A RhoA and Rnd3 cycle regulates actin 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 cytoskeleton 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 protein 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 human 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 homology 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 zebrafish (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 myosin 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 CELL BIOLOGY 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, myosins 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 proteins (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