Chiharu Higashida

ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632–induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632–induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.

Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis

Although Rho regulates cytokinesis, little was known about the functions in mitosis of Cdc42 and Rac. We recently suggested that Cdc42 works in metaphase by regulating bi-orient attachment of spindle microtubules to kinetochores. We now confirm the role of Cdc42 by RNA interference and identify the mechanisms for activation and down-regulation of Cdc42. Using a pull-down assay, we found that the level of GTP-Cdc42 elevates in metaphase, whereas the level of GTP-Rac does not change significantly in mitosis. Overexpression of dominant-negative mutants of Ect2 and MgcRacGAP, a Rho GTPase guanine nucleotide exchange factor and GTPase activating protein, respectively, or depletion of Ect2 by RNA interference suppresses this change of GTP-Cdc42 in mitosis. Depletion of Ect2 also impairs microtubule attachment to kinetochores and causes prometaphase delay and abnormal chromosomal segregation, as does depletion of Cdc42 or expression of the Ect2 and MgcRacGAP mutants. These results suggest that Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis.

Actin turnover–dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing

Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s−1, respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.

Rotational movement of the formin mdia1 along the double helical strand of an actin filament

Visualization of formin protein rotating along an actin filament gives insight into how it promotes actin assembly. Formin homology proteins (formins) elongate actin filaments (F-actin) by continuously associating with filament tips, potentially harnessing actin-generated pushing forces. During this processive elongation, formins are predicted to rotate along the axis of the double helical F-actin structure (referred to here as helical rotation), although this has not yet been definitively shown. We demonstrated helical rotation of the formin mDia1 by single-molecule fluorescence polarization (FLP). FLP of labeled F-actin, both elongating and depolymerizing from immobilized mDia1, oscillated with a periodicity corresponding to that of the F-actin long-pitch helix, and this was not altered by actin-bound nucleotides or the actin-binding protein profilin. Thus, helical rotation is an intrinsic property of formins. To harness pushing forces from growing F-actin, formins must be anchored flexibly to cell structures.

F- and G-actin homeostasis regulates mechanosensitive actin nucleation by formins

Physical force evokes rearrangement of the actin cytoskeleton. Signalling pathways such as tyrosine kinases, stretch-activated Ca2+ channels and Rho GTPases are involved in force sensing. However, how signals are transduced to actin assembly remains obscure. Here we show mechanosensitive actin polymerization by formins (formin homology proteins). Cells overexpressing mDia1 increased the amount of F-actin on release of cell tension. Fluorescence single-molecule speckle microscopy revealed rapid induction of processive actin assembly by mDia1 on cell cortex deformation. mDia1 lacking the Rho-binding domain and other formins exhibited mechanosensitive actin nucleation, suggesting Rho-independent activation. Mechanosensitive actin nucleation by mDia1 required neither Ca2+ nor kinase signalling. Overexpressing LIM kinase abrogated the induction of processive mDia1. Furthermore, s-FDAPplus (sequential fluorescence decay after photoactivation) analysis revealed a rapid actin monomer increase on cell cortex deformation. Our direct visualization of the molecular behaviour reveals a mechanosensitive actin filament regeneration mechanism in which G-actin released by actin remodelling plays a pivotal role.

Cdc42 and Rac Family GTPases Regulate Mode and Speed but Not Direction of Primary Fibroblast Migration during Platelet-Derived Growth Factor-Dependent Chemotaxis

ABSTRACT Cdc42 and Rac family GTPases are important regulators of morphology, motility, and polarity in a variety of mammalian cell types. However, comprehensive analysis of their roles in the morphological and behavioral aspects of chemotaxis within a single experimental system is still lacking. Here we demonstrate using a direct viewing chemotaxis assay that of all of the Cdc42/Rac1-related GTPases expressed in primary fibroblasts, Cdc42, Rac1, and RhoG are required for efficient migration towards platelet-derived growth factor (PDGF). During migration, Cdc42-, Rac1-, and RhoG-deficient cells show aberrant morphology characterized as cell elongation and cell body rounding, loss of lamellipodia, and formation of thick membrane extensions, respectively. Analysis of individual cell trajectories reveals that cell speed is significantly reduced, as well as persistence, but to a smaller degree, while the directional response to the gradient of PDGF is not affected. Combined knockdown of Cdc42, Rac1, and RhoG results in greater inhibition of cell speed than when each protein is knocked down alone, but the cells are still capable of migrating toward PDGF. We conclude that, Cdc42, Rac1, and RhoG function cooperatively during cell migration and that, while each GTPase is implicated in the control of morphology and cell speed, these and other Cdc42/Rac-related GTPases are not essential for the directional response toward PDGF.

G-actin regulates rapid induction of actin nucleation by mDia1 to restore cellular actin polymers.

mDia1 belongs to the formin family of proteins that share FH1 and FH2 domains. Although formins play a critical role in the formation of many actin-based cellular structures, the physiological regulation of formin-mediated actin assembly within the cell is still unknown. Here we show that cells possess an acute actin polymer restoration mechanism involving mDia1. By using single-molecule live-cell imaging, we found that several treatments including low-dose G-actin-sequestering drugs and unpolymerizable actin mutants activate mDia1 to initiate fast directional movement. The FH2 region, the core domain for actin nucleation, is sufficient to respond to latrunculin B (LatB) to increase its actin nucleation frequency. Simulation analysis revealed an unexpected paradoxical effect of LatB that leads to a several fold increase in free G-actin along with an increase in total G-actin. These results indicate that in cells, the actin nucleation frequency of mDia1 is enhanced not only by Rho, but also strongly through increased catalytic efficiency of the FH2 domain. Consistently, frequent actin nucleation by mDia1 was found around sites of vigorous actin disassembly. Another major actin nucleator, the Arp2/3 complex, was not affected by the G-actin increase induced by LatB. Taken together, we propose that transient accumulation of G-actin works as a cue to promote mDia1-catalyzed actin nucleation to execute rapid reassembly of actin filaments.

An order of magnitude faster AIP1-associated actin disruption than nucleation by the Arp2/3 complex in lamellipodia

The mechanism of lamellipod actin turnover is still under debate. To clarify the intracellular behavior of the recently-identified actin disruption mechanism, we examined kinetics of AIP1 using fluorescent single-molecule speckle microscopy. AIP1 is thought to cap cofilin-generated actin barbed ends. Here we demonstrate a reduction in actin-associated AIP1 in lamellipodia of cells overexpressing LIM-kinase. Moreover, actin-associated AIP1 was rapidly abolished by jasplakinolide, which concurrently blocked the F-actin-cofilin interaction. Jasplakinolide also slowed dissociation of AIP1, which is analogous to the effect of this drug on capping protein. These findings provide in vivo evidence of the association of AIP1 with barbed ends generated by cofilin-catalyzed filament disruption. Single-molecule observation found distribution of F-actin-associated AIP1 throughout lamellipodia, and revealed even faster dissociation of AIP1 than capping protein. The estimated overall AIP1-associated actin disruption rate, 1.8 µM/s, was one order of magnitude faster than Arp2/3 complex-catalyzed actin nucleation in lamellipodia. This rate does not suffice the filament severing rate predicted in our previous high frequency filament severing-annealing hypothesis. Our data together with recent biochemical studies imply barbed end-preferred frequent filament disruption. Frequent generation of AIP1-associated barbed ends and subsequent release of AIP1 may be the mechanism that facilitates previously observed ubiquitous actin polymerization throughout lamellipodia.

A Possible Role of Homeostasis Between Monomeric and Filamentous Actin in Filament Nucleation Revealed by Pharmacokinetic Modeling

The currently prevailing method to elucidate molecular functions in vivo is to knock out or knock down gene expression and to observe the resultant phenotypic changes. For example, fibroblasts extend pseudopods called lamellipodia toward the source of the platelet-derived growth factor (PDGF) gradient. We now know tens of molecules, both cell signaling intermediates and cytoskeleton regulators, involved in such cell polarization and migration. They interact with each other in a complicated manner both spatially and temporally. Our laboratory has recently carried out a screening to examine the effect of knockdown of Rho family GTPases on PDGF-induced chemotaxis. Many of these molecules have been implicated in the regulation of the cytoskeleton. Depletion of several Rho family GTPases had profound and distinct effects on cell morphology during migrating (Monypenny, J. et al., submitted). But given the complexity of the molecular interplay, it often remains unclear whether the induced phenotype is a direct or an indirect consequence of individual treatment. Even with the data showing clear morphological phenotypes, the question as to whether individual molecules function locally or globally, for example, is not easy to answer. How, then, can we investigate such fast and complex mechanisms in detail? Currently, we are applying several approaches to solve this question. One is directly capturing biochemical reactions quantitatively in living cells using “singlemolecule observation of cytoskeletal reorganization.” The second is “fast perturbation of the system using small compounds.” Drug treatment provides useful information in combination with time-lapse imaging as it enables us to capture initial effects of perturbation. Then, we apply “systematic configuration of biochemistry” to discover hidden connections between biochemical functions in the system. The actin system is one of the best characterized systems biochemically in