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Differential sensitivity of epithelial cells to extracellular matrix in polarity establishment.

Yonemura S - PLoS ONE (2014)

Bottom Line: In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel.In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens.The three lines also had similar cellular responses to ECM secreted by the cells themselves.

View Article: PubMed Central - PubMed

Affiliation: Electron Microscope Laboratory, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan; CREST, Japan Science and Technology Agency, Kobe, Hyogo, Japan.

ABSTRACT
Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D) culture systems rather than in two-dimensional (2-D) culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM) are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules), EpH4 cells (mouse mammary gland), and R2/7 cells (human colon) expressing wild-type α-catenin (R2/7 α-Cate cells). These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.

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Apical-basal polarity formation of epithelial spheroids in hanging drops.Spheroids of MDCK II, EpH4 and R2/7 α-Cate cells after 1.5–2 day culture in hanging drops. In this case, the ECM deposited by those cells is considered to be concentrated within the spheroids. Spheroids were stained for DNA (yellow), ZO-1 (blue), Scrib (red) and PKC zeta (green). Series of confocal optical sections were obtained and projected onto a single plane. In MDCK II cells PKC zeta accumulated on the surface of the spheroid (yellow arrowhead). A ZO-1 network was also found at the surface (yellow arrow). The epithelial cell polarity is opposite to that of cells in the ECM gels. EpH4 and R2/7 α-Cate cells showed similar protein distributions, and also showed similar polarities. Bar, 10 µm.
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pone-0112922-g005: Apical-basal polarity formation of epithelial spheroids in hanging drops.Spheroids of MDCK II, EpH4 and R2/7 α-Cate cells after 1.5–2 day culture in hanging drops. In this case, the ECM deposited by those cells is considered to be concentrated within the spheroids. Spheroids were stained for DNA (yellow), ZO-1 (blue), Scrib (red) and PKC zeta (green). Series of confocal optical sections were obtained and projected onto a single plane. In MDCK II cells PKC zeta accumulated on the surface of the spheroid (yellow arrowhead). A ZO-1 network was also found at the surface (yellow arrow). The epithelial cell polarity is opposite to that of cells in the ECM gels. EpH4 and R2/7 α-Cate cells showed similar protein distributions, and also showed similar polarities. Bar, 10 µm.

Mentions: To observe 3-D morphogenesis of epithelial cells dependent on the ECM they secrete themselves, we also tried the hanging drop culture method (Fig. 1C). In a hanging drop of culture medium, cells gather at the interface between the bottom of the drop and the surrounding air due to forces of gravity and then form an aggregate. The concentration of ECM secreted by the cells was considered to be high in the center of the aggregate and low in the culture medium. Cells were cultured for 2 days, and the resulting spheroids were collected, fixed, stained with several antibodies, and imaged by confocal microscopy (Fig. 5). As expected, the outer surfaces of the spheroids were judged to be apical membrane based on PKC zeta accumulation and the existence of tight junction networks in all cases, indicating that the direction of apical-basal polarity is opposite to that seen in spheroids formed when cultured in ECM gels. There was also no obvious interior space in the spheroids. EpH4 cells showed a clear apical-basal polarity similar to other two cell types with ECM found in the interior of the spheroid, indicating that ECM molecules secreted specifically by EpH4 cells, but not contained in Matrigel or collagen gel, are responsible for the determination of apical-basal polarity in EpH4 cells.


Differential sensitivity of epithelial cells to extracellular matrix in polarity establishment.

Yonemura S - PLoS ONE (2014)

Apical-basal polarity formation of epithelial spheroids in hanging drops.Spheroids of MDCK II, EpH4 and R2/7 α-Cate cells after 1.5–2 day culture in hanging drops. In this case, the ECM deposited by those cells is considered to be concentrated within the spheroids. Spheroids were stained for DNA (yellow), ZO-1 (blue), Scrib (red) and PKC zeta (green). Series of confocal optical sections were obtained and projected onto a single plane. In MDCK II cells PKC zeta accumulated on the surface of the spheroid (yellow arrowhead). A ZO-1 network was also found at the surface (yellow arrow). The epithelial cell polarity is opposite to that of cells in the ECM gels. EpH4 and R2/7 α-Cate cells showed similar protein distributions, and also showed similar polarities. Bar, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4231087&req=5

pone-0112922-g005: Apical-basal polarity formation of epithelial spheroids in hanging drops.Spheroids of MDCK II, EpH4 and R2/7 α-Cate cells after 1.5–2 day culture in hanging drops. In this case, the ECM deposited by those cells is considered to be concentrated within the spheroids. Spheroids were stained for DNA (yellow), ZO-1 (blue), Scrib (red) and PKC zeta (green). Series of confocal optical sections were obtained and projected onto a single plane. In MDCK II cells PKC zeta accumulated on the surface of the spheroid (yellow arrowhead). A ZO-1 network was also found at the surface (yellow arrow). The epithelial cell polarity is opposite to that of cells in the ECM gels. EpH4 and R2/7 α-Cate cells showed similar protein distributions, and also showed similar polarities. Bar, 10 µm.
Mentions: To observe 3-D morphogenesis of epithelial cells dependent on the ECM they secrete themselves, we also tried the hanging drop culture method (Fig. 1C). In a hanging drop of culture medium, cells gather at the interface between the bottom of the drop and the surrounding air due to forces of gravity and then form an aggregate. The concentration of ECM secreted by the cells was considered to be high in the center of the aggregate and low in the culture medium. Cells were cultured for 2 days, and the resulting spheroids were collected, fixed, stained with several antibodies, and imaged by confocal microscopy (Fig. 5). As expected, the outer surfaces of the spheroids were judged to be apical membrane based on PKC zeta accumulation and the existence of tight junction networks in all cases, indicating that the direction of apical-basal polarity is opposite to that seen in spheroids formed when cultured in ECM gels. There was also no obvious interior space in the spheroids. EpH4 cells showed a clear apical-basal polarity similar to other two cell types with ECM found in the interior of the spheroid, indicating that ECM molecules secreted specifically by EpH4 cells, but not contained in Matrigel or collagen gel, are responsible for the determination of apical-basal polarity in EpH4 cells.

Bottom Line: In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel.In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens.The three lines also had similar cellular responses to ECM secreted by the cells themselves.

View Article: PubMed Central - PubMed

Affiliation: Electron Microscope Laboratory, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan; CREST, Japan Science and Technology Agency, Kobe, Hyogo, Japan.

ABSTRACT
Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D) culture systems rather than in two-dimensional (2-D) culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM) are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules), EpH4 cells (mouse mammary gland), and R2/7 cells (human colon) expressing wild-type α-catenin (R2/7 α-Cate cells). These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.

Show MeSH
Related in: MedlinePlus