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Tight Junctions in Cell Types

Tight Junctions in Cell Types



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I took a quiz today on cell structure basics, which included cell junction types. I disagree with the "correct answer" according to the teacher.

Here is the question:

Which cell type would have the most tight junctions? a) Pancrease cells b) Ovarian cells c) Epithelial cells (in the intestine) d) Muscle cells (skeletal)

According to my teacher, muscle cells is the correct answer.

I argued that the answer should be epithelial cells, as muscles cells (according to the text) primarily use desmosomes for cell-to-cell adhesion, while epithelial cells rely on tight junctions primarily.

I searched it up quickly on Google, and from what I have found, epithelial cells is the correct answer.

Is my teacher incorrect?


Tight junctions are very specifically a structure found in epithelial cells, so your teacher is incorrect. Tight junctions are what allow epithelial cells to form a selective barrier between compartments. Fibroblasts can be induced to form tight junctions under certain experimental conditions, but I'm not aware of any case where a skeletal muscle cell expresses a tight junction. Regardless, it would not be normal physiology.

You can read more about tight junctions here, in the online Albert's Molecular Biology of the Cell. This figure from that Chapter illustrates the barrier function.

If you want to get into more of the details re: what we know about the various proteins involved in creating and regulating tight junctions, this is a good review.

One small correction to your reasoning, though, I wouldn't say the primary purpose of tight junctions is cell-to-cell adhesion, per se, it's selective permeability. These junctions may help the cells to hold together, but the tissues can resist shear stress in knock-out models. A knockout model without a necessary tight junction protein, however, will not maintain selective permeability.


Tight Junctions in Cell Types - Biology

Most animal cells release materials into the extracellular space. The primary components of these materials are proteins, and the most abundant protein is collagen. Collagen fibers are interwoven with carbohydrate-containing protein molecules called proteoglycans. Collectively, these materials are called the extracellular matrix (Figure 1). Not only does the extracellular matrix hold the cells together to form a tissue, but it also allows the cells within the tissue to communicate with each other. How can this happen?

Figure 1. The extracellular matrix consists of a network of proteins and carbohydrates.

Cells have protein receptors on the extracellular surfaces of their plasma membranes. When a molecule within the matrix binds to the receptor, it changes the molecular structure of the receptor. The receptor, in turn, changes the conformation of the microfilaments positioned just inside the plasma membrane. These conformational changes induce chemical signals inside the cell that reach the nucleus and turn “on” or “off” the transcription of specific sections of DNA, which affects the production of associated proteins, thus changing the activities within the cell.

Blood clotting provides an example of the role of the extracellular matrix in cell communication. When the cells lining a blood vessel are damaged, they display a protein receptor called tissue factor. When tissue factor binds with another factor in the extracellular matrix, it causes platelets to adhere to the wall of the damaged blood vessel, stimulates the adjacent smooth muscle cells in the blood vessel to contract (thus constricting the blood vessel), and initiates a series of steps that stimulate the platelets to produce clotting factors.


Tight Junctions

Epithelia are sheets of cells that provide the interface between masses of cells and a cavity or space (a lumen). The portion of the cell exposed to the lumen is called its apical surface. The rest of the cell (i.e., its sides and base) make up the basolateral surface. Tight junctions seal adjacent epithelial cells in a narrow band just beneath their apical surface. They consist of a network of claudins and other proteins. Tight junctions perform two vital functions:

  1. They limit the passage of molecules and ions through the space between cells. So most materials must actually enter the cells (by diffusion or active transport) in order to pass through the tissue. This pathway provides tighter control over what substances are allowed through.
  2. They block the movement of integral membrane proteins (red and green ovals) between the apical and basolateral surfaces of the cell. Thus the special functions of each surface, for example receptor-mediated endocytosis at the apical surface and exocytosis at the basolateral surface can be preserved.

The Epithelia of the Human Lung

A report by Vermeer, et al., in the 20 March 2003 issue of Nature provides a striking example of the role of tight junctions. The epithelial cells of the human lung express

  • a growth stimulant, called heregulin, on their apical surface and
  • its receptors on the basolateral surface. (These receptors also respond to epidermal growth factor (EGF), and mutant versions have been implicated in cancer.

As long as the sheet of cells is intact, there is no stimulation of its receptors by heregulin thanks to the seal provided by tight junctions. However, if the sheet of cells becomes broken, heregulin can reach its receptors. The result is an autocrine stimulation of mitosis leading to healing of the wound. Several disorders of the lung the chronic bronchitis of cigarette smokers, asthma, cystic fibrosis increase the permeability of the airway epithelium. The resulting opportunity for autocrine stimulation may account for the proliferation (piling up) of the epithelial cells characteristic of these disorders.


Tight Junctions

In a tight junction, a series of integral protein molecules in the plasma membranes of adjacent cells fuse together, forming an impermeable junction that encircles the cell. Tight junctions help prevent molecules from passing through the extracellular space between adjacent cells. For example, tight junctions between epithelial cells lining the digestive tract keep digestive enzymes and microorganisms in the intestine from leaking into the bloodstream (Note: some tight junctions may leak and allow certain ions to pass).

Desmosomes

An epithelial cell is shown joined to adjacent cells by three common types of cell junctions. (Note: Except for epithelia, it is unlikely that a single cell will have all three junction types.)

Desmosomes are anchoring junctions – mechanical couplings scattered like rivets along the sides of adjacent cells to prevent their separation. On the cytoplasmic face of each plasma membrane is a thickening called a plaque. Adjacent cells are held together by thin linker protein filaments that extend from the plaques and fit together like the teeth of a zipper in the intercellular space. Thicker keratin filaments extend from the cytoplasmic side of the plaque across the width of the cell to anchor to the plaque on the cell’s opposite side. In this way, desmosomes bind neighboring cells together and also contribute to a continuous internal network of strong wires.

This arrangement distributes tension throughout a cellular sheet and reduces the chance of tearing when it is subjected to pulling forces. Desmosomes are abundant in tissues subjected to great mechanical stress, such as skin and heart muscle.

Gap Junctions

A gap junction is a communicating junction between adjacent cells. At gap junctions the adjacent plasma membranes are very close, and the cells are connected by hollow cylinders called connexons, composed of transmembrane proteins. The many different types of connexon proteins vary the selectivity of the gap junction channels. Ions, simple sugars, and other small molecules pass through these water-filled channels from one cell to the next.


Results

Directed differentiation of small intestinal organoids

Cell histology and morphometrics

Bright-field images (Fig. 1a, top row) show the morphological complexity of organoids treated with Wnt and Notch modulators to direct their growth and development. ISC and PAN organoids typically have convoluted and narrow-diameter lumina and numerous buds, just like those of frequently studied TYP organoids (not shown, [20]), which contain disproportionately larger numbers of PANs and ISCs. In contrast, ENT and GOB organoids have larger luminal diameters and fewer, if any, buds. The average luminal diameters of ENT and GOB organoids are similar: 127 ± 17 and 123 ± 14 μm (n = 10), respectively. H&E staining of the various organoid types show organoid size and cellular complexity at both 20× (Fig. 1a, middle row) and 60× (bottom row) magnification. Nuclear chromatin, which stains blue to purple (basophilic), suggests a basolateral position of the nuclei in all cell types depicted, as would also be found in vivo. ISC organoids not only displayed a more basophilic nuclear stain, but also had significantly larger nuclei (by almost twofold, P < 0.05, Table 1) compared to those of other organoids. Virtually all cells in GOB organoids seemed to have mucus, and each GOB had numerous distinct white unstained granules, seemingly poised for secretion near the apical membrane (Fig. 1a, bottom row). PANs have apical secretory granules that were pink and hence more acidophilic than those of the GOB organoids. ENTs in ENT organoids were columnar, similar to ENTs in vivo. Like those in ISC organoids, the nuclei in PAN organoids were

33% larger compared to those in ENT organoids and GOB organoids, and occupy a significant amount of cell volume (Table 1 Fig. 1a, bottom row).

Bright-field and H&E images of ISC, ENT, GOB, and PAN organoids. a Images at 20× (top and middle) and 60× (bottom row). There were no differences in morphology of cells in two sets of organoids from different mice. Arrows depict secretory vesicles in GOBs and PANs. b Periodic acid-Schiff/Alcian blue stained images at 20× (top) and 60× (bottom). c Expression profile of biomarkers in TYP, ISC, ENT, GOB, PAN, intestinal crypts (CRY) and tissue homogenates (HOM). TYPs are undirected organoids with all cell types represented. The expression of each biomarker was normalized to that in TYP organoids (=1.0). i The biomarker for stem cells, leucine-rich-repeat-containing G-protein coupled receptor 5 (Lgr5), was highly expressed in ISC organoids (n = 4 for TYP and n = 3 for other organoids). ii PANs are marked by lysozyme (Lyz), which was very abundant in PAN organoids (see scale along ordinate axis) (n = 4 for TYP n = 3 for others). iii Mucin-2 (Muc2) was highly expressed in GOBs (n = 4 for TYP and n = 3 for others). iv The enteroendocrine biomarker chromogranin A (Chga) was expressed in GOBs and PANs (n = 4 for TYP and n = 3 for others). v ENTs were marked by sucrase isomaltase (SI) (n = 4 for TYP, ISC, and HOM, and n = 3 for others). vi Stem cells were marked by olfactomedin-4 (Olfm4) (n = 4 for TYP, ENT n = 3 for others). P a,b,c,d ≤ 0.05. d Immunofluorescence of biomarkers of ISCs, ENTs, GOBs, and PANs. Nuclei are blue. Organoids were stained with OLFM4 (red), lysozyme (LYZ green), mucin-2 (MUC2 green), sucrase isomaltase (SI red), or chromogranin A (CHGA green). 60× magnification bar = 25 μm. Supporting data sets are deposited in the figshare repository [50]. CHGA chromogranin A, CRY crypt, ENT enterocyte, GOB goblet cell, H&E hematoxylin and eosin stain, HOM homogenates of intestinal mucosa, ISC intestinal stem cell, L lumen, Lgr5 leucine-rich repeat containing G-protein-coupled receptor 5, LYZ lysozyme, MUC2 Mucin-2, OLFM4 Olfactomedin-4, PAN Paneth cell, SI sucrase isomaltase, TYP typical

PAS combined with Alcian blue staining, which indicates the presence of polysaccharides, glycoproteins, and glycolipids, demonstrates the relative paucity of these compounds in lightly stained ISC and ENT organoids (Fig. 1b, left columns). In contrast, there was significant staining of secretory carbohydrate-rich granules in GOBs enriched in GOB organoids. Interestingly, PAN granules stain PAS magenta near the apical membrane, indicating that their secretory granules are enriched in carbohydrate moieties [28] the basal region occupied mostly by nuclei remained opaque (Fig. 1b, right columns).

Interestingly, ENTs in ENT organoids were slightly shorter compared to cells in ISC, GOB, and PAN organoids (Table 1), while GOBs containing mucus granules in GOB organoids were significantly wider compared to individual cells in ISC, ENT, and PAN organoids. The length and diameter of ENTs in ENT organoids were similar to those of intestinal cells fractionated from the lower villus regions [29]. Previous work has observed that young GOBs near the villus base tended to be much larger, but their volumes decreased toward the upper villus regions, areas where mucin vesicles might already have been secreted [30]. Thus, these directed organoids are morphologically normal despite being exposed to growth factors and growth factor inhibitors, and likely comprise relatively younger cells, as would be expected since these organoids were collected just 2–3 days after differentiation from ISCs was initiated. The lifespan of ENT organoids is 5 days while that of GOB organoids is 4.5 days [20], similar to those of ENTs and GOBs in vivo. In contrast, ISC organoids, after reaching large sizes with numerous buds, can be dispersed and reseeded into new Matrigel, and thus theoretically, they are able to live (be passaged) indefinitely.

Biomarker distribution

The comparative expression levels of biomarkers among TYP, ISC, ENT, GOB, and PAN organoids, along with starting crypt material as well as proximal intestinal homogenate, confirmed that directed organoids were highly enriched in specific cell types (Fig. 1c) (P < 0.001 for all biomarkers tested). Levels of the stem cell biomarker leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) were highest in ISC organoids,

5-fold higher than TYP, which has proportionally a large number of ISCs [20, 21], and 300–500-fold higher than those of ENT and GOB organoids as well as crypt homogenates and intestinal mucosal homogenates. Lysozyme (Lyz, a PAN biomarker) was most highly expressed in PAN organoids, and expression levels were ≥ 20-fold higher than those of crypt samples, intestinal homogenates, and other organoids. GOB marker mucin-2 (Muc2) was most highly expressed in GOB organoids, about 800-fold higher than in ISCs, and five- to tenfold higher than in other samples. Levels of mRNA of the enteroendocrine biomarker chromogranin A (Chga), interestingly, were similar between GOB and PAN organoids, and both were 5- to 25-fold higher compared to those of other organoids, crypts, and homogenates. Expression of the ENT biomarker sucrase isomaltase (SI) was highest in ENT organoids whose expression levels were

3- to 15-fold greater than in those of other organoids, crypts, and homogenates. Similar results were obtained for alkaline phosphatase, another ENT biomarker (not shown). Finally, the mRNA levels of another stem cell biomarker, Olfm4, were greatest in ISC organoids.

Biomarker mRNA expression was confirmed with immunofluorescence of biomarkers in each organoid type (Fig. 1d). An antibody against OLFM4, whose mRNA expression pattern (Fig. 1c) mimics that of Lgr5, was used to mark ISCs by immunofluorescence, as antibodies against LGR5 do not seem to work. An additional stem cell biomarker, CD44, was also measured by immunofluorescence, and determined to be abundant in ISC organoids and present in PAN organoids (Additional file 3: Figure S1). OLFM4 was highly visible in the apical region of ISC organoids but was largely absent in ENT and GOB organoids (Fig. 1d). OLFM4 is secreted, and intense immunofluorescence in the closed lumen of ISC organoids reflects accumulated secretions. Its modest accumulation in the lumen of PAN organoids indicate the presence of some stem cells. ENT organoids were lined by large numbers of cells containing significant amounts of SI (

almost 90% SI-positive [20]), some MUC2 (

10%) and, rarely, CHGA. There were virtually no LYZ-positive cells. Virtually all cells in GOB organoids contained MUC2 (

90%), a few SI (15%) and some CHGA (

5% the total can exceed 100% as some cells co-express two different biomarkers), but, interestingly, virtually no OLFM4 or LYZ. Depending on the plane of focus, some cells that seem devoid of contents may actually contain MUC2 (Additional file 4: Figure S2). PAN organoids had high levels of LYZ (65% [20]) and CHGA (15%), some SI and OLFM4 (

5–10%), but no MUC2. Images of control sections incubated without primary antibodies are depicted in Additional file 4: Figure S2.

In summary, the predominant cell type in ISC organoids is stem cells at levels much higher than estimated in vivo (< 1%). In ENT organoids, the predominant cell type is ENTs at proportions similar to or greater than most published estimates in vivo (

80%). In GOB organoids, the predominant cell type is GOBs at levels much higher than in vivo (< 10%), and in PAN organoids, the predominant cell type is PANs at levels more than in vivo (

5%). As previously noted [21], PAN and GOB organoids both express significant numbers (

15%) of enteroendocrine cells as indicated by its biomarker, CHGA. Unfortunately, generating organoids, particularly secretory, with a single cell type is not yet possible, and we and others [21] have been unable to reduce the levels of enteroendocrine cells in organoids directed to contain primarily PANs or GOBs.

Tight junction proteins and the leak pathway

MRNA expression

Since there are numerous TJ proteins, we focused on ones that have been shown to be expressed significantly in the small intestine, to vary in expression along the crypt–villus axis, or potentially to regulate changes in paracellular permeability [1, 6]. mRNA levels of all organoid claudins were within an order of magnitude of those in isolated crypts or mucosal homogenates, suggesting that expression was similar to that in vivo. Claudin-1 (Cldn1) was most highly expressed (P < 0.0001 by one-way ANOVA) in secretory GOB and PAN organoids (

3- to 10-fold higher than that in TYP and ENT organoids and least expressed in ISC organoids Fig. 2a). In contrast, claudin-2 (Cldn2) expression was highest in ISC organoids, being

2.5- to 50-fold higher than that in crypt, mucosal homogenates, and all other organoids (P < 0.0001). Claudin-7 (Cldn7 Fig. 2a) was highest in CRY and in ENT organoids over all others (P < 0.0001). Claudin-15 (Cldn15) was the least heterogeneous, but was still most highly expressed in ENT and GOB organoids (P < 0.01). Expression of specific claudins in crude mucosal homogenates tended to vary depending on the levels of that claudin in the ENT organoids, reflecting the abundance of ENTs in the mucosa.

a Expression of claudins in different cell types normalized to that in TYP organoids (=1.0). i Cldn1 seemed greatest in GOBs and PANs (n = 4 ISC n = 3 others). ii Cldn2 was highest in ISCs (n = 4 ISCs n = 3 others). iii Cldn7 was high in ENTs (n = 4 ENTs n = 3 others). iv Cldn15 was relatively homogenous (n = 4 ENTs n = 3 others). P a,b ,c,d ≤ 0.05. b,c Expression of TJ proteins in different cell types. b,i Occludin (Ocln) was higher in ISCs and PANs (n = 5 ISCs n = 3 others). ii ZO-1 (Tjp1) was similar among organoids (n = 4 for ISCs, ENTs n = 3 others). iii Epithelial cadherin 1 (Cdh1) was relatively high in PANs (n = 4 ENTs n = 3 others). iv Myosin light chain kinase (Mylk) was lowest in ISCs (n = 3). c,i Tricellulin (Marveld2) was lowest in ENTs (n = 4 TYPs, ISCs, ENTs n = 3 others). ii Cingulin (Cgn) expression was high in PANs and CRYs (n = 5 ENTs n = 3 PANs, CRYs n = 4 others). iii Expression of junctional adhesion molecule-1 (Jam1) and iv JAM4 (Jam4) were lowest in ISCs (n = 5 ENTs n = 4 TYPs, ISCs, GOBs n = 3 others). d Immunofluorescence of representative TJ proteins in organoids probed with ZO-1 (green), ECAD (red), CLDN2 (CL2 red), CLDN7 (CL7 green), or OCLN (green) (60×, bars = 25 μm). e Cellular location. ZO-1 staining is mainly in the apical area and ECAD in the basolateral area. CLDN2 is found along the brush border of ISCs and PANs but not in ENTs and GOBs. CLDN7 seems basolateral but expressed less in ISCs. OCLN seems apical. Supporting data sets are deposited in the figshare repository [50]. Cdh or ECAD, E-cadherin, Cgn cingulin, Cldn or CL claudin, CRY crypt, ENT enterocyte, GOB goblet cell, HOM homogenates of intestinal mucosa, ISC intestinal stem cell, Jam, junctional adhesion molecule, L lumen, Marveld2 MARVEL domain containing 2, Mylk myosin light chain kinase, Ocln occludin, PAN Paneth cell, Tjp or ZO1 tight junction protein, TYP typical

Occludin (Ocln) expression among organoids tended to vary less than that of claudins (Fig. 2a,b). Ocln was expressed approximately twofold more in ISC and PAN organoids than in TYP, ENT, and GOB organoids (P < 0.0001). Variation among organoids in Tjp1 (ZO-1 Fig. 2b) expression was modest (P = 0.02), suggesting that ZO-1 expression may be ubiquitous and similar among cell types. There was a marked cell-type dependent variation in E-cadherin (Cdh1 or ECAD) (P < 0.0001) and in myosin light chain kinase (Mylk) (P < 0.0001). Mylk expression was highest in GOB and least in ISC organoids (eightfold less than that in GOBs). PAN organoids had the highest expression of Cdh1 relative to all other samples (> 2–4-fold).

Tricellulin (Marveld2) expression was

2- to 5-fold higher in ISC and PAN organoids than in TYP, ENT, and GOB organoids (P < 0.0001), like that of occludin (Fig. 2c). Variation among organoids in Cgn (cingulin) expression was modest (P = 0.02). There was also a modest cell-type dependent variation for junctional adhesion molecule-1 (JAM1 P = 0.05) and JAM4 levels (P < 0.0001), which are both expressed least in ISCs. JAM proteins are found at TJs and participate in the regulation of TJ integrity and permeability, while cingulin interacts with many TJ proteins and modifies their function [4].

In summary, the expression of many important players in the regulation of TJ permeability often varies markedly among different small intestinal cell types.

Protein expression and localization

We examined using immunofluorescence the expression of CLDN2 and CLDN7, whose mRNA levels were strongly modulated by cell type, and also the expression of ZO-1, whose deletion disrupts TJs [13], OCLN, which regulates macromolecular TJ permeability [2, 31], and ECAD, a structural protein associated mainly with adherens junctions [1]. ZO-1 was expressed sharply in virtually all cells of ISC, ENT, GOB, and PAN organoids (Fig. 2d,e). ZO-1 staining formed a relatively clear, narrow, punctate line along the apical surface of an organoid (Fig. 2d,e). Likewise, ECAD proteins seemed ubiquitously expressed in all organoid types. In contrast to ZO-1, which was limited primarily to or near the apical membrane, ECAD clearly lined the basolateral membrane regions of all cells in ISC, ENT, GOB, and PAN organoids (Fig. 2d,e). In GOB organoids, ECAD also seemed to be expressed in the apical region.

The distribution of CLDN2 was clearly dependent on cell type. ISC organoids had high amounts of CLDN2 while ENT and PAN organoids had low amounts, and it was not found in GOB organoids. In contrast, CLDN7 seemed to be expressed in all cell types. CLDN7 clearly lined the basolateral membrane areas and was noticeably absent from the apical area of all organoids except ISCs. OCLN was apically oriented and seemed to be greater in ISC and PAN organoids, confirming the pattern of mRNA distribution.

In summary, the expression of OCLN seemed low in GOB organoids, that of CLDN2 was high in ISC organoids, and that of ZO-1 similar in all organoids. CLDN2 was apically oriented in ISC and PAN organoids while ECAD and CLDN7 were largely basolateral, with some apical staining present in certain cell types.

Dextran permeability of directed organoids

Since the distribution of TJ proteins was heterogeneous, we determined whether the leak pathway would likewise differ between crypt- and villus-dwelling cell types. Using two-way ANOVA, we found highly significant effects for cell type and molecular weight on dextran transport into the organoid lumen (Fig. 3a). We saw that 4 kDa dextran was transported into the lumen of organoids at rates approximately threefold greater than 10 kDa dextran, suggesting that macromolecules with a Stokes radius of 1.4 nm may be better than those of 2.3 nm at distinguishing cell-type differences in the leak pathway of small intestinal TJ. Moreover, 4 kDa dextran accumulated in ENT and GOB lumen at amounts much higher (

2.5- to 4-fold) than those in ISC and PAN organoids. A similar pattern was observed for 10 kDa dextran accumulated fluorescence. Organoids were not permeable to 40 kDa dextrans [24], hence larger molecular weights were not tested. There was a strong statistical interaction, suggesting that cell type affected the effect of molecular size on dextran transport from serosa to lumen.

a Effects of cell type and dextran size on macromolecular permeability. A serosa to lumen gradient of 1.25 μM 4 or 10 kDa dextran was imposed on TYP, ISC, ENT, GOB, and PAN organoids for 30 min. i Representative images clearly depict the higher dextran accumulation in TYP, ENT, and GOB organoids. ii Levels of net fluorescence in all organoids were then analyzed as described in the text and normalized to that in 4 kDa TYP organoids (=1.0). (n = 4 for ISCs, ENTs, GOBs, and PANs, and n = 2 for TYPs, for both 4 and 10 kDa). Using two-way ANOVA, the serosal to luminal flux of both dextran levels was higher in ENT and GOB organoids, and 10 kDa dextran permeability was lower. b Effects of AT1002 (an active fragment of ZO toxin) and larazotide on permeability to 4 kDa dextran. ENT and ISC organoids were incubated overnight in 10 μg/mL AT1002 (+Z) or 12.5 mM larazotide acetate (+L) or both (Z + L) and then permeability to 4 kDa dextran was determined (i). Using two-way ANOVA, the dextran flux was higher in ENTs and greatest in AT1002-treated organoids (ii). (For ENTs, n = 3 each for CONs, +Z, +L, and n = 2 for + Z + L for ISCs, n = 3 for CONs, and n = 2 for + Z, +L and + Z + L). Supporting data sets are deposited in the figshare repository [50]. CON control cell, ENT enterocyte, FITC fluorescein isothiocyanate, GOB goblet cell, ISC intestinal stem cell, PAN Paneth cell, TYP typical

In summary, the macromolecular flux into ENT and GOB organoids is higher than that into ISC and PAN organoids, suggesting that villus-dwelling cell types may have leak pathways more permeable than those of crypt-dwelling cell types.

AT1002 increases dextran permeability of ENT and ISC organoids

We used 4 kDa dextrans to evaluate the effect of AT1002, which is known to disassemble TJ proteins and lead to marked increases in small intestinal permeability, and that of the TJ-regulator larazotide [25]. Larazotide is a novel therapeutic agent targeting TJ regulation in patients with celiac disease [27]. Using two-way ANOVA, we found dramatic effects for cell type and AT1002 treatment (P < 0.001). With AT1002, both ENT and ISC organoids exhibited a dramatic increase in 4 kDa dextran permeability (11- and 7-fold, respectively) compared to untreated ENT and ISC organoids (Fig. 3b). Larazotide by itself had no effect on dextran permeability. However, when organoids were incubated in AT1002 in the presence of larazotide, permeability was similar to that of control, untreated levels. Thus, TJ sensitivity to AT1002 in organoids mimics that in vivo, suggesting the similar mechanisms of AT1002 disruption are addressable by larazotide treatment. Although dextran permeabilities are different between ISC and ENT organoids, these were similarly susceptible to AT1002.

Dextran accumulation over time

After 10 min of incubation, ENT organoids had accumulated significant amounts of 4 kDa dextran (Fig. 4a). Dextran levels were similar throughout the organoid lumen, as images from Z-stacks of the same organoid exhibited similar fluorescence intensities relative to the background. Similar observations were made for 5 or 6 ENT organoids. Relative to the 10-min incubation, dextran levels increased by 68 ± 12% after 30 (n = 6) and by 65 ± 10% after 60 (n = 6) min of incubation. When incubated over the same duration, PAN (Fig. 4b) and ISC (Fig. 4c) organoids accumulated much less dextran, confirming the results in Fig. 3a. The paucity of dextran levels was evident throughout the lumen of the numerous PAN and ISC organoids examined, even after 60 (Fig. 4b,c) and 80 min (not shown). Since similarly shaped and sized ENT and PAN organoids accumulated dextrans at vastly different rates, while differently shaped and sized PAN and ISC organoids both accumulated dextrans at similarly limited rates, these findings also suggest that the shapes of organoids likely do not affect the accumulation of dextran, and by extension, of our estimates of permeability of the leak pathway.

Dextran accumulation in ENT, PAN, and ISC organoids over time. ENT, PAN, and ISC organoids were each exposed to 4 kDa dextran for 10, 30, and 60 min, washed, and then imaged with an inverted confocal DMi8 microscope equipped with a CSU-W1 spinning disk (see text). Then, 5-mm Z-stacks were acquired above and below the vertical midpoint of the organoid. Representative images from above (i, ii, iii) and below (iv, v, vi) the midpoint of ENT (a), PAN (b), and ISC (c) organoids at 10, 30, and 60 min intervals (n = 5 or 6 organoids). Dextran accumulation was clearly greater in ENT organoids at all time points. Luminal dextran levels increased with incubation time. Supporting data sets are deposited in the figshare repository [50]. ENT enterocyte, ISC intestinal stem cell, PAN Paneth cell

Dedifferentiation and TJ plasticity

Morphometrics and biomarker expression

Since cell type and thus, differentiation from stem to secretory and absorptive progenies affected TJ protein levels and permeability, we devised a method to dedifferentiate mature cell types [20]. We assessed the plasticity of TJ protein expression and dextran permeability in differentiated ENT organoids, and examined whether the acquisition of ISC-like biomarkers in dedifferentiated ENT organoids is accompanied by a return to an ISC-like TJ composition and permeability.

H&E and PAS staining of ENT and dENT organoids showed no remarkable morphological (Fig. 5a) or morphometric changes caused by dedifferentiation, compared to those depicted in Fig. 1a,b and Table 1, which show marked differences between ENT and ISC organoids. The percentage of GOBs in ENT organoids (

15% [20]) was similar to that of dENT organoids (14.7 ± 0.8, n = 6). The cell length was similar between ENT (Table 1) and dENT (14.0 ± 0.5 μm, n = 6) organoids. Despite similar morphometrics and histological morphology, ENT and dENT organoids exhibited marked differences in levels of the ENT biomarker SI and the stem cell biomarker OLFM4. SI staining in dENTs was significantly reduced compared to that of ENT organoids (Fig. 5a). In fact, only the lumen (likely containing cells exfoliated prior to dedifferentiation) had SI immunoreactivity while the ISC organoids had no SI at all, as would be expected. In contrast, OLFM4 levels were almost nonexistent in ENT organoids but then staining increased markedly in the dENT lumen where OLFM4 was secreted, indicating that organoids were indeed in the process of dedifferentiation, and were beginning to look the same as ISCs. Moreover, mRNA expression of stem cell biomarkers increased while that of ENT biomarkers decreased from ENT to dENT organoids [20]. Thus, while ENT and dENT organoids shared similar structure and cell composition, the activation of Wnt (via CHIR) and of Notch (via VPA) pathways induced the loss of markers of differentiation and the reacquisition of markers of stemness, as previously shown by us.

Effect of dedifferentiation of ENT organoids on expression of biomarker immunofluorescence and histological staining. From ISC precursors, ENT organoids were cultured for 3 days with C59 + valproic acid to full differentiation as indicated by the peak expression of biomarkers. To force dedifferentiation, the Wnt inhibitor C59 was removed from the medium, and ENT organoids were then exposed for 36 h to 6 μM CHIR (dENT). a ENT and dENT organoids were stained with H&E as well as PAS. The morphology of and cell dimensions within dENT organoids were similar to those of ENT organoids. However, the immunofluorescence of the biomarkers indicated there was a significant loss of ENT-marker sucrase isomaltase (SI) staining in dENT organoids as well as an increase in secretion of stem cell marker OLFM4. b The expression of TJ mRNA in dENT organoids. Cldn2 increased with dedifferentiation (n = 4 for ENTs, n = 3 for dENTs and ISCs). Cldn7 decreased slightly with dedifferentiation (n = 4), while Ocln levels did not change (n = 5 for ENTs, n = 3 for dENTs, and n = 4 for ISCs). Tjp1 (ZO-1) tended to increase (P = 0.09) with dedifferentiation (n = 5 for ENTs, n = 4 for dENTs, and n = 3 for ISCs). P a,b,c ≤ 0.05. c Immunofluorescence staining of CLDN2, OCLN, and ZO-1 in dedifferentiated cells. Nuclei are stained blue. Organoids were stained with the TJ protein CLDN2 (CL2 red), OCLN (green), and ZO-1 (green). Representative organoids are shown at 60× magnification. Bars are 25 μm. d Functional changes due to dedifferentiation. 4 kDa dextran permeability decreased in dENT organoids, P a,b,c ≤ 0.05 (n = 4 for ENTs, 3 for dENTs, and 5 for ISCs). Supporting data sets are deposited in the figshare repository [50]. Cldn claudin, dENT dedifferentiated enterocyte, ENT enterocyte, FITC fluorescein isothiocyanate, H&E hematoxylin and eosin stain, ISC intestinal stem cell, L lumen, Ocln occludin, OLFM4 olfactomedin-4, PAS periodic acid-Schiff stain, SI, sucrase isomaltase, TJ tight junction, Tjp or ZO-1 tight junction protein

TJ protein expression

Previously observed differences in mRNA expression of Cldn2, Cldn7, Ocln, and Tjp1 between ENTs and ISCs in Fig. 2a,b were similar to the pattern of differences between ENTs and dENTs (Fig. 5b). Expression of Cldn2 (P < 0.001), Ocln (P < 0.20), and Tjp1 (P < 0.0001) in dENT organoids increased from ENT levels and became similar to those of ISC organoids, while expression of Cldn7 decreased to ISC levels (P < 0.005). Thus, mRNA expression of these TJ components changed during dedifferentiation from that of ENTs to dENTs, to reflect ISC levels.

Immunocytochemistry

CLDN2 was present in ISC but not ENT organoids (Fig. 5c), as shown previously (Fig. 2d,e). CLDN2, however, was not highly expressed in the apical membrane of dENT organoids and thus, it did not follow the pattern of changes in Cldn2 mRNA. OCLN and ZO-1 were expressed along the apical membrane in a punctate manner in both ISC and ENT organoids as before, and were also expressed in dENTs.

Dextran permeability

Relative to ISC organoids, ENT organoids were more permeable (Fig. 5d) to 4 kDa dextran as previously observed (Fig. 3a). dENT organoids had a 70% reduction (P < 0.001) in permeability compared to ENT organoids, so that dENT permeability became similar to that of ISC organoids. This reduction in dextran permeability back to ISC levels suggests TJs exhibit functional plasticity, which may correlate with changes in the expression or location of unidentified TJ proteins whose levels or location become similar to those of ISC organoids. Here, similarly shaped and sized ENT and dENT organoids have markedly different levels of luminal dextran, suggesting that dextran accumulation is not significantly affected by organoid form.

Effect of time after induction of differentiation from ISC

Relative to the effects of cell type, the effects of organoid age or time after induction of differentiation from ISCs were much more modest. In ENT organoids, TJ-related genes were altered due to post-differentiation time and generally reflect a transition from ISC to ENT cell-type composition, which stabilized, based on the time course of biomarker expression, 2–3 days after transition from ISCs [20]. Cldn2 (P < 0.0001) and Ocln (P < 0.002) decreased significantly over time, while Cldn7 (P < 0.05) increased by twofold (P < 0.01). Recall that Cldn7 expression is typically low in ISCs. There was a modest effect (P = 0.05) of post-differentiation time on Tjp1 expression (Fig. 6a).

Effect of time after directed differentiation from ISC on mRNA expression of TJ proteins from ENT (a) and GOB (b) organoids. Within each gene, all treatments are normalized to day 1 (D1) post-differentiation for both ENT and GOB organoids. Data were analyzed using one-way ANOVA (see text for P values). a Cldn2 (n = 3 for ENTs D1 to D3), Cldn7 (n = 3 for D1 and n = 2 for D2 as well as D3), Ocln (n = 3 for D1 as well as D3, and n = 2 for D2), and Tjp1 (ZO-1) (n = 3 for all). b Cldn2 (n = 3 for GOBs D1 to D3), Cldn7 (n = 3 for D1 and n = 2 for D2 as well as D3), Ocln (n = 3 for all), and Tjp1 (ZO-1) (n = 3 for GOB D1 as well as D3, and n = 2 for D2). Trends in mRNA expression of TJ proteins during directed differentiation from ISC to ENT or GOB organoids matched those depicted in Fig. 2a,b. Supporting data sets are deposited in the figshare repository [50]. Cldn claudin, D day, ENT enterocyte, GOB goblet cell, ISC intestinal stem cell, Ocln occludin, TJ tight junction, Tjp or ZO-1 tight junction protein

In GOB organoids, TJ-related genes were also altered due to the post-differentiation time. Cldn2 (P < 0.0001) and Ocln (P < 0.025) expression decreased significantly by day 3 of differentiation to GOB organoids (Fig. 6b). In contrast, Cldn7 expression increased (P = 0.025), while that of Tjp1 did not change (P = 0.10). When differentiation is triggered from ISC to ENT or GOB organoids, the post-differentiation time course would reflect the in vivo journey of postmitotic cells from villus base to tip, and migration would be accompanied by generally small changes in expression of genes coding for TJ proteins. In fact, claudin differentiation may result in several physiologically distinct TJs within the lifetime of the same cell [14].


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Adherens Junctions

  • They hold cardiac muscle cells tightly together as the heart expands and contracts.
  • They hold epithelial cells together.
  • They seem to be responsible for contact inhibition.
  • Some adherens junctions are present in narrow bands connecting adjacent cells.
  • Others are present in discrete patches holding the cells together.
  • cadherins &mdash transmembrane proteins (shown in red) whose
    • extracellular segments bind to each other and
    • whose intracellular segments bind to

    We synthesize some 80 different types of cadherins. In most cases, a cell expressing one type of cadherin will only form adherens junctions with another cell expressing the same type. This is because molecules of cadherin tend to form homodimers not heterodimers.

    Inherited mutations in a gene encoding a cadherin can cause stomach cancer. Mutations in a gene (APC), whose protein normally interacts with catenins, are a common cause of colon cancer.


    Functions of TJs

    The classical functions of TJs are the regulation of paracellular permeability and the formation of an apical-basolateral intramembrane diffusion barrier that helps to maintain cell-surface polarity. More recently, TJs have been linked to various signalling mechanisms that guide gene expression, proliferation and differentiation. TJ components also form complexes with the cellular machinery that regulates basolateral cell-surface transport (the Sec6-Sec8 complex) however, this does not appear to be an exclusive property of TJ proteins because these complexes also contain adherens-junction components, such as E-cadherin and nectin 2 (Yeaman et al., 2004). The actual structure that forms the intramembrane diffusion barrier is poorly understood hence, we limit our discussion to the role of TJs in the regulation of paracellular permeability, as well as in signalling during epithelial proliferation and differentiation.

    Paracellular permeability

    TJs allow the passive selective diffusion of ions and small hydrophilic molecules through the paracellular pathway across epithelia and endothelia. The molecular mechanisms that are responsible for selective ion permeability and for diffusion of small hydrophilic molecules are distinct, as many manipulations and regulatory mechanisms specifically affect only one of the two processes, or downregulate one while activating the other (Aijaz et al., 2006). For example, the overexpression of occludin or certain occludin mutants in cultured epithelial cells stimulates the paracellular diffusion of small hydrophilic molecules but increases transepithelial electrical resistance (a measure of transepithelial ion permeability) (Aijaz et al., 2006).

    Occludin, tricellulin and the claudins are the main TJ membrane components that are involved in paracellular permeability. On the basis of observations in human disease, mouse models and cultured cell lines, it has been suggested that the claudin composition of TJs is a major determinant of the permeability properties of a tissue (Furuse and Tsukita, 2006 Van Itallie and Anderson, 2006). Absence of specific claudins can cause organ-specific defects, such as neurological, reproductive and renal defects. For example, mice that lack claudin 1 die after birth because of water loss across the skin, and the absence of claudin 5 causes leakage of small tracers across the brain endothelium (Furuse and Tsukita, 2006). Experiments with epithelial cell lines further suggest that different claudins favour paracellular diffusion of specific ions and, as mentioned above, these have lead to a model in which claudins form homo- and hetero-oligomers that engage in intercellular interactions to form paracellular aqueous pores. The ion-selectivity of these pores is determined by their claudin composition (Krause et al., 2008 Van Itallie and Anderson, 2006).

    The process that mediates the paracellular diffusion of small hydrophilic molecules is less well understood and the actual mechanism by which such molecules permeate the junction is not known. However, the process is regulated by RhoA signalling and seems to require actinomyosin-driven processes (Aijaz et al., 2006 McKenzie and Ridley, 2007 Nusrat et al., 2000 Utech et al., 2006). It is therefore possible that dynamic rearrangements of intramembrane strands lead to paracellular diffusion. Occludin has been linked to the regulation of paracellular permeability of small hydrophilic molecules across cultured epithelial monolayers (Balda and Matter, 2000 Schneeberger and Lynch, 2004) it is thought that this regulatory mechanism involves phosphorylation events as well as the actin cytoskeleton because the C-terminal domain of occludin binds to protein kinases and lipid kinases, as well as to actin filaments and cytoskeletal linkers (Aijaz et al., 2006 Schneeberger and Lynch, 2004). Strikingly, live-cell-imaging experiments using GFP-tagged occludin have recently suggested that occludin diffuses within the junction, suggesting that occludin dynamics might contribute to paracellular diffusion (Shen et al., 2008). However, because the experiment involved an N-terminal GFP-tag and blocking the N-terminus is known to interfere with anchoring of occludin within the junction, it is not clear whether the observed dynamic properties indeed reflect physiological occludin behaviour (Huber et al., 2000).

    Cell proliferation, polarity and differentiation

    The regulation of cell proliferation and polarisation is crucial for the development of differentiated tissues. Several studies have linked TJs to the regulation of cell proliferation and cell polarity. Similar to adherens junctions, TJs function in the suppression of proliferation (Gonzalez-Mariscal et al., 2007 Matter and Balda, 2007). Occludin suppresses oncogenic Raf-1 signalling (Wang et al., 2005) and ZO-1 interacts with ZONAB, thereby regulating gene expression, cell proliferation and epithelial morphogenesis (Matter and Balda, 2007 Sourisseau et al., 2006). ZO-2 localises to the nucleus and interacts with the DNA-binding protein scaffold attachment factor B (SAFB) as well as with several transcription factors (Gonzalez-Mariscal et al., 2007 Huerta et al., 2007 Traweger et al., 2003). TJs have also been linked to the regulation of RhoA-dependent proliferation through the junction-associated guanine-nucleotide exchange factor GEF-H1 and the junctional scaffolding protein cingulin, which binds to and inhibits GEF-H1 (Aijaz et al., 2005 Guillemot and Citi, 2006). It remains to be determined, however, whether and how these different TJ-associated signalling mechanisms are connected with each other and by which transmembrane proteins they are regulated. Strikingly, deficiencies in ZO-1 or ZO-2 expression are embryonic lethal in mice, which suggests that the two TJ proteins are important for development (Katsuno et al., 2008 Xu et al., 2008). Whether any of the signalling mechanisms that have been identified in cell culture experiments contribute to these phenotypes in vivo, however, is not clear.

    Two evolutionarily conserved cell-polarity signalling pathways reside at TJs. Similar to D. melanogaster, the CRB3-Pals1-PATJ pathway regulates junction assembly and biogenesis of the apical membrane in vertebrate epithelial cells (Shin et al., 2006). The signalling pathway downstream of the complex has not yet been elucidated. The second conserved signalling module is the PAR3-PAR6-aPKC complex, which was originally described as a regulator of cytoplasmic partitioning in the early embryo of Caenorhabditis elegans. PAR3 associates with the cytoplasmic domain of JAMs, which results in the recruitment of the PAR3-PAR6-aPKC complex to cell-cell junctions (Bradfield et al., 2007 Ebnet et al., 2004 Weber et al., 2007). The complex functions as an effector of Cdc42, a Rho-family GTPase that is essential for epithelial-cell polarity and becomes activated during junction formation – binding of the complex to GTP-bound Cdc42 stimulates activation of aPKC and, consequently, the formation of mature TJs (Ebnet et al., 2004 Shin et al., 2006). In agreement with these observations, Cdc42 is crucial for the establishment of epithelial polarity during early mammalian development, and the aPKC isoforms PAR3 and PAR6 are necessary for the formation of the epidermal barrier (Helfrich et al., 2007 Wu et al., 2007). Interestingly, PAR3 also suppresses the activation of Rac1, another member of the RhoGTPase family, during junction formation by binding the guanine nucleotide exchange factor TIAM1 (Chen and Macara, 2005). This function, however, does not require PAR6 or aPKC, which suggests that PAR3 can act independently of PAR6 and aPKC.


    Mucosal Vaccines: An Overview

    Prosper N. Boyaka , . Jiri Mestecky , in Mucosal Immunology (Third Edition) , 2005

    Zonula occludens toxin

    Zonula occludens toxin (Zot) is produced by toxigenic strains of Vibrio cholerae and has the ability to reversibly alter intestinal epithelial tight junctions , allowing the passage of macromolecules through the mucosal barrier. Nasal immunization of mice with a protein antigen and recombinant Zot, either alone or fused to the maltose-binding protein (MBP-Zot), induced high antigen-specific IgA antibody titers in plasma, as well as in vaginal and intestinal secretions ( Marinaro et al., 1999a ). Moreover, Zot as adjuvant induced antigen-specific IgG subclasses that consisted of IgG1, IgG2a, and IgG2b antibodies and resembled the pattern induced by LT ( Marinaro et al., 1999 ). Zot was recently shown to also act as adjuvant for rectal immunization ( Marinaro et al., 2003 ). These studies illustrate the importance of increasing the permeability of mucosal tissues for induction of mucosal immunity to vaccines.


    Tight Junctions and Cell Polarity

    AbstractThe tight junction is an intracellular junctional structure that mediates adhesion between epithelial cells and is required for epithelial cell function. Tight junctions control paracellular permeability across epithelial cell sheets and also serve as a barrier to intramembrane diffusion of components between a cell's apical and basolateral membrane domains. Recent genetic and biochemical studies in invertebrates and vertebrates indicate that tight junction proteins play an important role in the establishment and maintenance of apico-basal polarity. Proteins involved in epithelial cell polarization form evolutionarily conserved multiprotein complexes at the tight junction, and these protein complexes regulate the architecture of epithelia throughout the polarization process. Accumulating information regarding the regulation of these polarity proteins will lead to a better understanding of the molecular mechanisms whereby cell polarity is established.