H. Th'ng, X. Sun, and M.J. Hendzel, manuscript in preparation). Indicating that although the structural dynamics of speckles and the subnuclear distribution of ASF are dependent upon protein phosphorylation or ongoing RNA polymerase II transcription, the overall mobility of ASF is not.
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Quantitative Analysis of Fluorescence Recovery in Nuclear Speckles and the Nucleoplasm
Although we cannot resolve individual molecules of ASF, we can detect and measure their mean mobility. If ASF maintained high-affinity associations in speckles and the population of dispersed ASF molecules was freely diffusible, then we would expect a rapid uniform recovery of the nucleoplasm followed by the slower recovery of speckles. Instead, we observe a progressive front of GFP signal recovery that moves gradually toward the pole of the bleached half of the nucleus (Fig 5 B) with signal recovery in bleached speckles occurring concomitantly with signal recovery in the surrounding nucleoplasm. This indicates that the dispersed population of ASF molecules does not behave differently, with respect to mobility, than sites of enrichment.
We quantitatively analyzed recovery from nuclear speckles and nucleoplasmic regions by simultaneously bleaching two 1.5-µm spots aligned in the y axis of the image scan. Bleaching time was 200 ms. Because
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Second, after photobleaching half of the cell nucleus, we compared the rate of fluorescence draining by monitoring the relative intensities of speckles in an unbleached region of the nucleus. To do this, in unbleached regions of the nucleus, the ratio of the speckle maximum to the neighboring nucleoplasmic minimum was normalized to one and plotted over time. If a higher affinity binding site existed in nuclear speckles, we would expect an initial increase in the ratio of speckle signal to dispersed nucleoplasmic signal (ratio >1.0), reflecting the loss of the more mobile nucleoplasmic factor into bleached regions. We have observed this behavior with other GFP fusion proteins that are currently under investigation (unpublished observations). Fig 7 shows the ratio of speckles relative to adjacent nucleoplasmic regions in an unbleached region during recovery. Consistent with the FRAP results, this experiment shows that the relative intensity remains at 1.0, indicating that the change in speckle intensity equals that measured for the nucleoplasm. Thus, even though the spatial organization of ASF is not uniform, the mobility of ASF is not significantly different between the speckle compartment and the nucleoplasm. Both of these experiments support the conclusion that the association between ASF and binding sites in nuclear speckles and the nucleoplasm is qualitatively similar.
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Connectivity between Pools of ASF Associated with Immobile Binding Sites in Speckles and the Nucleoplasm
FLIP experiments can be used to demonstrate the connectivity between compartments. Our observations of FRAP recovery, particularly those where the large half-nucleus regions are bleached, suggest that the nucleoplasmic and speckle compartments constantly exchange molecules with each other. This was confirmed by FLIP analysis. A region of the cell nucleus was cyclically bleached and allowed to recover until the fluorescence intensity of the cell nucleus was near the detection limit in the experiment. If a significant population of molecules remained bound in nuclear speckles, we would expect that it would not be possible to drain the fluorescence within these regions upon repeated photobleaching of a nucleoplasmic region. Fig 8 shows that repeated photobleaching of a region within the nucleus results in overall loss of nuclear fluorescence. Thus, the ASF molecules, regardless of their nuclear location during the initial line scan, are in constant flux and move throughout the entire nucleus.
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Discussion |
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ASF-enriched Domains Do Not Behave as Structures that Are Free within the Nucleoplasm
The observation that ASF-enriched domains are able to move in a directed manner from nuclear speckles to sites of transcription (
The Movement of the ASF-splicing Factor through Nucleoplasmic Space
An implication of the mobility properties of speckles and smaller domains enriched in splicing factors is that splicing factor availability is tightly controlled in a spatiotemporal fashion. This could be mediated by association with putative architectural elements of the nucleus and tracking along this karyoskeleton as large multicopy domains. It has also been reported that there are significant quantities of splicing factors that are dispersed throughout the nucleoplasm (
In this study, we demonstrate that in the nucleoplasm splicing factors display a similar overall mobility to the speckle-associated ASF. It is possible that higher resolution techniques, such as fluorescence correlation spectroscopy, will reveal multiple populations that produce an average mobility that is reflected in our experiments. Certainly, over very short distances, this is probably the case. Interestingly, in one cell line, HISM, staining of the fibrillar centers or the dense fibrillar components of the nucleolus was occasionally observed. When cells containing this population were examined and the nucleoplasmic or speckle recovery was compared with nucleolar recovery, the nucleolar population recovered approximately twice as slowly as the extranucleolar populations. Because we suspected that nucleolar association was an artefact of overexpression, such cells were not included in our primary FRAP analysis. However, this observation, and the results of
An important observation in this study is that the movement of the population of ASF molecules occurs much more quickly than the time required for ASF-enriched structures, or speckles themselves, to move through nuclear space. This is evident in the comparison of rates of movement of structures in the 3-D time-lapse experiments versus the movement of molecules in the FRAP experiments. It is further evidenced in experiments where cells have been treated with transcription or kinase inhibitors. Under these conditions, the translocation of ASF-enriched structures from speckles is minimized (
Although splicing factors are capable of movement through the nucleus independently of the shuttling of factor-rich structures, this mobility is not representative of a freely mobile state. We observe both the dispersed and speckle-associated ASF to move at a rate significantly slower than other proteins that exhibit free mobility, such as HDAC4GFP (unpublished observations) and GFP itself. Essentially identical mobility results have recently been reported by 1% of that of a freely mobile protein in the nucleoplasm and slower than the measured mobility of diffusing poly(A) RNA (
The concept of a large pool of molecules or complexes that move freely through the cell nucleus until they associate with their substrate does not accurately reflect the behavior of splicing factors. Instead, our results are consistent with the nucleus containing a large number of relatively immobile sites capable of binding ASF, thereby significantly reducing its overall mobility compared with freely mobile proteins. The reduced mobility of ASF, even under conditions where newly synthesized transcripts are blocked from forming, indicates that RNA contributes only a portion of the total nucleoplasmic binding sites. The significance of nascent RNA in retarding the mobility of ASF is difficult to assess, however, since transcriptional inhibition results in a redistribution of nuclear ASF molecules. Before transcriptional inhibition, HeLa speckles were enriched 1.8-fold over nucleoplasmic regions. After transcriptional inhibition, speckles were enriched 2.5-fold over nucleoplasmic regions. Thus, it may simply be that the increased mobility of ASF in the nucleoplasm reflects a redistribution of nonRNA-binding sites from the nucleoplasm to the speckles rather than the straightforward explanation that it is a direct consequence of the absence of cotranscriptional binding sites for ASF within the nucleoplasm.
We propose that a transient association of ASF with these putative immobile acceptor sites serves to retard the translational mobility of splicing factors within the cell nucleus and establishes a dynamic equilibrium between mobile and immobile populations (see
A Model for Regulating the Distribution of Splicing Factors in the Cell Nucleus
We propose that heterogeneity in splicing factor concentration throughout the nuclear volume reflects differences in the concentration of immobile karyoskeleton-associated binding sites. These putative binding sites are present in lower abundance within the nucleoplasm and enriched in distinct morphological components of the nucleus, such as speckles. The cell nucleus is able to dynamically reconfigure the karyoskeleton, or the organization of binding sites within the karyoskeleton, to reposition domains. This reorganization occurs by blebbing, fusing, and translocating assemblies that contain high concentrations of these sites. This allows the cell to respond to changing needs for transcription-processing machinery in the vicinity of transcriptionally active genes. As suggested by
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviations used in this paper: 2-, 3-, and 4-D, two-, three-, and four-dimensional; ASF, alternative splicing factor; DIC, differential interference contrast; FLIP, fluorescence loss in photobleaching; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent fusion protein; HDAC4, histone deacetylase 4.
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Acknowledgements |
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We wish to thank Dr. Judith Sleeman (University of Dundee) for kindly providing the ASFGFP expression vector. We also thank Dr. Tom Misteli (National Cancer Institute) for communicating results before publication and providing valuable suggestions. Maryse Fillion provided excellent technical assistance and Dr. X. Sun (Cross Cancer Institute Cell Imaging Facility) is thanked for suggestions on image collection and analysis. We wish to acknowledge the Cell Imaging Facility at the Cross Cancer Institute for the use of microscopes and analysis software.
M.J. Kruhlak was supported by a studentship award from University Technologies International (University of Calgary); Wolfgang Fischle was supported by a studentship award from the Boehringer Ingelheim Foundation, Germany. Research funding was provided by operating grants from the Medical Research Council of Canada to M.J. Hendzel and to D.P. Bazett-Jones.
Submitted: 16 November 1999
Revised: 17 May 2000
Accepted: 17 May 2000
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References |
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