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Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells.

Rouven Brückner B, Pietuch A, Nehls S, Rother J, Janshoff A - Sci Rep (2015)

Bottom Line: For this purpose, we carried out ezrin depletion experiments and also enhanced the number of active ezrin molecules at the interface.Mechanical properties were assessed by force indentation experiments followed by membrane tether extraction.PIP2 micelles were injected into individual living cells to reinforce the linkage between plasma membrane and actin-cortex, while weakening of this connection was reached by ezrin siRNA and administration of the inhibitors neomycin and NSC 668394, respectively.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physical Chemistry, University of Goettingen, Tammannstr. 6, 37077 Goettingen.

ABSTRACT
Plasma membrane tension is responsible for a variety of cellular functions such as motility, cell division, and endocytosis. Since membrane tension is dominated by the attachment of the actin cortex to the inner leaflet of the plasma membrane, we investigated the importance of ezrin, a major cross-linker of the membrane-cytoskeleton interface, for cellular mechanics of confluent MDCK II cells. For this purpose, we carried out ezrin depletion experiments and also enhanced the number of active ezrin molecules at the interface. Mechanical properties were assessed by force indentation experiments followed by membrane tether extraction. PIP2 micelles were injected into individual living cells to reinforce the linkage between plasma membrane and actin-cortex, while weakening of this connection was reached by ezrin siRNA and administration of the inhibitors neomycin and NSC 668394, respectively. We observed substantial stiffening of cells and an increase in membrane tension after addition of PIP2 micelles. In contrast, reduction of active ezrin led to a decrease of membrane tension accompanied by loss of excess surface area, increase in cortical tension, remodelling of actin cytoskeleton, and reduction of cell height. The data confirm the importance of the ezrin-mediated connection between plasma membrane and cortex for cellular mechanics and cell morphology.

No MeSH data available.


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Topography of MDCK II cells after ezrin knock-down via siRNA.(a) Confocal fluorescence micrograph of the xz plane of a confluent cell layer. The plasma membrane is stained with PKH67 Green Fluorescent Cell Linker, F-actin is labeled with Alexa Fluor 546-phalloidin. Cells without ezrin (arrows) were found to be reduced in height compared with not successfully transfected cells. (b) Phase contrast image. (c) Corresponding fluorescence micrograph showing the ezrin distribution. Ezrin is stained with ezrin mouse IgG primary and Alexa Fluor 488 labeled goat anti-mouse IgG secondary antibody. Some cells (green) were not successfully transfected with siRNA and serve as a control. (d) AFM deflection and height image (e) of the same spot shown in (b,c). (f) Height profile along the green/white dotted line. Cells lacking ezrin are substantially flattened. (Scale bar: 20 μm).
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f6: Topography of MDCK II cells after ezrin knock-down via siRNA.(a) Confocal fluorescence micrograph of the xz plane of a confluent cell layer. The plasma membrane is stained with PKH67 Green Fluorescent Cell Linker, F-actin is labeled with Alexa Fluor 546-phalloidin. Cells without ezrin (arrows) were found to be reduced in height compared with not successfully transfected cells. (b) Phase contrast image. (c) Corresponding fluorescence micrograph showing the ezrin distribution. Ezrin is stained with ezrin mouse IgG primary and Alexa Fluor 488 labeled goat anti-mouse IgG secondary antibody. Some cells (green) were not successfully transfected with siRNA and serve as a control. (d) AFM deflection and height image (e) of the same spot shown in (b,c). (f) Height profile along the green/white dotted line. Cells lacking ezrin are substantially flattened. (Scale bar: 20 μm).

Mentions: Enforcing the connection between plasma membrane and underlying actin cortex by adding PIP2 to the inner leaflet of the plasma membrane results in increased membrane tension, overall tension, and actin remodelling. In order to address the reverse effect, a reduction of the actin-membrane attachment sites, we performed three different experiments: masking of PIP2 binding sites for ezrin through microinjection of the antibiotic neomycin (Fig. 4), inhibition of ezrin by adding of NSC 668394 (Fig. 5) and knock-down of ezrin expression by using siRNA (Fig. 6). Neomycin binds to PIP2 and thereby uncouples the membrane from the cytoskeleton42. After microinjection of the antibiotic (Fig. 4a), the cell showed no signal for the ezrin immunostaining (Fig. 4b). AFM images revealed flattening of the cell compared to adjacent neighbours by approximately 1 μm (Fig. 4c/e). Tether pulling from the plasma membrane was perfomed with AFM tips functionalised with Concanavalin A to enable a strong binding to the plasma membrane during retraction of the cantilever. Based on the results described above we expected a significant lower tether rupture force Ftether due to the loss of attachment sites for ezrin. After microinjection of neomycin Ftether drops slightly from (58.5 ± 1.2) pN to (50.0 ± 1.8) pN (Fig. 7c, Supplementary Fig. S4), indicating a weakened connection between plasma membrane and underlying cytoskeleton. Notably, controls shown in figure 7 comprise all cells of this study. Force indentation curves were modeled using an average base radius of R1 = 10 μm and a contact angle of ϕ = 12° to account for the altered cell morphology, i.e. flattening of the apical cap (Fig. 4e). 3 h after microinjection of neomycin we found an increasing overall tension (t0 = (0.65 ± 0.04) mN/m, control: t0 = (0.49 ± 0.01) mN/m) (Fig. 7a, Supplementary Fig. S2). The apparent area compressibility modulus was also found to be enlarged to (control: ) (Fig. 7b, Supplementary Fig. S3) indicating a loss of excess membrane area or detection of other contributions that stiffens the cell.


Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells.

Rouven Brückner B, Pietuch A, Nehls S, Rother J, Janshoff A - Sci Rep (2015)

Topography of MDCK II cells after ezrin knock-down via siRNA.(a) Confocal fluorescence micrograph of the xz plane of a confluent cell layer. The plasma membrane is stained with PKH67 Green Fluorescent Cell Linker, F-actin is labeled with Alexa Fluor 546-phalloidin. Cells without ezrin (arrows) were found to be reduced in height compared with not successfully transfected cells. (b) Phase contrast image. (c) Corresponding fluorescence micrograph showing the ezrin distribution. Ezrin is stained with ezrin mouse IgG primary and Alexa Fluor 488 labeled goat anti-mouse IgG secondary antibody. Some cells (green) were not successfully transfected with siRNA and serve as a control. (d) AFM deflection and height image (e) of the same spot shown in (b,c). (f) Height profile along the green/white dotted line. Cells lacking ezrin are substantially flattened. (Scale bar: 20 μm).
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Related In: Results  -  Collection

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f6: Topography of MDCK II cells after ezrin knock-down via siRNA.(a) Confocal fluorescence micrograph of the xz plane of a confluent cell layer. The plasma membrane is stained with PKH67 Green Fluorescent Cell Linker, F-actin is labeled with Alexa Fluor 546-phalloidin. Cells without ezrin (arrows) were found to be reduced in height compared with not successfully transfected cells. (b) Phase contrast image. (c) Corresponding fluorescence micrograph showing the ezrin distribution. Ezrin is stained with ezrin mouse IgG primary and Alexa Fluor 488 labeled goat anti-mouse IgG secondary antibody. Some cells (green) were not successfully transfected with siRNA and serve as a control. (d) AFM deflection and height image (e) of the same spot shown in (b,c). (f) Height profile along the green/white dotted line. Cells lacking ezrin are substantially flattened. (Scale bar: 20 μm).
Mentions: Enforcing the connection between plasma membrane and underlying actin cortex by adding PIP2 to the inner leaflet of the plasma membrane results in increased membrane tension, overall tension, and actin remodelling. In order to address the reverse effect, a reduction of the actin-membrane attachment sites, we performed three different experiments: masking of PIP2 binding sites for ezrin through microinjection of the antibiotic neomycin (Fig. 4), inhibition of ezrin by adding of NSC 668394 (Fig. 5) and knock-down of ezrin expression by using siRNA (Fig. 6). Neomycin binds to PIP2 and thereby uncouples the membrane from the cytoskeleton42. After microinjection of the antibiotic (Fig. 4a), the cell showed no signal for the ezrin immunostaining (Fig. 4b). AFM images revealed flattening of the cell compared to adjacent neighbours by approximately 1 μm (Fig. 4c/e). Tether pulling from the plasma membrane was perfomed with AFM tips functionalised with Concanavalin A to enable a strong binding to the plasma membrane during retraction of the cantilever. Based on the results described above we expected a significant lower tether rupture force Ftether due to the loss of attachment sites for ezrin. After microinjection of neomycin Ftether drops slightly from (58.5 ± 1.2) pN to (50.0 ± 1.8) pN (Fig. 7c, Supplementary Fig. S4), indicating a weakened connection between plasma membrane and underlying cytoskeleton. Notably, controls shown in figure 7 comprise all cells of this study. Force indentation curves were modeled using an average base radius of R1 = 10 μm and a contact angle of ϕ = 12° to account for the altered cell morphology, i.e. flattening of the apical cap (Fig. 4e). 3 h after microinjection of neomycin we found an increasing overall tension (t0 = (0.65 ± 0.04) mN/m, control: t0 = (0.49 ± 0.01) mN/m) (Fig. 7a, Supplementary Fig. S2). The apparent area compressibility modulus was also found to be enlarged to (control: ) (Fig. 7b, Supplementary Fig. S3) indicating a loss of excess membrane area or detection of other contributions that stiffens the cell.

Bottom Line: For this purpose, we carried out ezrin depletion experiments and also enhanced the number of active ezrin molecules at the interface.Mechanical properties were assessed by force indentation experiments followed by membrane tether extraction.PIP2 micelles were injected into individual living cells to reinforce the linkage between plasma membrane and actin-cortex, while weakening of this connection was reached by ezrin siRNA and administration of the inhibitors neomycin and NSC 668394, respectively.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physical Chemistry, University of Goettingen, Tammannstr. 6, 37077 Goettingen.

ABSTRACT
Plasma membrane tension is responsible for a variety of cellular functions such as motility, cell division, and endocytosis. Since membrane tension is dominated by the attachment of the actin cortex to the inner leaflet of the plasma membrane, we investigated the importance of ezrin, a major cross-linker of the membrane-cytoskeleton interface, for cellular mechanics of confluent MDCK II cells. For this purpose, we carried out ezrin depletion experiments and also enhanced the number of active ezrin molecules at the interface. Mechanical properties were assessed by force indentation experiments followed by membrane tether extraction. PIP2 micelles were injected into individual living cells to reinforce the linkage between plasma membrane and actin-cortex, while weakening of this connection was reached by ezrin siRNA and administration of the inhibitors neomycin and NSC 668394, respectively. We observed substantial stiffening of cells and an increase in membrane tension after addition of PIP2 micelles. In contrast, reduction of active ezrin led to a decrease of membrane tension accompanied by loss of excess surface area, increase in cortical tension, remodelling of actin cytoskeleton, and reduction of cell height. The data confirm the importance of the ezrin-mediated connection between plasma membrane and cortex for cellular mechanics and cell morphology.

No MeSH data available.


Related in: MedlinePlus