Merlin/ERM proteins establish cortical asymmetry and centrosome position

Abstract

The ability to generate asymmetry at the cell cortex underlies cell polarization and asymmetric cell division. Here we demonstrate a novel role for the tumor suppressor Merlin and closely related ERM proteins (Ezrin, Radixin, and Moesin) in generating cortical asymmetry in the absence of external cues. Our data reveal that Merlin functions to restrict the cortical distribution of the actin regulator Ezrin, which in turn positions the interphase centrosome in single epithelial cells and three-dimensional organotypic cultures. In the absence of Merlin, ectopic cortical Ezrin yields mispositioned centrosomes, misoriented spindles, and aberrant epithelial architecture. Furthermore, in tumor cells with centrosome amplification, the failure to restrict cortical Ezrin abolishes centrosome clustering, yielding multipolar mitoses. These data uncover fundamental roles for Merlin/ERM proteins in spatiotemporally organizing the cell cortex and suggest that Merlin's role in restricting cortical Ezrin may contribute to tumorigenesis by disrupting cell polarity, spindle orientation, and, potentially, genome stability.

Figure 1.

Restricted cortical distribution of Ezrin in single Caco2 cells. (A) Ezrin localizes to the cleavage furrow and nascent apical lumen in developing Caco2 cysts. Single Caco2 cells were embedded in Matrigel for 16 h (two-cell stage) or 6 d (cysts) and stained for Ezrin (green), actin (red), and DNA (blue). Unless otherwise indicated, all images shown are single confocal sections through the middle of each structure. Corresponding differential interference contrast (DIC) images are also shown. (B) Cortical Ezrin is progressively restricted into a cap-like structure (arrowheads) that colocalizes with markedly enriched cortical actin in single Caco2 cells. Cells were stained as in A and imaged using a wide-field fluorescence microscope. Images shown represent the stages of capping deduced from the analysis of hundreds of fixed samples processed at defined time points between 0 and 14 h after plating. (C) Apical polarity proteins are not concentrated in the Ezrin cap. Embedded Caco2 cells were stained for Ezrin (green), DNA (blue), and aPKC or Par-3 (red). Apical polarity is initiated following the first cell division (aPKC, two-cell). (D) The Ezrin cap forms prior to S phase. Embedded Caco2 cells were incubated with EdU for 14 h and stained for Ezrin (red), EdU (green), pHH3 (purple), and DNA (blue). Of the ∼53% of cells that had a mature Ezrin cap in this experiment, 85.1% stained positively for EdU and/or pHH3, and 14.9% stained negatively for EdU/pHH3. In contrast, cells with an immature Ezrin cap rarely stained positively for EdU/pHH3. Bars, 10 μm. This figure is related to Supplemental Figure S1.

Figure 2.

Regulation of Ezrin cap formation. (A) Ezrin cap formation is dependent on actin. Single embedded Caco2 cells were cultured with vehicle (DMSO), actin-disrupting compounds (cytochalasin D, jasplakinolide, or latrunculin A), myosin II inhibitors (blebbistatin), Rho kinase inhibitor (Y27632), or the microtubule-depolymerizing drug nocodazole and stained for Ezrin (green), actin (red), and DNA (blue). Note that the image of the blebbistatin-treated cell is intentionally overexposed to confirm blebbistatin activity. Cells were imaged using a wide-field fluorescence microscope. (B) The Ezrin cap contains active C-terminally phosphorylated ERMs (pERM). Single embedded Caco2 cells were fixed with TCA to preserve the pERM epitope and stained for Ezrin (green), pERM (red), and DNA (blue). (C) Neither MST4 nor the related kinases SLK and LOK are necessary for Ezrin cap formation. Cells infected with lentiviruses expressing control (scrambled; shSCR) or MST4, SLK, or LOK targeted shRNAs were subjected to immunoblotting analysis of Ezrin cap formation. (D) The pan-CDK inhibitor roscovitine prevents cortical Ezrin localization and cap formation. Single embedded Caco2 cells were cultured with 25 μM roscovitine for 14 h, then stained for Ezrin (green), actin (red), and DNA (blue). (E) CDK5 is required for Ezrin cortical localization and cap formation. Cells were stably infected with lentiviruses expressing control (shSCR) or two short hairpins targeting CDK5 (shCDK5.2 and shCDK5.3). Most shCDK5.2-expressing cells do not form Ezrin caps, while shCDK5.3-expressing cells exhibit a mild defect in cap formation, consistent with the weak knockdown achieved with shCDK5.3. Values shown are mean ± SEM. (**) P < 0.01. Bars, 10 μm. This figure is related to Supplemental Figure S2.

Figure 3.

Merlin is required for Ezrin cap formation. (A) Merlin exhibits uniform cortical localization in cells harboring a mature Ezrin cap. Cells expressing Flag-tagged versions of wild-type mouse Merlin (mNf2^wt) or mNf2^18–595 were stained for endogenous Ezrin (green), Merlin (red), and DNA (blue). (B) Endogenous Merlin is required for Ezrin cap formation. Caco2 cells stably expressing control (shSCR) or NF2 targeted shRNAs (shNF2) were stained for Ezrin (green) and DNA (blue). The immunoblot confirms the loss of Merlin. (C) Ezrin capping was rescued by reintroduction of wild-type or membrane-tethered Merlin. CaCo2 cells stably expressing shSCR or shNF2 were infected with lentiviruses expressing shNF2-resistant wild-type (hNF2^wt) or myristoylated (hNF2^myr) human Merlin. The immunoblot confirms the loss of endogenous Merlin and expression of exogenous hNF2^wt and hNF2^myr. (D) mNf2^18–595 did not rescue Ezrin cap formation. Cells stably expressing shSCR or shNF2 were infected with lentiviruses expressing mouse mNf2^wt or mNf2^18–595. The immunoblot confirms loss of Merlin and expression of exogenous mNf2^wt and mNf2^18–595. (E) α-Catenin is required for Ezrin cap formation. Cells expressing control (shSCR) or α-catenin targeted (shα-cat) shRNAs were stained for Ezrin (green), actin (red), and DNA (blue) and monitored for Ezrin cap formation. The immunoblot confirms the loss of α-catenin protein. Values shown are mean ± SEM. (**) P < 0.01. Bars, 10 μm. This figure is related to Supplemental Figure S3.

Figure 4.

Cortical Ezrin controls centrosome position. (A) Centrosome position correlates with cortical Ezrin. Cells were stained for Ezrin (green), Pericentrin (red), and DNA (blue) and imaged using a wide-field fluorescence microscope. Images shown represent the stages of capping deduced from the analysis of hundreds of fixed samples processed at defined time points between 0 and 17 h after plating. (B) In single cells, one spindle pole always localizes beneath the Ezrin cap. Cells were stained for Ezrin (red), α-tubulin (green), and DNA (blue). The angles between the spindle axis (dashed line) and a line connecting the center of the Ezrin cap to the center of the spindle (solid line) were measured using Zen software and are depicted by the scatter plot at the left. (C) Centrosomes localize beneath ectopic cortical Ezrin. Control (shSCR) or shNF2-expressing cells were stained for Ezrin (green), Pericentrin (red), and DNA (blue). (D) Microtubules are required for the positioning of centrosomes beneath cortical Ezrin. Cells were treated with vehicle (DMSO) or nocodazole (20 μM) and stained for Ezrin (green), Pericentrin (red), or DNA (blue). (E) APC2 is required for the positioning of centrosomes beneath cortical Ezrin. Control (shSCR) or shAPC2-expressing cells were stained for Ezrin (green), Pericentrin (red), and DNA (blue). Identical results were obtained with a second shAPC2 hairpin (not shown). Bars, 10 μm. This figure is related to Supplemental Figure S4.

Figure 5.

Merlin/ERMs are required for the development of cysts containing a single central lumen. (A) Ectopic Ezrin persists after the first cell division in developing shNF2-expressing cysts. Two-cell stage cysts expressing shSCR or shNF2 were stained for Ezrin (green), Pericentrin (red), and DNA (blue). In contrast to controls, Ezrin is not restricted to the cleavage furrow/nascent lumen in the absence of Merlin. Two optical Z sections are shown so that centrosomes from both cells can be seen. The cell on the left lacks ectopic Ezrin and the centrosome positions normally beneath the nascent lumen; the centrosome in the cell on the right is closely associated with ectopic cortical Ezrin. (B) Ectopic cortical Ezrin is associated with aberrant spindle orientation. shSCR- and shNF2-expressing cells were stained for actin (red), α-tubulin (green), Pericentrin (purple), and DNA (blue). (Schematic) Confocal images spanning both spindle poles were used to measure the angle between the spindle axis and a line connecting the apical surface of the cyst to the center of the spindle using Zen software. (C) Loss of Merlin yields cysts with multiple lumens. Cysts were stained for Ezrin (green), actin (red), aPKC (purple), and DNA (blue). Control cells (shSCR) developed into cysts with a single lumen, but cells expressing shNF2 developed into cysts with multiple lumens. The immunoblot confirms the loss of Merlin expression. (D) Dominant-negative Ezrin interferes with capping, centrosome positioning, and cyst formation. Cells infected with control or Flag-Ezr^Δact were cultured for 14 h (one- and two-cell structures) or 6 d (cysts). Single cells were stained for Flag-Ezr^Δact (green), actin (red), Pericentrin (purple), and DNA (blue), while two-cell and cyst structures were stained for Ezrin (green; detects Ezrin and Flag-Ezr^Δact) or Flag-Ezr^Δact (green), Pericentrin (red), and DNA (blue). Ezr^Δact-expressing cysts fail to form lumens and localize Ezr^Δact, actin and aPKC, which are normally apical, to the outer membrane that contacts the Matrigel. The immunoblot confirms the expression of Ezr^Δact. Bars: one- to two-cell structures, 10 μm; cysts, 20 μm. (E, top) Schematic showing how the restriction of cortical Ezrin (green) positions the interphase centrosome (red) and ensuing mitotic spindle in single control cells and in the derivative cyst with a single lumen. (Bottom) In the absence of Merlin, ectopic cortical Ezrin yields mispositioned centrosomes, misoriented spindles, and cysts with multiple lumens. Values shown are mean ± SEM. (**) P < 0.01. This figure is related to Supplemental Figure S5.

Figure 6.

Ectopic Ezrin localization in Nf2-deficient tissues in vivo. (A) In contrast to the adult mouse epidermis, where Ezrin normally concentrates at the apical surface of certain basal cells (arrowhead), Ezrin often prominently localizes to lateral boundaries between basal cells of the Nf2-deficient skin (arrowhead). (B) Ezrin is apically restricted in wild-type proximal tubules of the kidney but is mislocalized prior to the formation of renal adenomas in the Nf2^−/− kidney. (C) After repair of chemically induced damage, Ezrin localization is restored to the apical surface in the wild-type but not in the Nf2^−/− colonic epithelium, which exhibits ectopic Ezrin localization and becomes progressively multilayered. This figure is related to Supplemental Figure S6.

Figure 7.

Loss of Merlin prevents centrosome clustering. (A) Loss of Merlin yields multipolar spindles in Caco2 cells grown in 3D culture (top) or 2D culture (bottom). Cells were stained for α-tubulin (green), Pericentrin (red), or DNA (blue). The dotted line marks the cyst apical surface. (B) Loss of Merlin yields multipolar spindles in BT-549 mammary tumor cells. BT-549 cells grown in 2D cultures were stained for α-tubulin (green), Centrin-2 (red), and DNA (blue). (C) IST-MES1 human mesothelioma cells do not express full-length Merlin protein and exhibit frequent multipolar spindle formation; re-expression of full-length NF2^wt but not the control vector significantly reduces multipolar spindle formation in these cells. For all experiments, centrosome clustering was also scored, with essentially identical results (not shown). Bars, 10 μm. n = number of mitoses scored. Values shown are mean ± SEM. (**) P < 0.01; (*) P < 0.05. This figure is related to Supplemental Figure S7.