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Fluid shear triggers microvilli formation via mechanosensitive activation of TRPV6.

Miura S, Sato K, Kato-Negishi M, Teshima T, Takeuchi S - Nat Commun (2015)

Bottom Line: Here we demonstrate that fluid shear stress (FSS), an external mechanical cue, serves as a trigger for microvilli formation in human placental trophoblastic cells.We further reveal that the transient receptor potential, vanilloid family type-6 (TRPV6) calcium ion channel plays a critical role in flow-induced Ca(2+) influx and microvilli formation.TRPV6 regulates phosphorylation of Ezrin via a Ca(2+)-dependent phosphorylation of Akt; this molecular event is necessary for microvillar localization of Ezrin in response to FSS.

View Article: PubMed Central - PubMed

Affiliation: Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

ABSTRACT
Microvilli are cellular membrane protrusions present on differentiated epithelial cells, which can sense and interact with the surrounding fluid environment. Biochemical and genetic approaches have identified a set of factors involved in microvilli formation; however, the underlying extrinsic regulatory mechanism of microvilli formation remains largely unknown. Here we demonstrate that fluid shear stress (FSS), an external mechanical cue, serves as a trigger for microvilli formation in human placental trophoblastic cells. We further reveal that the transient receptor potential, vanilloid family type-6 (TRPV6) calcium ion channel plays a critical role in flow-induced Ca(2+) influx and microvilli formation. TRPV6 regulates phosphorylation of Ezrin via a Ca(2+)-dependent phosphorylation of Akt; this molecular event is necessary for microvillar localization of Ezrin in response to FSS. Our findings provide molecular insight into the microvilli-mediated mechanoresponsive cellular functions, such as epithelial absorption, signal perception and mechanotransduction.

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FSS-induced microvilli formation in trophoblastic cells.(a–c) Scanning electron microscopy surface images of BeWo cells cultured under static or flow conditions. Cells were seeded in the chamber area of the device and cultured overnight with or without medium perfusion (5 μl min−1) in both channels. For the flow-exposed cells, images were captured at the centre (low FSS) or inlet (high FSS) area of the chamber. The boxed areas in a–c are magnified in a′–c′, respectively. Scale bars, 20 μm (a–c) or 5 μm (a′–c′). (d) Quantification of microvilli. Total length of microvilli per field was measured from the SEM images (700 μm2, five fields) as described in the Methods. The data represent the mean±s.d.; ***P<0.001, analysis of variance. Representative of three independent experiments. (e,f) Time course analysis of FSS-induced microvilli formation. BeWo cells were cultured under FSS (5 μl min−1) for 12 h and then cultured without medium perfusion for additional 4 h. Culture medium was perfused only in the maternal channel/chamber (solid diamonds) or only in the fetal channel (open diamond). The cells were fixed at the indicated time points, and total length of microvilli per field (2,800 μm2, five fields) was analysed (e). The data represent the mean±s.d. The experiment was repeated twice with similar results. Note that numerous microvillar sprouts were observed 1 h after medium perfusion (f). Scale bar, 5 μm. (g–i) SEM images of HVTs cultured overnight with or without medium perfusion (2 μl min−1) in both channels. Representative images (g,h) were captured at the inlet area of the chamber, and total length of microvilli per field (11,200 μm2, five fields) was measured (i). Scale bar, 20 μm. The data represent the mean±s.d.; ***P<0.001, Student's t-test.
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f2: FSS-induced microvilli formation in trophoblastic cells.(a–c) Scanning electron microscopy surface images of BeWo cells cultured under static or flow conditions. Cells were seeded in the chamber area of the device and cultured overnight with or without medium perfusion (5 μl min−1) in both channels. For the flow-exposed cells, images were captured at the centre (low FSS) or inlet (high FSS) area of the chamber. The boxed areas in a–c are magnified in a′–c′, respectively. Scale bars, 20 μm (a–c) or 5 μm (a′–c′). (d) Quantification of microvilli. Total length of microvilli per field was measured from the SEM images (700 μm2, five fields) as described in the Methods. The data represent the mean±s.d.; ***P<0.001, analysis of variance. Representative of three independent experiments. (e,f) Time course analysis of FSS-induced microvilli formation. BeWo cells were cultured under FSS (5 μl min−1) for 12 h and then cultured without medium perfusion for additional 4 h. Culture medium was perfused only in the maternal channel/chamber (solid diamonds) or only in the fetal channel (open diamond). The cells were fixed at the indicated time points, and total length of microvilli per field (2,800 μm2, five fields) was analysed (e). The data represent the mean±s.d. The experiment was repeated twice with similar results. Note that numerous microvillar sprouts were observed 1 h after medium perfusion (f). Scale bar, 5 μm. (g–i) SEM images of HVTs cultured overnight with or without medium perfusion (2 μl min−1) in both channels. Representative images (g,h) were captured at the inlet area of the chamber, and total length of microvilli per field (11,200 μm2, five fields) was measured (i). Scale bar, 20 μm. The data represent the mean±s.d.; ***P<0.001, Student's t-test.

Mentions: To examine whether FSS induces microvilli formation in placental trophoblastic cells, we cultured BeWo cells under static or fluid flow conditions using our microfluidic device. In the absence of FSS, microvilli were observed at the cell–cell contact sites, but most of the cells had sparse microvillar surfaces (Fig. 2a). In contrast, after the overnight medium perfusion in both of the channels, all cells in the maternal chamber formed microvilli over the entire cell surface (Fig. 2b,c). At the centre of the chamber, where the FSS was low (∼0.001 dyn cm−2), the microvillar protrusions were long (> 2 μm); however, they were shortened (<2 μm) in the area with high FSS (at the inlet or outlet of the chamber, FSS: ∼0.1 dyn cm−2). To quantify the microvilli formation induced by FSS, we measured total length of microvilli/field from the scanning electron microscopy (SEM) images for each FSS condition. Microvilli were increased 10.8-fold at the low-FSS area and 5.6-fold at the high-FSS area compared with the static culture conditions. The measured lengths of microvilli were significantly different between under high- and low-FSS condition (Fig. 2d).


Fluid shear triggers microvilli formation via mechanosensitive activation of TRPV6.

Miura S, Sato K, Kato-Negishi M, Teshima T, Takeuchi S - Nat Commun (2015)

FSS-induced microvilli formation in trophoblastic cells.(a–c) Scanning electron microscopy surface images of BeWo cells cultured under static or flow conditions. Cells were seeded in the chamber area of the device and cultured overnight with or without medium perfusion (5 μl min−1) in both channels. For the flow-exposed cells, images were captured at the centre (low FSS) or inlet (high FSS) area of the chamber. The boxed areas in a–c are magnified in a′–c′, respectively. Scale bars, 20 μm (a–c) or 5 μm (a′–c′). (d) Quantification of microvilli. Total length of microvilli per field was measured from the SEM images (700 μm2, five fields) as described in the Methods. The data represent the mean±s.d.; ***P<0.001, analysis of variance. Representative of three independent experiments. (e,f) Time course analysis of FSS-induced microvilli formation. BeWo cells were cultured under FSS (5 μl min−1) for 12 h and then cultured without medium perfusion for additional 4 h. Culture medium was perfused only in the maternal channel/chamber (solid diamonds) or only in the fetal channel (open diamond). The cells were fixed at the indicated time points, and total length of microvilli per field (2,800 μm2, five fields) was analysed (e). The data represent the mean±s.d. The experiment was repeated twice with similar results. Note that numerous microvillar sprouts were observed 1 h after medium perfusion (f). Scale bar, 5 μm. (g–i) SEM images of HVTs cultured overnight with or without medium perfusion (2 μl min−1) in both channels. Representative images (g,h) were captured at the inlet area of the chamber, and total length of microvilli per field (11,200 μm2, five fields) was measured (i). Scale bar, 20 μm. The data represent the mean±s.d.; ***P<0.001, Student's t-test.
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f2: FSS-induced microvilli formation in trophoblastic cells.(a–c) Scanning electron microscopy surface images of BeWo cells cultured under static or flow conditions. Cells were seeded in the chamber area of the device and cultured overnight with or without medium perfusion (5 μl min−1) in both channels. For the flow-exposed cells, images were captured at the centre (low FSS) or inlet (high FSS) area of the chamber. The boxed areas in a–c are magnified in a′–c′, respectively. Scale bars, 20 μm (a–c) or 5 μm (a′–c′). (d) Quantification of microvilli. Total length of microvilli per field was measured from the SEM images (700 μm2, five fields) as described in the Methods. The data represent the mean±s.d.; ***P<0.001, analysis of variance. Representative of three independent experiments. (e,f) Time course analysis of FSS-induced microvilli formation. BeWo cells were cultured under FSS (5 μl min−1) for 12 h and then cultured without medium perfusion for additional 4 h. Culture medium was perfused only in the maternal channel/chamber (solid diamonds) or only in the fetal channel (open diamond). The cells were fixed at the indicated time points, and total length of microvilli per field (2,800 μm2, five fields) was analysed (e). The data represent the mean±s.d. The experiment was repeated twice with similar results. Note that numerous microvillar sprouts were observed 1 h after medium perfusion (f). Scale bar, 5 μm. (g–i) SEM images of HVTs cultured overnight with or without medium perfusion (2 μl min−1) in both channels. Representative images (g,h) were captured at the inlet area of the chamber, and total length of microvilli per field (11,200 μm2, five fields) was measured (i). Scale bar, 20 μm. The data represent the mean±s.d.; ***P<0.001, Student's t-test.
Mentions: To examine whether FSS induces microvilli formation in placental trophoblastic cells, we cultured BeWo cells under static or fluid flow conditions using our microfluidic device. In the absence of FSS, microvilli were observed at the cell–cell contact sites, but most of the cells had sparse microvillar surfaces (Fig. 2a). In contrast, after the overnight medium perfusion in both of the channels, all cells in the maternal chamber formed microvilli over the entire cell surface (Fig. 2b,c). At the centre of the chamber, where the FSS was low (∼0.001 dyn cm−2), the microvillar protrusions were long (> 2 μm); however, they were shortened (<2 μm) in the area with high FSS (at the inlet or outlet of the chamber, FSS: ∼0.1 dyn cm−2). To quantify the microvilli formation induced by FSS, we measured total length of microvilli/field from the scanning electron microscopy (SEM) images for each FSS condition. Microvilli were increased 10.8-fold at the low-FSS area and 5.6-fold at the high-FSS area compared with the static culture conditions. The measured lengths of microvilli were significantly different between under high- and low-FSS condition (Fig. 2d).

Bottom Line: Here we demonstrate that fluid shear stress (FSS), an external mechanical cue, serves as a trigger for microvilli formation in human placental trophoblastic cells.We further reveal that the transient receptor potential, vanilloid family type-6 (TRPV6) calcium ion channel plays a critical role in flow-induced Ca(2+) influx and microvilli formation.TRPV6 regulates phosphorylation of Ezrin via a Ca(2+)-dependent phosphorylation of Akt; this molecular event is necessary for microvillar localization of Ezrin in response to FSS.

View Article: PubMed Central - PubMed

Affiliation: Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

ABSTRACT
Microvilli are cellular membrane protrusions present on differentiated epithelial cells, which can sense and interact with the surrounding fluid environment. Biochemical and genetic approaches have identified a set of factors involved in microvilli formation; however, the underlying extrinsic regulatory mechanism of microvilli formation remains largely unknown. Here we demonstrate that fluid shear stress (FSS), an external mechanical cue, serves as a trigger for microvilli formation in human placental trophoblastic cells. We further reveal that the transient receptor potential, vanilloid family type-6 (TRPV6) calcium ion channel plays a critical role in flow-induced Ca(2+) influx and microvilli formation. TRPV6 regulates phosphorylation of Ezrin via a Ca(2+)-dependent phosphorylation of Akt; this molecular event is necessary for microvillar localization of Ezrin in response to FSS. Our findings provide molecular insight into the microvilli-mediated mechanoresponsive cellular functions, such as epithelial absorption, signal perception and mechanotransduction.

Show MeSH
Related in: MedlinePlus