RhoA inactivation is crucial to manganese-induced astrocyte stellation

Actin depolymerization through Rho GTPases or exogenous mechanical tension has been suggested as a key determinant for the formation of astrocyte stellation. Rho GTPases function as switching molecules to converge both extracellular and intracellular signals in regulation of cytoskeletal organization. Their involvement in manganese-induced astrocyte stellation was assessed. The disruption of cytoskeletal architecture by manganese indicated the decreased activity of RhoA. Pharmacological and biochemical approaches revealed the inactivation of RhoA by manganese. This inactivation was partly through the down-regulation of guanine nucleotide exchange factor phosphorylation. Furthermore, the dephosphorylation of myosin light chain and cofilin through the inactivated RhoA effectors synergistically destabilized actin stress fibers. We conclude that manganese regulates cytoskeletal organization in astrocytes by modulating the activity of p115RhoGEF and RhoA.     

Myosin light chain kinase and acto-myosin contractility modulate activation of the ERK cascade downstream of oncogenic Ras

The actin cytoskeleton is recognized as an important component of both adhesion- and growth factor-dependent signaling, but its role in oncogene-dependent signaling has received much less attention. In this study, we investigated the role played by the acto-myosin cytoskeleton and its main regulators, i.e., myosin light chain kinase and Rho kinase, in oncogenic Ki-Ras-induced signaling. We found that activation of the ERK cascade by Ras is dependent on acto-myosin contractility, under the regulation of myosin light chain kinase but not Rho kinase. Inhibition of myosin II or myosin light chain kinase caused a complete loss of ERK phosphorylation in a time- and dose-dependent manner, but proved dispensable for activation of the PI3K pathway. We also provide evidence that the target of myosin light chain kinase lays at the level of Raf activation. Since myosin light chain kinase is a target of ERK, these results suggest a previously uncharacterized signaling pathway involving Ras-mediated alterations of the actin cytoskeleton, which might play a critical role in ERK activation by the Ras oncogene and contribute to aberrant signaling and enhanced cell motility. In addition, restoration of stress fibers following ectopic expression of tropomyosin 2 resulted in reduced levels of ERK phosphorylation. Finally, these studies suggest that myosin light chain kinase but not Rho kinase plays an essential role in the generation of ERK signaling in transformed cells and indicate distinct cellular roles for Rho-kinase and myosin light chain kinase-dependent functions involving the regulation of acto-myosin contractility.     

Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading

GTPases of the Rho family regulate actinomyosin-based contraction in non-muscle cells. Activation of Rho increases contractility, leading to cell rounding and neurite retraction in neuronal cell lines. Activation of Rac promotes cell spreading and interferes with Rho-mediated cell rounding. Here we show that activation of Rac may antagonize Rho by regulating phosphorylation of the myosin-II heavy chain. Stimulation of PC12 cells or N1E-115 neuroblastoma cells with bradykinin induces phosphorylation of threonine residues in the myosin-II heavy chain; this phosphorylation is Ca2+ dependent and regulated by Rac. Both bradykinin-mediated and constitutive activation of Rac promote cell spreading, accompanied by a loss of cortical myosin II. Our results identify the myosin-II heavy chain as a new target of Rac-regulated kinase pathways, and implicate Rac as a Rho antagonist during myosin-II-dependent cell-shape changes.     

Smurf1 regulates tumor cell plasticity and motility through degradation of RhoA leading to localized inhibition of contractility

Rho GTPases participate in various cellular processes, including normal and tumor cell migration. It has been reported that RhoA is targeted for degradation at the leading edge of migrating cells by the E3 ubiquitin ligase Smurf1, and that this is required for the formation of protrusions. We report that Smurf1-dependent RhoA degradation in tumor cells results in the down-regulation of Rho kinase (ROCK) activity and myosin light chain 2 (MLC2) phosphorylation at the cell periphery. The localized inhibition of contractile forces is necessary for the formation of lamellipodia and for tumor cell motility in 2D tissue culture assays. In 3D invasion assays, and in in vivo tumor cell migration, the inhibition of Smurf1 induces a mesenchymal-amoeboid-like transition that is associated with a more invasive phenotype. Our results suggest that Smurf1 is a pivotal regulator of tumor cell movement through its regulation of RhoA signaling.     

Myosin‐II negatively regulates minor process extension and the temporal development of neuronal polarity

The earliest stage in the development of neuronal polarity is characterized by extension of undifferentiated “minor processes” (MPs), which subsequently differentiate into the axon and dendrites. We investigated the role of the myosin II motor protein in MP extension using forebrain and hippocampal neuron cultures. Chronic treatment of neurons with the myosin II ATPase inhibitor blebbistatin increased MP length, which was also seen in myosin IIB knockouts. Through live‐cell imaging, we demonstrate that myosin II inhibition triggers rapid minor process extension to a maximum length range. Myosin II activity is determined by phosphorylation of its regulatory light chains (rMLC) and mediated by myosin light chain kinase (MLCK) or RhoA‐kinase (ROCK). Pharmacological inhibition of MLCK or ROCK increased MP length moderately, with combined inhibition of these kinases resulting in an additive increase in MP length similar to the effect of direct inhibition of myosin II. Selective inhibition of RhoA signaling upstream of ROCK, with cell‐permeable C3 transferase, increased both the length and number of MPs. To determine whether myosin II affected development of neuronal polarity, MP differentiation was examined in cultures treated with direct or indirect myosin II inhibitors. Significantly, inhibition of myosin II, MLCK, or ROCK accelerated the development of neuronal polarity. Increased myosin II activity, through constitutively active MLCK or RhoA, decreased both the length and number of MPs and, consequently, delayed or abolished the development of neuronal polarity. Together, these data indicate that myosin II negatively regulates MP extension, and the developmental time course for axonogenesis. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Guanine Nucleotide Exchange Factor-H1 Regulates Cell Migration via Localized Activation of RhoA at the Leading Edge

Cell migration involves the cooperative reorganization of the actin and microtubule cytoskeletons, as well as the turnover of cell–substrate adhesions, under the control of Rho family GTPases. RhoA is activated at the leading edge of motile cells by unknown mechanisms to control actin stress fiber assembly, contractility, and focal adhesion dynamics. The microtubule-associated guanine nucleotide exchange factor (GEF)-H1 activates RhoA when released from microtubules to initiate a RhoA/Rho kinase/myosin light chain signaling pathway that regulates cellular contractility. However, the contributions of activated GEF-H1 to coordination of cytoskeletal dynamics during cell migration are unknown. We show that small interfering RNA-induced GEF-H1 depletion leads to decreased HeLa cell directional migration due to the loss of the Rho exchange activity of GEF-H1. Analysis of RhoA activity by using a live cell biosensor revealed that GEF-H1 controls localized activation of RhoA at the leading edge. The loss of GEF-H1 is associated with altered leading edge actin dynamics, as well as increased focal adhesion lifetimes. Tyrosine phosphorylation of focal adhesion kinase and paxillin at residues critical for the regulation of focal adhesion dynamics was diminished in the absence of GEF-H1/RhoA signaling. This study establishes GEF-H1 as a critical organizer of key structural and signaling components of cell migration through the localized regulation of RhoA activity at the cell leading edge.     

Coordination of actin filament and microtubule dynamics during neurite outgrowth

Although much evidence suggests that axon growth and guidance depend on well-coordinated cytoskeletal dynamics, direct characterization of the corresponding molecular events has remained a challenge. Here, we address this outstanding problem by examining neurite outgrowth stimulated by local application of cell adhesion substrates. During acute outgrowth, the advance of organelles and underlying microtubules was correlated with regions of attenuated retrograde actin network flow in the periphery. Interestingly, as adhesion sites matured, contractile actin arc structures, known to be regulated by the Rho/Rho Kinase/myosin II signaling cascade, became more robust and coordinated microtubule movements in the growth cone neck. When Rho Kinase was inhibited, although growth responses occurred with less of a delay, microtubules failed to consolidate into a single axis of growth. These results reveal a role for Rho Kinase and myosin II contractility in regulation of microtubule behavior during neuronal growth.     

Myosin II and mechanotransduction: a balancing act

Adherent cells respond to mechanical properties of the surrounding extracellular matrix. Mechanical forces, sensed at specialized cell-matrix adhesion sites, promote actomyosin-based contraction within the cell. By manipulating matrix rigidity and adhesion strength, new roles for actomyosin contractility in the regulation of basic cellular functions, including cell proliferation, migration and stem cell differentiation, have recently been discovered. These investigations demonstrate that a balance of forces between cell adhesion on the outside and myosin II-based contractility on the inside of the cell controls many aspects of cell behavior. Disturbing this balance contributes to the pathogenesis of various human diseases. Therefore, elaborate signaling networks have evolved that modulate myosin II activity to maintain tensional homeostasis. These include signaling pathways that regulate myosin light chain phosphorylation as well as myosin II heavy chain interactions.     

Central involvement of Rho family GTPases in TNF-alpha-mediated bovine pulmonary endothelial cell apoptosis

In our recent studies, we defined a critical role for increased levels of myosin light chain (MLC) phosphorylation, a regulatory event in the interaction between actin and myosin in TNF-alpha-induced pulmonary endothelial cell actomyosin rearrangement and apoptosis. The Rho GTPase effector, Rho kinase is an important signaling effector governing levels of MLC phosphorylation which contributes to plasma membrane blebbing in several models of apoptosis. In this study, we directly assessed the role of Rho kinase in TNF-alpha-induced endothelial cell microfilament rearrangement and apoptosis. Inhibition of RhoA GTPase activity by the overexpression of dominant negative RhoA attenuates TNF-alpha-triggered stress fiber formation, consistent with Rho activation as a key event in TNF-alpha-induced cytoskeletal rearrangement. Furthermore, pharmacologic inhibition of Rho kinase as well as dominant negative RhoA overexpression dramatically reduced TNF-alpha-induced bovine endothelial apoptosis reflected by nucleosomal fragmentation as well as caspase 7, 3, and 8 activation. These results indicate that Rho kinase-dependent cytoskeletal rearrangement is critical for early apoptotic events, possibly in the assembly of the death-inducing signaling complex leading to initiator and effector caspase activation, and suggest a novel role for Rho GTPases in endothelial cell apoptosis.     

Regulation of rho family GTPases is required to prevent axons from crossing the midline

Rho family GTPases are ideal candidates to regulate aspects of cytoskeletal dynamics downstream of axon guidance receptors. To examine the in vivo role of Rho GTPases in midline guidance, dominant negative (dn) and constitutively active (ct) forms of Rho, Drac1, and Dcdc42 are expressed in the Drosophila CNS. When expressed alone, only ctDrac and ctDcdc42 cause axons in the pCC/MP2 pathway to cross the midline inappropriately. Heterozygous loss of Roundabout enhances the ctDrac phenotype and causes errors in embryos expressing dnRho or ctRho. Homozygous loss of Son-of-Sevenless (Sos) also enhances the ctDrac phenotype and causes errors in embryos expressing either dnRho or dnDrac. CtRho suppresses the midline crossing errors caused by loss of Sos. CtDrac and ctDcdc42 phenotypes are suppressed by heterozygous loss of Profilin, but strongly enhanced by coexpression of constitutively active myosin light chain kinase (ctMLCK), which increases myosin II activity. Expression of ctMLCK also causes errors in embryos expressing either dnRho or ctRho. Our data confirm that Rho family GTPases are required for regulation of actin polymerization and/or myosin activity and that this is critical for the response of growth cones to midline repulsive signals. Midline repulsion appears to require down-regulation of Drac1 and Dcdc42 and activation of Rho.     

Activation of myosin in HeLa cells causes redistribution of focal adhesions and F-actin from cell center to cell periphery

Activation of actomyosin II by phosphorylation of its regulatory light chain is one of the main factors involved in the regulation of cytoskeletal dynamics. Phosphorylation of myosin regulatory light chain may be mediated directly and indirectly by several kinases including myosin light chain kinase (MLCK) and kinases activated by small GTP-binding proteins. Most of the myosin kinases, including PAK, can also interact with other proteins through binding sites located outside of their catalytic domains. In an attempt to study the effects due only to phosphorylation of myosin light chain, we expressed the constitutively active catalytic domain of ameba PAK in HeLa cells. The catalytic domain phosphorylates myosin light chain in vitro with high specific activity but has none of the sequences that target mammalian PAK to other proteins and membranes. Expression of the catalytic domain caused disassembly of focal adhesions and stress fibers in the cell center and accumulation of focal adhesions and F-actin at the cell periphery. There was a twofold increase in the phosphorylation level of endogenous myosin light chain and changes in cell shape consistent with enhanced cell contractility. The phenotype was independent of MLCK, ROCK, MEK, Rac, and Rho activities but was abolished by blebbistatin, a specific inhibitor of myosin II activity. Our data are consistent with myosin being directly phosphorylated by the expressed catalytic domain of ameba PAK with the induced phenotype resulting from cell retraction driven by contraction of peripheral actomyosin. The phenotype induced by expression of the catalytic domain is reminiscent of that caused by expression of active mammalian PAK, suggesting that myosin phosphorylation may play an important role in PAK-induced cytoskeletal changes. The catalytic domain of ameba PAK may be a useful tool for studying the effects of myosin light chain phosphorylation in other cells.     

Rho signaling in Entamoeba histolytica modulates actomyosin-dependent activities stimulated during invasive behavior

Interaction of Entamoeba histolytica trophozoites with target cells and substrates activates signaling pathways in the parasite. Phosphorylation cascades triggered by phospho-inositide and adenyl-cyclase-dependent pathways modulate reorganization of the actin cytoskeleton to form structures that facilitate adhesion. In contrast, little is known about participation of Rho proteins and Rho signaling in actin rearrangements. We report here the in vivo expression of at least one Rho protein in trophozoites, whose activation induced actin reorganization and actin-myosin interaction. Antibodies to EhRhoA1 recombinant protein mainly localized Rho in the cytosol of nonactivated amoebae, but it was translocated to vesicular membranes and to some extent to the plasma membrane after treatment with lysophosphatidic acid (LPA), a specific agonist of Rho activation. Activated Rho was identified in LPA-treated trophozoites. LPA induced striking polymerization of actin into distinct dynamic structures. Disorganization of these structures by inhibition of Rho effector, Rho-kinase (ROCK), and by ML-7, an inhibitor of myosin light chain kinase dependent phosphorylation of myosin light chain, suggested that the actin structures also contained myosin. LPA stimulated concanavalin-A-mediated formation of caps, chemotaxis, invasion of extracellular matrix substrates, and erythrophagocytosis, but not binding to fibronectin. ROCK inhibition impaired LPA-stimulated functions and to some extent adhesion to fibronectin. Similar results were obtained with ML-7. These data suggest the presence and operation of Rho-signaling pathways in E. histolytica, that together with other, already described, signaling routes modulate actomyosin-dependent motile processes, particularly stimulated during invasive behavior.     

p190-RhoGAP as an integral component of the Tiam1/Rac1-induced downregulation of Rho

The Rho family of small GTPases plays a central role in intracellular signal transduction, particularly in reorganization of the actin cytoskeleton. Rho activity induces cell contractility, whereas Rac promotes cellular protrusion, which counteracts Rho signaling. In this regard, the reciprocal balance between these GTPases determines cell morphology and migratory behavior. Here we demonstrate that Tiam1/Rac1 signaling is able to antagonize Rho activity directly at the GTPase level in COS-7 cells. p190-RhoGAP plays a central regulatory role in this signaling pathway. Interfering with its activation by Src-kinase-dependent tyrosine phosphorylation or its recruitment to the membrane through interaction with the SH2 domains of p120-RasGAP blocks the Tiam1-mediated rapid downregulation of Rho. This process is mediated by Rac1, but not by Rac2 or Rac3 isoforms. Our data provide evidence for a biochemical pathway of the reciprocal regulation of two related small GTPases, which are key elements in cell migration.     

Cdc42-MRCK and Rho-ROCK signalling cooperate in myosin phosphorylation and cell invasion

Actomyosin contractility is a mechanism by which cells exert locomotory force against their environment. Signalling downstream of the small GTPase Rho increases contractility through Rho-kinase (ROCK)-mediated regulation of myosin-II light chain (MLC2) phosphorylation. Cdc42 signalling has been shown to control cell polarity. Tumour cells can move through a three-dimensional matrix with either a rounded morphology characterized by Rho-ROCK dependence or with an elongated morphology characterized by Rho-ROCK independence. Here we show that contractility necessary for elongated morphology and invasion can be generated by Cdc42-MRCK signalling. MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) cooperates with ROCK in the maintenance of elongated morphology and invasion and either MRCK or ROCK is sufficient for MLC2 phosphorylation, through the inhibitory phosphorylation of myosin phosphatase. By contrast, in rounded ROCK-dependent movement, where MLC2 phosphorylation is higher, MRCK has a smaller role. Our data show that a Cdc42-MRCK signal mediates myosin-dependent cell motility and highlight convergence between Rho and Cdc42 signalling.     

PAK4 phosphorylates myosin regulatory light chain and contributes to Fcγ receptor-mediated phagocytosis

Phagocytosis of immunoglobulin G-opsonised particles takes place via Fcγ receptor ligation, leading to uptake through an actin-dependent mechanism. Myosin regulatory light chains have previously been reported to control contractility during uptake through the Fcγ receptor. In this study, we show that p21-activated kinase 4 contributes to Fcγ receptor-mediated uptake downstream of actin cup formation by regulating phosphorylation of myosin regulatory light chain. siRNA-mediated knockdown of p21-activated kinase 4 leads to reduced myosin regulatory light chain phosphorylation at Serine 19, with a corresponding reduction in phospho-myosin regulatory right chain localised to bound immunoglobulin G-opsonised red blood cells. p21-activated kinase 4 phosphorylates myosin light chain 9 at Serine 19 in vitro and RNA interference against myosin light chain 9 implicates this isoform, but not myosin light chain 12A or 12B, in Fcγ receptor-mediated uptake. Taken together, these data indicate that p21-activated kinase 4 regulates regulatory myosin light chain phosphorylation and myosin contractility during FcγR-mediated phagocytosis.     

Rho proteins: their function in neurons

Neurons possess a polarized morphology. In general, each neuron has several dendrites but only one axon. Such morphology is the basis for directionalized rapid signaling, information flowing from the short dendrites to the long axon. The mechanisms involved in the establishment of the neuronal polarity remain largely unknown. However, recently, members of Rho family proteins have been implicated in the regulation of neuronal morphology especially development of neuronal polarity, axon outgrowth and guidance, dendritic tree elaboration and synapse formation. Moreover, the Rho GTPases have been reported to be directly or indirectly involved in some neurological conditions such as X-linked mental retardation as well as Alzheimer's and Parkinson's diseases. These findings demonstrate the importance of Rho GTPases in the development, maintenance and function of the nervous system.     

RHO GTPase Signaling for Axon Extension: Is Prenylation Important?

Many lines of evidence indicate the importance of the Rho family guanine nucleotide triphosphatases (GTPases) in directing axon extension and guidance. The signaling networks that involve these proteins regulate actin cytoskeletal dynamics in navigating neuronal growth cones. However, the intricate patterns that regulate Rho GTPase activation and signaling are not yet fully defined. Activity and subcellular localization of the Rho GTPases are regulated by post-translational modification. The addition of a geranylgeranyl group to the carboxy (C-) terminus targets Rho GTPases to the plasma membrane and promotes their activation by facilitating interaction with guanine nucleotide exchange factors and allowing sequestering by association with guanine dissociation inhibitors. However, it is unclear how these modifications affect neurite extension or how subcellular localization alters signaling from the classical Rho GTPases (RhoA, Rac1, and Cdc42). Here, we review recent data addressing this issue and propose that Rho GTPase geranylgeranylation regulates outgrowth.     

Targeting by myosin phosphatase-RhoA interacting protein mediates RhoA/ROCK regulation of myosin phosphatase

Vascular smooth muscle cell contractile state is the primary determinant of blood vessel tone. Vascular smooth muscle cell contractility is directly related to the phosphorylation of myosin light chains (MLCs), which in turn is tightly regulated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase. Myosin phosphatase is the principal enzyme that dephosphorylates MLCs leading to relaxation. Myosin phosphatase is regulated by both vasoconstrictors that inhibit its activity to cause MLC phosphorylation and contraction, and vasodilators that activate its activity to cause MLC dephosphorylation and relaxation. The RhoA/ROCK pathway is activated by vasoconstrictors to inhibit myosin phosphatase activity. The mechanism by which RhoA and ROCK are localized to and interact with myosin light chain phosphatase (MLCP) is not well understood. We recently found a new member of the myosin phosphatase complex, myosin phosphatase-rho interacting protein, that directly binds to both RhoA and the myosin-binding subunit of myosin phosphatase in vitro, and targets myosin phosphatase to the actinomyosin contractile filament in smooth muscle cells. Because myosin phosphatase-rho interacting protein binds both RhoA and MLCP, we investigated whether myosin phosphatase-rho interacting protein was required for RhoA/ROCK-mediated myosin phosphatase regulation. Myosin phosphatase-rho interacting protein silencing prevented LPA-mediated myosin-binding subunit phosphorylation, and inhibition of myosin phosphatase activity. Myosin phosphatase-rho interacting protein did not regulate the activation of RhoA or ROCK in vascular smooth muscle cells. Silencing of M-RIP lead to loss of stress fiber-associated RhoA, suggesting that myosin phosphatase-rho interacting protein is a scaffold linking RhoA to regulate myosin phosphatase at the stress fiber.     

Non-Muscle Myosin II Regulates Neuronal Actin Dynamics by Interacting with Guanine Nucleotide Exchange Factors

Background

Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family.

Principal Findings

NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II–GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II–GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation.

Conclusion

Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF–Rho GTPase signaling pathway.     

Correlation of conformation and phosphorylation and dephosphorylation of smooth muscle myosin

The rate of phosphorylation and dephosphorylation of smooth muscle myosin by myosin light chain kinase and by two myosin light chain phosphatases (gizzard phosphatase IV and aorta phosphatase) are measured in various conditions; the relationship between the rate of phosphorylation and dephosphorylation of myosin and the myosin conformation is also studied. The rate of dephosphorylation of myosin was completely inhibited in the presence of 1 mM MgCl2 and ATP at low ionic strength where phosphorylated myosin forms a folded conformation. The inhibition was released when myosin formed either an extended monomer or filaments. The rate of phosphorylation of myosin was also affected by the conformation of myosin. The rate for a folded myosin was slower than those for an extended monomer and filamentous myosin. The phosphorylation and dephosphorylation of heavy meromyosin, subfragment-1, and the isolated 20,000-dalton light chain are not inhibited at low ionic strength, and the rate of phosphorylation and dephosphorylation was decreased with increasing ionic strength. KCl dependence of the rate of phosphorylation and dephosphorylation of myosin was normalized by using KCl dependence of subfragment-1, and it was found that the marked inhibition of the rate of phosphorylation and dephosphorylation of myosin is closely related to the change from an extended to a folded conformation of myosin.