XB-ART-52993J Cell Sci 2017 Jan 15;1302:420-428. doi: 10.1242/jcs.194704.
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RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells.
Homeostatic replacement of epithelial cells from basal precursors is a multistep process involving progenitor cell specification, radial intercalation and, finally, apical surface emergence. Recent data demonstrate that actin-based pushing under the control of the formin protein Fmn1 drives apical emergence in nascent multiciliated epithelial cells (MCCs), but little else is known about this actin network or the control of Fmn1. Here, we explore the role of the small GTPase RhoA in MCC apical emergence. Disruption of RhoA function reduced the rate of apical surface expansion and decreased the final size of the apical domain. Analysis of cell shapes suggests that RhoA alters the balance of forces exerted on the MCC apical surface. Finally, quantitative time-lapse imaging and fluorescence recovery after photobleaching studies argue that RhoA works in concert with Fmn1 to control assembly of the specialized apical actin network in MCCs. These data provide new molecular insights into epithelial apical surface assembly and could also shed light on mechanisms of apical lumen formation.
PubMed ID: 28089989
PMC ID: PMC5278671
Article link: J Cell Sci
Species referenced: Xenopus
Genes referenced: fmn1 mcc mtor rhoa tuba4b utrn
Morpholinos: fmn1 MO2
Article Images: [+] show captions
|Fig. 1. Active RhoA localizes to the apical domain of emerging MCCs. (A) Schematic of MCC morphogenesis. Upon specification of an MCC progenitor (1), a nascent MCC radially intercalates (2, 3) and docks at the tricellular junction to finally integrate with the pre-existing epithelium by expanding its apical surface (3, 4, apical emergence). (B) Image sequence of an active RhoA biosensor (rGBD, green) throughout apical emergence of a single unmanipulated cell. (C)Corresponding image of an apicallyemergingMCC(visualized using an actin marker, utrophin,GFP–UtrCH, gray, driven by anMCC-specific α-tubulin promoter). (D) Representative plot of medial actin and active RhoA dynamics during apical emergence. (E) Values of the intensity of active RhoA for consecutive categories of apical domain sizes, categorized by the binnedmean radius in controls. Data representmean and s.e.m., n=4 cells fromfour embryos. Actin and rGDB intensities were normalized by dividing the mean intensities of medial actin and/or rGBD by the mean intensities of actin and/or rGBD within the cortical region. Scale bars: 10 μm.|
|Fig. 2. RhoA controls the dynamics of MCC apical emergence. (A) Image sequence of an apically emerging MCC (visualized using an actin marker, utrophin, GFP–UtrCH, gray, driven by an MCC-specific α-tubulin promoter) in controls, (B) upon expression of a constitutively active RhoA construct (CA-RhoA) and (C) upon expression of dominant negative RhoA (DN-RhoA). CA-RhoA and DN-RhoA were expressed in MCCs under an MCC-specific α-tubulin promoter, see Fig. S1. Quantification of MCC apical surface area (e.g. dotted yellow line in A) is indicated in μm2 in the bottom left corner of each panel. Note that for a given apical domain surface, an apical domain of an MCC expressing DN-RhoA has a more polygonal shape compared to that of controls and to an MCC expressing CA-RhoA. (Note that an image in Fig. 2A is replicated in Fig. 4B; see Fig. 4 legend for details). The time after the start of imaging is indicated. (D) Dynamics of the apical domain area of the MCCs shown in A–C; controls, black; MCC expressing CA-RhoA, green; MCC expressing DN-RhoA, pink. (E) Expansion rate of an MCC apical domain in controls (black) and upon expression of CA-RhoA (green) or of DN-RhoA (magenta). The expansion ratewas determined by measuring the slope of the linear fit of the surface area data between 50 μm2 and 150 μm2. (F) The final apical domain areas of MCCs under the conditions described in E. Boxes extend from the 25th to 75th percentiles, with a line at the median. Whiskers represent the minimum and maximum values. ***P<0.001; n.s., not significant (Mann–Whitney U test). n>5 embryos; n values on the graph represent the number of cells analyzed. Scale bar: 10 μm.|
|Fig. 3. RhoA controls force balance in the apical surface during MCC emergence. (A) Schematic of forces acting on an MCC apical domain. Effective 2D pressure, δP (orange arrows), and junctional pulling forces, Λ (blue arrows), acting against cortical tension, γ (black arrows), and elasticity from the surrounding cells, E (red arrows). (B) Kurtosis values for the consecutive categories of apical domain sizes, categorized by binned mean radius, in controls and cells expressing DN-RhoA or CA-RhoA. Data represent mean±s.e.m. ***P<0.0005; n.s., not significant (two-tailed unpaired Student’s t-test). n=10 cells, n>5 embryos. (C) Simulations of apical domain shapes upon expression of DN-RhoA or CA-RhoA. The simulations assumed that only pressure or both pressure and cortical tension were impacted upon expression of the different RhoA constructs. The final shapes of the apical domains are each represented by a different color.|
|Fig. 4. RhoA controls the rate of MCC apical actin network assembly. (A) Schematic of the medial and cortical regions within an apical domain of an MCC. (B) A representative image sequence of an automatically segmented cortical and medial region within an apically expanding MCC (visualized using α-tubulin-promoter-driven GFP– UtrCH, gray). Note that the image here is replicated from Fig. 2A as an example. The time after the start of imaging is indicated. Scale bar: 10 μm. (C) Apical actin concentration (ratio of mean medial to mean cortical actin) dynamics in a control cell (black), an MCC expressing CA-RhoA (green) and an MCC expressing DN-RhoA (magenta). Circles, data points; lines, data smoothed with a moving average of 5. (D) Apical actin concentration as a function of apical area in control cells, in MCCs expressing CA-RhoA and MCCs expressing DN-RhoA; n>10 cells from n>5 embryos. Data represent mean and s.e.m. The slope for MCCs expressing DN-RhoA (pink dotted line) is significantly higher than that for control (P<0.05, Z-test for correlation coefficients), whereas the slope for MCCs expressing CA-RhoA is not significantly different from that of control.|
|Fig. 5. RhoA, FMN1 and Arp2/3 control the rate of turnover of actin in the MCC apical actin network. (A) Image sequence of an MCC apical domain in a control cell and (B) in a cell expressing DN-RhoA, visualized using monomeric YFP–actin upon photobleaching of a region of interest (dotted yellow line) within the medial portion of the apical domain. 0 s indicates the time of photobleaching. (C) Normalized actin recovery after photobleaching (FRAP) of monomeric actin within the medial portion of an apical domain of MCCs: in control cells (black), cells expressing CA-RhoA (green), cells expressing DN-RhoA (red), FMN1-knockdown cells (FMN1 KD; blue) and upon Arp2/3 inhibition with CK666 (orange). Data are mean±s.d. (D) Fluorescence half-time recovery after photobleaching of medial actin in controls (black), MCCs expressing CA-RhoA (green), MCCs expressing DN-RhoA (magenta), FMN1-knockdown cells (blue) and upon Arp2/3 inhibition with CK666 (orange). (E) The mobile fraction measured upon FRAP analysis of medial actin under the conditions described in D. Boxes extend from the 25th to 75th percentiles, with a line at the median. Whiskers indicate minimum and maximum values. *P<0.05; ***P<0.001; ns, not significant (Mann–Whitney U test). n>5 embryos. Scale bars: 10 μm.|
|Fig. 6. Different modes of apical emergence depend on RhoA activity. (A) Upon knockdown of Fmn1 (FMN1 KD), the apical domain collapses as a consequence of low pushing forces and high cortical tension. (B) Expression of DN-RhoA decreases both pushing forces and cortical tension. Depending on the net contribution of these forces, the apical domain (i) expands in an angular manner to an overall smaller size, (ii) undergoes non-periodic oscillations, (iii) collapses. (C) Expression of CA-RhoA does not change the dynamics of apical emergence.|
|Figure S1. RhoA regulates apical emergence dynamics. (A) Image sequence of an apically emerging MCC upon expression of CA-RhoA-RFP specifically in MCC using a MCC-specific -tubulin promoter. (B) Image sequence of an apically emerging MCC upon expression of DN-RhoA-RFP specifically in MCC using a MCC-specific -tubulin promoter. Scale bar, 10 μm.|
|Figure S2. Expression of DN-RhoA impairs the localization and dynamics of formin 1. (A) Image sequence of fluorescently labelled Fmn1 (green) during apical emergence in a control cell. (B) Image sequence of an apically emerging control cell visualized with an actin marker, UtrCH (grey). (C) Image sequence of fluorescently labelled Fmn1 (green) during apical emergence upon expression of DN-RhoA. (D) Image sequence of an apically emerging cell visualized with actin marker, UtrCH (grey) upon expression of DN-RhoA. (E) Kymograph of FMN1 signal during apical expansion of a control cell. (F) Kymograph of actin signal (visualized by UtrCH) in a control cell. (G) Kymograph of FMN1 signal upon expression of DN-RhoA. (H) Kymograph of actin signal (visualized by UtrCH) upon expression of DN-RhoA. (I) Formin 1 and actin (visualized by UtrCH) fluorescence intensity profile along the apical domain diameter (yellow dotted line, starts at 0 and ends at 1) in controls and upon expression of DN-RhoA (J). (K) Dynamics of Fmn1 and actin in controls and upon expression of DN-RhoA (L). Scale bar, 10 μm.|
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