1 Gastrointestinal Cell Biology Research, Division of Gastroenterology and Nutrition, Harvard Digestive Disease Center, Children's Hospital, Harvard Medical School, Boston 02115; and 2 Department of Immunology, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
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An
important feature of enterocyte maturation is the asymmetrical
distribution of cellular functions including protein localization. mRNA
sorting is one mechanism for establishment and maintenance of this
process in other systems, and we have previously demonstrated differential localization of mRNAs in human enterocytes. To study regulation of mRNA sorting, we established a model in polarized Caco-2
cells. Proxy cDNA constructs containing -galactosidase (
-gal)/green fluorescence protein (GFP) and the 3'-untranslated region (3'-UTR) of either human sucrase-isomaltase or villin were transfected transiently or stably. A control construct contained poly-A
sequence in place of 3'-UTR. Expression of GFP was observed by confocal
microscopy; intracellular location of the construct mRNA was imaged by
in situ hybridization. The sucrase-isomaltase mRNA proxy localized to
an apical position in Caco-2 cells as in native enterocytes; the villin
mRNA proxy did not show significant localization. The control construct
was not localized and was found diffusely throughout the cell. Proxy
GFP proteins tended to localize with their mRNA proxies, but with less
precision. This study establishes a valuable model for the
investigation of mRNA localization in intestinal epithelial cells.
Mechanisms controlling asymmetrical distribution of intestinal mRNAs
can be now be elucidated.
enterocyte; Caco-2 cells; sucrase-isomaltase; villin
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INTRODUCTION |
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AN IMPORTANT FEATURE OF ENTEROCYTE maturation is the differentiation of cell structure and function and the asymmetrical distribution of cellular components. While mechanisms that control this process are poorly understood, it is known that mRNA sorting is one mechanism for establishment and maintenance of cell polarity. mRNA sorting was first described in 1983 (11) in oocytes in which asymmetrical distribution of maternal mRNA was thought to contribute to the divergent differentiation of daughter cells that occurs during embryogenesis. Subsequently, mRNA sorting has been described in a variety of polarized cell types, including fibroblasts (7, 12), oligodendrocytes (6), and intestinal epithelial cells (20). Localization of different mRNAs to various intracellular domains helps to maintain mRNA and protein gradients that contribute to cell shape and differentiation. Disruption of mRNA sorting leads to an altered cellular phenotype in fibroblasts (13), implying an important physiological role for this process.
Investigations in other cell types, such as oocytes and fibroblasts, have revealed that the sequences that drive mRNA sorting are predominantly located within the 3'-untranslated region (3'-UTR) of the mRNA. Complexes formed of cellular proteins, mRNA, and ribosomes are translocated along the cytoskeleton and anchored at the final destination of the mRNA in the cell (13).
We (2, 15, 16, 20) previously demonstrated specific mRNA localization in intact adult intestinal epithelial cells and suggested that localization of mRNAs in enterocytes might function as a mechanism for protein sorting and maintenance of cellular polarity. The study of this process has been hampered by the lack of an appropriate model. Because small intestinal epithelial cells cannot be maintained more than a few days in primary culture, enterocyte-like cell lines have become the standard substitute. Of the few available, only Caco-2 cells demonstrate a polarized phenotype that includes a well-developed cytoskeleton, transcellular transport, and apical expression of marker proteins, such as sucrase-isomaltase (SI), typical of native enterocytes (8-10, 19, 23).
Very little is known about the mechanisms that drive mRNA sorting within the enterocyte, and advances in this field require a reliable in vitro model system that will reproduce observations in intact intestine. The model must also permit transfection of proxy mRNA constructs, mutational analysis, and mapping of sequences responsible for mRNA sorting in cultured polarized cells. Therefore, the goal of the present study was to develop a reliable in vitro model system for investigating mRNA sorting in intestinal cells.
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MATERIALS AND METHODS |
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Vectors.
Fusion reporter genes were synthesized to contain -galactosidase
(
-gal) and enhanced green fluorescence protein (GFP) linked upstream
to the 3'-UTR of the mRNA of interest and were cloned into vector
pCA13 (Microbix, Toronto, Ontario, Canada), under the control
of the cytomegalovirus promoter (PCMV). A polyadenylation signal (PAS) was inserted downstream to ensure mRNA
stability. Results were confirmed by vector sequencing (Tufts University Sequencing Core Facility, Department of Physiology, Tufts
University School of Medicine, Boston, MA). Three expression vectors
were constructed: for SI mRNA,
PCMV/
-gal/GFP/SI-3'-UTR/PAS/pCA13; for villin mRNA,
PCMV/
-gal/GFP/villin-3'-UTR/PAS/pCA13; and for the
control, PCMV/
-gal/GFP/PAS/pCA13.
-Gal is a large
protein that in short-term culture remains at the site to which its
mRNA is targeted in fibroblasts (13). The linked GFP
provided a fluorescent tag that could be analyzed by confocal
microscopy. We further hypothesized that the addition of
-gal to GFP
as a large fusion protein might enhance its capacity as a proxy marker
for the position of its mRNA.
Transient transfections. Caco-2 cells were chosen due to their capacity to express a phenotype similar to differentiated absorptive enterocytes (8-10, 19, 23). Preconfluent cells were transfected by using electroporation (14) with 10 µg of expression plasmid (40 µg total DNA) and plated at 100% confluence on fibrillar-collagen cell culture inserts (Becton-Dickinson, Bedford, MA) in DMEM supplemented with 10% FCS (GIBCO-BRL, Rockville, MD). Cells were maintained at 37°C under 5% CO2 with 100% humidity. Media were changed three times per week during the indicated culture periods.
At the indicated times after transfection, cells were washed twice with PBS and then fixed and permeabilized in a 40:40:20 solution of acetone/methanol/water for ~15 s. After cells were washed twice again with PBS, orientation within the cell was elucidated by staining nuclei with 2 µg/ml propidium iodide (Sigma, St. Louis, MO) and staining the microvillus membrane of the cells with either 0.33 µM rhodamine-conjugated or Texas red-conjugated phalloidin (Molecular Probes, Eugene, OR) for 1 h at room temperature. Cells were then rinsed with PBS, mounted on glass slides with SlowFade Light mounting medium (Molecular Probes), coverslipped, and sealed with nail polish.Stable transfections. The SI, villin, and control inserts described above were cut out of the pCA13 vectors and cloned into the backbone vector pEGFP-N1 (containing a neomycin-resistance cassette; Clontech, Palo Alto, CA) in place of the GFP coding sequence at the SalI and BamHI sites.
Preconfluent Caco-2 cells were transfected with 2 µg plasmid by using Effectene (Qiagen, Valencia, CA) and maintained in DMEM/10% FBS + 500 µg/ml G418 for ~2 wk until untransfected controls were dead. Cells were then trypsinized and selected by fluorescence-activated cell sorting for high levels of GFP expression. One thousand cells per line were collected and replated in DMEM/10% FBS + 500 µg/ml G418. Cells were split on reaching confluence and replated three times per week thereafter, with continuing maintenance in 500 µg/ml of G418.In situ hybridization. The mRNAs for the marker proteins were detected by using in situ hybridization. Antisense oligonucleotides (30 nt) to the 3'-UTR of either SI or villin were synthesized and end labeled with a Cy-5 fluor (Tufts University Sequencing Core Facility). Probe sequences were as follows: antisense probes for SI, 5'-GGGCTATTCAAATTTTGTTAAATATGCCTT-3' and 5'-AATAACTTTTCGATGTTATGAA- AGCTATAT-3'; sense probe for SI, 5'-AAGGCATATTTAACAAAATTTGAATAGCCC-3'; antisense probes for villin, 5'-GGTGAGAAAATGAGACCCTACAATCAGGGT-3' and 5'-AAAACTGGCATTTGCCACAGAAGTTTGTGC; sense probe for villin, 5'-GCACAAACTTCTGTGGCAAATGCCAGTTTT-3'.
Cells were hybridized by using a variation of the method developed by R. Singer (http://singerlab.aecom.yu.edu/protocols). After fixation and permeabilization as described above for transient transfections, cells were washed twice with 2× SSC and then incubated at room temperature in 2× SSC/40% formamide for 5 min. The insert membranes were then cut in half with RNase-free scissors, and each half was then hybridized overnight at 42°C in 100 µl of hybridization solution containing 2× SSC, 40% formamide (Fisher Scientific, Fair Lawn, NJ), 10 µg/µl tRNA (Sigma), 0.02% RNase-free BSA (Sigma), 1% RNasin (Promega, Madison, WI), and 30 µg of labeled oligo probe. If multiple probes against different sequences were used, equal amounts of each were used to reach the microgram total amount. Inclusion of dextran sulfate in the hybridization buffer created large amounts of background as the dextran sulfate appeared to absorb the fluorescent stains and furthermore interfered with equilibration of the SlowFade mounting medium, resulting in rapid quenching of the signal under the laser. Removal of the dextran sulfate from the hybridization buffer recipe and incubating the membranes in a double-humidified chamber to guard against evaporation in the absence of the dextran sulfate yielded satisfactory images. Interestingly, the chosen sense probes demonstrated an intermittent level of nonspecific organelle binding that made them unacceptable for this purpose. Because we were previously unable to detect native expression of the villin and SI mRNAs in the Caco-2 cells, the probe for SI was used as a negative control for the transfected villin construct and vice versa. On day 2, membranes were rinsed briefly with 2× SSC/40% formamide and then washed as follows: 2× SSC/20% formamide, 15 min, room temperature; 2× SSC/10% formamide, 15 min, room temperature; 2× SSC, 15 min, room temperature; 1× SSC, 15 min, 42°C. Cells were then counterstained and coverslipped as described above for transient transfections.Imaging. Visualization of the marker GFP after transient transfections was accomplished by using a Bio-Rad Zeiss fluorescent laser scanning confocal microscope. To determine the intracellular location of the GFP marker proteins, monolayers were selected at random when their apical and basal positions were clearly defined, a signal was present, and the control at the same intensity and contrast settings did not display a detectable signal (n = 85). Once the top and bottom positions of the monolayer were established, the monolayer was optically sectioned into 0.5-µm slices on an x-z plane by using the appropriate laser channel for each stain and merging all channels to create the final image. These z-section monolayer images were scored on a blinded basis by two of the investigators for apical, perinuclear, or basal location of the GFP protein in each cell visualized in the selected monolayer. After the scoring was performed, results were correlated with the transfected proxy construct and expressed as the percentage of specifically localized GFP in each region. Statistical analysis was performed by using a two-tailed ANOVA (GraphPad Instat Software, San Diego, CA), and significance was assigned as P < 0.05.
For detection of GFP in stable transfection experiments, horizontal fluorescent images were obtained every 0.5 or 1.0 µm on an Odyssey XL laser scanning confocal microscope (Noran), and the images were recorded with Noran Intervision 2D/3D Image Analysis software. Intracellular location of the GFP was established by determining the apical and basal membranes of randomly selected single cells (n = 33) containing observed GFP and recording the 0.5-µm intervals at which the GFP was visualized. Control monolayers were imaged by using the same contrast and intensity settings to verify that no detectable signal was present. The data were expressed as the percentage of GFP localized in the intracellular compartment of interest. Statistical analysis was conducted by using two-tailed ANOVA (GraphPad Instat Software), and significance was assigned at P < 0.05. For in situ hybridization experiments, transfected construct mRNA was detected by confocal microscopy on the Bio-Rad Zeiss laser scanning confocal microscope as described above. Laser intensity and contrast levels were set by using the negative controls (as described above) to establish background. The image was kept if it met the following selection criteria: a monolayer of intact nuclei, visible microvillus membrane staining, and visible probe. The Bio-Rad .pic file format was converted to a .tif format, and 47 individual cells that displayed a visible probe were digitally cut out of the image by using PaintShop Pro 7 (Jasc Software, Eden Prairie, MN) and pasted into an unlabeled collage. The intracellular position of probe within each cell in the collage was then assessed by two separate investigators on a blinded basis and scored as follows: 0 = no sorting; 1 = mostly apical; 2 = apical/perinuclear; 3 = mostly perinuclear; 4 = perinuclear/basal; 5 = mostly basal.Statistical analysis. Statistical significance was determined by using a coded, nonparametric contingency table, with the null hypothesis assigned as equal distribution into each intracellular region (GraphPad Instat Software). Significance was defined as P < 0.05.
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RESULTS |
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Morphology of monolayer.
Under the conditions used in these experiments, we initially
established that Caco-2 cells grown on plastic were not sufficiently or
reproducibly polarized to allow assessment of mRNA sorting (data not
shown). In contrast, cells grown on fibrillar-collagen inserts
demonstrated a polarized appearance including a well-developed microvillus membrane at approximately day 3 postconfluence,
as shown also by others (8-10, 19, 23). This
morphology persisted at all subsequent time points studied (Fig.
1). When grown on other substrates,
collagen I, fibronectin, or laminin, the Caco-2 cells demonstrated
distinctly sparser microvilli and a shorter cell height, resulting in a
squat, almost squamous appearance, compared with cells grown on the
fibrillar-collagen matrix at the same time points. Accordingly, all
subsequent experiments were conducted with the
fibrillar-collagen-coated inserts.
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Transient transfections. It had been shown previously that SI mRNA expression levels in Caco-2 cells peak at approximately day 10 postconfluence, plateau for ~5 days, and then decline thereafter (5, 23). Assuming that the subcellular machinery for sorting mRNAs would be operational at the time of maximum expression, we initially chose 10 days postconfluence as the time point for examining the sorting of SI and villin mRNAs. However, once the Caco-2 cells became differentiated, they proved entirely refractory to transfection by either electroporation or lipid techniques; therefore, it was necessary first to transfect preconfluent cells and then to culture them to the appropriate level of differentiation. By using this approach, optimal expression of GFP after transient transfection by either electroporation (10 µg) or Effectene (2 µg; Qiagen) occurred between 3 and 8 days postconfluence, with some fluorescence remaining after 14 days (data not shown). Because electroporation yielded a greater percentage of cells transfected, this method was chosen as the vehicle of choice for all further transient tranfection experiments. Interestingly, in contrast to the ease of detection of transfected mRNA proxies, the abundance of native SI and villin mRNA per cell was lower than the detection limit by using in situ hybridization (data not shown).
Localization of the GFP proxy within the cell was determined by optical sectioning of the cells in 0.5-µm slices from the apical membrane through the basal surface and then scoring masked images to establish the intracellular location of the GFP. The data are shown in Fig. 2. Most of the SI proxy protein (GFP) appeared to sort to the apical portion of the cell in concordance with the in vivo expression (n = 32) (2). The villin proxy protein, on the other hand, appeared predominantly perinuclear and basal (n = 30). Due to variability in the intracellular localization of the GFP, there was no statistically significant difference in the intracellular location of any of the construct proteins. The control construct protein was found evenly distributed within the cells (n = 23).
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Stable transfections.
In an effort to control for the variation in location of GFP, and to
attempt to increase expression, stably transfected Caco-2 cell lines
were created for the SI and villin constructs. Cells were imaged as
described above from days 3 through 7 postconfluence. Images obtained by using cells at 3 days
postconfluence (n = 33 cells) are shown in Fig.
3. These data demonstrate that the
predominant sorting of the SI mRNA construct tended to be apical
and perinuclear and that the predominant localization of the villin
proxy mRNA tended to be perinuclear and basal. However, these
differences were not statistically significant. No significant
differences were noted in GFP sorting when additional time points were
compared.
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In situ hybridization.
Because of the variability in the localization of GFP seen in Figs. 2
and 3, it was possible that the GFP/-gal fusion protein might not
anchor at the final destination of the mRNA as reported by others
(13). Accordingly, detection of transfected construct mRNA
was accomplished by using transient transfection techniques followed by
in situ hybridization to localize the proxy mRNAs within single cells
on a reconstructed x-z axis.
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DISCUSSION |
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This study demonstrates that Caco-2 cells in culture achieve and maintain sufficient polarity to allow visualization of intracellular events, including differential sorting of transfected mRNAs to specific intracellular locations. The value of this cell line as an in vitro model for native enterocytes has been well established (8-10, 19, 23). Furthermore, the model system described in this study displays distinct sorting patterns for SI and villin mRNA proxy constructs. This provides a means for investigating the control of mRNA sorting and its potential role in development and in the maintenance of intestinal epithelial cell polarity.
Investigations in other cell types have identified similarities in some of the mechanisms of mRNA sorting. Trans-acting, sequence-specific regions in the 3'-UTR of sorted mRNAs bind specific proteins that then complex with motor proteins to form a translocation particle. These particles move along the cytoskeletal apparatus until they reach their final intracellular destination, in which they anchor by an as yet incompletely understood process (17). The cytoskeletal components involved may be either microtubules, microfilaments, or both at alternate points in the sorting process (4). The differential sorting displayed in the current experiments by the SI and villin proxy constructs will facilitate a delineation of the mechanisms responsible for localization of these mRNAs in intestinal epithelial cells.
In the present experiments, the SI proxy mRNA localized apically within
the cell, mimicking the pattern of SI mRNA localization seen in native
human enterocytes (2, 15). The detection of the 3'-UTR
sequence apically in the cells may indicate that the mRNA was anchored
at its site of function. The apical localization of this mRNA
presumably facilitates translation as well as the insertion of the
mature SI enzyme into the apical membrane. It is important to note that
the SI construct mRNA was more distinctly localized than that of the SI
proxy protein GFP. This was most likely due to intracellular diffusion
of the GFP synthesized during posttransfection culture of the Caco-2
cells. Accordingly, GFP cannot be used as a marker for SI localization
in this model, unlike the findings of Kislauskis et al.
(12) in fibroblasts in which -gal could be used as a
proxy marker for
-actin mRNA in short-term culture. Having
established a reliable model system, we have now begun mutational
analysis and mapping of the SI 3'-UTR to determine the specific
sequences necessary for this localization.
In contrast to native enterocytes, in which villin mRNA is predominantly localized basally (15), in Caco-2 cells the villin proxy mRNA is localized less precisely. There are two possible explanations for this observation. First, there might be altered sorting mechanisms in Caco-2 cells in culture compared with enterocytes in vivo. Bacchi and Gown (1) found that, although villin protein was usually expressed apically in colonic adenocarcinomas, cytoplasmic and basolateral mislocalization was also identified. Because Caco-2 cells derive originally from colon carcinoma, this might contribute to a sorting pattern for the villin mRNA that is distinct from that seen in vivo in small intestine (15). Alternatively, a second possible explanation is that villin may be one of the rare genes whose sequences that govern sorting and/or anchoring of mRNA at the intracellular destination do not lie completely within the 3'-UTR (18). Experiments are currently underway to clone other portions of the mRNA into the villin proxy construct to determine whether this is the case.
An important component of the mRNA sorting system is the
trans-acting mRNA binding protein(s) that mediate(s)
attachment of the mRNA to the motor assembly (18). These
proteins have been identified and cloned for several mRNA species with
the use of electrophoretic mobility shift assays. Investigations in
other genes have shown that the sequences that govern sorting are often multipartite (13) and widely spaced within the 3'-UTR,
implying that folding and secondary structure of the mRNA is critical
in protein binding to these sequences. Further support for this theory comes from the proteins involved in sorting -actin in fibroblasts and Veg1 mRNA in Xenopus oocytes (21, 22).
These proteins display a significant homology to one another but bind
to different primary sequences in their respective mRNAs. However,
analysis of these mRNAs by computational folding programs predicts a
similar stem-loop structure within each of the mRNAs at the sequences shown to be necessary for their localization (3). As a
consequence of the present study, it will now be possible to establish
which proteins and elements of the cytoskeleton (microtubules and/or microfilaments) are involved in the sorting mechanisms for these intestinal mRNAs and to elucidate the predicted protein intermediates (likely, the cytoskeletal motor proteins), which may also mediate the
translocation of these mRNAs.
By extending the current findings, it may also be possible to identify the role of mRNA sorting in the establishment and maintenance of intestinal epithelial cell polarity during differentiation and in diseases that alter enterocyte shape, such as gluten-sensitive enteropathy. The model system we report here is a crucial first step in investigating this important process.
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ACKNOWLEDGEMENTS |
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The authors thank Drs. Stephen D. Krasinski and Marian R. Neutra for constructive discussions; Lauren Dowling for technical support; Drs. Ira Herman and Robert Wilson, Douglas Jefferson, and Anne Kane, GRASP Digestive Disease Center (Tufts University, Boston) for imaging and cell culture support and plasmid preparations, respectively; and Dr. Susan Hagen and Daniel Brown, Harvard Digestive Disease Center, for confocal microscopy support. Sequencing of molecular probes was performed in the Sequencing Core of the Department of Physiology, Tufts University School of Medicine (Michael Berne, Director).
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FOOTNOTES |
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This study was supported by National National Institute of Diabetes and Digestive and Kidney Diseases Grants R37-DK-32658, P30-DK-34928, P30-DK-34854, and T32-DK-07471.
Address for reprint requests and other correspondence: R. J. Grand, Division of Gastroenterology and Nutrition, Children's Hospital, Harvard Medical School, 300 Longwood Ave., Boston, MA 02115 (E-mail: richard.grand{at}tch.harvard.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 18, 2002;10.1152/ajpgi.00458.2002
Received 25 October 2002; accepted in final form 2 December 2002.
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