A RhoA and Rnd3 cycle regulates actin reassembly during membrane blebbing

Aoki, Kosuke; Maeda, Fumiyo; Nagasako, Tomoya; Mochizuki, Yuki; Uchida, Seiichi; Ikenouchi, Junichi

doi:10.1073/pnas.1600968113

Cited by 56 publications

(70 citation statements)

References 39 publications

(39 reference statements)

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“…This is in line with our finding that RhoA and RhoB controlled different aspects of 3D amoeboid leukaemia cell migration, with RhoA being important for cell rear retraction, while RhoB controlled membrane blebbing. This contrasts with previous studies that have suggested a role for RhoA in cell membrane blebbing (Gadea et al , ; Godin & Ferguson, ; Cartier‐Michaud et al , ; Tozluoglu et al , ; Takahara et al , ; Bang et al , ; Aoki et al , ). However, in most of these studies, experimental methods (e.g.…”

Section: Discussioncontrasting

confidence: 82%

KIF 13A‐regulated RhoB plasma membrane localization governs membrane blebbing and blebby amoeboid cell migration

et al. 2018

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Membrane blebbing‐dependent (blebby) amoeboid migration can be employed by lymphoid and cancer cells to invade 3D‐environments. Here, we reveal a mechanism by which the small GTPase RhoB controls membrane blebbing and blebby amoeboid migration. Interestingly, while all three Rho isoforms (RhoA, RhoB and RhoC) regulated amoeboid migration, each controlled motility in a distinct manner. In particular, RhoB depletion blocked membrane blebbing in ALL (acute lymphoblastic leukaemia), melanoma and lung cancer cells as well as ALL cell amoeboid migration in 3D‐collagen, while RhoB overexpression enhanced blebbing and 3D‐collagen migration in a manner dependent on its plasma membrane localization and down‐stream effectors ROCK and Myosin II. RhoB localization was controlled by endosomal trafficking, being internalized via Rab5 vesicles and then trafficked either to late endosomes/lysosomes or to Rab11‐positive recycling endosomes, as regulated by KIF13A. Importantly, KIF13A depletion not only inhibited RhoB plasma membrane localization, but also cell membrane blebbing and 3D‐migration of ALL cells. In conclusion, KIF13A‐mediated endosomal trafficking modulates RhoB plasma membrane localization to control membrane blebbing and blebby amoeboid migration.

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Section: Discussioncontrasting

confidence: 82%

KIF 13A‐regulated RhoB plasma membrane localization governs membrane blebbing and blebby amoeboid cell migration

et al. 2018

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“…21 Taken together, we propose that the interlocked feedback system between Rnd3 and RhoA rapidly switches during membrane expansion to retraction in blebbing membrane. At the expanding plasma membrane, Rnd3 and RhoGAP are present at the plasma membrane and inhibit RhoA-ROCK activity.…”

Section: Rhoa-rock-rnd3 Feedback Loop Regulates the Expansion And Retmentioning

confidence: 59%

“…21 In the retracting phase of membrane blebs, we found that RhoA is activated at the plasma membrane. RhoA GTP-form and ROCK are recruited to the plasma membrane at the onset of bleb retraction.…”

Section: Rhoa-rock-rnd3 Feedback Loop Regulates the Expansion And Retmentioning

confidence: 81%

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Membrane bleb: A seesaw game of two small GTPases

Ikenouchi

Aoki

2016

Small GTPases

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The plasma membrane is generally associated with underling actin cytoskeleton. When the plasma membrane detaches from actin filaments, it is expanded by the intracellular pressure and the spherical membrane protrusion which lacks underlying actin cortex, termed bleb, is formed. Bleb is widely used for migration across species; however, the molecular mechanism underlying membrane blebbing remains largely unknown. Our recent study revealed that 2 small GTPases, Rnd3 and RhoA, are important regulators of membrane blebbing. In the expanding blebs, Rnd3 is recruited to the plasma membrane and inhibits RhoA activity by activating RhoGAP. On the other hand, RhoA is activated at the retracting membrane and removes Rnd3 from plasma membrane by the activity of ROCK (Rho-associated protein kinase). ROCK is also important for the rapid reassembly of actin cortex and retraction of membrane blebs by activating Ezrin. We propose that a Rnd3 and RhoA cycle underlies the core machinery of continuous membrane blebbing. The plasma membrane is generally associated with underling actin cytoskeleton. The interaction between plasma membrane and actin cytoskeleton is mediated by some anchor proteins such as ezrin/radixin/moesin (ERM) family proteins. When the plasma membrane detaches from actin filaments, spherical membrane protrusion is formed by the intracellular pressure. [1][2][3] This membrane protrusion termed bleb is observed during cytokinesis 4 and cell migration. 5 Some cancer cells use membrane blebbing as a mode of motility during metastasis. 6 When cancer cells are cultured on rigid substrates, cells move with forming actin-rich plasma membrane protrusions, such as lammellipodia and filopodia. On the other hand, cells migrate by forming membrane blebs when embedded in the 3D extracellular matrix such as collagen gels. [7][8][9] Recently, several groups reported that cancer cells start to use membrane blebbing-associated cell migration in response to the physical changes of extracellular conditions. Down-regulation of cell adhesion to the extracellular matrix and up-regulation of physical confinement induce the formation of large polarized blebs in cancer cells. [10][11][12] This bleb-based migration is the faster mode of migration as compared to the migration using lammellipodia and filopodiaThe blebbing-associated cell migration is not only observed in cancer cells, but also under physiological conditions. For example, primordial germ cells (PGCs) migrate with forming membrane blebs in zebrafish 13 and Drosophila melanogaster embryos. 14 Dictyostelium also use membrane blebbing for migration during chemotaxis. 15 Thus, membrane blebbing is widely used for migration across species. The regulation of actin cytoskeleton during membrane blebbingThe process of expansion and retraction of blebs is largely depends on actin cytoskeleton organized underneath plasma membrane. Bleb forms when plasma membrane is detached from actin cytoskeleton. This is caused by the local increase of intracellular pressure or by the local ru...

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“…The knockout of ORP2 disrupted the F-actin organization of HuH7 cell and hampered cell adhesion, migration, and spreading of polarized lamellipodia. Instead of lamellipodia, the KO cells formed cell surface blebs, which may arise as a result of defective F-actin-plasma membrane interactions (41,42). The lamellipodia and migration defects in the ORP2-KO cells provide a plausible explanation for the growth mode of the KO cells, as the defects may result in an inability of the dividing cells to detach from one another and migrate, thus forming islet-like cell patches upon culture.…”

Section: Discussionmentioning

confidence: 99%

Analysis of ORP2‐knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation

et al. 2018

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ORP2 is implicated in cholesterol transport, triglyceride metabolism, and adrenocortical steroid hormone production. We addressed ORP2 function in hepatocytes by generating ORP2-knockout (KO) HuH7 cells by CRISPR-Cas9 gene editing, followed by analyses of transcriptome, F-actin morphology, migration, adhesion, and proliferation. RNA sequencing of ORP2-KO cells revealed >2-fold changes in 579 mRNAs. The Ingenuity Pathway Analysis (IPA) uncovered alterations in the following functional categories: cellular movement, cell-cell signaling and interaction, cellular development, cellular function and maintenance, cellular growth and proliferation, and cell morphology. Many pathways in these categories involved actin cytoskeleton, cell migration, adhesion, or proliferation. Analysis of the ORP2 interactome uncovered 109 putative new partners. Their IPA analysis revealed Ras homolog A (RhoA) signaling as the most significant pathway. Interactions of ORP2 with SEPT9, MLC12, and ARHGAP12 were validated by independent assays. ORP2-KO resulted in abnormal F-actin morphology characterized by impaired capacity to form lamellipodia, migration defect, and impaired adhesion and proliferation. Rescue of the migration phenotype and generation of typical cell surface morphology required an intact ORP2 phosphoinositide binding site, suggesting that ORP2 function involves phosphoinositide binding and transport. The results point at a novel function of ORP2 as a lipid-sensing regulator of the actin cytoskeleton, with impacts on hepatocellular migration, adhesion, and proliferation.-Kentala, H., Koponen, A., Kivelä, A. M., Andrews, R., Li, C., Zhou, Y., Olkkonen, V. M. Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation.

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A RhoA and Rnd3 cycle regulates actin reassembly during membrane blebbing

Cited by 56 publications

References 39 publications

KIF 13A‐regulated RhoB plasma membrane localization governs membrane blebbing and blebby amoeboid cell migration

KIF 13A‐regulated RhoB plasma membrane localization governs membrane blebbing and blebby amoeboid cell migration

Membrane bleb: A seesaw game of two small GTPases

Analysis of ORP2‐knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation

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