Formation Of Circular Ruffles Research Articles

Drink or drive: competition between macropinocytosis and cell migration.

The cytoskeleton is utilized for a variety of cellular processes, including migration, endocytosis and adhesion. The required molecular components are often shared between different processes, but it is not well understood how the cells balance their use. We find that macropinocytosis and cell migration are negatively correlated. Heavy drinkers move only slowly and vice versa, fast cells do not take big gulps. Both processes are balanced by the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3). Elevated PIP3 signalling causes a shift towards macropinocytosis and inhibits motility by redirecting the SCAR/WAVE complex, a major nucleator of actin filaments. High resolution microscopy shows that patches with high levels of PIP3 recruit SCAR/WAVE on their periphery, resulting in circular ruffle formation and engulfment. Results shed new light on the role of PIP3, which is commonly thought to promote cell motility.

A growth factor signaling cascade confined to circular ruffles in macrophages

SummaryThe formation of macropinosomes requires large-scale movements of membranes and the actin cytoskeleton. Over several minutes, actin-rich surface ruffles transform into 1–5 µm diameter circular ruffles, which close at their distal margins, creating endocytic vesicles. Previous studies using fluorescent reporters of phosphoinositides and Rho-family GTPases showed that signals generated by macrophages in response to the growth factor Macrophage Colony-Stimulating Factor (M-CSF) appeared transiently in domains of plasma membrane circumscribed by circular ruffles. To address the question of how signaling molecules are coordinated in such large domains of plasma membrane, this study analyzed the relative timing of growth factor-dependent signals as ruffles transformed into macropinosomes. Fluorescent protein chimeras expressed in macrophages were imaged by microscopy and quantified relative to circular ruffle formation and cup closure. The large size of macropinocytic cups allowed temporal resolution of the transitions in phosphoinositides and associated enzyme activities that organize cup closure. Circular ruffles contained transient and sequential spikes of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2), phosphatidylinositol (3,4,5)-trisphosphate (PIP3), diacylglycerol, PI(3,4)P2, PI(3)P and the activities of protein kinase C-α, Rac1, Ras and Rab5. The confinement of this signal cascade to circular ruffles indicated that diffusion barriers present in these transient structures focus feedback activation and deactivation of essential enzyme activities into restricted domains of plasma membrane.

H‐Ras and PI3K are required for the formation of circular dorsal ruffles induced by low‐power laser irradiation

The formation of circular dorsal ruffles upon growth factor stimulation facilitates the static cells for subsequent motility. Low-power laser irradiation (LPLI) has been shown to exert some promotive effects on migration and proliferation in various cell types. It is unclear whether LPLI could induce the formation of circular ruffles. In this study, using confocal fluorescence microscope, we for the first time demonstrated that LPLI could induce the production of circular ruffle structures in COS-7 cells. These structures were proved to be actin-based and originated from membrane microdomains enriched in cholesterol. Ras was shown to be activated by LPLI and expression of YFP-H-Ras (N17), a dominant negative H-Ras, blocked the generation of circular ruffles induced by LPLI. Wortmannin, PI3K inhibitor, potently suppressed the formation of LPLI-induced circular ruffles in a dose-dependent manner. However, blocking the activation of PKC, which was activated during LPLI-induced cell proliferation in our previous study, had no effect on the formation of circular ruffles. Thus, both H-Ras and PI3K were required for the formation of circular ruffles induced by LPLI and the generation of circular ruffles provides new information for the mechanisms of biological effects of LPLI. J. Cell. Physiol. 219: 535-543, 2009. (c) 2009 Wiley-Liss, Inc.

Polycomb Group Protein Ezh2 Controls Actin Polymerization and Cell Signaling

Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. Here, we report the existence of the cytosolic Ezh2-containing methyltransferase complex and tie the function of this complex to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes.

Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases

Rab5 is a small GTPase involved in the control of intracellular trafficking, both at the level of receptor endocytosis and endosomal dynamics. The finding that Rab5 can be activated by receptor tyrosine kinases (RTK) raised the question of whether it also participates in effector pathways emanating from these receptors. Here we show that Rab5 is indispensable for a form of RTK-induced actin remodelling, called circular ruffling. Three independent signals, originating from Rab5, phosphatidylinositol-3-OH kinase and Rac, respectively, are simultaneously required for the induction of circular ruffles. Rab5 signals to the actin cytoskeleton through RN-tre, a previously identified Rab5-specific GTPase-activating protein (GAP). Here we demonstrate that RN-tre has the dual function of Rab5-GAP and Rab5 effector. We also show that RN-tre is critical for macropinocytosis, a process previously connected to the formation of circular ruffles. Finally, RN-tre interacts with both F-actin and actinin-4, an F-actin bundling protein. We propose that RN-tre establishes a three-pronged connection with Rab5, F-actin and actinin-4. This may aid crosslinking of actin fibres into actin networks at the plasma membrane. Thus, we have shown that Rab5 is a signalling GTPase and have elucidated the major molecular elements of its downstream pathway.

Phosphoinositide-3-kinase-independent contractile activities associated with Fcgamma-receptor-mediated phagocytosis and macropinocytosis in macrophages.

Previous studies have shown that Fcgamma receptor (FcR)-mediated phagocytosis and macropinocytosis in macrophages consist of two dissociable activities: a phosphoinositide 3-kinase (PI3K)-independent extension of phagocytic cups and a PI3K-dependent contractile mechanism that closes phagosomes and ruffles into intracellular organelles. Here, we identify an additional contractile activity that persists in the presence of the PI3K inhibitor wortmannin. ML-7, an inhibitor of myosin-light-chain kinase (MLCK), inhibited FcR-mediated phagocytosis, macropinocytosis and cell movements associated with ruffling. Scanning electron microscopy demonstrated a striking difference in morphology between phagocytic cups in the different inhibitors: whereas phagocytic cups of control cells and wortmannin-treated cells conformed closely to particles and appeared to have constricted them, the phagocytic cups in cells treated with ML-7 were more open. Video microscopy of macrophages expressing green-fluorescent-protein (GFP)-actin fusions revealed that bound IgG-opsonized erythrocytes were squeezed during phagosome formation and closure. In ML-7, GFP-actin-rich protrusions extended outward but failed to squeeze particles. Moreover, in contrast to the effects of PI3K inhibitors, ML-7 markedly reduced ruffle movement, and perturbed circular ruffle formation. These PI3K-independent myosin-II-based contractile activities that squeeze phagocytic cups and curve ruffles therefore represent a third component activity of the actin cytoskeleton during phagocytosis and macropinocytosis.