ARTICLE |
Correspondence to: Ivan R. Nabi, Département dAnatomie, Université de Montréal, Pavillon principal, R-816, 2900 Edouard Montpetit, Montréal, Québec, Canada H3T 1J4.
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Summary |
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Autocrine motility factor receptor (AMF-R) is localized to an intracellular microtubule-associated membranous organelle, the AMF-R tubule. In well-spread untransformed MDCK epithelial cells, the microtubules originate from a broad perinuclear region and AMF-R tubules extend throughout the cytoplasm of the cells. In Moloney sarcoma virus (mos)-transformed MDCK (MSV-MDCK) cells, microtubules accumulate around the centrosome, forming a microtubule domain rich in stabilized detyrosinated microtubules. AMF-R tubules are quantitatively associated with this pericentriolar microtubule domain and the rough endoplasmic reticulum and lysosomes also co-distribute with the pericentriolar mass of microtubules. The Golgi apparatus is closely associated with the microtubule organizing center (MTOC) within the juxtanuclear mass of AMF-R tubules, and no co-localization of AMF-R tubules with the Golgi marker ß-COP could be detected by confocal microscopy. After nocodazole treatment and washout, microtubule nucleation occurs exclusively at the centrosome of MSV-MDCK cells, and only after microtubule extension to the cell periphery does the microtubule cytoskeleton reorganize to generate the pericentriolar microtubule domain after 30-60 min. AMF-R tubules dispersed by nocodazole treatment concentrate in the pericentriolar region in parallel with the reorganization of the microtubule cytoskeleton. MSV transformation of epithelial MDCK cells results in the stabilization of a pericentriolar microtubule domain responsible for the concentration and polarized distribution of AMF-R tubules. (J Histochem Cytochem 45:1351-1363, 1997)
Key Words: autocrine motility factor receptor, epithelial transformation, microtubule cytoskeleton, membrane tubule, Madin-Darby canine kidney
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Introduction |
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The polarization of the microtubule cytoskeleton, combined with directional microtubule-associated motors, regulates the cytoplasmic distribution and interactions of the membranous organelles of the cell (
AMF-R mediates transduction of the AMF motility signal via a pertussis toxin-sensitive G-protein, inositol phosphate production, protein kinase C activation, and production of the lipoxygenase metabolite 12-HETE (
The microtubule cytoskeleton of fibroblasts radiates from the centrally located microtubule organizing center (MTOC) to the cell periphery. The minus ends of the microtubules are associated with the centrosome, the site of microtubule nucleation, and the plus (growing) ends of the microtubules are located at the periphery of the cell. In polarized epithelial cells, microtubules are oriented vertically and extend from the apical (minus end) pole to the basal (plus end) pole of the cells (
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Materials and Methods |
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Cells and Antibodies
MSV-MDCK (DoCl1 cells; from ATCC, Rockville, MD) and MDCK II cells were grown in Dulbecco's minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), glutamine, nonessential amino acids, vitamins, penicillin, and streptomycin (Gibco; Burlington, Ontario, Canada). Cells were cultured at 37C in a humidified atmosphere with 5% CO2.
Monoclonal antibodies against AMF-R (-tubulin was from ICN (Mississauga, Ontario, Canada). Secondary antibodies conjugated to either fluorescein or Texas Red were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). The fluorescent antibodies were designated for use in multiple labeling studies and no interspecies crossreactivity was detected. To detect anti-AMF-R, secondary antibodies specific for the µ-chain of rat IgM were used. Except where otherwise indicated, all chemicals were purchased from ICN.
Immunofluorescence
Intracellular immunofluorescent labeling was essentially as previously described (
Confocal Microscopy
Confocal microscopy was performed with the x60 Nikon Plan Apochromat objective of a dual-channel BioRad 600 laser scanning confocal microscope equipped with a krypton/argon laser and the corresponding dichroic reflectors to distinguish fluorescein and Texas Red labeling. To generate a composite image of the complete cell depth for cells double labeled for AMF-R (Texas Red) and tubulin (fluorescein), Z-series of optical sections collected at 1-µm steps encompassing the major portion of the immunofluorescent label were projected using BioRad COMOS software, in which the most intense value for each pixel is presented. Merging of the fluorescein and Texas Red channels generated the green-red double labeling of AMF-R and tubulin. Confocal images were printed using a Polaroid TX 1500 video printer.
The degree of concentration of microtubule and AMF-R labeling within the pericentriolar region was quantified from images collected in one session from MSV-MDCK and MDCK cells double immunofluorescently labeled in parallel for -tubulin and AMF-R. Cells were imaged under conditions of equivalent pinhole and black level settings, and the gain was adjusted for each image to ensure that the pixel values in each section were not maximal and therefore not saturating. Z-series of optical sections at 1-µm steps encompassing the majority of the immunofluorescently labeled portion of the cell (from two or three sections) were projected using the Comos summation function, in which intensity values for each pixel are averaged over the selected sections. The region of intense juxtanuclear microtubule labeling (about 10-20% of the total cell area) was circumscribed, as was the equivalent juxtanuclear region in the AMF-R image, and average pixel intensity values for the circumscribed region and the total cell area obtained. To discount the contribution of the nonspecific nuclear AMF-R labeling (
Nocodazole Washout
MSV-MDCK cells were treated with 20 µM nocodazole in culture medium for 30 min at 37C, rinsed five times with warm DMEM, and then reincubated with prewarmed culture medium for the indicated periods of time at 37C. To visualize microtubule nucleation after nocodazole treatment, cells were fixed immediately after the five DMEM washes.
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Results |
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Juxtanuclear Concentration of AMF-R Tubules in MSV-MDCK Cells
The relationship between AMF-R tubule distribution and the microtubule cytoskeleton in MDCK and MSV-MDCK cells was assessed by double immunofluorescent microscopy for AMF-R (the red signal) and tubulin (the green signal) (Figure 1). The microtubules of MDCK cells have been previously described to extend to the cell periphery from a broad perinuclear region (
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Quantification of the juxtanuclear AMF-R tubule distribution in MSV-MDCK cells was performed on composite images representing the average pixel intensity of confocal optical sections encompassing the complete cell height. Average pixel intensity of both tubulin and AMF-R labeling was determined in the juxtanuclear region defined by increased microtubule labeling, which ranged from 10 to 20% of the total cell area in both MDCK and MSV-MDCK cells, and in the complete cell. These values generate an indicator of the proportion of AMF-R labeling located in the juxtanuclear region of the cell relative to the distribution of tubulin (see Materials and Methods). Interpretation of data obtained by this semiquantitative approach must be performed with caution, and conclusions as to the differential microtubule concentration in the juxtanuclear region of MDCK or MSV-MDCK cells cannot be inferred. The distribution of microtubules in the two cell types does, however, serve as an internal control, and the proportion of AMF-R labeling in this juxtanuclear region, relative to that of tubulin, was significantly increased in MSV-MDCK cells relative to MDCK cells (Figure 1C). These results clearly demonstrate an increased density of AMF-R tubules associated with the juxtanuclear microtubule dense region of MSV-MDCK cells compared to MDCK cells.
To assess whether the formation of the pericentriolar microtubule domain is associated with microtubule stabilization, we labeled MDCK and MSV-MDCK cells with antibodies to detyrosinated tubulin (-tubulin (Figure 2C and Figure 2D). The pericentriolar microtubule domain of MSV-MDCK cells therefore contains a distinct population of microtubules, including an increased number of stable microtubules. Detyrosinated microtubules co-distribute with AMF-R tubules in the same pericentriolar region (Figure 2E and Figure 2F). We were unable to identify co-alignment between AMF-R tubules and detyrosinated microtubules in the cell periphery.
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Optical sectioning by confocal microscopy of MSV-MDCK cells double immunofluorescently labeled for AMF-R and tubulin revealed the presence of a distinct focal MTOC within the juxtanuclear tubulin label of MSV-MDCK cells (Figure 3). Sections near the substrate revealed the extension of microtubules to the cell periphery from a central site adjacent to the cell nucleus (Figure 3B). Sections at a further distance of 0.8 µm from the substrate clearly show the site of origin of the microtubule fibers or the MTOC (Figure 3D). Confocal sections permit the visualization of distinct AMF-R labeled structures within the dense juxtanuclear AMF-R label which exhibit neither the linear elongation nor the peripheral orientation of AMF-R tubules located in the cell periphery (Figure 3A and Figure 3C). In sections close to the substrate, AMF-R tubules are also visualized in peripheral regions of the cell, where their orientation towards the cell periphery is similar to that of the microtubules (Figure 3A and Figure 3B). At increasing distances from the substrate, the AMF-R label is restricted to a region around the focal MTOC (Figure 3C). The dense juxtanuclear AMF-R labeling in MSV-MDCK cells (Figure 1B) is therefore a three-dimensional accumulation of AMF-R-labeled tubules and vesicles that co-distributes with microtubules to one side of the nucleus around the site of a focal MTOC in MSV-MDCK cells.
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The Pericentriolar Microtubule Domain and the Distribution of Other Membrane Organelles
The localization of the Golgi apparatus at the MTOC is microtubule-dependent (
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Double immunofluorescent labeling of MSV-MDCK labels with antibodies to LAMP-2, a marker for lysosomes and late endosomes (
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Concomitant Reorganization of the Microtubule Cytoskeleton and Redistribution of AMF-R Tubules to the Pericentriolar Region
Microtubules of MDCK cells are nucleated at acentriolar sites in spreading isolated cells (
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Treatment of MSV-MDCK cells with nocodazole and loss of microtubule integrity resulted in the dispersion of AMF-R-labeled tubules throughout the cell (Figure 7A and Figure 7B). Similar to the effect of nocodazole disruption on MDCK cells (
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Discussion |
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Epithelial to mesenchymal cell transitions occur during normal development but also following epithelial cell transformation to generate invasive, motile carcinoma cells (
Microtubules in epithelial cells do not radiate from a central MTOC, as in motile fibroblasts, and microtubule nucleation occurs at sites other than the centrioles (
In MDCK cells, AMF-R tubules are distributed throughout the cell, and the extension and peripheral orientation of AMF-R tubules are dependent on microtubule integrity (
Other cellular organelles, including the Golgi apparatus, endosomes, and lysosomes, cluster in the pericentriolar region of the cell in a microtubule-dependent manner (
The pericentriolar localization of the Golgi apparatus is due to the dynein-mediated movement of Golgi fragments to the minus ends of microtubules (
The microtubule association of AMF-R tubules resembles that of the ER and tubular lysosomes, whose extension to the cell periphery is microtubule-dependent (
The increased density of AMF-R labeling relative to that of tubulin in a defined pericentriolar region after MSV transformation of MDCK cells is indicative of the selective association of AMF-R tubules with a subpopulation of microtubules in MSV-MDCK cells. Microtubule dynamics have been shown to drive the formation of ER membranes into tubular membrane networks (
Selective stabilization of localized regions of microtubules has been proposed to be implicated in cell polarization and morphogenesis (
AMF-R mediates motility stimulation of tumor cells by the cytokine AMF (
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Acknowledgments |
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Supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society and from the Medical Research Council of Canada, and by an establishment award from the Fonds de la Recherche en Santé du Québec. DS was the recipient of a studentship from FCAR.
We thank Jennifer Lippincott-Schwartz, Steve Doxsey, Gregg Gundersen, and John Bergeron for kindly providing antibodies. We are particularly grateful to Nicole Leclerc for helpful suggestions concerning both the experimental work and the manuscript. We also thank Diane Gingras and Dale Laird for assistance with the confocal microscopy. The photographic reproductions were the work of Jean Léveillé.
Received for publication January 24, 1997; accepted April 24, 1997.
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