A RhoA and Rnd3 cycle regulates reassembly PNAS PLUS during membrane blebbing

Kana Aokia, Fumiyo Maedaa,1, Tomoya Nagasakob,1, Yuki Mochizukia, Seiichi Uchidab, and Junichi Ikenouchia,c,d,2

aDepartment of Biology, Faculty of Sciences, Kyushu University, Fukuoka 819-0395, Japan; bDepartment of Advanced Information Technology, Kyushu University, Fukuoka 819-0395, Japan; cPrecursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan; and dAMED-PRIME, Japan Agency for Medical Research and Development, Tokyo 100-0004, Japan

Edited by Thomas D. Pollard, Yale University, New Haven, CT, and approved February 12, 2016 (received for review January 21, 2016) The actin usually lies beneath the plasma membrane. membrane blebs is promoted by epidermal growth factor receptor When the membrane-associated actin cytoskeleton is transiently kinase substrate 8 (Eps8) and ezrin, and regulated by a RhoA– disrupted or the intracellular pressure is increased, the plasma Rho-associated kinase (ROCK)–Rnd3 feedback loop. membrane detaches from the cortex and protrudes. Such protruded membrane regions are called blebs. However, the molecular mech- Results and Discussion anisms underlying membrane blebbing are poorly understood. This Membrane Blebs Retract from Multiple Sites. We used the study revealed that epidermal growth factor receptor kinase sub- colon carcinoma cell line DLD1 to observe membrane blebbing. strate 8 (Eps8) and ezrin are important regulators of rapid actin When cultured in 2D conditions, DLD1 cells did not exhibit reassembly for the initiation and retraction of protruded blebs. Live- membrane blebbing (Fig. 1A, Left). However, DLD1 cells ac- cell imaging of membrane blebbing revealed that local reassembly tively formed membrane blebs when embedded in a type I col- of actin filaments occurred at Eps8- and activated ezrin-positive foci lagen gel (Fig. 1A, Right). To visualize the process of actin cortex of membrane blebs. Furthermore, we found that a RhoA–ROCK– reassembly in protruded membrane blebs, we established DLD1 Rnd3 feedback loop determined the local reassembly sites of the cells stably expressing both a filamentous actin marker, Lifeact– actin cortex during membrane blebbing. RFP, and a membrane marker, the pleckstrin domain of phospholipase Cδ (PLCδ–PH) tagged with green fluorescent membrane bleb | Rnd3 | Eps8 | actin cortex | cell migration protein (GFP) (Fig. 1B). After expansion of a cytoskeleton-free membrane, actin is ctin filaments usually lie beneath the plasma membrane. progressively recruited to the membrane during the retraction AWhen the plasma membrane detaches from actin filaments, phase. By repeated and careful observations, we observed that spherical protrusions of the membrane, termed blebs, form. the recovery of actin filaments occurred locally from multiple Several lines of recent evidence suggest membrane blebs are independent regions of membrane blebs (Fig. 1C, arrowheads) used for cell migration under both physiological and pathological and actin filaments grew from these sites to cover the entire conditions. For example, migrating primordial germ cells (PGCs) blebbing membrane (Movie S1). We did not detect continuous use membrane blebs to migrate in (1). Similarly, ac- expansion of the actin cortex from the edge of the membrane tively migrating PGCs exhibit membrane blebbing in Drosophila detachment site. These observations are in good agreement with melanogaster embryos (2). Dictyostelium use membrane blebbing those of a previous report, which showed that mDia1, an es- during chemotaxis (3, 4). Thus, membrane blebbing is widely sential actin nucleator for the regrowth of actin filaments at used as a driving force of motility across species (4, 5). In ad- membrane blebs, localizes to the cortex of blebs in a speckle dition, cancer cells use a membrane blebbing-associated mode of pattern (13). We next examined the localization of reg- motility in metastasis (6). Cancer cells migrate without degrading ulatory light chain 1 (MRLC1) tagged with GFP (GFP–MRLC1) (Fig. 1D). GFP–MRLC1 also accumulated at the initiation sites

the matrix by protruding membrane blebs in 3D extracellular matrixes (5, 7). Recently, cell physical confinement, down-reg- of actin cortex reassembly. These data suggest that the retraction ulation of cell adhesion to the extracellular matrix, and up-reg- of blebbing membranes begins from multiple regions of the pro- ulation of intrinsic cortical contraction forces were identified as truded membrane. key conditions for the induction of membrane blebbing-associ- ated cell migration in many cell types including invasive cancer Significance cells (8–10). However, the molecular mechanisms underlying membrane The plasma membrane and the underlying actin cortex show blebbing remain to be elucidated. Membrane blebs are initiated dynamic interactions. When the plasma membrane detaches as a rapid protrusion of the plasma membrane, which is driven from the actin cortex, the plasma membrane protrudes. The either by a change in intracellular hydrostatic pressure after local protruded membrane is called a membrane bleb and is often disruption of membrane–actin cortex interactions or by a local observed during cell migration or cytokinesis. In the present breakdown of actin filaments. Thereafter, actin filaments poly- study, we determined the molecular mechanisms involved in merize beneath the protruded membrane to halt bleb expansion. the reassembly of the actin cortex in membrane blebs using When actin filaments cover the protruded membrane, live-cell imaging. are recruited to these filaments (11). It is unknown how actin cortex reassembly is triggered during these processes (11, 12), Author contributions: J.I. designed research; K.A. and F.M. performed research; T.N. and S.U. contributed new reagents/analytic tools; K.A., F.M., T.N., Y.M., and S.U. analyzed although there are at least two possibilities. One possibility is that data; and J.I. wrote the paper. actin filaments constitutively form beneath the plasma membrane The authors declare no conflict of interest. and the plasma membrane stops extending when actin filaments are This article is a PNAS Direct Submission. sufficiently reconstructed. The other possibility is that actin cortex 1F.M. and T.N. contributed equally to this work. reassembly is triggered by the activation of unidentified signaling 2To whom correspondence should be addressed. Email: [email protected] when the cell senses a cytoskeleton-free plasma membrane region. u.ac.jp. In the present study, we addressed this issue using live-cell This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. imaging and revealed that the rapid recovery of actin filaments at 1073/pnas.1600968113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1600968113 PNAS | Published online March 14, 2016 | E1863–E1871 Downloaded by guest on September 28, 2021 Fig. 1. Eps8 locally accumulates at the initial phase of membrane retraction in the membrane bleb. (A) DLD1 cells exhibit membrane blebbing when cultured in a type I collagen matrix (Right). (Scale bar, 20 μm.) (B and C) Membrane blebbing of DLD1 cells transfected with Lifeact–RFP and GFP-tagged PLCδ–PH. Timing relative to the first image is indicated in white text. Actin cortex reassembly started from multiple sites of the protruded membrane (arrowheads). (Scale bar: B,5μm; C,2μm.) (D) Localization of GFP–MRLC1 during membrane bleb expansion and retraction. MRLC1 accumulates at multiple regions of membrane blebs (arrowheads). (Scale bar, 1 μm.) (E) Localization of GFP-tagged Eps8 in membrane blebs of DLD1 cells. Eps8 accumulates in multiple foci at the protruded membrane (arrowheads). (Scale bar, 2 μm.) (F and G) Kymographs showing actin localization (red) with respect to actin cytoskeleton-related (green) during bleb retraction. Bleb extension is shown on the horizontal axis, and time is shown on the vertical axis. Eps-8 (F, green) localizes to the protruded membrane before actin filaments. MRLC1 (G,green) is recruited after actin filaments. (H) Timing of arrival of Eps8 and MRLC1 relative to that of actin filaments (t = 0 s). Data are the mean ± SD.

E1864 | www.pnas.org/cgi/doi/10.1073/pnas.1600968113 Aoki et al. Downloaded by guest on September 28, 2021 PNAS PLUS CELL BIOLOGY

Fig. 2. The end-capping and actin-bundling activities of Eps8 are required for continuous membrane blebbing. (A) Expression of Eps8 is greatly reduced in Eps8-KD DLD1 cells. (B) Eps8-KD DLD1 cells are spherical and do not exhibit membrane blebbing when cultured in a type I collagen matrix. (Scale bar, 10 μm.) (C) Exogenous expression of GFP-tagged mouse Eps8 restores membrane blebbing (arrow) in Eps8-KD DLD1 cells. (Scale bar, 10 μm.) (D) Schematic drawings of mutant Eps8 constructs. The number of amino acid residues of Eps8 is shown. (E) Total cell lysates of DLD1 cells expressing each construct separated by SDS/ PAGE and immunoblotted with an anti-GFP mAb. (F) The percentages of GFP-positive cells showing membrane blebbing relative to the total number of GFP- positive cells in a given field were calculated. In each experiment, the total cell number was 100 (n = 3). Data are the mean ± SD. *P < 0.05 (Student’s t test). (G) Localization of GFP-tagged mutant Eps8 in Eps8-KD DLD1 cells. Expression of the GFP-tagged Eps8 mutant lacking the proline-rich region (GFP–Eps8ΔPR) or the GFP-tagged Eps8 mutant lacking the SH3 domain (GFP–Eps8ΔSH3) restores membrane blebbing in Eps8-KD DLD1 cells (arrows). (Scale bar, 10 μm.)

Aoki et al. PNAS | Published online March 14, 2016 | E1865 Downloaded by guest on September 28, 2021 Eps8 Localizes at the Initiation Sites of Actin Cortex Reassembly. DLD1 cells (Fig. S1 E and H). These data suggest that both the Considering that actomyosin complexes locally reassembled at actin-bundling and actin-capping activities of Eps8 are essential multiple sites during retraction of membrane blebs, we next for the formation and/or stabilization of cytoplasmic actomyosin sought to identify molecules that are involved in the regulation of cables and for the generation of continuous membrane blebbing. the local accumulation of actin filaments. We examined the distributions of actin cytoskeleton-related proteins during mem- Ezrin Is Activated in Multiple Regions of the Protruded Membrane. brane blebbing. Among the proteins tested, Eps8 was recruited to What recruits Eps8 to specific foci of membrane blebs? Ezrin/ the blebbing membrane and showed a unique distribution (Fig. 1E radixin/moesin (ERM) family proteins were recently reported to and Movie S2). Eps8 is an actin barbed-end capping protein with bind to Eps8 directly and regulate its functions (24). Furthermore, actin bundling activity (14, 15); however, its roles and dynamic Eps8 and ERM proteins were recently reported to colocalize at behavior in membrane blebbing have not been well characterized. the plasma membrane of membrane blebs (25). Therefore, we Therefore, we focused on the functional analysis of Eps8 in analyzed the functional relationship between ERM family proteins membrane blebbing in the following experiments. Eps8 accumu- and Eps8 in membrane blebbing in detail. lated in multiple foci at the blebbing membrane (Fig. 1E and As already reported (11), GFP–ezrin did not show a restricted Movie S2). Eps8-positive domains accumulated earlier than the distribution pattern but localized uniformly at the blebbing mem- actin cytoskeleton. The delay between the recruitment of actin and brane (Fig. 3A). However, ezrin adopts an active open conforma- that of Eps8 was visualized on two-color kymographs (Fig. 1 F and tion when phosphorylated at Thr567. Therefore, we next examined H). On the other hand, myosin was recruited to the plasma the distribution of phosphorylated ERM during blebbing. Because membrane after actin cortex reassembly (Fig. 1 G and H). Thus, GFP–ezrin uniformly localized at all membrane blebs, an anti-total endogenous Eps8 marked regions where the recovery of actin ERM antibody stained all protruded membrane blebs (Fig. 3B). filaments was initiated during the retraction of membrane blebs. Interestingly, an anti-phosphorylated ERM antibody stained some membrane blebs more strongly, suggesting that ERM is Eps8 Is Required for Continuous Membrane Blebbing. Next, to elu- activated only in expanding or retracting blebs (Fig. 3B). Phos- cidate the functional importance of Eps8 in membrane blebbing, phorylated ERM colocalized with Eps8 and actin filaments, in- we knocked down Eps8 expression using short hairpin RNA in dicating that ERM was activated only during membrane bleb DLD1 cells (Fig. 2A). Given that Eps8 was recruited to retracting retraction (Fig. 3 C and D). membrane blebs, we hypothesized that depletion of Eps8 would Next, we examined the effect of ERM depletion on the locali- lead to defects in membrane bleb retraction. Unexpectedly, Eps8- zation of Eps8 in membrane blebs. In DLD1 cells, only ezrin was knockdown (KD) cells had a spherical morphology and did not expressed among ERM family proteins (Fig. 3E). Therefore, we exhibit membrane blebbing (Fig. 2B). This phenotype was re- knocked out the ezrin using the CRISPR/Cas9 system (Fig. versed by exogenous expression of Eps8 (Fig. 2C and Fig. S1A). 3E). In ezrin-knockout (KO) DLD1 cells, Eps8 was no longer This finding suggests that Eps8 is required for the initiation of recruited to retracting membrane blebs and remained in the cyto- membrane blebs. As the up-regulation of intracellular pressure plasm (Fig. 3F). Furthermore, actin reassembly at the protruding and generation of membrane blebs is dependent on the contractile membrane was severely retarded in ezrin-KO DLD1 cells in com- actomyosin cortex in the , we next examined whether parison with wild-type DLD1 cells (Movies S3 and S4). We then formation of the actomyosin cortex covering the entire cell pe- performed histogram analyses of bleb expansion and retraction riphery is impaired in Eps8-KD cells (Fig. S1B). In Eps8-KD cells, velocities in wild-type DLD1 cells and ezrin-KO cells (Fig. 3H). The the amount of actomyosin cortex covering the entire cell periphery speed of the retraction phase of membrane blebs in ezrin-KO cells was greatly reduced, as judged by phalloidin staining (Fig. S1 B– was slower than that in wild-type DLD1 cells (Fig. 3H). Slow re- D). Furthermore, the level of the phosphorylated myosin light traction of blebs decreased the frequency of blebs and the formation chain was also significantly decreased in Eps8-KD cells (Fig. S1 F of larger blebs in ezrin-KO cells (Fig. 3 I and J). These analyses and G). Therefore, cytoplasmic actomyosin cortex depletion in the strongly support the notion that the retraction of membrane blebs cytoplasm may decrease the contractile activity required for the was severely retarded in ezrin-KO cells. generation of membrane blebs in Eps8 KD cells. On the other hand, the expanding speed of membrane blebs Eps8 regulates actin barbed-end capping activity and mediates was not significantly different between ezrin-KO cells and wild- actin bundling (16–19). In addition, Eps8 interacts with a number type cells (Fig. 3H). Although Eps8 KD cells did not show any of proteins, including Sos (16), IRSp53 (17), Src (20), Abi (21), membrane blebbing, ezrin-KO cells showed continuous slow and EGFR (22), through its proline-rich (PR) region and SH3 membrane blebbing. This difference in phenotype between Eps8 domain. Therefore, we examined which function of Eps8 un- KD and ezrin-KO cells may be partly because the cytoplasmic derlies its involvement in the regulation of membrane blebbing. pool of Eps8 remained intact in ezrin-KO cells. In good agree- We performed a mutational analysis of Eps8 (Fig. 2 D and E) ment with this assumption, the cytoplasmic actomyosin cortex of (23) and tested whether these mutants could rescue membrane ezrin-KO cells was similar to that of wild-type cells (Fig. S1 I and blebbing when transiently expressed in Eps8-KD DLD1 cells J). Therefore, we conclude that loss of ezrin only affected the (Fig. 2 F and G). Among the tested mutants, a GFP-tagged Eps8 retraction phase of membrane blebbing, not the expanding phase. mutant lacking the PR region (GFP–Eps8ΔPR) and a GFP- Exogenous expression of wild-type ezrin, but not the phos- tagged Eps8 mutant lacking the SH3 domain (GFP–Eps8ΔSH3) phorylation-defective mutant (T567A) of ezrin, rescued the lo- rescued membrane blebbing in Eps8-KD DLD1 cells (Fig. 2 F calization of Eps8 at retracting membrane blebs in ezrin-KO and G). On the other hand, expression of an actin filament- DLD1 cells (Fig. S2 A and B). As previously reported (11), the bundling–deficient mutant (GFP–Eps8ΔBundle) and an actin- phosphor-mimic mutant of ezrin (T567E) suppressed the for- capping–deficient mutant of Eps8 (GFP–Eps8ΔCap) did not mation of membrane blebs. Eps8 accumulated uniformly along reverse the phenotype of Eps8-KD DLD1 cells (Fig. 2 F and G). the plasma membrane in ezrin-KO cells overexpressing the Furthermore, in Eps8-KD DLD1 cells, expression of GFP– T567E mutant (Fig. S2A). These findings also support the notion Eps8ΔPR or GFP–Eps8ΔSH3 reestablished the formation of that activation of ezrin is required for the recruitment of Eps8 to contractile actin cables and the level of myosin light chain (MLC) the retracting blebs and for the reassembly of actin filaments at phosphorylation (Fig. S1 E and H). On the other hand, expression the protruded membranes. of GFP–Eps8ΔBundle or GFP–Eps8ΔCap failed to rescue the We next examined whether the retardation of actin reassembly reduction in the amount of actin filaments covering the entire affected the speed of cell migration using the Boyden chamber cell periphery and the level of MLC phosphorylation in Eps8-KD assay. In a Transwell migration assay, ezrin-KO DLD1 cells

E1866 | www.pnas.org/cgi/doi/10.1073/pnas.1600968113 Aoki et al. Downloaded by guest on September 28, 2021 PNAS PLUS CELL BIOLOGY

Fig. 3. Activation of ezrin occurs at retracting membranes and is required for the rapid retraction of membrane blebs. (A) Membrane blebbing in DLD1 cells transfected with GFP–ezrin. GFP–ezrin localizes uniformly at the protruded membrane. (Scale bar, 2 μm.) (B) DLD1 cells were fixed and stained with an anti– phospho-ERM antibody (red) and an anti-total ERM antibody (green). Nuclei were stained with DAPI (blue). The asterisk indicates a membrane bleb in which ERM proteins were not activated. (Scale bar, 2 μm.) (C) DLD1 cells were fixed and stained with an anti–phospho-ERM antibody (green) and an anti-Eps8 antibody (red). The arrowheads indicate the colocalization of Eps8 and phosphorylated ERM proteins. (Scale bar, 2 μm.) (D) DLD1 cells were fixed and stained with an anti–phospho-ERM antibody (green) and Alexa 594–phalloidin (red). The boxed area shows the membrane blebs with regrowing actin filaments. High-magnification image of the boxed area is shown in the right panels. The asterisks indicate a membrane bleb covered with actin cortex. (Scale bar, 10 μm.) (E) Total cell lysates of wild-type DLD1 cells and ezrin-KO DLD1 cells separated by SDS/PAGE and immunoblotted with an anti-total ERM antibody, an anti- ezrin antibody, an anti-Eps8 antibody, and an anti–α- antibody. (F) Membrane blebbing of wild-type DLD1 cells and ezrin-KO DLD1 cells transfected with Lifeact–RFP and GFP-tagged Eps8. (Scale bar, 10 μm.) (G) Tricolor map of membrane blebs in wild-type DLD1 cells and ezrin-KO DLD1 cells. Angular coordinates are shown on the horizontal axis, and time is shown on the vertical axis. Red zones represent expansion, blue zones represent retraction, and white zones represent no movement. (H) Histogram of bleb expansion and retraction velocities in wild-type DLD1 cells and ezrin-KO DLD1 cells. (I) The frequencies of membrane blebs in wild-type DLD1 cells and ezrin-KO DLD1 cells during 10 min were quantified. **P < 0.01 (Student’s t test). (J) The sizes of membrane blebs in wild-type DLD1 cell and in ezrin-KO DLD1 cells during 10 min were quantified. **P < 0.01 (Student’s t test).

Aoki et al. PNAS | Published online March 14, 2016 | E1867 Downloaded by guest on September 28, 2021 Fig. 4. The RhoA–ROCK–Rnd3 feedback loop determines the actin reassembly sites of retracting membranes. (A)GFP–ROCK-1 is recruited to the retracting protruded membrane in DLD1 cells. (Scale bar, 2 μm.) (B)GFP–ROCK is recruited to the retracting protruded membrane in ezrin-KO DLD1 cells (arrowheads). (Scale bar, 2 μm.) (C) Membrane blebbing of DLD1 cells transfected with Lifeact–RFP and GFP-tagged AHD. (Scale bar, 2 μm.) (D) Membrane blebbing of DLD1 cells transfected with Lifeact–RFP and GFP-tagged Rnd3. Rnd3 accumulation gradually disappears upon the initiation of membrane blebbing retraction (t = 25 s). (Scale bar, 2 μm.) (E) Kymographs showing the localization of actin (red) with respect to that of Rnd3 (green) during bleb retraction. Bleb extension is shown on the horizontal axis, and time is shown on the vertical axis. (F) Kymographs showing the localization of Rnd3 (red) with respect to that of Eps8 (green) during bleb retraction. Bleb extension is shown on the horizontal axis, and time is shown on the vertical axis. (G)GFP–p190B–Rho–GAP localizes only at expanding blebs that lack the actin cortex. The membrane localization of p190B Rho–GAP is gradually lost upon the initiation of actin cortex recovery. (Scale bar, 2 μm.)

E1868 | www.pnas.org/cgi/doi/10.1073/pnas.1600968113 Aoki et al. Downloaded by guest on September 28, 2021 PNAS PLUS CELL BIOLOGY

Fig. 5. A model of Rnd3- and RhoA-mediated regulation of actin cytoskeleton during membrane-blebbing cycle. (A) Localization of Eps8 in DLD1 cells expressing GFP–wild type Rnd3 (Upper) and GFP–Rnd3 S240A mutant. (Scale bar, 10 μm.) (B) Tricolor map of membrane blebs in DLD1 cells expressing GFP– wild type Rnd3 or GFP–Rnd3 S240A mutant. Angular coordinates are shown on the horizontal axis, and time is shown on the vertical axis. Red zones represent expansion, blue zones represent retraction, and white zones represent no movement. (C) Histogram of bleb expansion and retraction velocities in DLD1 cells expressing wild-type Rnd3 or the Rnd3 S240A mutant. (D) The frequencies of membrane blebs in DLD1 cells expressing GFP–wild type Rnd3 or GFP–Rnd3 S240A mutant during 10 min were quantified. **P < 0.01 (Student’s t test). (E) The sizes of membrane blebs in DLD1 cells expressing GFP–wild type Rnd3 or GFP–Rnd3 S240A mutant during 10 min were quantified. *P < 0.05 (Student’s t test). (F) In the expansion phase of membrane blebbing, Rnd3 and p190B–Rho– GAP inhibit the activation of RhoA. As the protruded membrane areas become enlarged, the relative concentration of Rnd3 decreases. Sporadic activation of RhoA leads to ROCK phosphorylation of Rnd3 and removal of p190B–Rho–GAP from the membrane. Thus, RhoA activation is amplified and sustained by the positive-feedback loop. ROCK also phosphorylates ezrin and activated ezrin recruits, which leads to reassembly of the actin cortex.

showed reduced migratory activity in comparison with wild-type of the phosphorylation-defective mutant (T567A) mutant did not DLD1 cells (Fig. S2C). Reexpression of ezrin significantly res- enhance the migration of ezrin-KO DLD1 cells (Fig. S2C). Taken cued the phenotype of ezrin-KO DLD1 cells; however, expression together, these findings show that activation of ERM is required for

Aoki et al. PNAS | Published online March 14, 2016 | E1869 Downloaded by guest on September 28, 2021 recruitment of Eps8 to retracting membrane blebs, rapid reassembly activation of RhoA may be amplified and stabilized by RhoA– of the actin cortex in membrane blebs, and efficient cell migration. ROCK phosphorylation of Rnd3, subsequent sequestration of Rnd3 in the cytoplasm, and inactivation of p190–Rho–GAP. Activation of Ezrin Is Regulated by the RhoA–ROCK–Rnd3 Feedback Increased ROCK activity leads to the phosphorylation of ERM Loop. Finally, we examined the molecular mechanisms that reg- and the recruitment of Eps8 to the protruded membrane. The ulate activation of ezrin only at retracting membranes during activation of ERM and recruitment of Eps8 at the plasma mem- membrane blebbing. The RhoA–ROCK pathway is reportedly brane induces the rapid retraction of the protruded membrane by involved in the activation of ERM family proteins (26–30); promoting the reassembly of the actin cortex. In conjunction with therefore, we examined the distribution of ROCK in membrane activation of ROCK, active RhoA may also activate another RhoA blebs. When GFP-tagged ROCK-1 was expressed in DLD1 cells, effector protein, mDia1, which was recently reported to be essential it was recruited specifically to the retracting membrane (Fig. 4A). for the regrowth of actin filaments at the retracting membrane (13). GFP–ROCK-1 was recruited to the retracting membrane blebs Finally, we would like to discuss some unsolved issues in this even in ezrin-KO cells, indicating that the recruitment of ROCK study. In ezrin-KO cells or in cells overexpressing Rnd3 S240A, the is epistatic to the activation of ezrin (Fig. 4B). retraction of membrane blebs was severely retarded. However, it Then, we next examined the distribution of active RhoA in should be noted that, at the plasma membrane in ezrin-KO cells or membrane blebs. It was recently reported that the well-conserved in cells overexpressing Rnd3 S240A, actin filaments continued to C-terminal domain of anillin [anillin homology domain (AHD)] cover the entire protruded membrane, albeit more slowly, even in selectively binds to the GTP-bound form of RhoA and that GFP- the absence of Eps8. Therefore, another mechanism may be pre- tagged AHD is a useful biosensor for the detection of active RhoA sent that enables ezrin- and Eps8-independent reconstruction of (31). When GFP–AHD was expressed in DLD1 cells, it was the actin cytoskeleton at the protruded membrane. This molecular recruited to retracting membrane blebs where reassembly of the mechanism will require investigation in future studies. actin cortex occurred (Fig. 4C and Movie S5). In the present study, we proposed that the local positive- As for the molecular mechanisms involved in the selective feedback loop of RhoA activation and global suppression by activation of RhoA in membrane blebbing, there are at least two Rnd3 determines the actin reassembly foci in membrane blebs. possibilities. One possibility is that certain Rho–GEFs are recruited There may be unidentified molecules involved in these processes. to retracting membranes. The other possibility is that Rho–GAPs In terms of candidates, the STRIPAK complex components are recruited to expanding blebs to inhibit RhoA activation. We FAM40A, FAM40B, and STRN3 were recently reported to be tested these possibilities and found that Rnd3 and p190B–Rho– involved in the amoeboid migration of cancer cells by regulating GAP were recruited to expanding membrane blebs where reas- the activation state of ERM proteins (35). Therefore, it will be sembly of the actin cortex did not occur (Fig. 4 D–F). Rnd3 (also interesting to examine how the STRIPAK complex is involved in named RhoE) reportedly antagonizes RhoA signaling by acti- the regulation of membrane blebbing in future studies. vating p190–Rho–GAP (32). Interestingly, Rnd3 was recruited to In addition to understanding the molecular components in- expanding membrane blebs; however, this membrane localiza- volved in the regulation of membrane blebs, it is also important tion was gradually lost during the retraction of membrane blebs to clarify whether changes in the physical properties of the (Fig. 4 D and E, and Movie S6). When mCherry–Rnd3 and protruded membrane are involved in the regulation of mem- GFP–Eps8 were simultaneously expressed in DLD1 cells, Rnd3 brane blebs. Membrane tension reportedly changes during bleb and Eps8 were only detected in the expanding membrane and expansion (36, 37), suggesting that it may lead to the gating of retracting membrane, respectively (Fig. 4F and Movie S7). GFP– stretch-activated ion channels and evoke active signaling (38). p190–Rho–GAP also had a distribution pattern similar to that of On the other hand, membrane tension was recently reported to Rnd3 during membrane blebbing (Fig. 4G). modulate the activity of small (39). We showed that Rnd3 disappeared as the reassembly of the actin cortex pro- Rnd3 preferentially localizes to the cortex-free plasma mem- ceeded (Fig. 4E). However, when the actin cytoskeleton was brane of expanding membrane blebs (Fig. S3), but the molecular disrupted by Latrunculin B treatment, Rnd3 persisted at the mechanism remains to be clarified. In future studies, it will be protruded membrane, indicating that Rnd3 preferentially local- very interesting to determine how cells sense the actin cortex- izes to the cortex-free plasma membrane (Fig. S3). free membrane via mechanical and chemical signals. Rnd3 is a constitutively active GTP-binding Rho family protein, and its membrane localization is inhibited when Rnd3 is phos- Materials and Methods phorylated by ROCK (33). Membrane retention of Rnd3 was re- Reagents. DLD1 cells were grown in DMEM supplemented with 10% (vol/vol) cently reported to be regulated by ROCK phosphorylation of FCS. The following primary antibodies were used for immunofluorescence serine-240, which leads to the sequestration of Rnd3 in the cyto- microscopy and immunoblotting: mouse anti-Eps8 monoclonal antibody plasm via its binding to 14-3-3 protein (34). Interestingly, over- (mAb) (Becton Dickinson), mouse anti–α-tubulin mAb (Sigma), rabbit anti- expression of the Rnd3 S240A mutant, which is resistant to ezrin antibody, rabbit anti-ERM antibody, and rabbit anti–phospho-ezrin inhibition by ROCK, phenocopied the ezrin-KO bleb phenotype. (Thr567)/radixin (Thr564)/moesin (Thr558) mAb (41A3). cDNAs encoding full- length ezrin, MRLC1, Eps8, anillin, p190–Rho–GAP-B, Rnd3, and RhoA were (Fig. 5 A and B,andMovie S8). When Rnd3 S240A was overex- amplified by RT-PCR, fused to the sequence encoding enhanced GFP, and pressed, the membrane recruitment of Eps8 was retarded (Fig. 5A), ligated into the pCAGGS-neo vector. Expression vectors of GFP–Rnd3 S240A resulting in a slower membrane bleb retraction phase and the for- mutant or GFP–ROCK-1 was kindly provided by Dr. A. S. Yap (University of mation of larger blebs than in wild-type DLD1 cells (Fig. 5 C–E). Queensland, St. Lucia, Queensland, Australia) or Dr. M. Takeichi (CDB, Kobe, Taken together, these findings show that, although Rnd3 and Japan), respectively. Stable ezrin-KO clones were produced using the p190–Rho–GAP are predominant at expanding membrane blebs, CRISPR–Cas9 system (40). Oligonucleotides were phosphorylated, annealed, RhoA and ROCK are activated at retracting membrane blebs. and cloned into the BbsI site of PX330 according to protocols of the Feng We propose that this positive-feedback mechanism of RhoA Zhang laboratory (Massachusetts Institute of Technology, Cambridge, MA). underlies the switch between the expansion and retraction pha- ses of membrane blebbing (Fig. 5F). When the intracellular Immunofluorescence Microscopy, Live Imaging, Tricolor Map, and Histogram Analysis. Details about immunofluorescence microscopy, live imaging, and pressure increases, the plasma membrane protrudes. Rnd3 and – – the quantitative analyses of live-imaging data are provided in Supporting p190 Rho GAP are present at the plasma membrane and in- Information and Figs. S4 and S5. hibit RhoA activation at the expanding plasma membrane. When the protruded membrane area increases and the concentration of ACKNOWLEDGMENTS. We thank all the members of the laboratory of J.I. Rnd3 per surface area of the membrane decreases, sporadic (Department of Biology, Faculty of Sciences, Kyushu University) for helpful

E1870 | www.pnas.org/cgi/doi/10.1073/pnas.1600968113 Aoki et al. Downloaded by guest on September 28, 2021 discussions. This work was supported by grants from the Japan Science and of Education, Culture, Sports, Science and Technology (Grants 26112713, 25711012, PNAS PLUS Technology Agency (research area, “Design and Control of Cellular Functions”), the and 15KT0152), the Uehara Memorial Foundation, Inoue Science Research Award, AMED-PRIME from Japan Agency for Medical Research and Development, Ministry the Cell Science Research Foundation, and Hoyu Science Foundation (to J.I.).

1. Blaser H, et al. (2006) Migration of zebrafish primordial germ cells: A role for myosin 22. Castagnino P, et al. (1995) Direct binding of eps8 to the juxtamembrane domain of contraction and cytoplasmic flow. Dev Cell 11(5):613–627. EGFR is phosphotyrosine- and SH2-independent. Oncogene 10(4):723–729. 2. Jaglarz MK, Howard KR (1995) The active migration of Drosophila primordial germ 23. Hertzog M, et al. (2010) Molecular basis for the dual function of Eps8 on actin dy- cells. Development 121(11):3495–3503. namics: Bundling and capping. PLoS Biol 8(6):e1000387. 3. Zatulovskiy E, Tyson R, Bretschneider T, Kay RR (2014) Bleb-driven chemotaxis of 24. Zwaenepoel I, et al. (2012) Ezrin regulates microvillus morphogenesis by promoting Dictyostelium cells. J Cell Biol 204(6):1027–1044. distinct activities of Eps8 proteins. Mol Biol Cell 23(6):1080–1094. 4. Tyson RA, Zatulovskiy E, Kay RR, Bretschneider T (2014) How blebs and pseudopods 25. Logue JS, et al. (2015) Erk regulation of actin capping and bundling by Eps8 promotes cooperate during chemotaxis. Proc Natl Acad Sci USA 111(32):11703–11708. cortex tension and leader bleb-based migration. eLife 4:e08314. 5. Fackler OT, Grosse R (2008) Cell motility through plasma membrane blebbing. J Cell 26. Matsui T, et al. (1998) Rho-kinase phosphorylates COOH-terminal threonines of ezrin/ Biol 181(6):879–884. radixin/moesin (ERM) proteins and regulates their head-to-tail association. J Cell Biol 6. Sahai E (2005) Mechanisms of cancer cell invasion. Curr Opin Genet Dev 15(1):87–96. 140(3):647–657. 7. Charras G, Paluch E (2008) Blebs lead the way: How to migrate without lamellipodia. 27. Jeon S, et al. (2002) RhoA and Rho kinase-dependent phosphorylation of moesin Nat Rev Mol Cell Biol 9(9):730–736. at Thr-558 in hippocampal neuronal cells by glutamate. J Biol Chem 277(19): 8. Bergert M, et al. (2015) Force transmission during adhesion-independent migration. 16576–16584. Nat Cell Biol 17(4):524–529. 28. Li Y, et al. (2007) Phosphorylated ERM is responsible for increased T cell polarization, 9. Liu YJ, et al. (2015) Confinement and low adhesion induce fast amoeboid migration adhesion, and migration in patients with systemic lupus erythematosus. J Immunol of slow mesenchymal cells. Cell 160(4):659–672. 178(3):1938–1947. 10. Ruprecht V, et al. (2015) Cortical contractility triggers a stochastic switch to fast 29. Hébert M, et al. (2008) Rho-ROCK-dependent ezrin-radixin-moesin phosphorylation amoeboid cell motility. Cell 160(4):673–685. regulates Fas-mediated in Jurkat cells. J Immunol 181(9):5963–5973. 11. Charras GT, Hu CK, Coughlin M, Mitchison TJ (2006) Reassembly of contractile actin 30. Onoue N, et al. (2008) Increased static pressure promotes migration of vascular cortex in cell blebs. J Cell Biol 175(3):477–490. smooth muscle cells: Involvement of the Rho-kinase pathway. J Cardiovasc Pharmacol 12. Charras GT (2008) A short history of blebbing. J Microsc 231(3):466–478. 51(1):55–61. 13. Bovellan M, et al. (2014) Cellular control of cortical actin nucleation. Curr Biol 24(14): 31. Piekny AJ, Glotzer M (2008) Anillin is a scaffold protein that links RhoA, actin, and 1628–1635. myosin during cytokinesis. Curr Biol 18(1):30–36. 14. Croce A, et al. (2004) A novel actin barbed-end-capping activity in EPS-8 regulates 32. Wennerberg K, et al. (2003) Rnd proteins function as RhoA antagonists by activating apical morphogenesis in intestinal cells of Caenorhabditis elegans. Nat Cell Biol 6(12): p190 RhoGAP. Curr Biol 13(13):1106–1115. 1173–1179. 33. Chardin P (2006) Function and regulation of Rnd proteins. Nat Rev Mol Cell Biol 7(1): 15. Disanza A, et al. (2004) Eps8 controls actin-based motility by capping the barbed ends 54–62. of actin filaments. Nat Cell Biol 6(12):1180–1188. 34. Riou P, et al. (2013) 14-3-3 proteins interact with a hybrid prenyl-phosphorylation 16. Scita G, et al. (1999) EPS8 and E3B1 transduce signals from Ras to Rac. Nature motif to inhibit G proteins. Cell 153(3):640–653. 401(6750):290–293. 35. Madsen CD, et al. (2015) STRIPAK components determine mode of cancer cell mi- 17. Funato Y, et al. (2004) IRSp53/Eps8 complex is important for positive regulation of Rac gration and metastasis. Nat Cell Biol 17(1):68–80. and cancer cell motility/invasiveness. Cancer Res 64(15):5237–5244. 36. Dai J, Sheetz MP (1999) Membrane tether formation from blebbing cells. Biophys J 18. Goicoechea S, et al. (2006) Palladin binds to Eps8 and enhances the formation of 77(6):3363–3370. dorsal ruffles and podosomes in vascular smooth muscle cells. J Cell Sci 119(Pt 16): 37. Peukes J, Betz T (2014) Direct measurement of the cortical tension during the growth 3316–3324. of membrane blebs. Biophys J 107(8):1810–1820. 19. Disanza A, et al. (2006) Regulation of cell shape by Cdc42 is mediated by the synergic 38. Liu X, et al. (2008) Stretch-activated potassium channels in hypotonically induced actin-bundling activity of the Eps8-IRSp53 complex. Nat Cell Biol 8(12):1337–1347. blebs of atrial myocytes. J Membr Biol 226(1-3):17–25. 20. Maa MC, Lai JR, Lin RW, Leu TH (1999) Enhancement of tyrosyl phosphorylation and 39. Houk AR, et al. (2012) Membrane tension maintains cell polarity by confining signals protein expression of eps8 by v-Src. Biochim Biophys Acta 1450(3):341–351. to the leading edge during neutrophil migration. Cell 148(1-2):175–188. 21. Biesova Z, Piccoli C, Wong WT (1997) Isolation and characterization of e3B1, an eps8 40. FA, et al. (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc binding protein that regulates cell growth. Oncogene 14(2):233–241. 8(11):2281–2308. CELL BIOLOGY

Aoki et al. PNAS | Published online March 14, 2016 | E1871 Downloaded by guest on September 28, 2021