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22 pages, 5534 KiB

Open AccessArticle

From Microspikes to Stress Fibers: Actin Remodeling in Breast Acini Drives Myosin II-Mediated Basement Membrane Invasion

by Julian Eschenbruch, Georg Dreissen, Ronald Springer, Jens Konrad, Rudolf Merkel, Bernd Hoffmann and Erik Noetzel

Cells 2021, 10(8), 1979; https://doi.org/10.3390/cells10081979 - 4 Aug 2021

Cited by 8 | Viewed by 3323

Abstract

The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and [...] Read more.

The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and soluble EGF stimulation. Traction force microscopy quantified the mechanical BM stresses that invasion-triggered acini exerted on the BM–ECM interface. We demonstrate that acini use non-proteolytic actin microspikes as functional precursors of elongated protrusions to initiate BM penetration and ECM probing. Further, these microspikes mechanically widened the collagen IV pores to anchor within the BM scaffold via force-transmitting focal adhesions. Pre-invasive basal cells located at the BM–ECM interface exhibited predominantly cortical actin networks and actin microspikes. In response to pro-invasive conditions, these microspikes accumulated and converted subsequently into highly contractile stress fibers. The phenotypical switch to stress fiber cells matched spatiotemporally with emerging high BM stresses that were driven by actomyosin II contractility. The activation of proteolytic invadopodia with MT1-MMP occurred at later BM invasion stages and only in cells already disseminating into the ECM. Our study demonstrates that BM pore-widening filopodia bridge mechanical ECM probing function and contractility-driven BM weakening. Finally, these EMT-related cytoskeletal adaptations are critical mechanisms inducing the invasive transition of benign breast acini. Full article

(This article belongs to the Special Issue Epithelial Cell Mechanics: From Physiology to Pathology)

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18 pages, 5355 KiB

Open AccessFeature PaperArticle

Skin under Strain: From Epithelial Model Tissues to Adult Epithelia

by Robin Püllen, Jens Konrad, Rudolf Merkel and Bernd Hoffmann

Cells 2021, 10(7), 1834; https://doi.org/10.3390/cells10071834 - 20 Jul 2021

Cited by 9 | Viewed by 3069

Abstract

Formation of a barrier capable of protecting tissue from external damage, chemical factors, and pathogens is one of the main functions of the epidermis. Furthermore, upon development and during aging, mechanoprotective epidermal functions change dramatically. However, comparative studies between embryonic and adult skin [...] Read more.

Formation of a barrier capable of protecting tissue from external damage, chemical factors, and pathogens is one of the main functions of the epidermis. Furthermore, upon development and during aging, mechanoprotective epidermal functions change dramatically. However, comparative studies between embryonic and adult skin in comparison to skin equivalents are still scarce which is especially due to the lack of appropriate measurement systems with sufficient accuracy and long-term tissue compatibility. Our studies fill this gap by developing a combined bioreactor and tensile testing machine for biomechanical analysis of living epithelia. Based on this tissue stretcher, our data clearly show that viscoelastic and plastic deformation behavior of embryonic and adult skin differ significantly. Tissue responses to static strain compared to cyclic strain also show a clear dependence on differentiation stage. Multilayered unkeratinized epidermis equivalents, on the other hand, respond very similar to mechanical stretch as adult tissue. This mechanical similarity is even more evident after a single cycle of mechanical preconditioning. Our studies therefore suggest that skin equivalents are well suited model systems to analyze cellular interactions of epidermal cells in natural tissues. Full article

(This article belongs to the Special Issue Epithelial Cell Mechanics: From Physiology to Pathology)

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Figure 1
Tissue stretcher setup for tensile measurements. The test specimen is mounted between a stationary fixture and a moving grip that can be locked in position until the holding frame is placed in the chamber unit and the needle is hooked into a titanium wire loop. Afterwards, it is precisely moved by a linear step motor and resulting forces are measured by a load cell. Samples can be kept fully viable in a liquid and temperature-controlled environment during the whole experiment. A front glass plate of the incubation chamber allows low-magnification imaging during experiments. Full article ">Figure 2
Setup calibration: (A) Two different elastomer samples of defined geometry and known Young’s modulus were strained stepwise from 0–0.04, 0.04–0.08 and 0.08–0.12. The resulting forces were measured with a rate of 10 Hz; (B) Taking the sample’s dimensions into consideration, the linear correlation of stress and strain enables the calculation of expected Young’s moduli for every step (indicated in red, green and blue, respectively, in (A) and (B)). Stated here is the mean Young’s modulus of all steps; (C) The sensitivity of the setup was shown by measuring the very small buoyancy correction to the force. To this end, the instrument’s needle was raised in liquid without a sample mounted to the amplitudes used in our experiments. Full article ">Figure 3
Skin model systems to analyze dermal and epidermal mechanical properties. Three models of different maturation state and composition were comparably prepared: (A) Adult rat skin explants were excised from back skin and the test dimension used for analysis previously marked in situ. The mean dermo-epidermal thickness (exemplarily labeled with red line) was manually determined from tissue slices (n = 16). Underlying hypodermal tissue was omitted here; (B) Back skin from embryonic day 18 rats was cut into indicated test dimensions after isolation. Analysis length was set to 10 mm initial length using a C-shaped PVDF frame (gray). Mean tissue thickness was measured from 14 independent sections (n = 14); (C) Similar PVDF frames were used for transfer of multilayered, simplified epidermis equivalents (SEEs). Therefore, the 20 × 20 mm cell sheets were folded 2 times along one axis to obtain the 5 × 20 mm test dimension. The mean cell sheet thickness is indicated (n = 17); (A–C) Right images show mounted samples previous to start of the test protocol. The lateral white PVDF transfer frame in fetal skin and SEE samples were cut after mounting and had therefore no influence on subsequent force measurements. Scale: 2 mm. Red label illustrates the outline of SEE; (D) Viability assay of epidermal cells via immunofluorescence was performed with fetal rat skin after transfer and stretching for at least 4 h. Non-transferred but identically prepared samples served as controls. Fetal skin in positive controls were deliberately killed with azide. Scale: 50 µm. Full article ">Figure 4
Repetitive static stretch protocol uncovers viscoelastic properties and tissue strain recovery: (A) The test protocol provides four cycles of 1 h at strain position followed by 1 h at zero position; (B–D) The stress curves of one representative experiment for each model system are shown. Tissue stress was calculated from the estimated cross-sectional area after isolation and fixation (see <a href="#cells-10-01834-f003" class="html-fig">Figure 3</a>). Data was recorded every 0.1 s; (E) Images on the right were taken at indicated time points after moving back to zero position. The tissue dimension before and during stretch are indicated on the left. The visible bending and length recovery (indicated by red arrowheads) was reproducibly observed over several samples and all repetitive cycles. Full article ">Figure 5
Tissue stress relaxation during repetitive static stretch and rest cycles: (A) To compare tissue behavior for repetitive cycles, force values of every stretch cycle were normalized to its initial peak force; (B–D) The initial stress relaxation curves for each tissue system were plotted as mean in dark color and SD in bright color over time (fetal: n = 3, adult and SEE: n = 4). Full article ">Figure 6
Tissue force recovery upon repetitive static stretch and rest cycles: (A–C) The initial force decay for each tissue system was plotted as mean in dark color and SD in bright color at relative time scale (fetal: n = 3, adult and SEE: n = 4); (D) The tissue force recovery was calculated as mean and SD of the cycle’s peak force divided by the highest, first peak force value. Full article ">Figure 7
Static and cyclic strain affects tissues differently: (A) The different tissue models were subjected to either static stretch with a constant load or trapezoid-cyclic stretch with 0.3 Hz but equal strain velocity and amplitude; (B–D) Normalized force relaxation curves of individual experiments were plotted. Note that both stretch protocols were performed at two different amplitudes. For better visualization, only peak forces of cyclic stretch experiments are shown. Full article ">

15 pages, 4111 KiB

Open AccessArticle

Mechanical Adaptations of Epithelial Cells on Various Protruded Convex Geometries

by Sun-Min Yu, Bo Li, Steve Granick and Yoon-Kyoung Cho

Cells 2020, 9(6), 1434; https://doi.org/10.3390/cells9061434 - 9 Jun 2020

Cited by 6 | Viewed by 4929

Abstract

The shape of epithelial tissue supports physiological functions of organs such as intestinal villi and corneal epithelium. Despite the mounting evidence showing the importance of geometry in tissue microenvironments, the current understanding on how it affects biophysical behaviors of cells is still elusive. [...] Read more.

The shape of epithelial tissue supports physiological functions of organs such as intestinal villi and corneal epithelium. Despite the mounting evidence showing the importance of geometry in tissue microenvironments, the current understanding on how it affects biophysical behaviors of cells is still elusive. Here, we cultured cells on various protruded convex structure such as triangle, square, and circle shape fabricated using two-photon laser lithography and quantitatively analyzed individual cells. Morphological data indicates that epithelial cells can sense the sharpness of the corner by showing the characteristic cell alignments, which was caused by actin contractility. Cell area was mainly influenced by surface convexity, and Rho-activation increased cell area on circle shape. Moreover, we found that intermediate filaments, vimentin, and cytokeratin 8/18, play important roles in growth and adaptation of epithelial cells by enhancing expression level on convex structure depending on the shape. In addition, microtubule building blocks, α-tubulin, was also responded on geometric structure, which indicates that intermediate filaments and microtubule can cooperatively secure mechanical stability of epithelial cells on convex surface. Altogether, the current study will expand our understanding of mechanical adaptations of cells on out-of-plane geometry. Full article

(This article belongs to the Special Issue Epithelial Cell Mechanics: From Physiology to Pathology)

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Figure 1
The shape of epithelial tissue and fabricated convex geometric structure. (A) A schematic of flat and curved epithelium on the designed protruded geometry; triangle, square and circle shape, respectively. The height and diameter of triangle, square, and circle ring structure is fixed as 60 μm. For the cell culture, fibronectin was fully covered on both protruded structure and flat surface. (B) The schematics showing cell orientation (∆θ) on the structure. ∆θ were measured from main axis of cells from the center of each structure (Pa); triangle, square, circle geometry. When the cells are oriented from reference axis from Pa in longitudinal (L) or perpendicular (P) direction, ∆θ equals 0° or 90°, respectively. The scanning electron microscope (SEM) images of the fabricated structure are shown in the bottom panel. Full article ">Figure 2
Adaptive morphology of epithelial cells on convex geometric structure. (A) Representative fluorescence images of MDCK cell morphology on flat and protruded structures. F-actin (green) was stained with phalloidin-FITC. (B) Averaged cell alignments (∆θ) of the cells on corner of triangle structure (navy), square (orange), and circle (red) shape. (C) Averaged ∆θ of MDCK cells on corner and arm of triangle (navy) and square (orange) structure. (D) Averaged area of the cells on corner of triangle structure, square and circle shape. (E) Averaged area of MDCK cells on corner and arm of triangle and square structure. Number of analyzed cells (<math display="inline"><semantics> <mrow> <msub> <mi>N</mi> <mrow> <mi>c</mi> <mi>e</mi> <mi>l</mi> <mi>l</mi> </mrow> </msub> </mrow> </semantics></math>) for flat surface and corner of triangle, square, circle structure was 1014 and 212, 471, 749, respectively (N = 3). <math display="inline"><semantics> <mrow> <msub> <mi>N</mi> <mrow> <mi>c</mi> <mi>e</mi> <mi>l</mi> <mi>l</mi> </mrow> </msub> </mrow> </semantics></math> for arm of triangle and square structure were 412 and 580 (N = 3). Error bars in graph indicate the standard error of mean (S.E.M.). One-way ANOVA test with post-hoc Fisher’s least significant difference (LSD) was used. **** p < 0.0001; *** p < 0.001; * p < 0.05; NS, not significant. Full article ">Figure 3
Actin contractility controls the morphological adaptation of MDCK cells on convex geometric structure. (A) Representative fluorescence images of MDCK cells on the 3D geometry treated with CN03 and Lat-A. F-actin (green) and nucleus (blue) were stained with phalloidin-FITC and 4′,6-diamidino-2-phenylindole (DAPI), respectively. Averaged (B) ∆θ, and (C) area of the cells on corner of triangle (navy), square (orange), and circle (red) structure with CN03 and Lat-A. <math display="inline"><semantics> <mrow> <msub> <mi>N</mi> <mrow> <mi>c</mi> <mi>e</mi> <mi>l</mi> <mi>l</mi> </mrow> </msub> </mrow> </semantics></math> for CN03 treatment on the triangle, square and circle structure were 198, 364, and 430, respectively (N = 3). <math display="inline"><semantics> <mrow> <msub> <mi>N</mi> <mrow> <mi>c</mi> <mi>e</mi> <mi>l</mi> <mi>l</mi> </mrow> </msub> </mrow> </semantics></math> for Lat-A treatment on the triangle, square and circle structure were 287, 343, and 435, respectively (N = 3). Error bars in graph indicate S.E.M. One-way ANOVA test with post-hoc Fisher’s LSD was used. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; NS, not significant. Full article ">Figure 4
Enhanced vimentin expression on convex geometric structure. (A) Representative fluorescence images of vimentin (red) in MDCK cells on the flat and triangle, square, circle structure. (B) The box plots indicate the total intensity of vimentin per individual cells on corner of triangle (navy), square (orange) and circle (red) structure. The result from flat surface is plotted in gray color as a control (N = 3). (C) Mean relative vimentin intensity of the cells on the geometric structure compared to that on the flat surface. <math display="inline"><semantics> <mrow> <msub> <mi>N</mi> <mrow> <mi>c</mi> <mi>e</mi> <mi>l</mi> <mi>l</mi> </mrow> </msub> </mrow> </semantics></math> for flat surface and triangle, square, circle structure was 457 and 306, 473, 919, respectively (N = 3). Error bars in graph indicate S.E.M. One-way ANOVA test with post-hoc Fisher’s LSD was used. **** p < 0.0001; ** p < 0.01; NS, not significant. Full article ">Figure 5
Enhanced cytokeratin 8/18 expression on convex geometric structure. (A) Representative fluorescence images of cytokeratin 8/18 (CK8/18; red) in MDCK cells on the flat and triangle, square, circle structure. (B) The box plots indicate the total intensity of CK8/18 per individual cells on corner of triangle (navy), square (orange), and circle (red) structure. The result from flat surface is plotted in gray color as a control (N = 5). (C) Mean relative CK8/18 intensity of the cells on the geometric structure compared to that on the flat surface. <math display="inline"><semantics> <mrow> <msub> <mi>N</mi> <mrow> <mi>c</mi> <mi>e</mi> <mi>l</mi> <mi>l</mi> </mrow> </msub> </mrow> </semantics></math> for flat surface and triangle, square, circle structure was 4761 and 571, 799, 1324, respectively (N = 5). Error bars in graph indicate S.E.M. One-way ANOVA with post-hoc Fisher’s LSD was used. **** p < 0.0001; ** p < 0.01; NS, non-significant. Full article ">Figure 6
Disturbing vimentin intermediate filaments (IFs) networks influence cell growth and adaptation on convex structure. Representative fluorescence images of MDCK cells after 3 days of incubation without- and with 2mM acrylamide, causing collapse of vimentin filaments organization. F-actin (green) and nucleus (blue) were stained with phalloidin-FITC and DAPI, respectively. Full article ">Figure 7
Enhanced α-tubulin expression on convex geometric structure. (A) Representative fluorescence images of α-tubulin (red) in MDCK cells on the flat, triangle, square, and circle structure. (B) The box plots indicate the total intensity of α-tubulin per individual cells on corner of triangle (navy), square (orange), and circle (red) structure. The result from flat surface is plotted in gray color as a control (N = 6). (C) Mean relative α-tubulin intensity of the cells on the geometric structure compared to that on the flat surface. <math display="inline"><semantics> <mrow> <msub> <mi>N</mi> <mrow> <mi>c</mi> <mi>e</mi> <mi>l</mi> <mi>l</mi> </mrow> </msub> </mrow> </semantics></math> for flat surface and triangle, square, circle structure was 4893 and 603, 704, 1487, respectively (N = 6). Error bars in graph indicate S.E.M. One-way ANOVA with post-hoc Fisher’s LSD was used. **** p < 0.0001; NS, non-significant. Full article ">

12 pages, 3591 KiB

Open AccessArticle

Transcriptional and Ultrastructural Analyses Suggest Novel Insights into Epithelial Barrier Impairment in Celiac Disease

by Agnieszka Sowińska, Yasser Morsy, Elżbieta Czarnowska, Beata Oralewska, Ewa Konopka, Marek Woynarowski, Sylwia Szymańska, Maria Ejmont, Michael Scharl, Joanna B. Bierła, Marcin Wawrzyniak and Bożena Cukrowska

Cells 2020, 9(2), 516; https://doi.org/10.3390/cells9020516 - 24 Feb 2020

Cited by 11 | Viewed by 3547

Abstract

Disruption of epithelial junctional complex (EJC), especially tight junctions (TJ), resulting in increased intestinal permeability, is supposed to activate the enhanced immune response to gluten and to induce the development of celiac disease (CD). This study is aimed to present the role of [...] Read more.

Disruption of epithelial junctional complex (EJC), especially tight junctions (TJ), resulting in increased intestinal permeability, is supposed to activate the enhanced immune response to gluten and to induce the development of celiac disease (CD). This study is aimed to present the role of EJC in CD pathogenesis. To analyze differentially expressed genes the next-generation mRNA sequencing data from CD326+ epithelial cells isolated from non-celiac and celiac patients were involved. Ultrastructural studies with morphometry of EJC were done in potential CD, newly recognized active CD, and non-celiac controls. The transcriptional analysis suggested disturbances of epithelium and the most significant gene ontology enriched terms in epithelial cells from CD patients related to the plasma membrane, extracellular exome, extracellular region, and extracellular space. Ultrastructural analyses showed significantly tighter TJ, anomalies in desmosomes, dilatations of intercellular space, and shorter microvilli in potential and active CD compared to controls. Enterocytes of fetal-like type and significantly wider adherence junctions were observed only in active CD. In conclusion, the results do not support the hypothesis that an increased passage of gluten peptides by unsealing TJ precedes CD development. However, increased intestinal permeability due to abnormality of epithelium might play a role in CD onset. Full article

(This article belongs to the Special Issue Epithelial Cell Mechanics: From Physiology to Pathology)

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Figure 1
Multivariate visualization of the analyzed genes. (a) PCA scatter plot PCA plot showing variance between CD patients’ samples and non-celiac control samples (PC1), and heterogeneity between the five biological replicates in each group (PC2). (b) Volcano plot representing the results of the analysis. Each dot representing one gene, and the blue highlighted genes were significantly differentially expressed (p-value < 0.05) and log fold change cut-off 2. (c) Heatmap shows hierarchical clustering of genes on the left side and the clustering of the samples on the top. The histogram represents the expression data of the significant differentially expressed genes (green is the down-regulated and red is the up-regulated). Full article ">Figure 2
Results of functional enrichment analysis. The outer circle shows a scatter plot of each term, including their corresponding genes (blue dots down-regulated and red dot up-regulated). Z-score indicates the tendency to increase or decrease each of the gene ontology (GO) terms based on the ratio of the differentially expressed genes. Only the significant terms are displayed in the three main categories: (a) biological process, (b) cellular components, and (c) molecular function. Full article ">Figure 3
Ultrastructure of small intestine enterocytes from the non-CD control group, patients with potential and active CD (a). Cross-sections of non-CD enterocytes with anchoring filaments (AF), endosomes (e) and no intracellular dilatations between cells present in the control group. Enterocytes in potential and active CD exhibit numerous endosomes (e) and tubules of an apical canicular system (ACS), and dilated intercellular spaces (*). The length and width of the brush border microvilli (b). Measurements were done with the use of the morphometric iTEM program (Olympus) in 10 selected epithelial areas at a magnification of ×60,000, and at least 3 values/patient were obtained. All measurements are presented. Statistical analysis was performed with the use of one-way ANOVA with Tukey correction for multiple comparisons. * p < 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Full article ">Figure 4
The proximal region of the enterocytes with tight junctions (TJ), adherence junctions (AJ) and desmosomes (D) from the non-CD control group, patients with potential and active CD (a), and ultrastructural features of intercellular junctions (b,c). Desmosomes with an incorrect asymmetrical structure (D*) present in a patient with active CD. The widths (b) and lengths (c) of EJC were measured using the morphometric iTEM program (Olympus) at a magnification of ×60,000. Measurements were done in 10 selected epithelial areas through longitudinally sectioned intercellular junctions, and at least 5 values of each type of junction/patient were obtained. All measurements are presented. Statistical analysis was performed with the use of one-way ANOVA with Tukey correction for multiple comparisons. * p < 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. Full article ">

Review

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19 pages, 3758 KiB

Open AccessReview

How Mechanical Forces Change the Human Endometrium during the Menstrual Cycle in Preparation for Embryo Implantation

by Anna K. Sternberg, Volker U. Buck, Irmgard Classen-Linke and Rudolf E. Leube

Cells 2021, 10(8), 2008; https://doi.org/10.3390/cells10082008 - 6 Aug 2021

Cited by 19 | Viewed by 12729

Abstract

The human endometrium is characterized by exceptional plasticity, as evidenced by rapid growth and differentiation during the menstrual cycle and fast tissue remodeling during early pregnancy. Past work has rarely addressed the role of cellular mechanics in these processes. It is becoming increasingly [...] Read more.

The human endometrium is characterized by exceptional plasticity, as evidenced by rapid growth and differentiation during the menstrual cycle and fast tissue remodeling during early pregnancy. Past work has rarely addressed the role of cellular mechanics in these processes. It is becoming increasingly clear that sensing and responding to mechanical forces are as significant for cell behavior as biochemical signaling. Here, we provide an overview of experimental evidence and concepts that illustrate how mechanical forces influence endometrial cell behavior during the hormone-driven menstrual cycle and prepare the endometrium for embryo implantation. Given the fundamental species differences during implantation, we restrict the review to the human situation. Novel technologies and devices such as 3D multifrequency magnetic resonance elastography, atomic force microscopy, organ-on-a-chip microfluidic systems, stem-cell-derived organoid formation, and complex 3D co-culture systems have propelled the understanding how endometrial receptivity and blastocyst implantation are regulated in the human uterus. Accumulating evidence has shown that junctional adhesion, cytoskeletal rearrangement, and extracellular matrix stiffness affect the local force balance that regulates endometrial differentiation and blastocyst invasion. A focus of this review is on the hormonal regulation of endometrial epithelial cell mechanics. We discuss potential implications for embryo implantation. Full article

(This article belongs to the Special Issue Epithelial Cell Mechanics: From Physiology to Pathology)

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Figure 1
The plasticity and contractility of the human uterus require the constant adjustment of the endometrium to prepare and support embryo implantation and enable subsequent embryo development. The scheme highlights the multilayered wall structure of the uterus and menstrual cycle-dependent endometrial changes. The uterus is positioned in the pelvis below the peritoneal cavity. The inverted cone-shaped organ is connected through the Fallopian tube to the ovaries and opens into the vagina. It consists of three major layers: the outer connective tissue-rich perimetrium, the smooth muscle-containing contractile myometrium, and the inner endometrium. The endometrium can be further subdivided into the permanent stratum basale and the transitory stratum functionale, which is shed during desquamation and rebuilt during the proliferative phase, with changes in differentiation during the secretory phase to prepare for blastocyst implantation during the window of implantation. For further details, see Info Boxes 1 and 2 (<a href="#app1-cells-10-02008" class="html-app">Appendix A</a>). Full article ">Figure 2
Schematic representation of blastocyst adhesion to the endometrial epithelium and subsequent trophoblast invasion into the endometrium. The developing blastocyst—consisting of the fluid-filled blastocoel, the embryoblast encompassing the inner cell mass, and the outer trophoblast cell layer—adheres to the uterine surface epithelium. Adhesion induces processes in both the embryonic blastocyst and the maternal epithelial and stromal cell layers, which result in the transmigration of the trophoblast through the epithelial layer and the invasion of the endometrial stromal compartment. Subsequently, specialized single trophoblast cells, the extravillous trophoblasts, invade the decidua and erode endothelia and glandular epithelia from their respective basal sides. For further details, see Info Box 3 (<a href="#app1-cells-10-02008" class="html-app">Appendix A</a>). Full article ">Figure 3
The schemes highlight endometrial changes affecting endometrial biomechanics during the menstrual cycle in preparation for blastocyst adhesion and invasion. Key features of the endometrium during the proliferative phase are depicted at the top, and key features during the window of implantation are depicted at the bottom. Note the differences in the endometrial epithelial cell layer including cell height, apical surface specializations (e.g., microvilli and pinopodes), cytoskeletal organization, nuclear morphology and position, distribution of lateral cell–cell junctions, and basolateral plasma membrane invaginations. Additionally note the changes in the connective tissue compartment with a reduced fiber content and altered fibroblast morphology during the window of implantation, thus providing a different biomechanical environment for the epithelial cell layer. The adhering blastocyst impacts the epithelium from the apical side. The red arrows indicate different types of forces that act in all spatial directions within and on the endometrial epithelium. They include shear stress from the uterine lumen, hydrostatic pressure from the adhering blastocyst, tensile forces within the epithelium, traction forces resulting from the epithelial–stromal interphase, and intracellular contractile forces. Epithelial viscoelasticity and extracellular matrix stiffness have impacts on force distribution. Full article ">Figure 4
The lateralization of desmosomal cell–cell adhesions coincides with the window of implantation and polarization of endometrial epithelial cell lines. The confocal fluorescence micrographs (inverse presentation; single focal planes are shown in (a–c); the projections of 8 consecutive focal planes shown in (d–f)) reveal anti-desmoplakin (Dsp) reactivity that detects punctate desmosomes in the endometrial epithelial cell layer of the human endometrium obtained at different days of the menstrual cycle (a–c) and in gland-like spheroids (d–f) derived from endometrial adenocarcinoma cell lines with high polarity (HEC-1-A), intermediate polarity (Ishikawa), and low polarity (RL95-2). Note the different distributions of desmoplakin-positive desmosomes along the basolateral plasma membrane (arrows). Lu, lumen. Scale bars: 20 μm (a–c), 10 μm (d–f). The images were modified from [<a href="#B27-cells-10-02008" class="html-bibr">27</a>,<a href="#B31-cells-10-02008" class="html-bibr">31</a>]. Full article ">

20 pages, 1453 KiB

Open AccessFeature PaperReview

Cell Mechanics in Embryoid Bodies

by Kira Zeevaert, Mohamed H. Elsafi Mabrouk, Wolfgang Wagner and Roman Goetzke

Cells 2020, 9(10), 2270; https://doi.org/10.3390/cells9102270 - 11 Oct 2020

Cited by 34 | Viewed by 11508

Abstract

Embryoid bodies (EBs) resemble self-organizing aggregates of pluripotent stem cells that recapitulate some aspects of early embryogenesis. Within few days, the cells undergo a transition from rather homogeneous epithelial-like pluripotent stem cell colonies into a three-dimensional organization of various cell types with multifaceted [...] Read more.

Embryoid bodies (EBs) resemble self-organizing aggregates of pluripotent stem cells that recapitulate some aspects of early embryogenesis. Within few days, the cells undergo a transition from rather homogeneous epithelial-like pluripotent stem cell colonies into a three-dimensional organization of various cell types with multifaceted cell–cell interactions and lumen formation—a process associated with repetitive epithelial-mesenchymal transitions. In the last few years, culture methods have further evolved to better control EB size, growth, cellular composition, and organization—e.g., by the addition of morphogens or different extracellular matrix molecules. There is a growing perception that the mechanical properties, cell mechanics, and cell signaling during EB development are also influenced by physical cues to better guide lineage specification; substrate elasticity and topography are relevant, as well as shear stress and mechanical strain. Epithelial structures outside and inside EBs support the integrity of the cell aggregates and counteract mechanical stress. Furthermore, hydrogels can be used to better control the organization and lineage-specific differentiation of EBs. In this review, we summarize how EB formation is accompanied by a variety of biomechanical parameters that need to be considered for the directed and reproducible self-organization of early cell fate decisions. Full article

(This article belongs to the Special Issue Epithelial Cell Mechanics: From Physiology to Pathology)

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Figure 1
Schematic representation of commonly used methods for the formation of embryoid bodies. (A) EBs are commonly produced from monolayer cultures of pluripotent stem cells using enzymatic treatment to fragment and detach cell colonies from the culture well. (B) Seeding single pluripotent stem cells in hanging drops facilitates the generation of homogeneous EBs. Alternatively, single cells can be spun down in (C) U-shaped bottom wells or (D) AggreWellsTM to facilitate homogeneous cell aggregation. (E) Bioprinting using cell-laden biological ink allows better control over the composition of EBs. Finally, (F) hydrogels provide an elastic 3D environment which allows the spontaneous formation and differentiation of EBs. (G) Exemplary phase contrast images of EBs produced by enzymatic treatment and (H) EBs produced by a Spin-EB assay starting with a specific cell number (D0) to generate aggregates that are more uniform in size (D1). Full article ">Figure 2
Gene expression and morphological changes during the course of EB development. (A) Gene expression patterns of embryoid bodies show simultaneous upregulation of germ layer-specific markers (Brachyury, PAX6, GATA6) and downregulation of pluripotency-related genes (OCT4, NANOG, SOX2) over the course of their differentiation. (B) Morphologically, EBs transition from a dense mass of cells into fluid-filled cavitated structures that can later develop additional appendages. Full article ">Figure 3
Cellular patterning during embryoid body formation. Around day 2, cells on the surface of the EB form a layer of primitive endoderm which exhibits an epithelial morphology with an increased expression of E-cadherin and a sensitive response to FGF signaling. These cells deposit a basement membrane which is rich in laminin and collagen IV. Cells adjacent to the basement membrane receive survival signals, whereas cells without contact undergo caspase-dependent apoptosis. Cavitation inside the EB forms a yolk sac-like structure and results in the formation of a columnar epithelium. Cells between the primitive endoderm and the columnar epithelium are highly responsive to different signaling pathways, including Wnt, Nodal, and BMP signaling. Full article ">

31 pages, 1435 KiB

Open AccessReview

The Intestinal Barrier and Current Techniques for the Assessment of Gut Permeability

by Ida Schoultz and Åsa V. Keita

Cells 2020, 9(8), 1909; https://doi.org/10.3390/cells9081909 - 17 Aug 2020

Cited by 259 | Viewed by 24124

Abstract

The intestinal barrier is essential in human health and constitutes the interface between the outside and the internal milieu of the body. A functional intestinal barrier allows absorption of nutrients and fluids but simultaneously prevents harmful substances like toxins and bacteria from crossing [...] Read more.

The intestinal barrier is essential in human health and constitutes the interface between the outside and the internal milieu of the body. A functional intestinal barrier allows absorption of nutrients and fluids but simultaneously prevents harmful substances like toxins and bacteria from crossing the intestinal epithelium and reaching the body. An altered intestinal permeability, a sign of a perturbed barrier function, has during the last decade been associated with several chronic conditions, including diseases originating in the gastrointestinal tract but also diseases such as Alzheimer and Parkinson disease. This has led to an intensified interest from researchers with diverse backgrounds to perform functional studies of the intestinal barrier in different conditions. Intestinal permeability is defined as the passage of a solute through a simple membrane and can be measured by recording the passage of permeability markers over the epithelium via the paracellular or the transcellular route. The methodological tools to investigate the gut barrier function are rapidly expanding and new methodological approaches are being developed. Here we outline and discuss, in vivo, in vitro and ex vivo techniques and how these methods can be utilized for thorough investigation of the intestinal barrier. Full article

(This article belongs to the Special Issue Epithelial Cell Mechanics: From Physiology to Pathology)

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Graphical abstract
Full article ">Figure 1
A schematic drawing of the intestinal barrier and passage routes across the epithelium. Solutes can pass the intestinal epithelium via either the (A) paracellular route (larger hydrophilic solutes); (B) transcellular route (small hydrophilic and lipophilic solutes) (C); transcellular route via aqueous pores (small hydrophilic solutes) or active carrier-mediated absorption (nutrients); or (D) endocytosis, followed by transcytosis and exocytosis (larger particles, peptides and proteins). The barrier constitutes of (1) the lumen, bacteria and antigens are degraded by biliary juices, gastric and pancreatic acids and the colonization of pathogens is inhibited by commensal bacteria producing antimicrobial substances; (2) the microclimate; unstirred water layer, glycocalyx, bacterial adhesion is prevented by mucus and IgA secretion; (3) the epithelial cells; luminal content is transported while noxious stimuli is impeded by chloride secretion and production of antimicrobial peptides (AMP), junctional complexes between the cells regulate permeability, for details see right panel; (4) the lamina propria; immunoglobulins and cytokines are secreted from cells of both the innate and acquired immunity with direct or indirect effects on permeability, interactions with the endocrine and enteric nervous system. TAMP: tight junction-associated-MARVEL proteins including occludin, tricellulin and Marvel D3; JAM: junctional adhesion molecule; MLC: myosin light chain; MLCK: MLC of myosin II kinase. Full article ">Figure 2
Overview of the different techniques used to measure intestinal barrier function. It is important to consider the aim of the study and the resources in the laboratory prior choosing the methodology. For a thorough assessment of intestinal barrier function the techniques are preferably combined, for example in vivo and ex vivo techniques can be combined with in vitro studies for a more mechanistic approach. TER: transepithelial resistance. Full article ">

27 pages, 1685 KiB

Open AccessReview

Ciliary Genes in Renal Cystic Diseases

by Anna Adamiok-Ostrowska and Agnieszka Piekiełko-Witkowska

Cells 2020, 9(4), 907; https://doi.org/10.3390/cells9040907 - 8 Apr 2020

Cited by 22 | Viewed by 8762

Abstract

Cilia are microtubule-based organelles, protruding from the apical cell surface and anchoring to the cytoskeleton. Primary (nonmotile) cilia of the kidney act as mechanosensors of nephron cells, responding to fluid movements by triggering signal transduction. The impaired functioning of primary cilia leads to [...] Read more.

Cilia are microtubule-based organelles, protruding from the apical cell surface and anchoring to the cytoskeleton. Primary (nonmotile) cilia of the kidney act as mechanosensors of nephron cells, responding to fluid movements by triggering signal transduction. The impaired functioning of primary cilia leads to formation of cysts which in turn contribute to development of diverse renal diseases, including kidney ciliopathies and renal cancer. Here, we review current knowledge on the role of ciliary genes in kidney ciliopathies and renal cell carcinoma (RCC). Special focus is given on the impact of mutations and altered expression of ciliary genes (e.g., encoding polycystins, nephrocystins, Bardet-Biedl syndrome (BBS) proteins, ALS1, Oral-facial-digital syndrome 1 (OFD1) and others) in polycystic kidney disease and nephronophthisis, as well as rare genetic disorders, including syndromes of Joubert, Meckel-Gruber, Bardet-Biedl, Senior-Loken, Alström, Orofaciodigital syndrome type I and cranioectodermal dysplasia. We also show that RCC and classic kidney ciliopathies share commonly disturbed genes affecting cilia function, including VHL (von Hippel-Lindau tumor suppressor), PKD1 (polycystin 1, transient receptor potential channel interacting) and PKD2 (polycystin 2, transient receptor potential cation channel). Finally, we discuss the significance of ciliary genes as diagnostic and prognostic markers, as well as therapeutic targets in ciliopathies and cancer. Full article

(This article belongs to the Special Issue Epithelial Cell Mechanics: From Physiology to Pathology)

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