A Fluorescence images for ZO Mechanical properties are inferred from AFM-based indentation experiments that provide information about cortical strength, membrane tension and excess membrane area. Interestingly, although the cells are visibly separated from each other, the mechanical parameters are only slightly altered by ZO-1 depletion Fig. The biggest effect is an increased cortical strength after weakening of tight junctions.
A Overall tension comprising cortical tension and membrane tension. B Apparent area compressibility modulus. C Membrane tension. Box plots extend from the 25 th to 75 th percentile, whiskers from the 10 th to the 90 th.
Grey dots show data extracted from force-indentation curves recorded on untreated cells ctrl , red ones were recorded on cells without zonula occludens ZO-1 siRNA. A rank-sum test was performed to test the null hypothesis that the data of the indicated datasets are from populations with equal medians. Asterisks indicate that the null hypothesis was rejected at the 0. In order to exclude mechanical alterations due to the transfection procedure control measurements using non-targeting siRNA are shown in the supplementary information suppl.
S1 Applying the transfection protocol shifts mechanical parameters to slightly lower values, i. In summary, ZO-1 is extremely important for the formation of a dense cell layer, which shields underlying cells. The morphology of ZO-1 lacking cells is changed tremendously but the overall stiffness of the cells increases only slightly compared to well-connected cells. Viewed from the apical side, adherens junctions are placed beneath the tight junctional complex.
It has been shown that intact disulfide bridges play a pivotal role for functional cell-cell adhesion of epithelial cells We used this chemical instead of siRNA or administration of antibodies since we could easily remove the agent to examine the reversibility of opening of adherens junctions. Moreover, this procedure was highly reproducible. Notably, it is known that DTT might also activate integrins and thereby generates side effects Since we are only interested in the apical cortex it is reasonable to assume that contributions from adherens junctions dominate for the mechanical investigation.
In control cells, E-cadherin is clearly enriched at the cell-cell boundaries in the central focal plane Fig. Within this time interval no visible changes in the E-cadherin distribution are observed Fig. DTT interferes with E-cadherin mediated cell-cell junctions. A Phase contrast images. The focus was set to the apical D and basal F cell site and to the focal place corresponding to the E-cadherin channel E. Phase contrast imaging shows no changes in the cellular structure during the entire observation period Fig.
In contrast to ZO-1 silenced cells, the cell monolayer stays intact after incubation with DTT-containing medium, also tight junctions are not affected by DTT treatment Fig. The distribution of actin on the apical cell side remains unchanged Fig.
Interestingly, in the central focal plane Fig. In contrast, stress fibre formation at the basal cell side is not significantly affected by disruption of the adherens junctions Fig. The intracellular E-cadherin is known to be connected to the actin cytoskeleton via catenins and we found that the actin distribution at the lateral cell side is severely perturbed by DTT treatment Fig. The images shown in A and B are recorded at the same focal plane.
AFM imaging confirms the presence of an intact cell monolayer during the whole experimental procedure Fig. However, a detailed inspection of the topography revealed that the morphology of the connected cells is altered by DTT incubation.
A Untreated cells. Next, we asked for the impact of DTT treatment and recovery from the treatment on the mechanical properties of the cells. Fundamental changes of the mechanical parameters are observed during the exposure to DTT and recovery from the drug treatment.
In summary, we found that opening of adherens junctions by DTT leads to a considerable softening of the individual cells. Both, cortical and membrane tension drop and either the bending modulus of the composite shell is reduced or the excess membrane area is enlarged depending on the interpretation of the apparent area compressibility modulus. This holds true both for structure and mechanics. Cells reach their initial values by going through a state of higher stiffness as initially found for control cells overshoot.
In the present study we examined how different cell-cell junctions contribute to the mechanical properties of individual epithelial cells and how the cells maintain tension homeostasis after selectively destroying the junctions.
It is clear that cell-cell junctions are important for providing efficient and selective barriers against the environment. This also includes a stable mechanical connection between adjacent cells that permits the cell layer to respond to external mechanical stress without losing its integrity. There are seminal studies addressing questions like, which forces are necessary within cells to establish strong cell-cell binding complexes 29 , 30 , how different types of junctions influence each other 31 , 32 , and how forces are transmitted within a cell layer 33 , 34 , 35 , However, it is still not entirely clear if and to what extent cell-cell junctions impact the mechanical behaviour of individual cells of a confluent monolayer.
When one of the major players of tight junctions, ZO-1, is depleted, we found that cells are visibly separated from each other. AFM imaging reveals that only a few finger-like connections between the cells remain Fig. Surprisingly, however, these significant alterations of the cell morphology have only a small effect on the mechanical behaviour of the individual cells that are still connected via adherens junctions. Slightly elevated overall tension and area compressibility moduli Fig.
This is in good agreement with results from Fanning et al. An enhanced myosin II level and higher actomyosin contractility may also contribute to higher tension values. Contrary to Fanning et al. This may be due to the knock down of only ZO-1 and thereby explain the only moderately enhanced tension in our case.
Compared to their work, we also found less pronounced stress fibre formation, when the cells are separated from each other Fig. Ikenouchi et al. Actin on the lateral cell side is separated between individual cells In our experiments cells undergo substantial morphological changes upon ZO-1 depletion vide supra. The membrane-cytoskeleton attachment describing membrane tension T t , however, remains largely at the same level that we found for control cells.
This supports the work from Fanning et al. In contrast to ZO-1 depletion, E-cadherin interference has dramatic consequences for the mechanical properties of MDCK II cells, while the impact on the morphology of the cell monolayer is small. Our AFM studies confirm this result, the removal of adherens junctions results in a substantial rounding of the cells indicative of an interfacial tension release.
Considering that contractile cortical tension is balanced by the presence of cell-cell contacts it is expected that cells bulge into a more roundish shape after losing the cell-cell counter force.
Changes in this protein distribution provoke a reduction of the overall tension and the area compressibility modulus since actin is no longer connected to the lateral cell membrane. A tension generating actin belt is partially degraded, as shown by confocal fluorescence microscopy at the central focal plane Fig. Studies on Xenopus revealed that cadherins influence the actin architecture Thus, the mechanical stability provided by the actin belt cannot be maintained after E-cadherin disruption.
Additionally, we were able to show that recovery of the original E-cadherin distribution after DTT removal is accompanied by a recovery of the initial cell topography Fig. At this time point E-cadherin is already partly located at the cell-cell junctions. Other studies put the cadherin-catenin-actin complex as the only cadherin-cytoskeleton connection way into question However, there is evidence in the literature that force generation within the cell is necessary to establish cadherin mediated cell-cell contacts 41 , 42 , In our experiments cells start to rebuild adherens junctions after DTT removal through a temporary increase overshoot of the measured mechanical parameters, essentially forming stiffer cells than initially present.
Eventually the cells restore E-cadherin mediated cell-cell junctions Figs 5B4 and 6A4. After AJ degradation we found a small decrease in membrane tension followed by an enhanced T t level during E-cadherin reassembly. It is indisputable that membrane tension is largely governed by the attachment of the cytoskeleton to the plasma membrane In studies with C.
This linker protein is then available for enhancement of the apical membrane-cytoskeleton connection leading to higher T t values More pinning points to the membrane also increase the level of cross-linking of the actomyosin network that also allows the cells to adopt a higher cortical tension through motor activity. Tension homeostasis of epithelial cells depends on many complex mechanisms in single cells and the whole tissue.
Our study shows that intact lateral cell junctions are not solely responsible for control of mechanical integrity. Tight junctions enable a sealed epithelial monolayer with a controlled perijunctional flux. The impact on mechanical stability of single cells of cell sheets is, however, small. The presence of intact tight junctions is synonymous with softer cells, maybe by decoupling the apical from the basolateral part of the polarised cell.
In contrast, loss of functional adherens junctions results in a severe softening of cells that is, however, fully reversible. Medium additionally contained penicillin 0.
Fluorescence images are shown in false colours. Cells were prepared as desired and fixed by incubation with 2. Image processing was performed using software provided by the AFM manufacturer. Force indentation cycles were recorded on the above-mentioned setup. This enables strong interaction between the cantilever and the plasma membrane during indentation and results in pulling out membrane tethers upon retraction of the indenter.
The exact spring constant of each cantilever was determined using the thermal noise method Cells were indented up to a force of 1 nN. After a dwell time of 0. These force indentation experiments were performed while scanning laterally across the sample, referred to as force mapping Force indentation curves were analysed as described in our previous studies The advantage of this simplified model over more sophisticated ones, where the geometry of the cell is considered 47 , is its analytical tractability and that it only considers the area increase due to the presence of the indenter.
It has been shown by Discher and co-workers that a polynomial fit linear and cubic term serves well to describe the experimental data Membrane tension T t was calculated from force steps F tether according to:. An example of a force indentation and retraction cycle and the fitting procedure is shown in the supplementary information suppl. The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.
Balda, M. Tight junctions at a glance. Cell Sci. Harris, T. Adherens junctions: from molecules to morphogenesis. Cell Biol. Hatzfeld, M. Cold Spring Harb. This was also true of the junctional molecules claudin-1 and TJP1 Fig. Claudin-1 and JAM3 staining was most intense in the intermediate, parabasal, and basal epithelium and also around dermal papillae. Staining for TJP1 was less intense and localized to the parabasal and basal epithelium Fig.
TJP1 distribution was more punctate than the other junctional molecules studied and could also be visualized within the cytoplasm of the epithelial cells. This is in concordance with the known function of TJP1 as an intracellular scaffolding molecule. Expression of occludin was diffuse, and the intensity was highest in the parabasal epithelium Fig. Because adhesion molecule expression patterns were identical in the ectocervical and vaginal epithelia, only images for the ectocervix are shown.
Junctional molecules of the ectocervix and vagina visualized by immunocytochemistry Cy3-labeled antibodies appear red; DAPI-stained nuclei appear blue.
All junctional molecules studied were detected in both ectocervical and vaginal tissues, displaying a spiderweb-like pattern throughout the epithelium. Ectocervical staining patterns are shown here. Cy3-labeled IgG was excluded by the tight junctions of the endocervical single-cell columnar epithelium Fig.
In contrast, fluorescent Cy3-labeled IgG penetrated the superficial layers of the ectocervical mucosal epithelium Fig. As noted above, these apical layers are composed of cornified epithelial cells that do not appear to have exclusionary epithelial adhesion junctions. Below these cells, approximately three or four cellular layers from the lumen, epithelial junctions restricted the diffusion of labeled IgG between epithelial cells.
Cervical permeability to Cy3-labeled red IgG. The endocervical epithelium provided a robust barrier to penetration of Cy3-tagged IgG, whereas the most apical layers of the ectocervix were permeable.
A Endocervical tissue incubated with Cy3-labeled IgG. No diffusion is seen into the epithelium past the tight junctional complex left arrow. The arrow on the right indicates a compromised epithelial cell. B Cy3-labeled IgG penetrated the apical cellular layers of ectocervical epithelium.
Cervicovaginal epithelial barrier integrity is maintained by intercellular junctions that prevent the invasion of microbes, with the exception of certain pathogenic organisms that have developed strategies to breech the epithelial barrier.
Many other factors further fortify this barrier. Mucus produced by cervical and vaginal epithelial cells forms a glycocalyx on the epithelial surface that retains immunological mediators, including immunoglobulins and antimicrobial peptides [ 20 — 23 ].
Furthermore, a variety of leukocytes migrate into and through the epithelium to conduct immunosurveillance [ 23 , 24 ]. The purpose of the present study was to characterize cervical and vaginal epithelial junctions to better understand their role in STI pathogenesis and immune defense of the lower female genital tract. Our electron-microscopy studies indicate that classical tight junctions comprise the principal intracellular junctions between epithelial cells in the endocervix, in accordance with the current knowledge of the structure of simple columnar epithelia [ 25 ].
These tight junctions formed a barrier that was impermeable to Cy3-labeled IgG. In contrast, the uppermost layers of the stratified squamous ectocervical epithelium were devoid of organized intracellular junctions, and the apical layers were permeable to Cy3-labeled IgG.
Exclusionary junctions were observed directly beneath this layer, and IgG did not penetrate beyond this point. The present study also surveyed the expression of discrete junctional molecules representing the different types of intracellular junctions in both the columnar epithelium of the endocervix and the stratified squamous epithelium of the ectocervix and vagina.
The results from this investigation indicate that F11R, E-cadherin, occludin, claudin-1, and TJP1 are abundant junctional molecules in the human endocervix. JAM3, a desmosomal junctional molecule, was not detected in the endocervix.
Claudin-1 expression was found in distinct foci, whereas the other junctional molecules were expressed uniformly at sites of cellular contact throughout the epithelium. Previous studies of cervical cancer biomarkers have described select junctional proteins in human cervical tissue. E-cadherin was previously described in the endocervical mucosa [ 26 ], and images published by Lee et al. All of the intracellular junctional proteins surveyed in the present study, including JAM3, were detected in ectocervical and vaginal stratified squamous epithelia.
The ectocervix is structurally a part of the vaginal wall and shares a continuous, morphologically identical mucosal layer with vaginal tissue [ 19 ]. Each of these molecules displayed a spiderweb-like distribution in the basal and suprabasal layers consistent with their functions as mediators of cell-cell adhesion. Little or no staining was observed in the most apical layers, where the epithelial cells gradually lose cell-cell contacts and are eventually sloughed into the lumen.
Claudin-1, occludin, TJP1, and E-cadherin expression has been previously described in the human ectocervix [ 14 , 27 ]. Claudin-4 and claudin-7 have been also detected in ectocervical and vaginal epithelial cells [ 14 ]. JAM3 expression has not been well studied in stratified squamous epithelia and has not been detected previously in the female mucosal epithelium, but it has been observed in the endothelium and retinal epithelium [ 18 , 28 ].
The structure and distribution of adhesion molecules in the endocervical columnar epithelium and cervicovaginal squamous epithelium, as revealed by the present study, are diagrammed in Figure 6. Localization of selected interepithelial adhesion molecules in the female lower genital tract.
A The endocervical epithelium contains classic tripartite junctions. The tight junctions are located near the apical surface; they seal the epithelium and maintain cellular polarity. Adherens junctions are located directly below the tight junctions and are primarily responsible for cell-cell adhesion. E-cadherin is the common transcellular component of all epithelial adherens junctions and is anchored to the actin cytoskeleton by vinculin and alpha and beta catenins.
Desmosomes are the most basal adhesion structure and endow the tissue with mechanical resistance and strength. The major extracellular components of the desmosome are desmoglein and desmocollin, which connect to intermediate filaments of adjacent cells through an intracellular scaffolding network. B The most robust junctions in the stratified squamous epithelium of the ectocervix and vagina lie in the parabasal epithelium, just above the basal layer in contact with the basement membrane.
Adherens junctions are particularly abundant. The integrity of the junctions progressively lessens as epithelial cells are pushed toward the apical surface, where they become cornified, lose all cellular contacts, and are sloughed into the lumen. Some pathogens are known to affect the integrity of epithelial junctions to facilitate transmission across the mucosal surface. In the context of the female reproductive tract, Nazli et al. This correlated with increased production of the proinflammatory cytokine tumor necrosis factor-alpha and disruption of the tight junctional molecules TJP1, occludin, and claudin-1, -2, and -4 [ 16 ].
F11R is a known ligand of lymphocyte-associated antigen 1 expressed on T cells, macrophages, and neutrophils; it may provide a foothold for migratory leukocytes [ 17 , 29 ]. Similarly, E-cadherin is a receptor for the lymphocyte adhesion molecule alphaEbeta7 integrin on T cells [ 30 ].
In contrast to tight and adherens junctions, little evidence indicates that desmosomal structure is altered by pathogen invasion or inflammation [ 31 ]. However, the desmosomal molecule JAM3 is a ligand for the macrophage-1 receptor on macrophages and neutrophils, and JAM3 regulates the influx of leukocytes, particularly neutrophils, in response to inflammatory stimuli [ 18 ]. For a leukocyte to migrate between epithelial cells, the epithelial junctional bonds must be disrupted.
Permeability to infiltrating leukocytes is largely regulated by secreted proinflammatory cytokines and chemokines [ 32 ]. A definitive understanding about the composition of cervical and vaginal epithelial junctions also provides an important foundation for future studies on pathogen transmission.
For infections such as HIV, preventing epithelial barrier breach by cell-free or cell-associated virus is of the utmost importance [ 33 ]. Results from the present study indicate that the uppermost layers of the stratified squamous epithelium covering the vagina and ectocervix may not comprise a physical barrier against STIs but, rather, a potential zone for interactions with immunological mediators that may be retained at this site.
Mapping the normal expression of key molecular regulators of barrier resistance is an important first step in elucidating how microbes take advantage of these mechanisms to infect a host. Centers for Disease Control and Prevention. Sexually Transmitted Disease Surveillance Atlanta, GA: U. Department of Health and Human Services; World Health Organization. Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom.
Eur J Cell Biol ; 81 : — Google Scholar. Epithelial barriers in homeostasis and disease. Annu Rev Pathol ; 5 : — Meng W , Takeichi M. Adherens junction: molecular architecture and regulation. Cold Spring Harb Perspect Biol ; 1 : 1 — Desmosomes: new perspectives on a classic.
J Invest Dermatol ; : — Gorodeski GI. Estrogen decrease in tight junctional resistance involves matrix-metalloproteinasemediated remodeling of occludin. Endocrinology ; : — Molecular physiology and pathophysiology of tight junctions. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Tight junctions and human diseases. Med Electron Microsc ; 36 : — The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state.
Although it sounds like a bad thing, apoptosis — or the process of programmed cell death — is an essential aspect of development. Without it, repair and replenishment processes would overrun tissues with new cells.
The orderly demise of a certain proportion of cells is therefore necessary for normal tissue turnover and maintenance of homeostasis. Apoptosis is distinct from necrosis , a messier form of cell death that causes cells to literally swell and burst.
Necrotic cell death is not programmed; rather, it occurs in response to trauma or injury. A range of extracellular and intracellular signals can trigger either cell growth or apoptosis. When cells receive these signals from their neighbors or from other aspects of the external environment, they carefully weigh them against each other before choosing a course of action.
For instance, signals that indicate a lack of nutrients or the presence of toxins would likely stall cell growth and promote apoptosis. Within the cell, damage to the DNA or loss of mitochondrial integrity might also result in programmed cell death. Cells self-destruct cleanly and quickly during apoptosis, thanks to the activation of a variety of enzymes — proteases and nucleases — that break down proteins and nucleic acids, respectively.
In fact, scientists look for a characteristic pattern of fragmentation and nuclear condensation within tissues as evidence that apoptosis has occurred. This page appears in the following eBook. Aa Aa Aa. Cell Adhesion and Cell Communication. Figure 2: The different types of cell junctions. Tight junctions blue dots between cells are connected areas of the plasma membrane that stitch cells together. Figure 3: A gap junction. In a gap junction, the lipid bilayer of adjacent cells is pierced through by proteins called connexons.
Some cell signaling occurs on a local level, such as when cells interact with the surrounding extracellular matrix or with their immediate neighbors. This type of signaling is especially important to the structure and function of tissues. Various signaling molecules allow the cells within a tissue to share information about internal and external conditions. This information helps the cells arrange themselves, coordinate their functions, and even know when to grow and when to die.
Some of these signaling molecules also function in an adhesive capacity — not just relaying messages between the cells in a tissue, but physically joining these cells to one another. Cell Biology for Seminars, Unit 4.
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