Epithelial Barrier Function In Vivo Is Sustained Despite Gaps in Epithelial Layers
2005; Elsevier BV; Volume: 129; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2005.06.015
ISSN1528-0012
AutoresAlastair J.M. Watson, Shaoyou Chu, Leah K. Sieck, Oleg V. Gerasimenko, Tim F Bullen, Fiona Campbell, Michael McKenna, Tracy L. Rose, Marshall H. Montrose,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoBackground & Aims: Epithelial cells of the small intestine migrate to the tip of the villus at which they are shed. It is not understood how the intestinal barrier is maintained during this high cell turnover. The aim of this study was to use high-resolution in vivo light microscopy to investigate the mechanism of epithelial shedding and the site of the permeability barrier during cell shedding. Methods: A laparotomy was performed on anesthetized mice, and a segment of small intestine was opened. The exposed epithelial surface of the intestine was imaged by multiphoton microscopy. Nuclei, cytosol, and cell membranes were imaged using the dyes Hoescht 33258, BCECF, a transgenically expressed fluorescent protein, and the membrane dye DiI. The fluorescent caspase substrate PhiPhiLux was used to detect apoptosis. Results: In the epithelial monolayer, gaps were observed that lacked nuclei or cytosol but appeared to be filled with an impermeable substance. Studies with membrane impermeant fluorophores (Lucifer Yellow and Alexa-dextran) showed that the impermeable substance completely fills the void left by the absent cell. Only a fraction of gaps have either ZO-1 staining or cytoplasmic extensions from neighboring cells at the basal pole. Time-lapse studies reveal that cell shedding results in genesis of a gap and that shedding usually occurs prior to detectable cellular activation of caspase 3 or nuclear condensation. Conclusions: Results suggest that epithelial barrier function is sustained at the apical pole of the epithelial layer, despite discontinuities in the cellular layer. Background & Aims: Epithelial cells of the small intestine migrate to the tip of the villus at which they are shed. It is not understood how the intestinal barrier is maintained during this high cell turnover. The aim of this study was to use high-resolution in vivo light microscopy to investigate the mechanism of epithelial shedding and the site of the permeability barrier during cell shedding. Methods: A laparotomy was performed on anesthetized mice, and a segment of small intestine was opened. The exposed epithelial surface of the intestine was imaged by multiphoton microscopy. Nuclei, cytosol, and cell membranes were imaged using the dyes Hoescht 33258, BCECF, a transgenically expressed fluorescent protein, and the membrane dye DiI. The fluorescent caspase substrate PhiPhiLux was used to detect apoptosis. Results: In the epithelial monolayer, gaps were observed that lacked nuclei or cytosol but appeared to be filled with an impermeable substance. Studies with membrane impermeant fluorophores (Lucifer Yellow and Alexa-dextran) showed that the impermeable substance completely fills the void left by the absent cell. Only a fraction of gaps have either ZO-1 staining or cytoplasmic extensions from neighboring cells at the basal pole. Time-lapse studies reveal that cell shedding results in genesis of a gap and that shedding usually occurs prior to detectable cellular activation of caspase 3 or nuclear condensation. Conclusions: Results suggest that epithelial barrier function is sustained at the apical pole of the epithelial layer, despite discontinuities in the cellular layer. The intestinal epithelium presents a permeability barrier to the luminal contents that prevents undesirable solutes, microorganisms, and luminal antigens from entering the body.1Montrose M.H. The future of GI and liver research: editorial perspectives: I. Visions of epithelial research.Am J Physiol Gastrointest Liver Physiol. 2003; 284: G547-G550Crossref Scopus (28) Google Scholar, 2Clayburgh D.R. Shen L. Turner J.R. A porous defense the leaky epithelial barrier in intestinal disease.Lab Invest. 2004; 84: 282-291Google Scholar However, it remains mysterious how the intestinal barrier is sustained during the high rate of normal cellular turnover in the epithelium. Epithelial cells of the mammalian small intestine arise from stem cells at the base of the crypt and migrate up to the villus at which they are shed. In the mouse, ∼1400 cells are shed from each villus per 24 hours.3Potten C.S. Loeffler M. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt.Development. 1990; 110: 1001-1020Crossref Google Scholar It is remarkable that, despite this loss of ∼1 cell/minute, the functional permeability pore size on the villus is less than .6 nm, some 20,000 times less than the diameter of a villus epithelial cell.4Fihn B.M. Sjoqvist A. Jodal M. Permeability of the rat small intestinal epithelium along the villus-crypt axis effects of glucose transport.Gastroenterology. 2000; 119: 1029-1036Abstract Full Text Full Text PDF Scopus (123) Google Scholar The simplest explanation is that mechanisms of cell shedding somehow do not disturb the epithelial barrier in healthy tissue. Tight junctions are unequivocally a site and structure that restricts paracellular flux between adjacent epithelial cells. Furthermore, regulation of the tight junctional complex is a well-established mechanism controlling passive fluxes during sodium and glucose absorption,5Turner J.R. Rill B.K. Carlson S.L. Carnes D. Kerner R. Mrsny R.J. Madara J.L. Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation.Am J Physiol. 1997; 273: C1378-C1385Google Scholar and altered tight junctional structure has been observed in several diseases displaying increased intestinal permeability (for review see Clayburgh et al2Clayburgh D.R. Shen L. Turner J.R. A porous defense the leaky epithelial barrier in intestinal disease.Lab Invest. 2004; 84: 282-291Google Scholar). It remains less clear whether loosening of tight junctions is solely responsible for the increased intestinal permeability that has been implicated in the pathogenesis of inflammatory bowel disease, celiac disease, graft vs host following bone marrow transplantation, and the response to intestinal pathogens.6Pearson A.D. Eastham E.J. Laker M.F. Craft A.W. Nelson R. Intestinal permeability in children with Crohn’s disease and coeliac disease.Br Med J (Clin Res Ed). 1982; 285: 20-21Google Scholar, 7Katz K.D. Hollander D. 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Increased intestinal permeability precedes the onset of Crohn’s disease in a subject with familial risk.Gastroenterology. 2000; 119: 1740-1744Abstract Full Text Full Text PDF Scopus (225) Google Scholar, 13Berkes J. Viswanathan V.K. Savkovic S.D. Hecht G. Intestinal epithelial responses to enteric pathogens effects on the tight junction barrier, ion transport, and inflammation.Gut. 2003; 52: 439-451Google Scholar Deficiency in heparin sulfate at the basolateral surface of enterocytes has been implicated in protein-losing enteropathy and lowered transepithelial resistance.14Bode L. Eklund E.A. Murch S. Freeze H.H. Heparan sulfate depletion amplifies TNF-α-induced protein leakage in an in vitro model of protein-losing enteropathy.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G1015-G1023Google Scholar Abnormality of cell shedding has been invoked in the pathogenesis of serrated polyps in the colon.15Jass J.R. Whitehall V.L. Young J. Leggett B.A. Emerging concepts in colorectal neoplasia.Gastroenterology. 2002; 123: 862-876Abstract Full Text Full Text PDF Scopus (428) Google Scholar One hypothesis is that deranged cell shedding could contribute to dysfunction of the epithelial barrier in a variety of intestinal disorders. Our lack of understanding about the process of in vivo villus cell shedding limits our ability to test this hypothesis. The mechanisms of intestinal epithelial cell shedding remain highly controversial.16Mayhew T.M. Myklebust R. Whybrow A. Jenkins R. Epithelial integrity, cell death and cell loss in mammalian small intestine.Histol Histopathol. 1999; 14: 257-267Google Scholar Apoptosis has been suggested as one mechanism of cell death at the villus tip.17Iwanaga T. Han H. Adachi K. Fujita T. A novel mechanism for disposing of effete epithelial cells in the small intestine of guinea pigs.Gastroenterology. 1993; 105: 1089-1097Google Scholar, 18Hall P.A. Coates P.J. Ansari B. Hopwood D. Regulation of cell number in the mammalian gastrointestinal tract the importance of apoptosis.J Cell Sci. 1994; 107: 3569-3577Crossref Google Scholar In detached intestinal epithelial cells, apoptosis occurs with activation of the initiator caspases 2 and 9, with subsequent hierarchical activation of executioner caspases such as caspase 3.19Grossmann J. Walther K. Artinger M. Kiessling S. Scholmerich J. Apoptotic signaling during initiation of detachment-induced apoptosis (“anoikis”) of primary human intestinal epithelial cells.Cell Growth Differ. 2001; 12: 147-155Google Scholar However, it is not clear whether apoptosis is a cause or consequence of the shedding process because apoptotic bodies are rarely observed within the villus epithelium.18Hall P.A. Coates P.J. Ansari B. Hopwood D. Regulation of cell number in the mammalian gastrointestinal tract the importance of apoptosis.J Cell Sci. 1994; 107: 3569-3577Crossref Google Scholar, 20Marshman E. Ottewell P.D. Potten C.S. Watson A.J. Caspase activation during spontaneous and radiation-induced apoptosis in the murine intestine.J Pathol. 2001; 195: 285-292Google Scholar Interestingly, tissue culture models reveal parallels between the shedding of apoptotic cells and epithelial wound closure after physical removal of cells. In both cases, a coordinated purse-string contraction of actin filaments surrounding the monolayer defect helps to close the gap and restore barrier function.21Rosenblatt J. Raff M.C. Cramer L.P. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism.Curr Biol. 2001; 11: 1847-1857Google Scholar, 22Nusrat A. Delp C. Madara J.L. Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells.J Clin Invest. 1992; 89: 1501-1511Google Scholar, 23Bement W.M. Forscher P. Mooseker M.S. A novel cytoskeletal structure involved in purse string wound closure and cell polarity maintenance.J Cell Biol. 1993; 121: 565-578Google Scholar A major regulator of this contraction is the phosphorylation of myosin II regulatory light chain by myosin light chain kinase and rho-associated kinase.24Turner J.R. Angle J.M. Black E.D. Joyal J.L. Sacks D.B. Madara J.L. PKC-dependent regulation of transepithelial resistance roles of MLC and MLC kinase.Am J Physiol. 1999; 277: C554-C562Google Scholar, 25Clayburgh D.R. Rosen S. Witkowski E.D. Wang F. Blair S. Dudek S. Garcia J.G. Alverdy J.C. Turner J.R. A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability.J Biol Chem. 2004; 279: 55506-55513Google Scholar Tight junctions may also play a role to maintain the epithelial barrier during physiologic cell shedding. It has been observed by electron microscopy that neighboring cells form a tight junction beneath the extruding cell.26Madara J.L. Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium physiological rearrangement of tight junctions.J Membr Biol. 1990; 116: 177-184Google Scholar Finally, a role for subepithelial myofibroblasts has been proposed, wherein they contract following epithelial cell loss or injury, thereby restoring epithelial continuity.27Moore R. Carlson S. Madara J.L. Villus contraction aids repair of intestinal epithelium after injury.Am J Physiol. 1989; 257: G274-G283Google Scholar The contribution of each of these elements to the maintenance of barrier function during physiologic cell shedding remains speculative.21Rosenblatt J. Raff M.C. Cramer L.P. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism.Curr Biol. 2001; 11: 1847-1857Google Scholar, 28Florian P. Schoneberg T. Schulzke J.D. Fromm M. Gitter A.H. Single-cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions.J Physiol. 2002; 545: 485-499Google Scholar Studies to date give only limited insight into barrier maintenance during cell shedding in vivo because investigators have been limited to study of either fixed tissue, cells collected from the intestinal lumen, or cell culture models. We have combined fluorescent probes with confocal and multiphoton microscopy to allow real-time study of epithelial architecture, cell shedding process, and barrier function in living mice having an intact circulation to the gut mucosa.29Chu S. Tanaka S. Kaunitz J.D. Montrose M.H. Dynamic regulation of gastric surface pH by luminal pH.J Clin Invest. 1999; 103: 605-612Google Scholar, 30Baumgartner H.K. Kirbiyik U. Coskun T. Chu S. Montrose M.H. Endogenous cyclo-oxygenase activity regulates mouse gastric surface pH.J Physiol. 2002; 544: 871-882Google Scholar, 31Dunn K.W. Sandoval R.M. Kelly K.J. Dagher P.C. Tanner G.A. Atkinson S.J. Bacallao R.L. Molitoris B.A. Functional studies of the kidney of living animals using multicolor two-photon microscopy.Am J Physiol Cell Physiol. 2002; 283: C905-C916Google Scholar Results suggest that the previously identified mechanisms cannot fully explain the observed maintenance of epithelial barrier function. Surgical procedure was a modification of published in vivo procedures for rodent stomach.29Chu S. Tanaka S. Kaunitz J.D. Montrose M.H. Dynamic regulation of gastric surface pH by luminal pH.J Clin Invest. 1999; 103: 605-612Google Scholar Mice (ICR) were housed in a standard 12-hour light/dark cycle with lights on at 0600 hours. Experiments were performed routinely between 1300 and 2000 hours. Mice were anesthetized with thiobutylbarbital 100–150 mg/kg intraperitoneally (IP) (Inactin; Sigma Chemical Co, St. Louis, MO). A tracheotomy was performed to facilitate breathing. A mid-abdominal incision (1–1.5 cm) was made, and a segment of small intestine was exteriorized, flushed with saline, and opened longitudinally along the antimesenteric border by cutting cautery. The anesthetized animal was placed supine on a custom-built chamber on the stage of a multiphoton microscope (Zeiss LSM 510 NLO; Ziess, Jena, Germany) that was heated to 37°C by a circulating water bath. All subsequent drug treatments were made on the anesthetized mice. The opened intestinal segment was placed, luminal surface down, on the inverted microscope stage while bathed in 0.9% NaCl and the villus epithelium imaged with a 40× C-Apo objective. Muscular contraction of the intestine was minimized by application of xylazine directly on the gut. At the end of the experiment, the animal was humanely killed. All procedures were approved by the animal care and use committee of Indiana University. All images were collected at 20–50 μm below the villus tip in anesthetized animals by a combination of confocal and 2-photon imaging. Autofluorescence images were collected with 2-photon excitation (710 nm) and 380–650 nm emission wavelengths. Nuclear staining was achieved by IP injection of 2 mg/kg Hoescht 33258 (Molecular Probes, Eugene, OR),31Dunn K.W. Sandoval R.M. Kelly K.J. Dagher P.C. Tanner G.A. Atkinson S.J. Bacallao R.L. Molitoris B.A. Functional studies of the kidney of living animals using multicolor two-photon microscopy.Am J Physiol Cell Physiol. 2002; 283: C905-C916Google Scholar and images were collected with 800-nm excitation and 435–485-nm emission. 2′,7′-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM; 10 μmol/L in saline; Molecular Probes) was loaded into intestinal epithelial cells by direct application to the gut mucosal surface for 15 minutes and confocally imaged at 488-nm excitation and >505-nm emission. Cytosolic YFP fluorescence was imaged at 514-nm excitation and 530–630-nm emission. Plasma membranes were stained by exposure of the gut mucosal surface 15 minutes to 1,1′-dilinoleyl-3,3,3′,3′,-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (DiI; 5 μL Vybrant cell stain, Molecular Probes) and imaged with 543-nm excitation and 565–615-nm emission. Lucifer Yellow (100 μmol/L in the luminal fluid; Molecular Probes) was used as a membrane impermeable marker of the luminal compartment, imaged with 800-nm, 2-photon excitation and 530–650-nm emission. Dextran MW 10,000 conjugated to Alexa Fluor 647 (2 mg/mL, Molecular Probes) was injected intravenously to image blood vessels and to act as a permeability marker for the extracellular space beneath intestinal epithelial cells. Exposure of the luminal mucosal surface to PhiPhiLux (10 μmol/L; Oncoimmunin, Gaithersburg, MD) identified caspase-3-like activity, imaged at 543-nm excitation and 565–615-nm emission. Confocal reflectance images were collected by reflecting excitation light wavelengths to a confocal detector. Murine small intestine was gently flushed with physiologic saline, placed in 10% neutral-buffered formalin saline for 6 hours, processed through to paraffin blocks, and 4-μm sections cut. Great care was taken not to disturb the section of intestine to be sectioned so as to preserve fragile structures. Sections were stained with either alcian blue/diastase-periodic acid schiff for goblet cells or H&E. The human specimen was collected from a right hemicolectomy patient after informed consent was given. The specimen had minimal handling and warm ischemia before fixation. Ethical approval was given by the local research ethics committee at University of Liverpool (Study no. 03/09/182/C [A]). Tight junctional structures were identified in formalin-fixed paraffin-embedded sections (5 μm) of murine intestine. Sections were incubated with rabbit anti-ZO-1 antibody (Zymed catalog 61-7300). Prior to immunostaining, sections were subjected to proteolytic enzyme digestion for 60 minutes at 37°C, using 0.7% Trypsin (VWR International Ltd, Cat. No. 390414M) in Tris-buffered saline (TBS; 0.05 mol/L Tris, 0.12 mol/L sodium chloride, pH 7.6). After rinsing in tap water followed by deionized water, sections were transferred to an Autostainer (DakoCytomation, Denmask) for staining. Sections were incubated with primary antibody for 40 minutes at room temperature and washed with TBS-Tween (TBS with 0.05% Tween-20); the detection system was ChemMate EnVision HRP (DakoCytomation, Cat. No.K5007), which was used according to the manufacturer’s instructions. Sections were removed from the Autostainer, counterstained with Mayer’s haematoxylin, dehydrated through ethanol, cleared in xylene, and coverslipped using a resinous mountant. We have used multiphoton and confocal microscopy for real-time study of individual epithelial cell dynamics and barrier function in the small intestine of living mice.29Chu S. Tanaka S. Kaunitz J.D. Montrose M.H. Dynamic regulation of gastric surface pH by luminal pH.J Clin Invest. 1999; 103: 605-612Google Scholar, 30Baumgartner H.K. Kirbiyik U. Coskun T. Chu S. Montrose M.H. Endogenous cyclo-oxygenase activity regulates mouse gastric surface pH.J Physiol. 2002; 544: 871-882Google Scholar, 31Dunn K.W. Sandoval R.M. Kelly K.J. Dagher P.C. Tanner G.A. Atkinson S.J. Bacallao R.L. Molitoris B.A. Functional studies of the kidney of living animals using multicolor two-photon microscopy.Am J Physiol Cell Physiol. 2002; 283: C905-C916Google Scholar To observe epithelial architecture in living native tissue, initial studies evaluated cellular autofluorescence in villi of normal, anesthetized mice in response to 2-photon excitation (Figure 1A). Settings were used that had previously been established to measure NAD(P)H fluorescence in response to 2-photon excitation.32Rocheleau J.V. Head W.S. Piston D.W. Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response.J Biol Chem. 2004; 279: 31780-31787Google Scholar Villus epithelial cells displayed cytosolic, but not nuclear, autofluorescence, and occasional gaps were noted within the annulus of cells in villus cross-sections (Figure 1A, arrows). Simultaneous confocal reflectance imaging, used to identify physical structures that reflect 710-nm laser light,29Chu S. Tanaka S. Kaunitz J.D. Montrose M.H. Dynamic regulation of gastric surface pH by luminal pH.J Clin Invest. 1999; 103: 605-612Google Scholar reported that this apparently cytoplasm-free gap contained reflective (yet nonfluorescent) material (Figure 1B and 1C). Four vital stains were used to probe annular gaps. Tissue of anesthetized animals was loaded with the cytoplasmic dye BCECF and the nuclear DNA dye Hoechst 33258. Gaps in the annular ring of fluorescent nuclei occurred (Figure 1D, arrow), coincident with lack of cytoplasmic staining (Figure 1E), yet containing reflective material (Figure 1F and 1G). Using mice transgenic for a fluorescent protein (YC3.0 calcium chameleon transgenic mice), results also confirmed that nuclear gaps (Figure 1H, arrows) were coincident with lack of cytoplasmic fluorescent protein (Figure 1I), despite the presence of reflective material (Figure 1J and 1K). Staining with the membrane dye DiI33Gan W.B. Bishop D.L. Turney S.G. Lichtman J.W. Vital imaging and ultrastructural analysis of individual axon terminals labeled by iontophoretic application of lipophilic dye.J Neurosci Methods. 1999; 93: 13-20Google Scholar demonstrated that discontinuities in the apical brush border membrane (Figure 1M, arrow) occurred when nuclei were absent (Figure 1L), despite continued presence of reflective material (Figure 1N and 1O). Overall, 6 independent markers report that the small intestinal villus epithelium of living mice is a discontinuous layer, interrupted by gaps having coincident loss of apical brush border membrane, cytosol, and nucleus. In all analyses, serial optical sections at 1-μm intervals identified gaps that were approximately the same volume as an individual cell, using orthogonal views at the plane of the nuclei (Figure 2A). In contrast to epithelial wound healing models, cells neighboring gaps were not wider or flatter than epithelial cells distant from gaps.21Rosenblatt J. Raff M.C. Cramer L.P. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism.Curr Biol. 2001; 11: 1847-1857Google Scholar, 34Abreu M.T. Palladino A.A. Arnold E.T. Kwon R.S. McRoberts J.A. Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells.Gastroenterology. 2000; 119: 1524-1536Abstract Full Text Full Text PDF Scopus (123) Google Scholar Focusing in from the villus tip, subcellular resolution of villus epithelial cells was observed to 70-μm depth. This is equivalent to the top 20% of the villi (average length of the mouse villus is 350 ± 22 μm; n = 50 villi). Morphometric analysis in vivo showed that ∼3% of cell positions were gaps, equally distributed along the final 70 μm of the villus tip (Figure 2B). This may underestimate the frequency of gaps in some regions, as en face views of cytosolic fluorescent protein near the apical surface of epithelial cells (Figure 2C) show that the gap diameter is variable, often less than adjacent epithelial cells. In formalin-fixed sections, en face gaps were distinct from goblet cells (Figure 2D). Data demonstrate frequent discontinuities in the epithelial layer of living mice, implying that tight junctions at the apical pole of cells cannot be the only mechanism sustaining intestinal barrier function13Berkes J. Viswanathan V.K. Savkovic S.D. Hecht G. Intestinal epithelial responses to enteric pathogens effects on the tight junction barrier, ion transport, and inflammation.Gut. 2003; 52: 439-451Google Scholar and that another mechanism must be invoked within the gaps. It has been suggested that tight junction formation beneath shedding cells can sustain epithelial barrier function.26Madara J.L. Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium physiological rearrangement of tight junctions.J Membr Biol. 1990; 116: 177-184Google Scholar In live tissue studies using cells expressing a cytosolic fluorescent protein, we tested whether such a mechanism explained observations. In a population of 22 gaps, 45% had lamellipodia from neighboring cells that contacted each other under the epithelial gaps (Figure 3A shows CFP fluorescence of a representative cell, which is overlaid with confocal reflectance [red] in Figure 3B). In the remaining 55% of gaps, there was no discernable cytoplasm from neighboring cells in the gaps (Figure 3C and 3D). Using fixed tissue, we immunostained the tight junction protein ZO-1 to determine whether tight junctions were present under shedding cells. In a population of 73 shedding cells, 9% were observed to be bounded by neighboring cells contacting at the basal pole in a V-shaped formation with a spot of ZO-1 immunoreactivity at the apex of the V (Figure 3E). The remaining cells had no identifiable ZO-1 immunoreactivity near the basement membrane (Figure 3F). Thus, in both fixed and live tissue, evidence suggests that tight junctions from neighboring cells can only be used in a fraction of instances to help reseal the breach in the epithelial layer. To test whether gaps present a luminal permeability barrier, we added the cell impermeable dye Lucifer Yellow35Swanson J. Burke E. Silverstein S.C. Tubular lysosomes accompany stimulated pinocytosis in macrophages.J Cell Biol. 1987; 104: 1217-1222Google Scholar to luminal fluid and identified cell-free regions by lack of Hoescht-stained nuclei. The majority (229 of 235, 97%) of nucleus gaps (Figure 4C, arrow) did not permit entry of Lucifer Yellow (Figure 4A), presumably because of being filled with the reflective material (Figure 4B). In the remaining 3% of gaps (6 of 235), entry of Lucifer Yellow occurred (Figure 4A, arrowhead) but never extended deeper than the line of nuclei from adjacent cells. Deeper entry of Lucifer Yellow appeared limited by the confocal reflective substance (Figure 4B and 4D, arrowhead). We noted one additional category in which Lucifer Yellow fluorescence extended into the epithelial layer identified by confocal reflectance. In these cases, nuclei were present but were more centrifugally positioned than neighbors (Figure 4G, arrowhead), suggesting that the cells were in the process of being shed. Lucifer Yellow permeation (Figure 4E) extended into the space enveloping the exiting nuclei, suggesting that cell membrane integrity had been compromised during cell shedding. Thus, 45% of all Lucifer Yellow intrusions into the epithelial layer (5 of 11) were into regions with exiting nuclei. Conversely, no centrifugal nuclei were observed that sustained impermeability to Lucifer Yellow. To localize the permeability barrier relative to the basal pole of the epithelial layer, we injected a 10-kilodalton fluorescent dextran (conjugated to Alexa Fluor 647) intravenously into mice expressing a cytosolic fluorescent protein. As shown in Figure 5A, dextran fluorescence was observed in the lateral intercellular spaces between adjacent epithelial cells up to the level of the tight junctions (gap identified by lack of CFP fluorescence as shown in Figure 5B). The dextran also permeated into the perimeter of gaps but did not extend as far toward the apical membrane. As shown for 3 representative gaps in Figure 5C–E, this is most clearly seen in en face views near the apical surface, at which dextran fluorescence surrounding gaps is minimal compared with that observed in lateral spaces between adjacent cells. To monitor the biogenesis of cell-free gaps, we made time-lapse images of Hoechst 33258-stained villus tips every 1–3 minutes in anesthetized animals (Figure 6A). Nuclei were shed from the monolayer at a speed of 0.83 ± 0.06 μm/minutes (n = 53 cells), initiating departure at apparently random times (Figure 6B, upper graph). Time-lapse series revealed that resultant gaps were not fully filled in by migration of neighboring cells for at least 60 minutes (data not shown), explaining the heterogeneity of gap size noted earlier in Figure 2C. We sought to test whether intact cells or cell fragments were shed during the biogenesis of gaps. Apoptosis has been suggested as the mechanism of cell loss at the villus tip.18Hall P.A. Coates P.J. Ansari B. Hopwood D. Regulation of cell number in the mammalian gastrointestinal tract the importance of apoptosis.J Cell Sci. 1994; 107: 3569-3577Crossref Google Scholar, 19Grossmann J. Walther K. Artinger M. Kiessling S. Scholmerich J. Apoptotic signaling during initiation of detachment-induced apoptosis (“anoikis”) of primary human intestinal epithelial cells.Cell Growth Differ. 2001; 12: 147-155Google Scholar, 20Marshman E. Ottewell P.D. Potten C.S. Watson A.J. Caspase activation during spontaneous and radiation-induced apoptosis in the murine intestine.J Pathol. 2001; 195: 285-292Google Scholar Measurement of nuclear DNA fluorescence intensi
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